Handbook on Metalloproteins

’~ a g n e fResonance j~ Center University of Florence 1-50019 Sesto Fio~entino,Italy ‘institute of inorganic Chemist...

Author: Ivano Bertini | Astrid Sigel

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’~ a g n e fResonance j~ Center University of Florence

1-50019 Sesto Fio~entino,Italy

‘institute of inorganic Chemistry University of Basel CH-4056 Basel, Swit~erland

M A R C E L

MARCELDEKKER, INC. D E K K E R

-

NEWYORK BASEL

-3 The figure on the dustcover corresponds to the right-hand part of Figure 1 of Chapter 21 by Elena Babini and Maria Silvia Viexzoli. It shows the cx domain (C terminal) of human ~d~-me~allothionein. This book is printed on acid-free paper. ers Marcel Dekker Inc. 270 Madison Avenue, New York, IVY 10016 tel: 212-696-9000; fax: 212-685-4540

ion

aste

utgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-261-8482; fax: 4 1 - 6 1 ~ 2 6 1 - ~ ~ 9 6

or1 h t t p : / / m . dekker.com ublisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special SalesProfessional Marketing at the headquarters address above.

Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 10 9 8 7 6 5 4 3 2 1

In the 1950s, inorganic chemistry and coordination chemistry experienced a pronounced revival which left its marks on our current understanding of the ties of metal ions and their complexes. At about the same time, bioinorganic chemistry emerged and soon a mutual stimulation took place. The possibility to carry out crystal structure analyses, first of low-molecular-weight complexes and then of large molecules like proteins, created much excitement among the researchers in this area and the advent of NMR, allowing such structural determinations in solution, further fostered a rapid development which has led to a host of information collected in the Protein Data Bank. The increasing number of sequenced genomes-the first genome of a multiceltis was completed in 1998-makes available the lular organism ( ~ a e n o r ~ a b d ~elegans) proteins of those living organisms whose genomes are known and this allows studies not thought of before, The coordination spheres of metal ions in proteins can now be probed in subtle ways, eg., by substituting single amino acids. Therefore, we felt that it i s the appropriate time to summarize much of the information that has accumu~ ~ e t a l l o p r o ~ e i nwith s the aim of providing a lated and to prepare a H a n d ~ o oon significant tool for the development of biological inorganic chemistry and of also giving insights to those not in the field. This idea was met with enthusiasm by the cont~butingauthors and the publisher as well-for which we, the editors, are grateful. o o k of 23 chapters written by 43 experts from all over the The ~ ~ ~ d b consists world in close collaboration with the editors, With the exception of the introductory chapter and the final one, which gives a perspective to future research, the core 21 chapters are organized in a similar way, emphasizing the structure of the proteins and the coordination spheres of the metal ions as well as the corresponding structurefunction relationships, This analogous organization should help researchers from other fields such as general coordination Chemistry, biotechnology, biophysics, medicine or pharmacology to easily find access and hopefully answers to their questions. The Handbook focuses on the metals of life, ie., Na, K, Mg, Ca, V, Cr, Cu, Zn, Mo, and , and their roles in proteins, but metal ions like Rb+, Sr2+,Cd2 ', Pb", La", Sm'", or No:'+ are also considered since they are used as substitution probes in studies of metal ion-protein interactions.

iii

IV

PREFACE

It is the hope of the editors and authors that not only will this Haizdbook be useful due to Lhe wealth of information it provides but that it will also contribute to the advancement of' biotechnology and biological research in general and stimulate further studies in the exciting and rapidly expanding field o f metal ion-protein interactions at a time when bioinorganie chemistry will certainly benefit from genornic sequencing.

Astrid Sigel Helmut Sigel

ts

...

1ll

PREFACE CONTRI~UrrO~S

mi

Titles of Related Interest: xxv ~ D ~ O ON O TOXICITY K OF I N O R G ~ I CCOMPOUNDS ~ ~ ~ ON O METALS O K IN CLINICAL AND ANALYTICAL CHE XXV METAL IONS IN ~ I O L O ~ I C ASYSTEMS L (list of volumes) COLOR F I ~ ~ ~ S following page xxx r l AND USE OF THE ~ ~ O O K Iuano Bertini, Astrid Sigel, and Helmut Sigel 1. Scope o f the Handbook 2. Organization of the Handbook 3. Some Web Sites for Further Information 4. General Comments and Outlook References

ter I N T E ~ C T I O NOF SODIUM AND POTASSIUM WITH PROTEINS Todd M. Lnrsen and George H. Reed 1. Introduction 1.1. Bioinorganic Chemistry o f Naf and K' 1.2. Coordination Chemistry of Na' and K^' 2. EnzymesiFroteins with Known Structure 3. EnzymesiProteins with Unknown Structure 4. Structure-Function Relationships 4.1. Dialkylglycine Decarboxylase 4.2. Pyruvate Kinase 4.3. Diol Dehydratase 4.4. Hsc70 4.5. Class I1 Fructose-1,B-Bisphosphate Aldolase 4.6. ~ r u ~ t o s e - ~ , 6 - ~ i s p h o ~ p h a t a s e 4.7. Carbamoyl Phosphate Synthetase V

10 10 10 11 15 15 15 16 19 20 22 23 23

vi

CONTENTS

24 24 25 26 28 28 29 30 30 31 32 32 32

4.8. Cytochrome P450cam 4.9. S-AdenosylmethionineSynthetase 4.10. Tryptophan Synthase olzpz Complex 4.11. Tryptophanase 4.12. Tyrosine Phenol-Lyase 4.13. Ascorbate Peroxidase 4.14. ~ e t h i o n i n eArninopeptidase 4.15. Thrombin 4.16. a-Amylase 5. Perspectives Acknowl~~e~ts Abbreviations and Definitions References

N OF S

~

D

I

~

1. ~ntroduction 1.1. Ion Channels: Definition and Role in Excitable o Voltage-Gated Ion Channels Work? d Sodium Channel Proteins with Known Primary S t ~ c t u r e 2. 2.1. Potassium Channels 2.2. Sodium Channels e s Proteins and Protein omains Resolved to Dale 3. ~ t ~ c t of~Channel 3.1. The Pore of a Bacterial Potassium Channel an and Sodium vation Domains of ~ a ~ m a l i Potassium 3.2.

40 40 40 41 42 44 45 45

5. Perspectives ~ c ~ o w l ~tsd ~ e n reviations and Definitions rences

47 48 48 50 53 53 54 54

ACTIVATE^ ENZYME S Y S T E ~ S d Louis T. J. Delbaere 1. Introduction 1.1. Chemistry of M ~ e s i u m 1.2. ~ a ~ e s i u m - L i g a n Chemistry d 1.3. ~etabolismof Magnesium within

60 60 61 62

4. Structure-Function Relationships 4.1. Selectivity and Permeation 4.2. Gating: Activation and Inactivation

vii

CONTE~TS

Known Structures in Magnesium-Activated Enzyme Systems 2.1. Bi-MgZ+-Bound Structures 2.2. Kinases $+-Induced Conformational Changes in Proteins 3. Unknown Structures 3.1. ~ a ~ e s i u ~ - P r o ~ o p o rIX p hChelatase ~in 3.2. Sphingomyelinase 3.3. Nycobacterial GDP-Mannose Pyrophosphorylase 3.4. N1-(5'-Phos ibosyl)adenosine-5'-monophosphate Cyclohydrolase 4. Structure-Functio 4.1. Kinases 4.2. DNA Polymerases 4.3. p21"" 4.4. Ribozymes 4.5. Isocitmte Dehydrogenase 4.6. Xylvse Isomerase 5. Conclusions Acknowledgments Abbreviations References

2

AND rrs Andreas Muranyi and Bryan E. Finn 1. Introduction 1.1. Aims and Scope 1.2. Coordination Chemistry of Ca" 1.3. Bioinorganic Role of C&'+ 1.4. stasis and Metabolism 1.5. ution of Ca2+-BindingProteins 1.6. Introduction to ms Chosen for Discussion 2. E n ~ ~ m e s ~ ~ r ~ wt e i n s 2.1. EF-Hand Proteins 2.2. Annexins 2.3. @2 Domains 2.4. EGF-Like Modules 2.5. Lectins 3, EnzymesProteins with Unknown Structure 4. Structure-Function Relationships 5. Perspectiges 6. Ca" and Protein-Related Internet Resources bbreviations and Definitions eferences ~~~~~~

64 71 71 72 72 72 73 73 73 74 74 77

78 79 80 81

82 82 82

84

93 94 94 95 95 95 97 99 200 100 115 121 128 133 139 140 140 141 141 142

Chapter 6 VANADIUM IN PROTEINS AND ENZYMES 3153 Alison Butler, J u y m N. Curter, and Matthew T.Simpson 1. lntroduction 154 1.1. Coordination Chemistry of Vanadium 154 1.2. Bioinorganic Role of Vanadium 155 2. Vanadium Enzymes with Known Structure: Vanadium Haloperoxidases 155 2.1. Vanadium Chloroperoxidase 157 2.2. Vanadium Bromoperoxidase 165 3. Vanadium Enzymes of Unknown Structure: Vanadium Nitrogenase 167 3.1. Occurrence and Biological Significance 167 3.2. Structural Considerations and Reactivity 168 4. Stmcture-Function Relationships 169 4.1. Vanadium Haloperoxidase Expression Systems 169 4.2. Comparative Aspects of the Vanadium Sites in V-3rPQ and V-C1PQ 170 4.3. Mechanistic Considerations of the Catalytic Cycle 172 5. Perspectives 173 Acknowledgments 174 Abbreviations and Definitions 174 References 175 ter 7

ARE THERE PROTEINS CONTAINING CHROMIUM?

281

R. Bruce Martin 1. Introduction 2. EnzymesProteins with Known Structure 3. Enzymes/Proteins with Unknown Structure 4. Perspectives Abbreviations References

181 185 186 187 188 188

~ h ~8 p t ~ MANGANESE-CONTA.INING ENZYMES AND PROTEINS David 6. Weatherburn 1. Introduction 1.1. Coordination Chemistry of Manganese 1.2. Manganese as an Oxidizinfleducing Agent 1.3. Bioinorganic Role of Manganese 1.4. Homeostasis and Metabolism 2. EnzymesProteins with Known Structure 2.1. Oxidoreductases 2.2. Transferases

193 196

196 197 198 198 201 201 207 212

CONT~~TS

2.4. Lyases 2.5. Isomerases 2.6. Ligases 2.7. Proteins Containing ound Manganese 3. Manganese Enzymes with Unknown Structure 3.1. O~idoreductases 3.2. Transferases 3.3. Hydrolases 3.4, Lyases 3.5, Isomerases 3.6, Ligases 3.7. Proteins Containing 4. Structure- unction Relatio escription of the Coordination Sphere of Manganese in Proteins escription of Reaction ~ e c h ~ n i s ~ s 5 . Perspectives and Outlook Acknowledgments Abbreviations References

EME AND R E ~ T E Paola Turano and Yi Lu 1. Introduction 1.1. Coordination 1.2. Biosynthesis 1.3. Bioinorganic Role 2. Enzymes and Proteins iiown Structure 2.1. Gytochromes 2.2. Globins 2.3. Nitrophorin 2.4. Heme-Based 2.5. Catalases 2.6. Peroxidases 2.7. Cytochrome P450 2.8. Nitric Oxide Synthase 2.9. Hydroxylamine Oxidoreductase 2.10. Nitrite Reductase 2.11. Bacterioferritins 2.12. Heme Oxygenase 3. Enzymes/Proteins with Unknown Structure 3.1. Guanylyl Gyclase 3.2. Cystathionine P-Synthase

ix

217 219 224 227 230 231 236 237 239 239 240 240 240 24 1 241 243 244 244 246

271 271 279 285 286 286 297 300 301 303 304 313 315 316 317 320 320 322 322 323

X

3.3. Indoleam~ne2,S-Dioxygenase and Tryptophan 2,3-Dioxygenase 4. S t ~ c t ~ e ~ F u n ~Relationships tion 4.1. Expression Systems 4.2. Detailed S t ~ c t ~ e - ~ n cRelatio~ships t~on erspectives and Outlook 5.1. Why Heme‘? Evolutionary Aspects 5.2. An Outlook A~~owled~ents A ~ ~ r e ~ a ts i o n Re€erences

ter 1 - S ~ PROTEINS ~ ~ ~ R tlef Bentrop, Fraancesco Capozzi, and Clnudio Luc~inat 1. ~ n t r o d u c t i o ~ 1.1. Coordination Chemistry of Iron in Iron-Sulfur Proteins 1.2. Overview of Consensus Sequences and Structural Classification 1.3. Bioinorganie Roles of Clusters th Known Structures Other Proteins with ulfur Clusters 2.3. 2Fe-2S Ferredoxins 2.4. Ferredoxins with Fe3S4and/or Fe4S4Clusters . High Potential Iron . Aconitase and Iron 2.7. Sif.oheme-Contaiiii~~ Proteins 2.8. Nitrogenme Iron Protein e4S4Cluster-Containiiig 2.10. Glutamine Phosphoribosylpyro.osphate hidotransferase 2.11. ~ ~ m e t h y l a m i n Dehydrogenase c ’ or “Meatball” Cluste

.14. Pyruvate:Ferredoxin Oxidoreductase

se and Related S edoxin:Thioredoxin Reducta 3.5, The SoxR Protein 4. ~ t ~ c ~ ~ Relationships ~ . ~ ? - F ~ n ~ ~ ~ ~ ~ The Role of the Cluster and of the rotein Moieties in Electron 4.1. Transfer by Iron-Sulfur Proteins

323 324 324 326 338 338 339 340 341 342

3 359 359 362 365 372

372 375 379 381 387 389 391 393 396 399 40 1 402 406 4-08

410 410 412 413 414 415 416 416

xi

CONTENTS

4.2. Fe3S41F’e4S4Interconversion 4.3. Fe4S4/Fe2S4Conversions 4.4. Fe-Only Hydrogenases: The H Cluster le of the Cluster in Folding and Stability of Proteins 5. Perspectives Aspects 5.1. Evolu ns 5.2. Open Acknowledgments Abbreviations and Definitions References

- F ~ N C T ~OFO NO^^ ~

421 424 425 428 429 429 442 445 446 447

EON COO

Par Nordlund 1. ~ n t r o d u c t i o ~ 1.1. Iron Homeosta i.2. Iron and Noiih 2. Iron-Qxygen/Nitrogen 2.1. Structural and stie Studies o f Iron-O~ygen/~itrogen Proteins 3. Structure and Mechanisms of Iron-Oxyg~n~~itrogen Proteins 3.1. Lipoxygenases 3.2. Tntradiol Dioxy 3.3. Pterin-Depen ases and Related E n z ~ ~ ~ ~

genases Containing Rieske Centers

4. An Emerging View of the S t ~ c ~ u r e - F u n c ~ Relationship ~on of Iron-Q~gen/NitrogenProteins 4.1. Effects of the Coordination Environment 4.2. Effects of Net Charge and Charge Distributions 4.3. Conformationa~F~exibilityand the Control of O2 4.4. Geometries of the Activated O2 Species Ackno~~ed~ents A ~ b r e ~ a t i and o~s References

463 463 465 468 470 471 471 478 484 s491 502 511 516 19 541 544 547 550 552 554 555 556 557 558 559

xii

AMD TRANSPORT PROTEINS Fubio Arnesan,o and Alessandro Provenzani 1. Introduction roteins with Known Structure 2.1. .@'erritins: Occurrence and Biologicdl Role 2.2. Ferritins: Structural Classification 2.3. Structures of Ferritins 2.4. Transfemins: Occurrence and Biological Role 2.5. Transferrins: Structural Classification 2.6. Structures of Transferrins 3. Proteins with Unknown Structure 3.1. The Iron Pathway 4. Structure-Function Relationships 4.1. Expression Systems 4.2. Ferritins: Structure-Function Relationships 4.3. Transferrins: Stru cture-Function Relationships 5. Perspectives Acknowledgments Abbreviations and Definitions eferences

VITAMIN Biz AND ITS ENZYMES John M. Pratt 1. Introduction 1.1. Oulline of Dates, Structures, and Reactions 1.2. Aims, Scope, and Organization of the Review 1.3. Nonienclature of Corrinoids 1.4. Basic Coordination Chemistry: Oxidation States, cis and trans Effects 1.5. Why Cobalt? 1.6. Why Corrin? 2. Enzymes with Known Structure: BY2-DependentMutases 2.1. Reactions Catalyzed by Mutases (Isomerases1 2.2. Available Structures of Mutases 2.3. Major Structural Features: Domains, Modular Construction, and Evolution 2.4. Structural Features Relevant to the Nlutasc eaction Mechanism 2.5. Summary of Main Points 3. ~ ~ ~ -~ n~z y ~~ e ns ~ rdo ~with i e i~nUnknown s~ ~ t ~ c t u ~ e ~ 3.1. Distribution and Biosynthesis 3.2. Absorption, Transport, and Transformation of Cobalt Corrinoids

572 5 73 573 573 575 578 578 579 584 584 585 585 586 590 593 593 594 594

603 605 605 610 610 611 613 615 618 6 19 622

623 627 630 631 631 632

xiii

ependeiit Methyltransfer~ses Known or Possible B12-Dependent Enzymatic Reactions 4. Models, Mechanisms, and Structure-Function Relationships of the Mutases 4.1. Fission of the CO-CBond: Mechanism for Applying Steric Distortion 4.2. The Co-Radical Charge-Transfer Complex 4.3. Rearrangement of the Substrate-Derived Radical 4. Summary of Main Points 5.1. Blz-Dependent tases: Precis of Present Results 5.2. Blz in the Remote Past: Insights into Evolution 5.3, Blz in the Near Future: Problems and Prospects Acknowledgments Abbreviations References

634 636 637 638 646 647

652 653 654 657 659 660 660 662

14 Stefuno Ciii,rEi and Stefaito Mangani 1. Introduction nic Role of Nickel s with Known Structure 2.1. Usease 2.2. Hydrogenase 2.3. MethyI-Coenzyme M 3. EnzymeslProteins with Unknown Structure 3.1. Carbon Monoxide ~ehydrogenase/Acetyl-CoenzymeA Synthase 3.2. Nickel Superoxide Dismutase 3.3. Nickel Chaperonins 4. Structure-Function Relationships 4.1. Urease 4.2. ~ N ~ ~ e 1 " H y ~ r ~ g e n a s e 4.3. Methyl-Coenzyme M Reductase 4.4. Carbon Monoxide e h ~ ~ r o g e n a s e l ~ c e t y l - C o eAn zSynthase ~e 4.5. Superoxide Dismutnse 5. Future Perspectives Abbreviations References

670 670 671 671 675 680 683 683 685 687 689 689 690 693 699 699 699 700 700

xiv

QTEINS IN THE TRANSPORT AND ACTNATION OF D I O ~ G EAND ~ , THE R ~ D U ~ T I OOF N I N Q ~ G MOL~CULES ~ I ~ Malcolm A. Hakrow, Peter F. Knowles, and Simon,E. V. Phillips 1. Introduction 1-1. Coordination Chemistry of Copper 1.2. Bioinorganic Role of Copper omeostasis ttnd MetaboIism 2. EnzpesProteins with Known Structure 2.1. Galactose Oxidase 2.2. Amine Oxidases 2.3. Peptidylglycine a-Hydroxylating Monooxygenase 2.4. Hexnocyanin 2,5. Catechol Oxidase 2.6. Cytoehrome c Oxidase 3. Structural Features of Copper Oxidative Enzymes of Unknown 3D Structure 3.1. Lysyl Oxidase 3.2. Dopamine P-Monooxygenase 3.3. Tyrosinase 3.4. Methane Monooxygenase and Ammonia Monooxygenme 3.5. Nitrous Oxide Reductase 4. $ t r u c ~ r e - ~ c ~ Relationships ion 4.1. Catalytic Mechanisms 4.2. Structurefinetion Comparisons Between Enzymes 5. P~rsp~ctives and Outlook eviations and Definitions

7

710 710 711 713 715 720 721 723 725 726 727 730 730 730 731 732 733

734 734 746 749 751 751

DASES 1. Introduction 1.1. Spectroscopic Classification of Copper Ions 1.2. The Cupredoxin Fold 1.3. The Overall Architecture of the Multi-Copper Oxidase Family own Structures in the Multi-Copper Oxidase Family 2.1. Ascorbate Oxidase 2.2. Laccase 3. Unknown Structures: Coagulation F 3.1. Roles of Factors V and VIII in

v and VIrI Coagulation

764 765 767 767 771

771 772 774 776 779 779

CO~T~NTS

3.2, Overall Configuration of Factor V and VIII Molecules 3.3. A Model for Factor VIII Based on Human Ceruloplasrnin 4. Structure-Function Relationships 4.1. The Copper Binding Sites 4.2. Organic Substrate Binding Sites in Ascorbate Oxidase and Laccase 4.3. Mechanism of Oxygen Reduction by the Trinuclear Copper Center 4.4. h i d e Inhibition in Ascorbate Qxidase and Human Ceruloplasrnin 4.5. Ceruloplasrnin and Ferroxidase Activity 4.6. Binding of Organic Substrates to Human Ceruloplasmin 4.7. Ceruloplasrnin and 5. Perspectives 5.1. Evolutionary Aspects of the Multi-Copper Oxidase Family 5.2. Open Questions Acknowledgments Abbreviations and Definitions References

xv 779 780 780 780

187 788 791 194 797 800 801 801 805 806 806 807

c c

17 IN ~ L E C T R O N ~ T Alejandro J. Vila and Claudio 0. Femandm 1. Introduction 2. Proteins with Known Structure 2.1. The ~ p r e d o x i nFold 2.2. Blue Copper Proteins 2.3. Blue Oxidases 2.4. Nitrite Reductase lear Cu, Site nknown Stmcture 3.1.. Blue Copper Proteins 3.2. Blue Qxidases 3.3. N 2 0 Reductase 4. Structure-Function Relationships 4.1. Description of the Coordination Sphere of the Metal Ions 4.2. Spectroscopic Studies 4.3. Mutagenesis Studies 4.4. Redox Potentials 4.5. Electron Transfer Mechani~ms 5. Perspectives Acknowledgments breviations and ferences

814 816 816 818 830 831 832 833 833 834 835 835 835 836 838 839 840 a41 842 842 843

xvi

OF VARIOUS FUNCTIONS CONTAINING COPPER Peter li: Lindley 1. Introduction 2. Known X-Ray Structures: Albumin and Copper,Zinc Superoxide Dismutase 2.1. Serum Albumin uperoxide Disrnutase tructures: Metallothioneins and Menkes’ Copper-Transporting ATPases 3.1. Metdothioneins 3.2. Menkes’ Cu-Transporting ATPases 4. Structure-Function Relationships 4.1. Copper Binding Sites in Serum Albumin 4.2. Structure-Function Relationships in Superoxide Dismutase 4.3. Coppcr Binding Sites in Metallothioneins 4.4. Copper Binding Site in the Menkes’ Cu-Transporting ATPase 5, Perspectives: The Copper Chaperones Acknowledgments Abbreviations and Definitions eferences

858 858 858 860 860 860 862 862 862 863 872 873 894 874 875 877

IN ~ ~ AND ~ ~ 1 , PA~~T Q~ T~ E ~ ~ ~~ 1 David S. Auld 882 1. Introduction 882 1.1, A Growing Awareness of Zinc in Biology 883 1.2. Zinc Chemistry: Ideal for its Varied Functions 884 2. Classification of Zinc Sites in Metalloenzymes and 885 2.1. Catalytic Zinc Sites 891 2.2. StrucLural Zinc Sites 893 2.3. Cocatalytic Zinc Sites 902 2.4. Protein Interface Zinc Sites 907 3. Tracking Zinc Enzymes through Their Putative Zinc Binding Sites 907 3.1. Determination of the Number of Zinc Enzymes 3.2. Zinc Binding Site Motifs as a Means fbr the 909 New Zinc Families 915 3.3. Mutagenesis as a Means of Detecting New Zinc Binding Sites 917 4. Structure-Function Relationships 917 4.1. Inactivation of Metalloenz-me Catalysis 924 4.2. Effect of Scaffolding on Catalytic Activity 931 4.3. Mechanistic Studies of Catalytic Zinc Sites 937 4.4. Mechanistic Studies of Cocatalytic Zinc Sites

CONTENTS

5. Concluding Remarks Abbreviations and Definitions eferences

ZINC! FINGER PROTEI Gert E. Folkers, Hiroyok

nzuwa, and Rolf Boelens 1, Introduction 1.1. Historical Perspectives 1.2. Definition o f Zinc Finger Domains 1.3. Focus of this Chapter 2. Zinc Finger Proteins with Known Structure 2.1. Zinc Finger ornains Containing One Zinc 2.2. Double-Zinc 23. ~embrane- in ding Double-Zinc-Finger Domains 3. Zinc Fingers with Unknown Structure 4. Structure-Function 4.1. ~ v o ~ u t i o n a ~ 5. Perspectives Abbrevkations eferences

OTHER ZINC INS: INS: M E ~ A L L O T ~ I O N E AND I ~ S INSULIN Elena Babini and Maria Siluia Viezzoli 1. Introduction 2. Proteins with Known Structure 2.1. ~ e ~ ~ l o t h i o ~ Occurrence eins: and Biological Role 2.2. ~etallothioneins:Structural Classification 2.3. Structure of an, Rabbit, Rat, and Mouse Metallot~i~neins 2.4. Structure of Grab Metallothionein 2.5. Structure of Purple Sea Urchin Metallothionein 2.6. Insulin: Occurrence and Biological Role 2.7. Insulin: Stiwtural Classification 2.8. Insulin: Structure 3. Proteins with Unknown Structure 3.1. The Human Neuronal Growth Inhibitory Factor (~~tallothionein-3) 3.2. Phytochelatins 3.3. Cod Insulin 3.4. Expression Systems 4. Structure-Func~ionRelationships 4.1. Metallothioneins

xvii

941 942 943

61 962 962 962 963 964 964 976 981 982 985 985 989 990 992

1001 1002 1002 1002 1003 1003 1006 1007 1007 1008 1009 1012 1012 1013 1013 1013 1014 1014

4.2. Insulin: Relationship between Confomational Transitions and iological Function 5. Perspectives 5.1. Metallothioneins 5.2. Insulin A b b r e ~ a t i o ~and s Definitions References

D PROTEINS C O N T ~ ~ I NMO G N C. ~ a v Garner, i ~ Russell Banhmrn, Serenu, J. Cooper, E. S t e p ~ e nDavies, and Lisa J. Stewart 1. ~ ~ t r o d u c t i o n 1.1. Perspective 1.2. factors 1.3. Molybdenum vs. Tungsten 1.4. ~oordinationChemistry of ~olybdenumand Tungsten Relevant to

3. Enzymes with Unknown Structure 4. ~ t r u c t u r e ~ ~ u n c tRelationships ion xpression Systems n Sphere of the Metal Ions

5. ~~breviation~ Ref~rence~

1015 1016 1016 1016 1016 1017

1024 1024 1025 1029 1030 1034 1034 1035 1038 1059 1063 1069 1069 1070 1071 1072 1072 1072 1074

1092 1092 1094 1098 1098 1099

2.3. Lessons for Enzymic Catalysis ocus on the Metal: Metal Specificity -Ligand Covalency and the Use of 4s Orbitals 3.1. Co(I1I) ILigands and Coulombic/Solvation 3.2. Fe(II) i3.3. Fe(I1) I- 02:Acceptor Ligands 3.4. Fe(I1) + CO:?? 3.5. Usage = Reactivity x Availability: Cr, Go, Cu 4. Conclusions Abb~eviati~ns References

S

1100 1103 1104 1106 1107 1108 1111 1113 1114

inside back inside back cover

This Page Intentionally Left Blank

S

Numbers in parentheses indicate the pages on which the authors’ contributions begin. Magnetic Resonance Center, University of Florence, Via L. Sacconi iorentino, Italy (<[email protected]>j (5711 Center for Biochemistry and Biophysical Sciences and Medicine and Department of Pathology, arvard Medical School, S. 6.Mudd Building, Room 123~4, Boston, MA 02115, USA ()(881)

mi Food Science and Technology Laboratory, University of Ravennate 1020, 1-47023 Cesena, Italy () (I

School of Chemistry, University of Nottingham, ~ o t t i n NG7 g ~ ~ ~ ussel 2RD, UK (1023) Department o f Physiology 11, University of Tubingen, -72074 Tiibingen, Germany ( c de~lef.bentrop~uni~tuebingen.de >) (357) agnetic Resonance Center, University of Florence, Via L. Sacconi 6 , rentino, Italy (FAX: +39-055-4574271; ) (1)

partment of NMR Spectroscopy, Bijvoet Center for Biomolecula~ University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands (FAX: +31-30-2537623; ) (961) er Department of Chemistry, University of California, Santa 10, USA (FAX: 4-1-805-893-4120; j(

~ e p ~ t r n e of n tChemistry, University of Galabria, Via Pietro Bucci, C15, 1-87036 Reiide (Cosenza), Italy (357) ~~~~

& ~ Department ~ ~ r o f Chemistry, University of‘ California, Santa 06-9510, USA (153)

i Department of ~ o ” E n v i r o n m ~ n t Science al and Technology, University of Bologna, Wale Bcrti Pichat 10, 1-40127 Bologna, Italy ([email protected] >) (669)

xxi

School of Chemistry, University of Nottingham, Nottingham, )

School of Chemistry, University of Nottingham, Nottingham, NG7 2RD, TJK (1023) Department of Biochemistry, University of Saskatchewan, 107 on, SK S7N 5E5, Canada (FAX: +1-306-966-4390; ) (59) er Department of Physiology 11, University of Tubingen, Ob dem Himmelreich 7, D-72074 "iibingen, Germany (FAX: +49-107147815; ) (39) k d e z LANAIS, RMN-F (CONICET - University of Buenos Aires), Junin 956, 61113 AAD Buenos Aires, Argentina (813)

Division of Physical Chemistry 2, Chemical Centre, Lund Uhiversity, 124, S-22 100 Lund, Sweden (FAX: i-46-46-222-4543; ) (93)

rs Department of NMR Spectroscopy, Bij et Center for Biomolecular cht University, Padualaan 8, NL-3584 C Utrrecht, The Netherlands (961)

School of Chemistry, University of Nottingham, Nottingham, 2467 : +44-115-951-3563; )(1023)

c

~ School o ~ of Chemistiy, University of Leeds, Leeds, LS2 9JT, UK

(<m ~ c o l m h ~ ~ c h e ~ i s t ~ . l e ~ d s (709) ,ac~uk>)

a Department of NMR Spectroscopy, Bijvoet Center for search, Utrecht University, Padualaan 8, NL-3684 CH Utrecht, The Netherlands (961)

wles Department of Biochemistry and Molecular Biology, University of

,LS2 9JT, UK () (709) Institute for Enzyme Research and Department of Biochemistry, s o n , WI 53705, USA (9) University of ~ i s c ~ ~ i n - ~ a d iMadison, European Synchrotron Facility (ESRF), BP 220, F-38043 Grenoble [email protected]>l (763, 857) of Chemistry, MC-712, University of Ellinois at UrbanaChampaign, A322 Chemical and Life Science Building, Box 8-6, 600 S. Matthews Ave., Urbana, IL 61801, USA (FAX: + 1-217-333-3685 ) (269)

Magnetic Resonance Center, University of Florence, Via L. Sacccmi 6,I-50019 Sesto Fiorentino, Italy (FAX: +39-055-2757555; >(357)

xxii i i Department of Chemistry, University of Siena, Via Aldo Moro, (<[email protected]>) (669)

Department of Chemistry, University of Virginia, ~ c ~ o r m ~ c Road, Charlottesvillc, VA 22903, USA () (181)

atte Department of Biochemistry, University of Saskatchewan, 107 5E5, Canada () (59) ision of Physical Chcmistry 2, Chemical Centre, Lund University, P.O. Box 124, 5-22100 Lund, Sweden (93) Department of iochernistry, Stockholm University, Svante , S-10691 StOCklhOl , Sweden (FAX: i-46-8-153679; <par(l?biokemi.s~i.ser)(461) rtment o f Biochemistry and Molecular 9sT, UK (709)

tt Department of Chemistry, Imperial College of Science, techno lo^ and Medicine, South Kensington, London SW7 ZAY, UK. Mail address: Mew Pond GU3 lIN, UK (<j.pratt@su~ey.ac.uk>) Farm, Mew Pond Road, ~ o m p t o nSurrey, , (603, 1091) sonance Center, University of' Florence, Via L. Saceoni 6, I-50019 Sesto Fiorentino, Italy (<[email protected]>) (571)

ess

Institute for Enzyme Research and Department of Biochemistry, University of Wiscon adison, Madison, WI 53705, USA ( ~ r e e d ~ x t a ~ l . e n z y m e . w i s c . e(9) du~)

1 Institute of Inorganic Chemistry, University of Basel, ~ ~ i t a l s t r a s51, se asel, Switzerland (FAX: i-41-61-267-1017; ) (1) 1 Institute of Inorganic Chemistry, University of Basel, Spitalstrasse 51, CH-4056 Basel, Switzerland (FAX: +4 1-61-267-1017; chelmut [email protected]>} t 1) Department of Chemistry, University of California, Santa , USA (153) of Chemistry, University of Nottingham, M o t t i ~ g hNGI ~,

2RD, UK (1023) an0 Magnetic Resonance Center and Department of Chemistry, Florence, Via Luigi Sacconi 6, 1-50019 Sesto Fiorentino, Florence, Italy (FA%: +39-055-4209253; )(269)

e z ~ Magnetic ~ 1 ~ Resonance Center, University of Florence, Via L. Sacconi 6,I-50019 Sesto Fiorentino, Italy () (1001) "Present uddress: Department of Biochemistry and Oxford Centre for Molecular Sciences, University of Oxford, South Farks Road, Oxford OX1 3QT, UK

xxiv

CQ~T~IB~TO

iophysics Section, University of Rosario, Suipacha 531, ntina ( (813)

>

erburnt School of Chemistry and hysical Sciences Victoria University of Wellington, P. 0. Box 600, Wellington, New Zealand (cd a ~ i ~ . w ~ a ~ h e r b u r n ~ ~>w) .(193) ac.nz

arcel Dekker, Inc. publications are of interest for any reader dealing in one way or another with metals: edited by Hans G. Seiler and Helmut SigeE, with Aslrid Sigel In 74 chapters, written bjr 84 intc?rnationalauthorities, this book covers the physiology, toxicity, and levels of tolerance, including prescriptions for detoxification, for all elements of the Periodic Table (up to atomic number 103). The book also contains short summary sections for each element, dealing with the distribution of the elements, their chemistry, technoloL4cal uses, and ecotoxicity as well as their analytical chemistry.

M edited by Hans 6. Sezler, Astrid Sigel, and Helmat Sigel This book is written by 80 international authorities and covers over 3500 references. The first part (15 chapters) focuses on sample treatment, quality control, etc., and on the detailed description o f the analytical procedures relevant for clinical chemistry. The second part (43 chapters) is devoted to a total of 61 metals and metalloids; all these contributions are identically organized covering the clinical relevance and analytical determination of each element as well as, in short summary sections, its chernistry, distribution, and technical uses. (list of volumes) edited by Astrid Sigel and Helnzut Sigel Volume 1. ~

~ COMPLEXES" M P ~

Volume 2. ~~~~~-~~~~~

~

C O ~ P L ~ X ~ S ~

~ O L ~ ~ U ~ OL ~~ ~RL ~ X E S ' ~ Volume 4:METAL IONS AS PROBES" Volume 5 . ~EACTIVITYOF ~ ~ O ~ D I ~~ OA ~~ PI O ~ N ~ ~ * IOLOGICAL A ~ ~ OF I METAL ~ N IONS" *Out of print

xxv

xxvi

Volume 7. IRON IN MODEL AND NATU

COMPOUND^*

CLEOTIDES AND D E R ~ A T I ~ THEIR S: LIGATING BIVALENCY Volume 9. AMINO ACIDS AND ~ ~ ~ V ~ r AS I VBIVALENT E S LIGANBS Volume 10. C~CINOGENICITYAND METAL IONS Volume 11. METAL COWLEXES AS ANTICANCE~AGENTS" Volume 12. ~ R O ~ E R T I XOF S COPPER Volume 13. COPPER PROTEINS" Volume 14. I N O R G ~ I CDRUGS IN DEFICIENCY AND DISXASX Volume 15. ZINC AND ITS ROLE IN BIOLOGY AND NUTRITION Volume 16. METHO S I ~ O L METAL ~ N IONS ~ AND C O ~ P L E X IN~ ~ CLINICAL CHEMISTRY Volume 17. C A L C I AND ~ ITS ROLE IN B I O L O G ~ Volume 18. C I ~ ~ U ~ T IOF O METALS N IN THE E N V I ~ O N ~ E N ~ Volume 19. ANTIBIOTICS AND THEIR C O M P L E ~ ~ Volume 20. CONCEPTS ON METAL, ION ' r O X ~ C ~ T ~ Volume 21.

IONS OF' NUCLEAR ~ G N E T I C NETIC SPECIES

Volume 22. ~ N D EPR, O ~ AND ~ ELECTRON SPIN ECHO COO~DINATIONSPHERES Volume 23. N I C ~ AND E ~ ITS ROLE IN BIOLOGY Volume 24. ~ U ~ I N UAND M ITS ROLE IN ~ I ~ L ~ G Y Volume 25. I N T ~ ~ R E ~ T I OAMONG NS METAL IONS, ~ N ~ EAND S ,GENE E~R~SSION Volume 26. ~ ~ ~ ~ ~ ~ONT~ S GX ~U M ~ ANTS S ITS I ROLE U ~ IN B I ~ L O ~ Y , NUTRITION, AND P ~ S I O L O ~ Y

Volume 27. ELECTRON TRANSFER REACTIONS IN M ~ T ~ ~ L O P R O ~ E I N ~ Volume 28. D ~ G ~ ~ A T IOF O EN~ I R O N ~ E N T AP LO L L U T ~ T SBY MIC~OORGAN~SMS AND THE^^ ~ E T A L L O E N ~ ~ S

Volume 29. ~ I O L ~ ~ I CPROPERTIES AL OF METAL ALMYL B E R ~ A ~ I ~ Volume 30. M ~ T A L L O E N Z ~ EI S ~ O L AMINO ~ N A~C I ~ - R E S I D UAND ~ RELATED RADICALS

"Out of print

xxvii

TITLES OF RELATED I N T ~ R ~ S T

S ROLE FOR LIFE

Volume 31. VANA

METAL IONS 'WITH NUCLEOTIDES, N ~ G ~ E I G ~ONSTITUENTS Volume 33. PRQBING OF NUGLEIC ACIDS BY METAL ION COMPLE L MQLECULES Volume 34. M E ~ G AN ~ R ITS ~ E ~ ~ E ON C ~E N S ~R~NMENT

IOLOGY

STOMGE IN ~ I C R O Q R G A ~ I S ~ S , EN FREE RADICALS AND ME IN LIFE PROCESSES Volume 37. ~

G

~

E AND S ITS E ROLE IN BIOLOGICAL PROCESSES

Volume 38. ~ R O B I ~OF G ROTEI INS BY METAL IONS AND THEIR LO ~ O L E C U ~ R - ~ I GCOMPLEXES HT Volume 39. M Q L ~ D E AND N ~ TUNGSTEN: THEIR ROLES IN BIO~OGIC PRO~ESSES(in preparation) Volume 40. THE: LANT THEIR I ~ T E R R E ~ T I O N S SYSTEMS (in preparation)

BIO-

e lit

rtini

Magnetic Resonance Center, University of Florence, Via L. Sacconi 6, 1-50019 Sesto Fiorentino, Italy

Institute of Inorganic Chemistry, University o f Basel, spitalstrasse 51, asel, Switzerland

1. ~

~ OF ~THE P

E

1.

ith the advent of the SO-c e pa& 40 yews, the signi e x interplay with pro I1 1. Several texibaoks have now become available on t nized that metal ions have a double role in the physiol i n ~ s p e ~ s afor b l normal ~ life [71, whereas most are t o 3, i.e., they adversely &ect the act toxic ones, such as

2

BERTINI, A. SIGEL AND

H. SlGEL

The properties of metal ions depend strongly on the ligating atoms present in their coordination sphere and, to a lesser extent, on the polarity of the medium, hydrophilic or hydrophobic, in which the complex resides. Previously, mostly low molecular weight complexes were studied, but the rapid progress in physico-chemical methodology, especially X-ray crystallography [231 and nuclear magnetic resonance spectroscopy C241, has allowed investigators to deal with high molecular weight complexes and thus to determine the structure and coordination sphere of metal ions in proteins in great detail. The sequencing of genomes now makes available to the scientific community all proteins of a living organism. One of the resulting targets is that of acquiring the structures of these proteins, which can then be compared among living organisms and among communities within the same living species. The next step comprises studies of their functions with regard to small molecules in catalytic processes and their interactions with other proteins or with DNA and

RNA. Many proteins need metal ions to function. For this reason, bioinorganic chemistry has a growing role in postgenomic research and, as far as the hnctioning of metalloproteins is concerned, understanding of their coordination chemistry is compulsory. Vwing to the considerable amount of information that has already been accumulated on the structure of metalloproteins, we feel that the time is appropriate for this Handbook on Metalloproteins, which was developed in collaboration with competent experts from all over the world and which stresses the importance of the molecular structure to our understanding of the function at the molecular level. However, to some extent, enzymes with unknown structures and functions, different from those with known structures, are also covered. The above reasonings prompted us to focus on metalloproteins and metalloenzymes from the point of view of their structures as deposited in the Protein Data Bank [251. Usually, for each class of enzymes representative examples are discussed in terms of structure-function relationship with an emphasis on mechanism, as far as possible. To facilitate an overview, the alkali and alkaline earth ions as well as the transition elements, of which proteins are described in detail in this Handbook, are shown in bold letters in the simplified periodic table given in Fig. 1. Of course, they correspond to the metal ions known to be essential for life [a]. Interestingly, several of these are now also employed in artificial peptidases and proteases C261. In addition, the symbols of those metal ions that are mentioned in the text but not covered by chapters in this book, because they are not the natural metals, are inserted in light print in Fig. 1; the information about these metal ions is best accessible via the Subject Index. This book is intended for advanced students and scientists in the field of bioinorganic chemistry, inorganic chemistry, coordination chemistry, biochemistry, biophysics, clinical chemistry, medicine, pharmacology, etc.; in other words, for people working in different areas and with different backgrounds. For this reason the contributors have made a special effort to write the chapters in such a way that

SCOPE AND USE OF TH

3

a

n=6 n=7

b

PIG. 1. Simplified version of the periodic table showing in bold type those metal ions of which rnetalloproteins and metalloeiizyrnes are covered in detail in this Handbook. The metals shown in light print are also mentioned in the Handbook; the corresponding text parts are best found via the Subject Index.

they may be generally understood and to define all terms and abbreviations that may not be in general use.

2. Chapters 2-22 are organized, as far as possible, in the same way to facilitate the use of the Handbook. In general the contents include the following sections: 1. Introduction 2. EnzymesProteins with Known Structure 3. EnzymesiProteins with Unknown Structure 4. Structui-+Function Relationships 5. Perspectives Abbreviations and Definitions (where appropriate) References In the Introduction. to the various chapters, the authors have attempted to summarize those coordination chemistry properties of the given metal ion that are relevant to its bioinorganic or biological role. Where appropriate and known, a few remarks regarding homeostasis and metabolism of the metal ion are included. The other headings given above are self-explanatory. Of course, these sections are subdivided by the authors in such a way as to organize the available information on the given topic in an optimal way. Chapters 2 to 5 are devoted to the four essential main-group metal ions; sodium, potassium, magnesium, and calcium. Chapters 6-22 deal with the transition metal

TINI. A.

4

I AM

EL

ions and their proteins. Here it was partly necessary to subdivide the material a v d able for a given metal ion, e.g., iron in hemes, iron-sulfur proteins, etc., to break down the lmge amount of existing data. In the concluding chapter, the author attempts to identify “emerging themes and patterns among metalloproteins” by using several metal ions and their proteins as examples. ndbooh terminates with a detailed Subject Index, and at the very end two tables are given: One lisb the one- and three-letter symbols for amino acids and the other provides the standard genetic code, i.e., the nucleotides that define a certain amino acid. The information in these two tables was collected from several textbooks 127-291.

TE

N

The inte~estedreader is referred to: ank (PDB): http://www.rcsb.or~/pdb/ 1. 2.

.ac.Wpromise or 3,

ctural Classification of Proteins ( S ~ ~ ~ ) :

5. 4%

7.

s ~ c a t ~ of o nprotein domains using ho~ologoussuperfa~ilies,topology

earch and retrieval system (I3

//m, mips.biochem.mpg.de/

10.

11.

~ t t p : / / ~ ~ a r t . eheidelberg.de/ m~l-

dbook, the various metal ions and thei ue to the nature of this ever, it is evident that in a b plexes appear in d i ~ e r e n t niic organization, i.e., the everything is interlinked turnover of metal ions and other metabolic substances. As a conse~uence,interdependencies between the various compounds occurring in metabolic processes exist, and this is also true for metal ions. This is demonstrated in Fig. 2, where some ~nterdependenc~es between essential and toxic elements regarding their toxicity are given 1301. The last ~ e n t i o n e din ications imply immediately that in the cell the traffic of transition metal ions, such as copper or iron must be regulated to mainta~nthe concentration necessary for biological function while avoiding toxic ex [31,321. Indeed, there are meta~ochaperones,i.e., transport proteins that protect metal ions within the cell, delivering them safely to the appropri receptor E331. For example, one such chaperone delivers copper to superox tase, a ~ o p ~ e r -enzyme ~ ~ n c [34-36], and as shown recently, this chaperone is active at very low copper conccntrations and directly inserts the metal ion into the enzyme [37] (see also E381). In fact, id was concluded that the upper limit o f so-called “free” copper is less than a single metal ion per cell [31,371 (owing to the high binding constant and the small volume of a cell). Unlil now it was a comm~nbelief that metal ions such as copper are in equilimay be rather brium with metallogroteins 1311; now it appears that the “~quilib~ia” between proteins, and it remains to be seen if this is applicable to other physiologically relevant transition metal ions as well. f course, there is considerable interest in general in the factors controlling the ~ l i y s i o ~ o ~concentrations ~cal of metal ions. Indeed, proteins have been ~scoveredthat facilitate the transport of ~ ~ ~ g a n e s 132,391, iron [32,40421, and zinc, as well as copper across the plasma m ~ ~ ~

FIG. 2. ~nte~de~endencies among 30 elements in mammals. An arrow from element A to B indicates that administration of element A may reduce toxicity due to element B, low levels of element A may heighten the toxicity of element B, or high levels of element salutary eEects of element A. ( ~ e p ~ o d u c eby d permission of R. Bruce Martin and Marcel Dekker, Inc. from Ref. 30.)

6

~ ~ R T I NA.I ,SIGEL AND H. SIGEL

E321; a bacterial metallochaperone that delivers nickel to the enzyme urease has also been reported 1433. Certainly, one of the future research trends will be t o unravel the interplay between DNA and the regulation of the expression of moleculeslproteins that are involved in met& ion uptake and release. The comments given above should make it clear that despite the vast amount of knowledge now available on metalloproteins, much of which is accumulated in this book, there is an extraordinarily bright future for further research on metalloproteins and ~etalloenzymes.It is the hope of the authors and editors that this Handbook will “catalyze” the advent of exciting new results that evidently are to be expected in this b i ~ i n o r g ~field. ic

R 1. A. Sigd and R. Sigel (eds.), Metal Ions in Biological Systems, New York; the first volume appeared in 1973 (Vol. 2). Since t have been published (see Fief. 261, and re are in preparat~on. 2. J. J. R. Frdusto da Silva and R. J. P. lliams, The Biological Chemistry of the Elements, Glarendon Press, Oxford, 1991, pp. 1-561. 3 . W. Kaim and €3. Schwederski, Bioanorganische Chemie, Teubner, Stuttgart (Germany), 1991, pp. 1-450. English version: Bioinorganic Chemistry, Wiley, Chichester (U.K.), 1994. 4. ertini, II. B. Gray, S. J. Lippard, and J. S. Valentine (eds.), Bioirtorganic istry, University Science Books, Mill Valley, CA, 1994, pp. 1-611. 5. S. J. Lippard and J. M. Berg, Principles of Bioinorgan,ic Chemistry, iversity Science Books, Mill Valley, CA, 1994, German version: anorganische Chemie, Spektrum Akad. Verlag, Berlin (Germany), 1995, pp. 1-429. 6. (a) R. J. P. Williams and J. J. R. Frausto da Silva, The Natural Selection of the Chemical Elements, Glarendon Press, Oxford, 1996, pp. 1-646. (b) R. J. P. Williams and J. J. R. Frausto da Silva, Bringing Chemistry to Life. From tler to Man, Oxford University Press, Oxford, 1999, pp. 1-548. 6, Seiler, A. Sigel, and H. Sigel (eds.), ~ a n d ~ on o o ~tals in Clinical and 7. Analytical Chemistv, Marcel Defier, New York, 1994, 8. H. G. Seiler, R, Sigel, and A. Sigel (eds.), ~ a n d on ~ Toxicity o ~ ~ of Inorganic mpounds, Marcel Dekker, New York, 1988, pp. 1-7 069. Sigel and A. Sigel (eds.), Compendium on Magnwium and 9. Logy, Nutrition, and Physiology, Vol. 26 of Systems, Marcel Dekkcr, New York, 1990, pp. 1-744. 10. H. Sigel and A. Sigel (eds.), Calcium, clnd Its Role in Biology, Vol. 17 oEMeta1 Tons RioZoc&al Svstems. Marcel Dekker, New York, 1984, pp. . .. in -.. .... - - 1-532. c1 u

11. I-I. Sigel and A. Sigel (eds.), Vanadium and Its Role in Life, Vol. 31 of Metal Ions in Biological Systems, Marcel Dekker, Ncw York, 1995, pp. 1-779. 12. A. Sigel and a.Sigel (eds.), Manganese and Its Role in Biological Processes, Vol. 37 of Metal Ions i n Biological Systems, Marcel Dekker, New York, 2000, pp. 1-761. . R. Crichton, Inorganic Biochemistry of Iron Melabolism, Ellis Horwood, 13. Ghichester (U.M.), 1991, pp. 1-263. 14. A. Sigel and H. Sigel (eds.), Iron Transport and Storage i n Microorgan,isms, Plants, and Animals, Vol. 35 of Metal Ions in Biological Systems, Marcel Dekker, New Uork, 1998, pp. 1-775. 15. B. Kr$iutler, D. Arigoni, and B. T. Golding (eds.), Vitamin B I Z and B I Z Proteins, Wiley-VCH, Weinheim, 1998, pp. 1-542. 16. H. Sigel and A. Sigel (eds.), Nickel and Its Role in Biology, Vol. 23 of Metal Ions i n Biological Systems, Marcel Dekker, New York, 1988, pp. 1-488. 17. H. Sigel and A. Sigel (eds.), Copper Proleins, Val. 13 of Metal Ions in Biological Systems, Marcel Dekker, New York, 1981, pp. 1-394. 18. N. Sigel and A. Sigel (eds.), Zinc and Its Role i n Biology and Nutrition, Vol. 15 of Metal lons in Biological Systems, Marcel Dekker, New York, 1983, pp. 1493. 19. I. Bcrtini, C.Lucliinat, W. Maret, and M. Zeppezauer (eds.), Zinc Enqymes, Birkhauser, Boston, 1986, pp. 1-640. 20. H. Sigel and A. Sigel (eds.), Concepts on Metal Toxicity, Vol. 20 of in Biological Systems, Marcel Dekker, New Uork, 1986, pp. 1-386. 21. H. Sigel and A. Sigel (eds.), Aluminum and Its Role in Biology, Vol. 24 o€ Metal Ions in Biological Systems, Marcel Dekker, New York, 1988, pp. 1-424. 22. A. Sigel and H. Sigel (eds.), Mercury and Its Effects on Environmen,t and Biology, Vol. 34 of Metal dons in Biological Systems, Marcel Dekker, New York, 1997, pp. 1-604. 23. H. M. Berman, J. Westbrook, 2. Feng, G. Gilliland, T. N. Bhat, I. N. Shindydov, and P. E. Bourne, “The Protein Data Bank”, Res., 28, 235-242 (2000). 24. I. Bertini and C. Luchinat, “NMR of Paramagnetic Substances”, Coord. Chem. Rev., 250, 1-292 (1996). 25. (a) h t t p : / / w ~ . r c s b . o r ~ p d b(b) ; H. Weissig and P. E. Bourne, “An Analysis of the Protein Data ank in Search of Temporal and Global Trends”, Bioinformatics, 15, 8 0 7 4 3 1 (1999); (c) see also Ref. 23. 26. A. Sigel and €3. Sigel (eds.), Probing of Proteinu by Metal Ions and Their LowMolecular- Weight Complexes, Vol. 38 of Metal dons in Biological Systems, Marcel Dekker, New York, 2001, pp. 1-690. 27. L. Stryer, Riochemie, Spektrum Akademischer Verlag, Heidelberg, 1991 (last page of the book). English version: Biochemistry, W. H. Freeman, New York, 1988.

8 28.

29. 30. 31. 32. 33,

34. 35. 36.

37. 38.

39,

BERTINI, A. SIGEL AND H. SIGEL

. Ram, ~ i v c h e ~ ~N.s Patterson t~, Publ., Burlington, NC,

1989 (second inside the dust cover). . Voet and J. 6 . Voet, Biochemistry, 2nd ed. John Wiley and Sons, New ork, 1995 (first page inside the dust cover). R. B. Martin, Bioinorganic Chernisiry of Toxicity, Chapter 2 in Ref. 8, p. 9, S. J. Lippard, Science, 284, 748-749 (1999). D. Radisky and J. Kaplan, J. Biol. Chem., 274, 4481-4484 (1999). (a>R. A. Pufahl, C. P. Singer, K. L. Peariso, S.4. Lin, P, J. Schmidt, C. Pahrni, V. 6. Culotta, J. E. Penner-Hahn, and T. V. O’Halloran, Science, 2 853-856 (1997). (b) T. V. O’Halloran and V. C. Culotta, J. BioE. Chem., 275, 25057-25060 (2000). V. C. Culotta, L. W. J. Klomp, J. Strai . L, B. Casareno, B. Krems, and J. . Gitlin, J. Biol. Chern., 272, 23469-2 . L. B. Casareno, D. Waggoner, and B i d . Chem., 27*3,2362523628 (1998). J. D. Rothstein, M. Dykes-Hoberg, L. B. Corson, M.Becker, I?. W. Cleveland, . E. Price, V, C. Culotta, and P. C. Wong, J. ~ e ~ r o c 72, ~ e422429 ~ . ~ 999). T. D. Rae, P. J. Schmidt, R. A. Pufahl, V. C , Culotta, and T. V. O’IlaIloran, kkience, 284, 805-808 (1999). T. J. Lyons, E. B. Gralla, and J. 23. ~ a l e ~ t i n e“Biologicd , Chemistry of C o ~ p e ~ - Superoxide ~i~c Dismutase and Its Link to ~ y o t r o p Lateral ~i~ 8cIerosis)’in A, &gel and H. &gel (ds.), Interrelations between Free Radicals tal Ions in B i o l o ~ i c aSystems, ~ 1 Ions in Life Processes, Vol, 36 of kker, New Yark, 1999, pp. 125-17 roorganisms, Chapter 2 of nganese Transpart in

S, A. hieong and G. W i ~ ~ o l ~e c z ~~Biology l a ~ l of ~Iron ~Trunsport ~ in~ ~ ~ n Chapter ~ i ,4 of Ref. 14, pp. 147-186. R. Byers and J. E. L. Arcenearn, Microbial 41. quisition by Pathogenic ~ ~ c ~ o ~ g aC n~ ~a s~2~~ se , r rial. Iron Transp raun, K. Hantke, and W. Koster, 42. netics, and ~ e ~ ~ l a tChapter ~on,3o J. Colpas, T. G. 43. 4 0 ~ 8 (299 ~ ~ g ~

40,

et

SSi

Institute for Enzyme Research and Department of Biochemistry, The University 01Wisconsin-Madison,

1.1. Bioinorganic Chemistry of NaC and K' 1.2. Coordination Chemistry of Na+ and K+ E ~ ~ ~ O T WITH E ~ N S

STRUCTURE

~~~

10 10 10 11

15 4. S T ~ U C T ~ ~ E - F U N C T I O ~E N~ T I O N S H I P S 4.1. ~ialkylglycineDecarboxylase 4.2. Pymvate Kinast? 4.3. Diol Dehydratase se70 4.4. 4.5. Class I1 ~~ctose-1,6-bisphosphate Adolase 4.6. Fruetose- 1,6-Bisphos~hat ase 4.7. hosphate Synthetase 4.8. Cytochrorne P450cam 4.9. ~ - ~ d ~ n o s y l rn e t h i o nSynthetase ine 4.10. T ~ t ~ p Synthase ~ a n cqJ3z Complex 4.11. Tryptophanase 4.12. Tyrosine Phenol-Lyase 4.13. Ascorbate Peroxidase 4.14. Methionine ~ i n o p e p t i ~ a s e 9

15 15 16 19 20 22 23 23 24 24 25 26 28 28 29

LARSEM AND REED

10

4.15. Thrombin 4.16. a-Amylase

30 30

5. ~ E ~ S P E C T I ~ ~

31 32

~ ~ ~ ~ ~ A T I Q M S

32

32

1. I 1.I.

~ i o j ~ o ~Chemistry ~ a n i ~ of Na' and K'

Ions of sodium and potassium represent the cationic electrolytes of living organisms. Of the two ions, K+ has the greater role in biology 111. The Cytosol in cells from animals, plants, and bacteria contains high concenti-ations of K+-typically 2 0.1 M. Plants require potassium for growth, and potassium is one of the three major inorganic ingredients of commercial fertilizers. On the other hand, plants and most microbes exhibit little or no requirement for sodium. However, sodium does have important roles in animals, where it is usually found in highest concentrations in extracellular fluids. In animals, an ATP-dependent extrusion or pumping of Na' from cells is coupled to the inward movement of K+. A substantial amount of ATP utilization by animal cells is devoted to operation of the Na"/'K-' ATPases, which are transmembrane proteins that are responsible €or this ion pumping I2,3]. At the same time, the gradient of [Na'l created by the active pumping facilitates the inward movement of sugars and amino acids by sodium-dependent cotransporters 141. The active pumping of K" into and Na' out of cells occurs against a hack-diffusion of the ions down their concentration gradients. The structure and function of channel proteins that control diffusion of the ions across biological membranes is a topic in Chapter 3 of this voluine. The roles of K' and Na+ as electrolytes in numerous physiological processes are of critical importance in many aspects of human health and, therefore, medicine [51. One expects NaC and K' to bind nonspecifically to proteins, nucleic acids, negatively charged head groups of phospholipids, and anionic metabolihes. However, in addition t o the Na'/K' ATPases and other membrane proteins involved in ion transport, various other enzymes and proteins exhibit specific requirements for K+ or Ma+ [61. This chapter deals with stmctural and functional aspects of the specific interactions of Naf and K+ with proteins.

1.2. ~oordi~ation Chemistry of Na' and

'M

Both ions have inert gas cores-Na+ {Ne} and K+ {Ar}-and present a single oxidation state, i-1,in their solution chemistries. The ions are hard Lewis acids, but they

are not appreciably electrophilic because of their unit positive charge and consequent low chargeiradlius ratio. The unit positive charge of the sodium and potassium ions can, however, attain greater potency in regions of low dielectric constant. Interactions ofthe ions with ligands are largely ionic and weak. Hydration numbers of the ions in solution are largely indeterminate, although there is some evidence that four water molecules compose the first hydration spheres. In crystalline solids, the coordination geometry and coordination numbers are variable between 4 and 12. The capacity of K+ and Na’ to bind simultaneously to four or more ligand groups and their concentrations in biological fluids gives them advantages over protons-another important species of monovalent cation. Selective complexing agents, such as the crown ethers and the naturally occurring ionophores, exploit chelation and differences in the size of the ions to achieve respectable affinities and selectivity. These same factors are no doubt used by proteins in providing binding sites for the ions. The ionic radii of the ions, determined from high-resolution crystallographic data [coordination number in { }: Kf (4) 1.51 A; (6) 1.52 A; ( 8 ) 1.65 A; {10)1.73 A; {12}1.78A; Na+ (4) 1.13 A; (6) 1.16 A; ( 8 ) 1.32A; (12) 1.53 A] 171, show that the radii inflate with increasing coordination number as expected from steric considerations. These values can be combined with those of common ligands [0(-2) 1.32 A; N(-3) 1.7’1A1 to predict the “regular” metal ligand distances. The fact that Na+ and K+ serve as essential cofactors of several enzymes [61 appears to be somewhat out o f character with the seemingly prosaic nature of the coordination chemistry of the ions. This apparent paradox has stimulated considerable curiosity regarding the roles played by these ions in the catalytic cycles of enzymes since the early discovery of a K’ requirement by pyruvate kinase I81. The success of recent X-ray crystallographic determinations of the positions of K+ and Na’ in complexes with proteins provides some clues to the specificity of the proteins for their monovalent cofactors and to the function of these inorganic cations in assisting in the biological activity of their host proteins.

WN STRUCTURE The Protein Data Bank (PDB) presenLly contains more than 30 coordinate files of 16 different proteins whose activities are influenced by monovalent cations and wherein K’ or Na’ have been assigned in the corresponding electron density maps. These proteins and PDB file names are listed in Table 1.In most instances, the functional requirements of the proteins for monovalent cations had been identified previously through traditional biochemical assays. These functional rcquirements typically cxhibit specificity in the sense that saturation of the binding site with a different species of monovalent cation results in significantly reduced activity. For some of the proteins, structures obtained with Na+ and K+ reveal differences that provide additional clues to the function and specificity of the proteins for their respective activator. For other proteins, the biochemical assays are more sensitive than structural methods in detect-

12

i

I

+)

2

a+)

Pa49

+ site 1 a+ site 1 a+ site 2

+

la511 laqf

+

INTERACTION OF Na AND K WITH PROTEINS

2

T

a N u ~in~~ r~ e ~ t ~is ethe s ~ @ ~ s bof etherrespective cation in the structure. bNumber in parentheses is the number of ligands present in the ex. file was not released when this c ~ ~was~ prepa t e ~ dThe ~ ~ r e s ~ ~~in ~ the e s PD~ @ ~ ~ g

L A ~ S AND ~ N BEE0

14

Enzymes Requiring Na’ or K+ That Do Not Rave Structures or Where the Monovalent Binding Site Has Not Been Assigned in the Structure Enzyme and EC number ( ~ ) - ~ i n o p r o p a ndehydrogenase ol 1.1.1.75 6-Phosphofructokinase 2.7.1.11 Acetate kinase 2.7.2.1 Acetate-CoA ligase 6.2.1.1 Adenosylomethionine cyclotransferase 2.5.1.4 Aldehyde dehydrogenase 1.2.1.5 AMP deaminase 3.5.4.6 Aspartate kinase 2.7.2.4 ATPase 3.6.3.17 Carbamate kinase 2.7.2.2 D-Alanine ligase 6.3.2.4 Dihydro~olatereductase 1.5.1.3 Formiminotetrahydrofolate cyclodeaminase 4.3.1.4 Forinyl~etra~iy drofo~at e synthctase 6.3.4.3 Fosfomycin resistance protein (FosA) Glutathione synthase 6.3.2.3 Glycerol dehydratase 4.2.1.30 Glycerol dehydrogenase 1.1.1.6 Homoserine dehydrogenase 1.1.1.13 Inosine monophosphate dehydrogenase 1.1.1.205 Ketohexokinase 2.7.1.3 Leucyl-tRNA ligase 6.1.1.4 L-Serine dehydratase 4.2.1.13 Malate dehydrogenase 1.1.1.38 Methylamine dehydrogenase Methylaspartate ammonia lyase 4.3.1.2 Methylenetetrahydrofolatedehydrogenase (NADP+) 1.5.1.5 synthase 6.3.5.1 Pantothenate synthetase 6.3.2.1 Phosphate acetyltransferase 2.3.1.8 Phosphodiesterase 1 3.1.4.1 Phosphoribosyl~inoimida~olec~boxa~ine fomyltransferase 2.1.2.3 Phosphor~bosylformy~~lycina~idase synthase 6.3.5.3 Porphobilinogen synthase 4.2.1.24 +-

Ref.

I N T ~ ~ A ~ OF T I Na O AND ~ K WITH PROTEINS

15

TABLE 2 (Continued) __

Enzyme and EC number Propionyl-CoA carboxylase 6.4.1.3 Protein kinase 2.7.1.37 (brain) Pyruvatk phosphate dikinase 2.7.9.1 Tartrate dehydrogenase 1.1.1.93 (Mono) Threonine deanzinase 4.2.1.16 Tyrosyl-tRNA synthetase 6.1.1.1 y- Glutamylcysteine synthetase 6.3.2.2

ing differences between complexes of Na+ and K'. All of these structures provide insight into the natural strategies for exploiting monovalent cations in achieving a partidax structure in a complicated heteropolymer.

S / ~ ~ O TWITH ~ ~ NU NSK OWN STWUGTU Many other enzymes are activated by or Na+. Table 2 provides a list of some of the enzymes in this category. Structures are available for some of the proteins listed in Table 2, but the monovalent cations have not yet been identified in the electron density maps. The Na"/I";+ ATPase is perhaps the most prominent member of this class. This ATPase is a trans-membrane protein for which there is a large body of biochemical and physiological data available. This information is sum~arizedin reviews and monographs [1-5,9-111.

Dialkylglycine decarboxylase (EC 4.1.1.16) belongs to the a family of PLP-dependent enzymes and, like several other PLP-dependent enzymes, is activated by K+ and inhibited by Na+ or Li+ [12,131. The catalytic cycle of the enzyme includes both a decarboxylation and a transamination reaction. For example, decarboxylation of amethylalanine yields acetone, 6 0 2 , and transfer of the amino group to pyruvate, producing L-alanine. The enzyme from Pseudom'onas cepacza exists as a tetramer, built up as a dimer of dimers [13,141.Each monomer consists of a large domain, which contains the PLP binding site, and a small domain. The catalytic site is located in a cleft near the

~ R S E AND N REED

16

interface between monomers of the dimeric unit, Residues from both subunits of the dimer participate in forming the active site. The 2.1-A resolution structure reveals the presence o f two sites for monovalent cations in each subunit, One site is close to the catalytic site, and this site shows selectivityfor K" .A second site, which is remote from the active site, prefers Na+ over K'. Two structures have been solved (Fig. 1):(1)Ktin site 1and Na" in site 2; ( 2 ) Naf in both sites. At site 1,K" coordinates to five protein ligands and a water, with an average metal-to-ligand distance of 2.7 A [143. The structure with Naf at site 1 shows three proteins ligands and two waters average met~-to-~gand distance of 2.3 A [141. Comparison of the two complexes reveals changes in the vicinity of site 1when Na' replaces Kf. These changes propagate to the active site and cause rearrangements in the positions of two residuesSerBO and Tyr301. A water molecule in the Na' complex replaces the hydroxyl oxygen of Ser80-a ligand to K+. This rearrangement changes the position of Tyr301, a residue in the active site. Sodium at site 2 binds at the C terminus of an a-helix where five ligands from the protein and a water compose the coordination sphere.

yruvate Kinase Pyruvate kinase (EC 2.7.1.40) catalyzes the final step in glycolysis-conversion of phos~hoenolp~v and a ~ADP to pyruvate and ATP. A proton is taken up in the reaction. Both substrates are trianions at neutral pH, and the inorganic cofactor r e q ~ r e ~ of~ the n t enzyme i s 2 Mj$+ and K'. Inclusion of the proton in charge balance reveals that the inorganic cofactors balance the charges on the substrates arrd products. Early on, assays of pyruvate kinase uncovered a requirement for K" in the assay cocktail-the first report of a specific requirement for K' in an enzymic reaction CSI. The 2.9-k resolution structure of pyruvate b a s e from rabbit muscle in its complex with Mnz+ and pyruvate revealed an electron density that was assigned to K' [lSl. In this complex, K" coordinates to oxygens of the side chain of N74, S76, and D112, and to the main-chain carbonyl oxygen of T113. A subsequent 2.7-Aresolution structure (Fig. 2, top) of the complex with L-phosphoIactate, an analog of phosphoenolp~vate,revealed that an oxygen from the phosphate group of the analog bound directly t o K+ 1161. A direct interaction between K+ and the y phosphate of ATP was present in the 2.1-A resolution structure of the complex of the enzyme with ATP and oxalate (Fig. 2, bottom) /171. Ml three inorganic cations coordinate to the y phosphate of ATP. The structure of the -oxalate complex with Na" was i n d ~ s t i n ~ i s ~from b l e that with K+. Activity of Na+ in the reaction is about 9% of that of K+,and the lack of structural diflerences is not too surprising given the appreciable activity of Na' in the reaction. A small number of bacterial pyruvate kinases lack the requirement for activation by K+ 118-223. Whereas the activity of pyruvate kinase from rabbit muscle is stimulat~ by 1.5 x lo3 by optimal concentrations of IC+ [291, the activities of these few bacterial pyruvate kinases are not influenced by monovalent cations. The

I N T E ~ A C T I ~OF N Na AND K WITH

17

FIG. 1. Stereo views of the coordination o f K' (top) and Na+ (bottom) to dialkylglycine decarboxylase. The figure was prepared from PDB files lDKA and 1DKB. The program MolScript [I261 was used to prepare this figure and Figs. 2 and 4 to 12.

18

LARSEN AND REED

L-P-lactiltit

1

PIG. 2. Stereo views of'the coordination of K' in the active site of pyruvate kinase. The top view is from the complex of the enzyme with Mg2+ and 1,-phospholactate,and the bottom view is from the complex of the enzyme with Mg'I-ATP and M$'-oxalate. The figure was prepared from PDB files la49 and laqf. See Figure 2.2 in the color insert.

INTE~ACTIONOF Na AND

K WITH PROTEINS

19

sequences of these proteins, however, reveal that the binding site for K+ is conserved except for substitution of Leu for Thr at position 113 [151. The main-chain carbonyl of T113 is the ligand for K’, so the Thr-to-Leu substitution at position 113 would appear to be conservative. The other conspicuous change in the sequences of the K+-independent enzymes was a Lys for Glu switch at position 117. The structure of the complex of pymvate kinase and pryuvate had revealed that the side chain of 6 1 ~ 1 1 7was near the binding site of Kt [15]. The E117K variant of rabbit muscle pyruvate kinase was constructed to test the hypothesis that the E ammonium from a Lys at position 117 could provide an “internal” monovalent cation, thereby eliminating the need for binding of an exogenous cation [231. Activity of E117K pyruvate kinase (12% of w.t.1 was independent of monovalent cations in the assay cocktail. The monovalent cation in pyruvate kinase interacts directly with the migrating phosphoryl group [16,171. An indirect interaction with other important active site residues, e.g., Arg72, also occurs through hydrogen bonding between the K+ ligand, D112, and the guanidinium moiety of Arg72. Arg72, in turn, interacts with the polyphosphate moiety of ATP. The direct interaction of K+ with substrates and, indirectly, with residues in the active site account for the dependence of the activity on K+.

Diol dehydratase (EC 4.2.1.28) is a bacterial enzyme that catalyzes an adenosylcobalamin- and K+-dependent dehydration of 1,2-diols to the corresponding aldehydes C241. In the catalytic cycle, the 5’-deoxyadenosyl radical, created by homolysis of the Co-C bond of the cofactor, abstracts a hydrogen atom from 6-1 of the substrate to generate a substrate radical intermediate. The radical rearranges to a product radical with transfer of the hydroxyl at C-2 to C-1 and the radical center to 6-2. The product radical acquires a hydrogen atom from the $-methyl of 5’-deoxyadenosiiie, leaving the hydrated aldehyde. Shibata et al. 1251 solved the three-dimensional structure of the complex of diol dehydratase in its complex with the cofactor analog, cyanocobalan-iin, 1,2-propanediol,and K+ /251. Diol dehydratase exists as a dimer of heterotrimers (aPy>z. The rr. subunit contains a (P/c& barrel motif. Cyanocobalamin x ) ~ motif, and 1,2-propanediolbinds in binds at the C terminal end of the ( ~ I c barrel ~ Potassium chelates to the vicinyl hydroxyls of the substrate (Fig. the ( P l z ) barrel. 3 ) . The protein contributes five ligands to K+, creating a seven-coordinate site without water ligands. The region of the active site containing the substrate and K+ is not solvent-accessible. Direct coordination of K+ to the substrate hydroxyls reveals the role of K” in substrate binding and in positioning the substrate within the active site cavity. This binding scheme also provides some space between the reactive radical intermediates and amino acid residues within the active site, perhaps protecting the protein from oxidation. Shibata et al. C251 suggest the possibility that K+ participates directly in the migration of the hydroxyl group of C-2 to C-1 of the radical form of 1,2-propanediol.

2

4.4. Hsc7 The bovine hcak-shock cognab protein (Hsc70) is a member of the 78-kd heat-shock prokin familys Wsc70 belongs to a dass of ce:Pllu_tarproteins, referred to as mokctdar chapesnes, These proteins bind to misfofded segments o f polypeptides and, in an A4TP-depa&mtpro~ess,releme ~ r segroents pmwicli ~ ng another ~ o p p o ~ u~nity €01-proper folding 126,271. The 7Ise70 protein has three domains: an m i n o teminal 44-kd KFPase domain; an 28-kd pepiide binding domain; and a curbnxylltemind 1O-kd domain. Potassium is required for r%TPhydrolysis and proper re,.ulation of peptide binding and release C28,N. The stm%ureof the ATPase domain has been determined J 30.311. The nucleotide binding site in the ATPase is at the base of a deep deft between the twu lobes ofthe domain [30,311, The structure of the ATPme domain in complex with Mg"ATP, Pi, and K ' (Fig. 4,top) L311 shows that two potassium ions am bound in the nucleotide binding site. One M ' c ~ o ~to ~ the 2-fi b d g e oxygen of ADP, a peripheral oxygen from the p phosphate of ADP, two ligands fx-om Ihe protein, and three water molecules. The second K ' coordinates to ax). oxygen froiri Pi, three ligands from the protein, m d a water molecub. The binding of rJlle potassium ions to 859P and to Pi auggests that these ciiticms play a role in positioning of thc nrrc'leotide [311.The structure of the Hsc70 ATPase fragment in eomplex with ~~~~A~~~P , and Na' (Fig. 5 ) 1301reveals that the coordination ofNa+ and K' are diffkent, and this Werenee likely accounts for the specificity of the reaction for R+. Site-directed mutagenesis of the ATPase &agment of EIsc70 was completed to assem the ability o f the I-:-aminogroup of a nonnative lysine chain to substitute for a ~~~~~~~~

4

FIG. 4. Stereo views showing the coordination of two potassium ions in the ATPase site of Hsc7O (top) and the site-specific mutant sc70 C206K (bottom) in the complex with ADP and phosphate. These views were prepared from PDB files llipm and 1baO.

monovalent cation in the active site ~321.The ~ 2 ~ 6 K - i n t r o d u ~ c-amino ed nitrogen occupied nearly the same position as the site 2 potassium ion (Fig. 4, bottom). The 6ammonium of K206 in the mutant protein was a good structural mimic of the potassium ion bound in the w.t. structure. Nucleotides bind with greatcr affinity to the lysine variant than to the w.t. protein. owever, D206K exhibits less than 5%ATPase

L

22

D2Q6

FIG. 5 . Stereo view of the coo~dinationof two sodium ions in the ATPase site of complex with NIP and phosphate. The view was prepared using PDB file lbup.

activity when compared to the w.t. enzyme. The 5% activity is imp~essive does show that the h y d r o l ~ i cactivity probably depends on subtle st properties that are not adequately mimic~edby the E a m ~ o n i u m

Fructose 1,~-bisphosphatealdolase (FBP aldolase) (EC 4.1.2.13) catalyzes the cleavage of fmctose 1,~-bisphosphateinto dihydroxyactetone phosphate and hyde 3-phosphate in glycolysis and the reverse reaction (conden gluconeogenesis. Class I1 F P aldolases are found in bacteria. These enzymes require Zn2+ and are activate by monovdent cations [33], whereas the class I aldolases of aldolase is a homodirner. higher organisms use LP as a cofactor. The class I1 F The subunits have the common (alP)s barrel motif. The stmcture of the enz was s complex with the transition state analog, phosp~oglycolohydrox~ate, ty in close association 2.0 resolution [341. assigned to the mono ement was performe ith the ~ s u r n p t i o n that the monovalent cation was Na', although the density could also be a mixture of Na' and +,In addition to the phosphate moiety of the i cation binds to four rn -chain carbonyl groups and a water molecule. suggest that the binding of the monovalent cation assists in formation of the necessary environment for binding the phosphate moiety of the inhi~itorand, by analogy, to the phosphate groups of the s ~ b s t r a t eand products.

A

IN

K WITH

s

3

hos 3.1.1.11) catalyzes the hydrolysis of frucand inorganic phosfructose 6-phosphate (F alent cations and a e enzyme requires two ctivity [36-381. FBPase i monovalent cation to a The monovalent cation with a subunit mole ) and inhibits at high concentrations ( in complex with the substrate -P2), and K' or TI+ 11391 rev ositions occupied by e probable that the mono ivation by mono s to Glu280 and from metal site 2. Villeret et al. 11391 suggest that Elf at site 1 is responsi~lefor mono~alentcation inhi~itionof the enzyme. Fructose 196-bisphosph

hos Carbamoyl phosphate synthetase (EC 6 , 3 . ~ . catalyzes ~) the synthesis of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of M$ is the first committed step i e b i o s ~ t h e t i pathways c for the production of arginine and p ~ i m i d i n e assium is required for all reactions catalyzed by the enzyme [41]. The pr an a,P heterodimer consisting of a small amidotrans~erasesubunit (about that is complexed to a large synthetase s ~ b u n i t it, which catalyzes the two phospho~lation ts: the c a r b o ~ h o s p h a t esynthetic component; the oligomerization ; the carbamoyl phosphate synthetic component; and the allosteric d o ~ a i n . carboxy phosphate synthetic component, the physix protein ligands, including a carboxylate Fig. 6 ) 11441. The other carboxylate oxygen - and 3'-hydroxyls of the ribosyl moiety of P. This cation- ind ding pocket is sphate synthetic component also residue number) bridges the potassium to the carboxylate group of a glutamate. There is an additional K+ in the carbamoyl phosmponent. At this latter site, K+ binds to two oxygens from the P

24

FIG. 6. Stereo view of the coordination of K" in Ihe carboxy phosphate synthetase site of carbamoyl phosphate synthetase. The view was prepared from PDB file lJDI3.

.

Cytochrome P450cam

Cytochrome P450's are heme-containing monooxygenases that catalyze the monooxygenation of a wide variety of hydrophobic substrates. Many of these enzymes are associated with membranes. Cytochrome P450cam (EC 1.14.15.1)is, however, a soluble enzyme that catalyzes hydoxylation of the substrate camphor. The topography of P450carn resembles z1 triangular prism [45]. Camphor is buried in an internal pocket. Camphor binds more readily to the active site in the presence o f K', thus providing a connection between K" and substrate 1461. The side chain hydroxyl group o f Tyr96 hydrogen-bonds with the substrate, camphor. A more recent structure of P450cam has defined the cation binding site 1471. Based on temperature factors and an average cation-ligand distance of about 2.7 it was determined that potassium is the cation. Potassium binds to four ligands from the protein, including the carbonyl oxygen of Tyr96, and two water molecules (Fig. 7). The cation appears to have an indirect, structural role, possibly in creating the proper oriention of Tyr96 [471.

A,

4.9.

S-Ademosylmethionine Synthetase

S-~denosylmethioninesynthetase (EC 2.5.1.6) catalyzes the formation of X-adenosylmethionine, pyrophosphate, and phosphate from ATP and t-methionine. The enzyme requires two divalent cations and one monovalent cation per active site for maximal adivity [48].The enzyme is a homotetramer. The two active sites in each dimer are

I N T ~ ~ A ~ T OF ION Na AND K WITH P

25

FIG. 7. Stereo view of the coordination of Kc to the enzyme cytochrome P45Ocarn. The view was prepared from PDB file 5CP4.

located at the interface between subunits. The structure of the enzyme complexed with ADP and P, revealed a K' ion located near the active site 1491. K" binds to the hydroxyl of Ser263 and to both carboxylate oxygens of Glu242. Potassium does not P or with Pi. A second potential site for binding of interact directly with located near the center of the dimer [491.

lex

4.10.

T ~ t o p h a nsynthase a2pz complex (EC 4.2.1.20) catalyzes the last two reactions in a n ono ova lent cation activation has been reported the biosynthesis of L - t ~ ~ o p h [SO]. p h ~ aaf3 for t ~ p t o p h a nsynthase p and for the t ~ ~ t o synthase flsubunit of t ~ ~ t o p h synthase an is a member of the p family The s t r u ~ t u of ~ ethe M . $ ~ c o ~ p l e xreve that the M. subunit is an eightfol~a-pbarrel and that the j3 subunit cQn~ists of two mains (Nand C) [541. The s t ~ c t u r shows e t sabout 25 A from the ac that the active site of the a s ~ b ~lies subunit and that they are connected by a hydrophobic tunnel. A st on the eEects of three m o n ~ v ~ ecations nt revealed that Na', C s', or K" in the B subunit (site 1)about from the phosphate of PLP. ~oordinatio includes three l i g ~ from n ~ th rotein and two water molecules. coordinates to the same three protein ligands and one water mole a ~ the d ~oordin~tion ~ sphere t o six l i g a ~ d sfrom the p~otein. carbony1 oxygens o pS~08are the ligands ~ o n o v ~ ecation nt su

A

26

LARSEN AND REED

FIG. 8. Stereo view ofthe coordination ofK+ to tryptophan synthase. The figure was prepared from PUB file 1TTQ.

does not play a direct role in binding of substrate or cofactor. The structural study 1541 did find that the substitution of K+ or Cs' for Na' produces local changes that affect the interactions between the 01 and P subunits, which in turn alters the conformation of the indole tunnel. An important difference of the three cations is the movement of the side chains of PPhe280 and pTyr279 from a position partially blocking the tunnel in the structure with Na+ t o a position out of the tunnel in the structures with K+ or Cs+ [541.

. Try~tophana~e Tryptophanase (EG 4.1.99.1) is a bacterial P ~ ~ - d e p e n d elyase n t that catalyzes the degradation of L-tryptophan to give indole, pyruvate, and ammonia r551. The enzyme belongs to the a family of PLP-dependent enzymes (561. N Rb' activates the enzyme [5'7], whereas Na+ inhibits it 1581. The structure of the enzyme was solved in the presence of Kf to 2.1-A resolution (Fig. 9, top). The enzyme is a homotetramer. Each subunit has an N-terminal arm and two a@ domains [59]. Pairs of subunits associate to form a catalykic dimer. The K' binding sites are located at the iiitersubunit interface in the catalytic dimer L591. Potassium coordinates to two ligands from subunit 1 of the protein, two Iigands from subunit 2, and three water molecules. Potassium is situated about 8 A from the nearest cofactor atom. The monovalent cation may influence the relative position of the domains in the subunit and may also provide the necessary alignment of catalytic groups at some stage of catalysis 1591.

I N T ~ ~ ~ ~ OF T I Na O N AND K WITH PROTEINS

27

N262

FIG. 9. Stereo views of' the coordination of K+ to the enzyme trygtophanase (top) and of the coordination of Cs+ to the enzyme tyrosine phenol-lyase (bottom). The figure was prepared from PDB files 1AX4 and 'LTPL.

L

N

4.1 Tyrosine phenol-lyase (4.1.99.21, a member of the G, family of ~ L P ~ d ~ p e T ienzymes de~~t to~ give pyruvate, phenol, and an ~ ~ oion. n The i catalyzes the cleavage of L - Q protein is structurally similar to t-ryptophanase and, like the latter, requires M+ for activity C561. The enzyme has four identical subunits that form a homotetramer. As in tryptophanase, pairs of dimers associate to form a catalytic dimer and the monovalent site is located at the interface of the catalytic diiner [56]. The structure of the enzyme was solved with Cs+ occupying the site for monovalent cations (Fig. 9, bottom) [601. Cesium binds to two ligands from the protein subunit 1, one ligand from protein subunit 2, and 3 water molecules. The monovalent cation lies about 6 A from the active site cleft 1601 and does not appear to play a direct role in substrate binding.

kcorbate peroxidases (EC 1.11.1.11) are plant enzymes that scavenge hydrogen peroxide using ascorbate as the reductant. Pea cytosolic ascorbate peroxidase is a dimer of homodimers 1611. The structure of the enzyme reveals that each monomer contains a ferric protoporp~yrin and a monovalent cation site 1611. A pocket in the protein, , contains His163, ~ p ~ Qand $ ,Trp179. His163 is located proximal to the heme directly below the heme group, whereas Asp208 forms hydrogen bonds with His163 179, thereby creating a hydrogen bond n ~ t w o from r ~ Trp149 to the heme group. In ascorbate peroxidase, a cation binding site is located about 8 from the 01 carbon of Trp179 [kill. Potassium was suggested to be the preferred cation, although in other p ses Ca" binds to a similar site C611. The protein contributes seven ligands to g. 10). The fcsnction of cations in peroxidrases is not entirely clear; however, the ions are thought to exert long-range electrostatic effects on the redox properties of the heme group, In the "compound I" intermediate state of ascorbate peroxidase, one oxidizing equivalent exists in the form of a porphyrin rc-cation radical 1631. In compound I of cytockrome c peroxidase, whose active site is very similar to that of ascorbate peroxidase, a cation radical on Trp191 is present in compound I. Trp193. i s believed to provide an efficient route for electron transfer from cytochrome e. The presence of the cation binding site, somewhat remote from the p-Trp triad, appears to be the major difference between the active sites of wo peroxidases. Hence, the cation was suspected of influencing Lhe distribution of oxidizing equivalents and the stability of the compound I intermediate staie in ascor~ateperoxidme. Site-directed mutagene~iswas used to engineer a cation binding site in cytochrome c peroxidase in a position analogous t o that in ascorbate poroxidase. In order to create a K+ binding site, five amino acid substitutiom194?X,T1996) and G201S-were inserted in the primary sequence [SZ]. A structure of the mutant enzyme at 1.5-A resolution confirmed the proper

29

FIG. 10. Stereo view o f the binding of R+ to the enzyme mcorbate peroxidaso. The figure was prepared from PDE file 1AXP.

folding of the variant and the presence of K+ in the engineered site [&?I. The activity of the mutant form of Gytochrome c peroxidase was inhibited by b i n d i ~ g of K+ at the engineered site. The characteristic electron paramagnetic resonance signal for the compound state was also suppressed when K" was present, These results support the hypothesis that the remote cation in peroxidase has a profound e s the heme group [61,62]. influence on the redox p ~ o ~ e r t i of

ethionine aminopeptidase (EC 3.4.ll.18) catalyzes the removal of the initiator Nterminal Met residue from newly synthesized proteins. The enzyme exhibits specificity toward proteins with small, uncharged residues at the penultimate position [63,641. The fold of E. eoli M e t ~ a ~ i n o p e p t i ~consists ase of an internal ~seudo~twofold symmetry that s t ~ c t u r a ~relates ly the first and second halves of the enzyme. The active site is near the face of a centrally located antiparallel fi sheet. A dinuclear metal center (2Co"f) in the active site is essential for activity. A monovalent cation site was found at the interface between the two subdomains of the protein approximately 13A from the dinuclear metal center [651. This monovalent cation appears to have a structural role.

LARSEN A N D REED

30

4.1 5. ~ h r o ~ b i ~ Thrombin (E6 3.4.11.181,a member of the serine protease family, is the filial enzyme of the coagulation cascade. The reaction converts fibrinogen to fibrin I and fibrin 11by limited proteolysis i66-691. Binding of Na+ enhances the catalytic activity of thrombin [70,711. The enzyme has a short A chain and a longer B chain. The A and B chains are cross-linked by one disulfide bond forming a molecule with a prolate ellipsoid shape [72]. Sodium binds near the middle of a cylindrical cavity of the P-chain 173J. The cavity crosses from the active site to the opposite side. Sodium coordinates to the main-chain carbonyl oxygens from K224 and R221 and four water molecules (Fig. 11) 1731. A structural and kinetic study has shown that the mutations of Y225 of human thrombin influence Na". binding and therefore the optimal activity in thrombin [74].

4.16.

a-Amylase

The cl-amylases (EC 3.2.1.1) catalyze the breakdown of starch and related polysaccharides while retaining the a-anomeric configuration in the products. Calcium is required for catalytic activity in a-amylases. The stability and enzymatic activity of a-amylases is enhanced by Na' I 75-781. The structure of a-amylase froin Bacillzcs licheniforinis shows that the protein has three discrete domains: a central, N-terminal A domain; a B domain; and a 6-terminal domain [791. The A domain contains a ( p / a ) g barrel that includes residues 1-101 and 203-396. The active site is located at

F1G. 11. Stereo view of the binding of Nai to the enzyme thrombin. The figure was prepared using MolScript [1261 and PDB file ZTHF.

INTERACTION OF Na A N D

K WITH PROTEINS

31

the C-terminal end of the barrel. The I3 domain (residues 102-2022), described as a protrusion from the A domain, is composed mostly of' sheet and a pronounced loop [79]. A unique Ca-Na-Ca trinuclear metal center appears at the stem of the loop in the R domain. Sodium coordinates to five carboxylate oxygens from Asp side chains and to a main-chain carbonyl from I201 (Fig. 12). The five carboxylate groups form bridges between Nai and the two calcium ions. Machius et al. [791 suggest that the Ca-Ma-Ca metal triad is an important factor in the high thermal stability of the B. Zicheniformis enzyme.

5. P

IV

The structures of complexes of Kf and Na ' with enzymes and other proteins reveal a variety of coordination schemes and functions. A preponderance of main-chain carbony1 groups as ligands, together with hydroxyls from Thr and Ser and carboxylates from Asp and Glu, is consistent with the known coordination tendencies of the ions. Direct coordination between the ions and substrates in several of the enzymes demonstrates an active role of the ions in binding of the substrates and positioning of these molecules for their reactions. In other cases, it appears that the ions have a critical role in establishing the correct structure of the proteins or in stabilizing a particular structure. It is also significant that in several enzymes the monovalent cations are found near divalent cations-often with bridging ligands. In some cases, differences in the structures upon substitution of Na+ for Kf reveal the source of specificity in the

FIG. 12. Stereo view of the Ca-Na-Ca triad in the enzyme a-arnyIase from Bacillus lzcheniformis. The figure was prepared from PDB file 1RLI.

32 activation of the enzyme. The structural and hnctional results with the peroxidases suggest that the positive charge of K+ may exert an electrostatic effect that can influence the redox properties of a distant center. The rapid accumulation o f structural data for proteins fuels expectations that the monovalent cation binding sites in many of the enzymes of Table 2, including Na+/Kt ATPase, shall be forthcoming.

The authors gratefully acknowledgethe contribution of Fig. 3 by Prof. Tetsuo Toraya. Work from the authors' laboratory was supported by NIM Grant GM 35752.

FBPase Hsc70

PLP w.t.

adenosine 5'-diphosphate adenosine 5'-triphosphate fructose l,~-bisphosphate fmctose 1,6-bisphosphatase heat-shock cognate protein r o o ~ a v e nProtein Data Bank pyridoxal phosphate wild type

s

EFE

I. 2.

3.

4. 5. 6. 7. 8.

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. Aikawa, ed.), C

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. 12, ~ 4 ~ (19$7). 1 5 ~ edersen and E. Carafoli, Trends i o c ~ @ ~Sei.,

INTE~ACTIONOF Na AND K WITH P

11. 12.

13. 14.

15.

16.

17.

20.

INS

33

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K WITH PROTEINS

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epartment of Physiology 11, University o f Tubingen, Ob dem Himmelreich 7, D-72074 Tubingen, Germany

1. INTRODUCTION 1.1. Ion Channels: Definition and Role in Excitable Membranes 1.2, How Do Voltage-Gated Ion Channels Work? 2. POTASSIUM AND SODIUM C W M E L PROTEINS WITH K N O W PRIMARY STRUCTURE 2.1. PoLassium Channels 2.2. Sodium Channels

3. STRUCTURES OF CHANNEL PROTEINS AND PROTEIN DOMAINS RESOLVED TO DATE 3.1. The Pore o f a Bacberial Potassium Channel 3.2. Inactivation Domains of Mammalian Potassium and Sodium Channels

40 40 40 41 42 44

45 45 47

4. S T ~ U ~ T U ~ E - F U N C ~TE~LOA~T I O N S ~ I P S 4.1. Selectivity and Permeation 4.2. Gating: Activation and Inactivation

48 48 50

5 . PERSPECTrVES

53

AC~OWLEDGM~~TS

53

ABBREVIATIONS AND DEFINITIONS

54

RE~ERENCES

54

39

FAKLE R

40

efinition and Role in Ion channels generate and orchestrate the electrical signals that drive heart beat, muscle contraction, perception of sensory stimuli and thinking in the brain. As m a c r o m o l e c ~ aprotein ~ tunnels, ion channels span the plasma membrane’s lipid bilayer and allow for flow of the inorganic ions sodium, potassium, calcium, and c ~ o r i d ein and out of the cell at rates greater than 106 per second. This high rate is considered a diagnostic feature distin~ishingchannels from other ion transport devices such as the Na”/Kf pump. In contrast to the latter, ionic flux through channels does not require immediate delivery of metabolic energy because it i s driven by electrochemical gradients f1,ZJ. p~oximatelyone-third of the energy generated in cells is expended to maintain such ionic gradients across the plasma membrane that make the cell a battery. The energy collected and stored by the cell is then spent in short bursts by the ion channels upon opening of t.heir pore. Generally, channel pores are opened (and closed) by specific biological stimuli, such as extracellular and intracellular ligands (ligandgated ion channels), transmembrane voltage (voltage-gated ion channels), or a combination of these factors. In addition, some channels are selective for one sort of ion, classifying them as Na’, Kf, Ca”, or Cl- channels; some of them are indiscriminant and therefore termed nonselective ion channels (for review, see 121 or rll). ~~o~lectively, these channels make complex temporal patterns, such as the action potential, which serves to transduce signals and coordinate Ca2’ entry into the cell. The action potential is a millisecond sequence of changes in membrane potential of about 100 mV: influx of Na’ through selective or nonselective channels depolarizes the membrane, whereas outflow of K+ mediated by K+ channels stops this “disturbance” and leads to repolarization. The “‘elementary event” of an action potential may illustrate the key role of ion channels for excitation and show that understanding the fun~tionalityof these proteins is fundamental to understanding the signal transduction in biological systems. Q

V Q ~ t a ~ ~ -ion ~ aChannels t~d

The functional behavior of ion channels is basically determined by two processes: permeation and gating [1721.Permeation denotes selectivity for and passing of ions through the pore, and gating describes the opening and closing of the channel that controls access of ions to the permeation pathway. To get a more vivid picture of such channel work, we shall consider gating and permeation in the voltage-gated K+ (Kv) channel, which has long served as the m,odel for channel biophysics [3-51. As shown In Fig. 1, the channel is closed at the normal resting potential (which is close to the Nernst potential for -70 to - ~ Q m VUpon ~ ~ depolarization, the channel first opens by “movement” of

41

Na AND K CHANNEL PR

I

gate

inactivation gate

PIG. 1. Gating of voltage-gated Kf and Naf channels. The scheme represents functioiial states of a channel with respect to positions of activation and inactivation gates: C i s the closed or activatable state (usually adopted in the resting state), 0 is the open or conducting state, and I i s the inactivated state, where the open pore is occluded by the inactivation gate (states 0 and I are adopted upon depolarization). (Modified from (‘7,831.)

the activation gate(s) and then enters a long-living nonconducting state, the inactivated state. Inactivation thereby results from transition of the inactivation gate, which occludes the open pore as long as the membrane is depolarized. On repolarization, the inactivation gate removes from the pore and allows for the next activation of the channel. Both activation and inactivation are driven by the membrane potential via a “domain” within the channel that, senses the transinembrane voltage and couples it to the “gating” machinery [6,71. Once the channel is open, the pore properties of selectivity and c o n ~ u c ~ ~ c govern the flow o f ions along their electrochemical gradients (21. Thus, all nels show a selectivity sequence o f K+ Rb+ > Cs+, whereas permeability for the smallest alkali ions Na+ and Li+ is immeasurably low. Thus, Kf is at least 104times more permeant than Na+ [21, implying that K+ channels can distinguish between Pauling radii of 0.95 Waf) and 1.33 (K+). Even more surprisingly, this high selectivity is managed at a throughput rate of 108K+ ions per second, which is close to the diffusion limit. This paradoxon as well as the nature of the channel gating has challenged biophysicists since the 1950s and propelled extensive work. The respective results will be summarized in the following sections, which focus on the structural understanding o f voltage-gated XI+ and Na+ channels.

A

2. PO KN

NEL PROTEINS WIT

The fist sequence informations on ion channel proteins became available in the middle and late 1980s upon cloning of the “pore-forming polypeptides” (asubunits)

42

FAKLER

of the Naf channel from the eel Electrophorus eEectricus TSl and the K+ channel from Drosophila m.elanogaster [9-111. Analysis of the hydropathy profile o f these proteins predicted that they are made up of a series of hydrophobic domains long enough to span the membrane as an a-helix (so-called transmembrane domains, TMs) and hydrophilic domains that link them together (Fig. 2). This first glimpse of channel topology was filled with structural and functional data in the 1990s using molecular biology together with Functional characterization of heterologously expressed channel proteins. From this machinery, molecular pictures of Na+ and K+ channel proteins evolved as described below.

otassium Channels The superfamily of genes coding for K+ channels exhibits significant variability and consists of a number of families and subfkmilies with distinct “structural” and functional properties. According to their membrane topology, they may roughly be divided into four superfamilies with two, four, six, and eight TMs as illustrated in the “pedigree” in Fig. 2 [12]. Thereby, the two- and six-segment channels are structural prototypes, whereas the four- and eight-segment channels might be regarded as variations of these two.

I 1 nmolmin-I mg-' protein. This transport system is ATP-

MAGNESIUM-ACTIVATED ENZYME SYSTEMS

63

independent, untilizing the membrane potential as a driving force. CorA also fimctions in MgZt efflux from cells, beginning function at an extracellular Mg2+ concentration o f about 1mM. Three additional genes, designated corB, core and corD, have been identified in S. typhimurium that mediate Mg2+ efflux but not influx [16]. CorA protein from S. typhimurium has a large N-terminal, periplasmic domain of about 240 amino acids followed by three membrane-spanning regions F171. There is only one negatively charged residue within the membrane-spanning segments, insufficient by itself to transport a M$+ ion. It has alternatively been suggested that CorA transports M g + using charge-lone pair interactions with carbonyl or hydroxyl groups on the protein. Not all CorA homologues may possess the same membrane topology as the protein from 5. typhimurium and could potentially even have additional functions. Within the membrane itself, CorA is postulated to have an oligomeric-type structure. In addition to the Corn system, E. coli and S. lyphimurium contain an inducible Mg2+ influx system that is activated at low (10pM) extracellular M8+ concentrations. This system, encoded by two genes designated mgtA and mgtB, is regulated by the extracellular Mg2+ concentration [IS]. Under conditions of limited Mg2+ (1lOpM), transcription of the m@A and mgtB genes is enhanced several thousand fold 1191. MgtA (95 kDa), like MgtB (101 m a ) , is a P-type ATPase with the two proteins showing only 50% overall amino acid sequence identity [19]: The MgtB system is the major inducible transporter for intracellular Mg2+. Both the mgtA and mgtB gene sequences indicate that the respective proteins are most homologous to eukaryotic Ca'+-ATPases 1201. MgtB shows 50% overall sequence similarity to sarcoplasmic reticular CaZC-ATPase,and only 10-30% similarity to prokaryotic Ptype ATPases. Analysis of'the membrane topology of the MgtB protein suggests the protein consists of 10 transmembrane segments, with both amino and carboxy termini localized within the cytosoll211. Recently, an additional gene of unknown function from E. coli, designated mgrB, has been identified as having an Mg2+-responsive promoter and is induced under conditions of Mg2+ limitation 1221. While extracellular MgL+obviously enhances the rate of transcription of mgtA and mgtB, there is apparently no direct Mg2+-mediatedmechanism. Rather, extracellular Mgz+ acts as a signal through the PhoPPhoQ virulence system of S. typhimurium. Under physiological concentrations of extracellular Mg2+, PhoP-activated genes are transcriptionally repressed. At low Mg2+ concentrations, the periplasmic domain of PhoQ, in the absence of bound Mg2+,undergoes a conformational change that activates PhoP and enhances transcription of PhoP-mediated genes [231. A 146amino-acid polypeptide corresponding to the periplasmic (sensing) domain of PhoQ undergoes altered tryptophan fluorescence upon binding Mg2+ 1241. The periplasmic domain of PhoQ binds both Mgz+ and Ca2' at distinct sites, although Mg2+is more potent at transcriptionally repressing PhoP-activated genes in vivo. As the PhoP1 PhoQ system of S. typkimurium has been implicated in the virulence of this bacterium, it can be concluded that response to M$+ can act as a virulence attenuation signal under appropriate environmental conditions.

64 1.3.3.

nesium Transport Systems in Eukaryotes

Much literature has accumulated on the physiological effects of i'+ on various cells, tissues, and organs in both lower and higher eukaryotes. owever, unlike the case with the bacterial magnesium transport systems, relative le is known about the molecular details of magnesium transport and homeostasis in eukaryotic cells. Within such cells, m a ~ e s i u mis c o m p a ~ m e n t ~ z ewithin d the endoplasmic and sarcoplasmic reticulum, the mitochondria, and the cell nuclei (reviewed in [141). Transport of m ~ n e s i u macross the plasma membrane of some cell types is apparently hormonally r e ~ l a t e dIncreases . in CAMPlevels resulting from stimulation by a-and p-adrenergic agonists or other agents result in efflux of Mg2' from some cell types [14]. Most data suggest that an Na+/Mg2+ exchange system is responsible for Mg2+ translocation across the plasma membrane, although this protein has not been identified definiu~ within eukaryotic tively. For more detailed information on m a ~ e s i homeostasis interested reader i s referred to reviews [13,14,25,261. ~ces are moleukaryotic organisms, only in the yeast S ~ ~ ~ h u r o m cereuisiae cular details of Mg2+ transport starting to emerge. Two genes, d e s i ~ a t e dALRl and ~ ~have 2 been identified , as components of the yeast Mg2' influx system 1271. Both proteins share weak homology with CorA protein from S. typh deletion results in a ma~esium-requiringphe~otype.Like CorA, ~redictedto have a C-terminal t r ~ s m e m b r a n edomain. While ALRl a 70% sequence identity, gene disruption of only ALRl was lethal. Add l2 or MgS04 to the medium increased the growth of U B 1 mutants,

s IN MA

TIV

YM

Many examples are now available of enzyme structures c o n t a i n i ~ bound magnesium. As ~ af themknown structures ~ is presented in Table 1.The interested reader is referred to the relevant PDB coordinates or the reference(s) listed to obtain more detailed information. ~ e v e r ageneral l comments should be made with reference to Table 1. First, in many insta~ces,the metal ion has been introduced as a consequen~eof cocrystalli~ation in the presence of m a ~ e s ~ uormsoaking of p r ~ ~ o w crystals n in solutions cont a i n i n ~m a ~ e s i at u ~some specified concentration. ~wever,this is not to say that endogenous m a ~ e s i u m already , bound to the prot , may not c o p u ~ f ywith the enzyme of interest and be present a priori. This also raises the possibility that the m rather some other electron density to be interpreted is in fact not m a ~ ~ e s i ubut metal, or even a water molecule. Both an g2* ion and a water molecule contain 10 e l e c t r o ~ sso ~ in terms of total scatterin aterial, they are essentially the same. ay signal and must be n", m a ~ e s has i ~essentially no anomalous factors. For these peabased on coordination geometry and tempe

65

M A G N ~ ~ I U M - A C T I V A TENZYM ~D

Crystal Structures of ~ a ~ e s i u m - A c t i v a t Enzymes ed and Proteins

Enzyme

C Number

PDB Resolution (A> code

5.4.2.9

1P

1.8

1 1

2.00 1.60

2.4.2.8 phosphoribosyltransferase Xanthine phosp~oribosy~- .4.2.22 transferase Adenine phosphori~osyltransferase

1

~

~1.80L 1.50 2.00

Glutmine p ~ o s ~ h o ~2.4.2.14 i~syltr~sfer~e Qrotate phosphoribosyitransferase .2.22

1ECC

1A95

2.90 2.00

1A49

2.70 2.10

1A5U

2.35

Pyruvate kinase

Casein kinase-1 dependen en^ pro~ein kinase

2.40 2.70 2.30

2.00 2.70 2.20 1.80 1.90

Ligandb)

ef,

Mg2+, oxdate

1281

I m m u c i l l i n ~ ~ , /291 PPi, ~ g ~ 1301 + rnP, P g)z So:-, MgZ+ 1311 A ~ ~ ~ icitrate, ne, Mg2+ AMP, citrate, M6p2+_

1321

1331

1341

MATTE AND ~ ~ L ~ A E R

66

TBLE 1 (continued) Enzyme

Phosphofm ctokinase Nucleoside diphosphate kinase

EC Number

2.7.1.11 2.7.4.6

PDB Resolution code (A,

5um

1.90

lPFK

2.4 2.30 2.00 2.00 2.20 2.00 2.00 2.5 2.5

1 1

lNLK 1 1

~hosphoglyceratekinase

2.7.2.3

lNUE 1B4S 13PK 1PRP 1QPG

1.65 2.40

lVPE

2.00

Adenylate kinase Shikimate kinase Adenosine kinase Arginine kinaso

2.7.4.3 2.7.1.71 2.7.1.20 2.7.3.2

2AKY 2SHK 1BX4 lBGO

1.96 2.60 1.50 1.86

Glycerol kinase

2.7.1.30

1B lGLC

3.00 2.65

Protein kinase CkZ/asubunit

2.7.1.37

1A60

2.10

Bovine heart ~ i t o c h o n ~ iF1al ATPase N-Ethylmalemide sensitive factor Myosin head piece

3.6.1.34

lBMF

2.85

N/A

1NSF

1.90

3.6.1.32

1 ~ N E 2.70

Adenylyl cyclase: GpGw,S

4.6.1.1

lAZS

icm

2.30 2.80

LigandM

Ref.

AD?, 659, AlF3, [46] Mg2+ ADP, FBP, Mg2+ [471 AZD, Mg2+ [481 dTDP, Mg2+ [491 ADP, Mg2+ 1501 P, Mg2 151I Mg2+, AlF, r52l GDP, Mg2' 1531 ADP, Mg", PO: [541 ADP, Mg2+, 3PG, 155,561

Po;ADP, Mg2+

[571 3PG, ~ 9 - P N P , [581 Mg2+ 3PG, ~ P - ~ N PI591 ,

arginine, nitrate ATF, Mg'+ 1641 ADP, G3P, Mg2+, [651 Zn"

ATP, Mggg,;"+

[@I

AMP-PNP, M$+, 1671 P, Mg2+ ATP, Mg2+

1681

Pyrophosphate, MgZ+ GSP, Mg2', FKP GSP, M g C , FQK,

1691

D

[701 [711

67

SIUM-ACTIVAT~DENZYME SYSTEMS

TABLE 1 (contirzued) Enzyme

EC Number

70-kDa cognate heatshock protein, ATPase domain (Hsc 70)

3.6.1.3

PDB Resolution code (A)

lCJv

3.00

lCJK

3.00

1ATR 1 3HSC

lBA0

2.34 1.70 1.90 1.90

GroEL-GroES complex Archaeal chaperonin (thermosome) Myosin motor

NIA N/A

1AON 1A6E

3.00 3.20

NIA

1L-m

1.90

Smooth muscle myosin motor domain (MD) Myosin head piece ( R761P, I762N) Is0 ses Acetohydroxy acid isomeroreductase D-Xylose isomerase

3.6.1.32

lBR4

3.62

L,3.6.1.32 1M

2.10

Ligand(s)

f.

GSP, Mg", Zn2+, FOK, D GSP, M[g2+,Mn2+, FOK, AGS ADP, Mg2*, PO:-, 1721 ATP, Mg2" BDP, Mg2+, PO:ADP, M g f , PO:-, Mg2+, ~ P - P N P 1731 ADP, Mg2+ [741 ADP, Mg2+,AIF, "751 MNT-ADP, MgZf, BeF; ADP, Mg2+, BeFg "771

ADP, Mg2+

1781

.

HIV-1 integrase Sarcoma virus integrase Bacteriophage H p l integrase

1.1.1.86

1.81 1.96 2.19

h10 Mg2+ glucose,+'@i 3-O-methylfructose, Mg2+

2.50 1.70 2.50

Jug2+ Mg2" Mg2+

2.06

Mg2'

3.1.26.4

2-80

Mg2+

3.1.22.4 2.1.7.7

2.50 2.20

Mn2 DG3, Mg2+, DNA

5.3.1.5

lXUA 1XYB lxYC

NIA 2.7.7.49 NIA

3.1.26.4 Escherichia coli RNase H1 E. coli RuvC resolvase T7 DNA polymerase

1.65

lTFR

+

[84 1

6 TAElLE 1 (continued) Enzyme

EC Number

Klenow fragment (E. eoli 2.7.7.7 DNA poly I, exonuclease domain) NA p o l y m e r ~ eI 2.7.7.7 Taq DNA polymerase

PDB Resolution code

(A)

lKFS

2.10 2.03

2K.F" 2BDP

2.7.7.7 1QSY

lQTM NA polymerase f3 Exonuclease III Flap endonuclea~e-1 (FEN-1) T5 5'-exonuclease EcoRV endonuclease capping enzyme ~RNA RecA protein HIV-1 reverse rans script ~e clease H domain)

2.7.7.7 3.1.11.2 NIA

1BPY 2BPF ""1AK0 1A76

3.1.11.3 *lEXN 3.1.21.4 1RVB 1RVC 2.7.7.50 lCKN 3.4.99.37 *lREA

DAD, Mg2+, DNA TTP, Mg2+, DNA DCT, Mg2+, DNA DCT, Mg2+, DNA Mn2+ Mn2+

2.50 2.10 2.10 2.50 2.70 2.40

Mn2+ NA, Mg2' DNA, MgZf GTP, Mn2' ADP, Mg2" Mn2+

2.50 2.10 1.60 1.26 1.35 2.03 2.50

NIA

aqua,~i~i~s) Giul

NIA

2.00 2.20 2.70

NIA s4:G(I Alphal) complex N/A NIA

2.30 2.80 2.50 2.50 2.40

/A

Ref.

Trinucleotide, Mg", Zn2+ $+,Mn2+,Zn2+ DNA, M$Lt

1.80 2.60 2.30 2.30 2.30 2.20 2.90 1.70 2.00

EF-TU ( T ~ W W L U S

protein (Rhoa)

Ligand(s)

1961 1971 r981 1991

e1001

GDP, All?,, Mg2+ [loll GDP, AF,, Mg2+ I1021 GYP, Mg24 [I031 GNP, Mg" [1041 GDP, Mg2' 11051 GNP, Mg2+ [1061

MA

69

LE 1 (continued) Enzyme

EC Number

PDB Resolution code

-ras-P21 (G12P) c-Rafl:RaplA complex Transducin-a

N NIA

1CLU lClP 1TND

1.70 2.20 2.20

1AUS 2RUS 9RUB

2.20 2.30 2.60

1PVD

2.30

3.1.3.11

1FBP

2.50

3.3.3.57

lINP

2.30

6.3.4.4

1JUY

2.60

2LGS 1A82 lDAK 5..1,1.11 lAMU

2.80 1.80 1.60 1.90

1.1.1.42

2.50 2.50

NIA

carboxylase oxygenase ~~h~dospirillu~i rubrum) Pymrvate decarboxylase 4.1.1.1

bisphosphatase Inositol p o l ~ h o s p h a t e l-phosphatase Adenylosuccin~te synthetase Glutamine synthetase Dethiobiotin synthet ase Gramicidin synthetase l-phenylalanine activating domain 1. ses Isocitrate dehydrogenase

(A)

6.3.1.2 6.3.3.3

lIDE 1BL5

LigandW

ef.

DABP-GTP, Mg2'

[1141 11151 11161

ATP,S, Mg2+

F6P, AMP, MgZf 11211

Mg"+

1122.1

ATP, Mg"' ADP, Mg2+ AMP, ~~~~, Phe

[125]

NAP 11281 2-oxyglutaric acid,

2.60

5IC 1AI3

CheY protein (kinase/

h o s ~ h a t ~~e a) l m o n e l ~ ~ ~ y p h i ~ u ~ i u ~

1 1 2

C

2.50 1.90

1.76 1.90 ~ 1.80 ~

[126]

11291 Isocitrate, Mg2+

11301 11311

70

MATTE AND DEL

(continuedj Enzyme Photosynthetic reaction center Cytochrome c oxidase: Fv complex 1 es P4-P6 domain of

EC Number

PDB Resolution code (A1

Ligand(s)

Ref.

NIA

lAIG

2.60

BCL, BPH, Mg2+, r135l U-10, Mg“+, Fe3+

1.9.3.1

lARl

2.70

Heme-a, Mg2+, [1361 l?e3+,~a’+, cu2+

NIA

lGID

2.50

Mg”, Co(NH&’

[1371

Tetrahymena thermophila group 1 intron Hepatitis virus genomic ribozyme

sons, structures in which both the apo and holo form of the enzyme are available are preferred, and can be particularly insightful when refined at high resolution. Fortunately, several o f the models in Table 1 fit these requirements. It cannot be overemphasized, however, that thesc are protein m,odels based on experimentd measuwements and therefore contain errors to a greater or lesser exlent that must be carefu1l.y considered when making bioEogica1 interpretations. For many enzymes, the metal binding properties have been characterized extensively by various biochemical means, and these data, when available, should be integrated with the structural information to present a coherent view of the metal site and its environment. The concentration of Mg2+used in crystal soaking experiments of cocrystallization deserves attention, Oftentimes a higher than physiological concentration will be employed to obtain high occupancy of the metal site. ile having enough metal ion at a biochemically important site is a desirable goal, such high concentrations sometimes result in binding of Mg2+ at additional sites that are not relevant to the function of the enzyme. A case in point is that found with the structure o f human adenosine kinase [621. Crystals of this protein were grown from ns containing gC12. A consequence o f this high concentration was 3 binding sites. Only one of these sites, near one of the two adenosine binding sites, is the biochemically relevant binding site r621. This example, as well as others, shows the importance of having supporting biochemical data in determining the “true” Mg’+ binding site when employing crystallographic methods. An interesting case in point can be made with the metal binding sites of ribonuclease H. Four acidic residues are known t o be conserved in the primary structure of all known bacterial and retroviral ribonuclease sequences. In the crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase soaked in 45 mM

MAGNESIUM-ACTIVATE E N Z ~ M SYSTEMS ~

71

MnC12, two metal ion binding sites are observed [loo]. However, only one divalent cation binding site is observed in E. coli RNase H. This apparent discrepancy can be rationalized through understanding the difference in crystal packing environment between the two proteins. In the crystals of E. coli RNase H, the cluster of Asp and Glu residues responsible for metal binding interacts with a Lys side chain of a symmetry-related molecule, preventing the binding o f a second divalent cation. The different packing environment of HN-1 rcversc transcriptase crystals allows two divalent cations to bind. This example aptly illustrates the sort of problem that can be encountered in deducing correct biochemical information using crystallographic analyses alone. In many instances, metals other than Mg2+have been used to characterize these binding sites by X-ray crystallography or NMR. Such metals include Mn2' [50,86,941, Ca", and Sm" , for example. Manganese has proven a particularly usehl probe ion for magnesium sites in enzymes, both for structural and biochemical studies. As noted previously, Mn2' is both considerably heavier than Mg2+ (23 electrons vs. 10) and has a good anomalous signal at its X-ray absorption edge (6.537 keV, see [1391, for example). Furthermore, in many cases Mn2' can function kinetically as well as or better than Mg"' for the same enzyme 1140,1411.In some instances, this has raised the question of whether a particular enzyme is really manganese- or magnesium-dependent or activated. In most biological systems, the physiological concentration of free Mg2+ is much higher than that of Mn2' and is therefore more likely to be physiologically relevant. As always, there are exceptions to this general rule. In those instances where the cvystal structure has been determined with either Mn2+ or Mg2+ bound at the same site, very little difference in structure has been noted [41,1411. 2.1.

Bi-Mg*'-Bound Structures

A number of enzymes use more than one Mg2' ion as part of their catalytic machinery. These enzymes can use two metal Mg2+ ions, or may employ mixed-metal clusters consisting of Mg2+ and a second metal such as Mn2+ or Zn2+. As with the biochemical data, there is in most instances a lack of direct structural information with both physiologically relevant metals bound from which to draw firm conclusions. However, the structural information that is available provides some insight into the possible role of dual metal ions in facilitating catalysis.

2.2. Kinases Almost without exception, Mg2+ is found bound to the protein and nucleotide substrate in one of a few arrangements. Commonly, the Mg2+ ion is coordinated directly to two protein side chains, to the p- and y-phosphoryl groups of the nucleoside triphosphate, and, through two water molecules to additional protein side chains, often Asp

72

residues 11421. 'When the nucleotide is the diphosphate as opposed to the triphosphate, the M 8 + coordination environment can be altered. In enzymes having a phosphate- ind ding loop motif (denoted as a -loop motif [1431), one of the protein side chains is usually a conserved Ser or Thr residue that is part of the amino acid sequence of a W ~ e r - Aconsensus sequence [1441. The Walker-A sequence was o r i ~ n ~ identified ly as part of the nucleotide binding site in ~ l ~ A T P a smyosin, e, and several kinases, and is used routinely to identify nucleotide-binding proteins a; this amino acid sequence is also called a kinase-la consensus enzymes having a P-loop motif, one of the protein side chains is usually a conserved Ser or Thr residue that is part of the Walker-A or kinase-la motif [143]. In some enzymes, such as adenylate kinase, there are few, if any, direct interac~ionsbetween protein side chains and the 2c ion [Sol. In this structure, three water molecules and the p- and ~ - p h o s p h o groups ~l of the pseudosubstrate adeno~ine-(phosphate)~-adenosine (Ap5A) make up the coordination sphere. This 2c coordination environment may be found in additional nucleoside monophosphate kinases that have a variation on the common Walker-A motif.

onfo~~ational Chan g!'+ in the presence of a cosubstrate, such as AT or GTP, may i significant changes in protein structure, as is the case in many kinases, there are few examples where the M?" cation itself has been showii to induce § t ~ c t u r ~ changes in proteins. An exception to this is found protein from E. coli in the presence and absence o as 10 A are detected upon Mg2' coordination to CheY, resulti turn of a helix and formation of a new p turn. These changes play a role in its activatio~and thus its role in bacterial signal ~ r a ~ s d u c t i o ~ .

chelatase cat~lyzesthe AT - d e ~ e n d ~ i~sertion nt of

IX in photosynthetic organisms. A consensus sequence,

M A G N E S I U M - A ~ T I V A TENZYME ~~ SYSTEMS

73

3.2. Sphingomy~linase Sphingomyelinase catalyzes the hydrolysis of sphingomyelin to phosphorylcholine and ceramide r1471. Sphingomyelinasefrom Bacillus cereus was shown to possess at least two Mg2' binding sites with high and low affinities; the low affinity site is required for catalytic activity 11481. A model for the tertiary structure of this enzyme has been proposed 11491, based on a protein fold recognition method; mutational studies of this enzyme are basically in agreement with this model. A catalytic mechanism of B. cereus sphingomyelinase has been proposed [1481 that does not involve the magnesium cation. The exact hnction of Mg2+ in this interesting enzyme awaits a complete structural analysis.

3.3. Mycobacterial GDP-Mannose Pyrophosphorylase Mycobacterial GDP-mannose pyrophosphorylase catalyzes the formation of GDPmannose from the reaction of a-o-mannose-1-phosphateand GTP. The GDP-mannose is used in mycobacteria to synthesize cell wall lipoarabinomannan [IS01 and various mannose-containing glycolipids and polysaccharides [1511. Mycobacterium srnegmatis GDP-mannose pyrophosphorylase was isolated and purified and shown to require Mg"+ for optimal activity Tl521. The enzyme was specific for a-D-mannose-1-phosphate and relatively specific for GTP in contrast t o the pig liver enzyme [1531.The exact function of Mg2+ in this enzyme is unknown.

3.4. N1-(5'-Phosphoribosyl)adenosine-5'-monophosphate yclohydrolase A unique metalloenzyme entitled N1-(5'-phosphoribosyl)adenosine-5'-monophosphate cyclohydrolase (PR-AMP cyclohydrolase) from Methanococcus uannielii has been purified [1541. This enzyme catalyzes the hydrolytic cleavage of N1(5'-phosphoribosyl)adenosine-5'-monophosphateto produce another intermediate in histidine biosynthesis. The isolated PR-AMP cyclohydrolase contains Zn2' bound to a high-afinity site. Kinetic analysis revealed an absolute requirement for Mg2+ bound to a lower affinity site on the enzyme to catalyze the reaction. Both metal cations are essential for enzyme activity. Structural studies are necessary to determine the exact functions of these metal ions in this unique enzyme in histidine biosynthesis.

74

MATTE AND ~ E L ~ A ~ R

U~CTIO RELATI ~

4.

Alkaline phosphatase is a nonspecific phosphomonoesterase that contains two Zn2+ ions and one Mg2+ion at the active site [1551; structure-function relationships of this enzyme are described in Chapter 19.

4.1.

kin as^^

4.1 .I. Single dependent Kinases Most kinases require a divalent metal ion, usually Mg2+, to facilitate phosphoryl transfer through its association with a nucleotide. In many instances M 2 ' binding to ATP proniotes its association with the enzyme, as well as increases the rate of catalysis. In some enzymes, such as ~ P / C M kinase P and possibly nucleoside diphosphate kinase, Mg2+ does not influence nucleotide binding to the enzyme, although it does play an essential role in the phosphoryl transfer reaction [44,501. While autophosphorylation of the active site histidine residue can be accomplished in the absence of Mg2+with nucleoside diphosphate kinases, Mg"+ is required for phosphoryl donation from the enzyme to the second substrate 1501. Coordination schemes for Mg2+ bound to kinases vary depending on both the nucleotide (nucleoside diphosphate vs. nucleoside triphosphate), the presence or absence of a second substrate if present, as well as the particular enzyme under scrutiny. With nucleoside triphosphates, the Mg2" is bound either by the p- and yphosphate, or by all three phosphoryl groups. In complexes with only a nucleoside R phosphate, the a-and 0-phosphates coordinate the Mg'". In those cases where an NDP molecule is present with a second, phosphorylated substrate, the M i 2 + coordination usually utilizes the p-phosphoryl group of the NDP and the phosphate group of the second substrate. This arrangement is observed in phosphoglycerate kinase with A D P / M ~ +and 3-phosphoglycerate [551,phosphofmctokinase bound to ADP/Mg2+ and 1,6~bisphosphate[47], and glycerol kinase bound to Ph4i2+and glycerol 1651. ithout exception, water molecules contribute signlfrcantly to the coordination ent of Mg2+in kinase structures. In lower resolution structures, where hexa coordination cannot be observed (see, €or example, 1551j, the missing ligands are almost certainly water molecules that are in turn hydrogen~bondedto conserved Asp or Glu residues. Most nucleotide-Mg2" complexes utilize some protein ligands directly as part of the Mg2+ coordination sphere. An exception to this i s fou nucleoside diphosphate kinase from Dictyostelium diseoideurn bound to ADPI511 or d T D P - M ~ +1491. Another example is found in ~ P / C kinase ~ P from D. diseoideurn bound to UP5A/Mg2+1441. In NDP kinase, the Mg2+ is coordinated by oxygen atoms from the a- and Bphosphates of ADP, as well as four water molecules that mediate all interactions to the protein. Carboxylate groups from two acidic residues (Asp-125and Glu-58) as well as the carbonyl 0 atom of Gly-123, hydrogm-bond with these Mg'+-coordinated

MAGNESIUM-ACT1VATED ENZYME SYSTEMS

75

water molecules, forming the second coordination sphere. As Mg2+ coordinates to CLand P-phosphoryl groups as well as fluorine from either AlFp, or BeF;, along with three water molecules in complexes with .ADP/M8+ 1521, it appears the Mg2+cation changes coordination with either NTP or NDP bound at the active site. Phosphotransfer reactions are thought to have either a dissociative transition state (SN1-like)dominated by bond breaking or an associative transition state (23~2like) dominated by bond formation [1421. In phosphotransfer enzymes that use primarily a dissociative mechanism of catalysis, such as nucleoside diphosphate kinase, the Mg2+ ion is expected to play a different role than in those enzymes that utilize primarily an associative mechanism. In the dissociative case, Mg2+likely contributes mainly through precise orientation of the terminal phosphoryl group, as well as through charge neutralization and possibly stabilization of the negatively charged transition state r1561. A major unresolved issue is the precise role of Mg2+ in formation of the transition state. Again, the answer to this question differs depending on whether a dissociative or an associative mechanism is being utilized. As water molecules frequently contribute significantly to the coordination sphere of the metal, and given t does not exchange water molecules efficiently, it follows that the ability of undergo changes in ligand environment during catalysis is limited. This is not to say that Mg" coordination does not change between enzyme-substrate and enzyme-product complexes. In some instances, as for example with yeast adenylate kinase, Mg2+ has been suggested to accompany the phosphoryl group during transfer [60].

ent 4.1.2. Dual M 2 ' - and ~ n 2 + - ~ e p e n ~Kinases 4.1.2.1. Phosphoenolpyruvate Carboxykinase As described above, the primary role of Mg2+ in kinases is in assisting phosphoryl transfer of the y-phosphoryl group t o an acceptor molecule (direct-displacementreaction) or an amino acid residue (double-displacementreaction; reviewed in 11421). Two enzymes, pyruvate kinase 11571 and phosphoenolpyruvate carboxykinase (PCK;summarized in [158l), have been documented to utilize both M 8 + and Mn2+ synergistically in achieving maximum rates of catalysis. The synergistic effect of both Mg2+and Mn2+ on the reaction catalyzed by PCK has been revealed through crystallographic studies with substrates and either Mg"+ [42] or both Mgzc and Mn2+ [43] bound at the active site (see Fig. 1).Both Mgz+ and MnZf are octahedrally coordinated, although the coordination sphere of the Mn2+ is slightly distorted from ideal geometry. Ligands Tor the Mg2+ cation include oxygen atoms of the 0- and y-phosphoryl groups of ATP,Thr-2550Y,and three water molecules. Two of the three water molecules are hydrogen-bonded to two aspartate residues, Asp-268 and Asp-269-residues found to be conserved in all PCK sequences. The Mn2+ coordination sphere consists of one oxygen atom from the y-phosphoryl H i ~ - 2 3 2 ~and ' ~ , two water group of ATP,an oxygen atom from Asp-269, Ly~-213*~, molecules. The distance between the MgZf and Mn2+ cations is 5.2 A.

76

FIG. 1. Stereodiagram of' the MgZi-Mn2 ' cluster at the active site of Escherichia coli phosphoenolpyruvate carboxykinase 1431. Both cations have octahedral coordination with Mgz+ being surrounded by six oxygen atoms and Mn" being surrounded by four oxygen atoms and two nitrogen atoms. ATP has the unusual baselsugar syn conformation as well as a high-energy eclipsed conformation for the p- and y-phosphoryl groups.

Both metal binding sites within the E. coli PCK active site are optimal for binding their respective metal cations. Magnesium clearly does not bind to the Mn2'-specific site in the presence of ATP and oxalate, as revealed in a crystallographic study 1421. Likewise, in the presence of substrates with 5 mM M&12 and 5 mM MnG12, Mn" is found to bind specifically at a single site [431. Binding at these respective sites can be rationalized based on the preferred ligand environments for either Mg2+ or Mn2'. Mg2-"-is a hard Lewis acid, and prefers hard Lewis bases for coordination, especially orryanions, Similarly, Mn2+, as a softer Lewis acid than &I$" prefers coordination environments that contain one or more softer Lewis bases, such as nitrogen. The two different ligand environments available for binding either Mg2+ or Mn2+therefore select the appropriate cation at the appropriate binding site within the PCK active site. m i l e the binding constants for M 8 + and Mn2+ at their respective sites are unknown in the case of the E. coli enzyme, maximal activity is obtained with milli-

ED ENZYME SYSTEMS

77

molar and micromolar concentrations of Mg2+ and Mn2+, respectively. While the Mg" ion only interacts with P and almost certainly plays a direct role in associative phosphoryl transfer, the function of the Mn2+ cation c be in promoting c~boxylatio~decarboxylation as well as phosphoryl transfer. Mg2+ and &In2'" likely contribute t o lowering the activation energy barrier to c is in a number of ways, including (1)precise geometrical positioning of substrates (orbital steering), (2) electrophilic catalysis via polarization of the terminal phosphorus-oxyge~ bond through direct coordination by both $+and &In2+, and (3) charge neutralization ghout formation o f the enolate anion interof the ground state complex and t mediate and transition state(s). Since Mn2+ exchanges water molecules at a much faster rate than Mg', Mn2+may undergo changes in ligand environment throughout the reaction coordinate, exchanging its waters for an inner sphere complex with the enolate anion intermediate I43I. 4.1.2.2. Pyravate Kinase Them are both similarities and differences in the dual divalent metal ion environment it muscle pyruvate kinase [381. M i l e Mn2+ has been demonstrated in d ESR studies to bind and coordinate to oxalate, a structural analogue of the enolate of pyruvate, two Mg2+ions are observed in the pyruvate kinase substrate complex. The distance between the two Mg2+ ions (5.0-5.1 is very similar to that observed between Mg"+ and &In2+in the E. coli PCK complex. One Mg2+ cation coordinates oxygen atoms from the a-,13- and y-phosphoryl groups of ATP, resulting in the ATP adopting a curled structure. The coordination sphere of the nucleotideassociated Mg2+ cation is completed by three water molecules. The second Mg2+ cation is coordinated by Glu-271, Asp-295, bidentate coordination to oxalate, as well as an oxygen atom from the y-phosphoryl group of ATP and a water molecule. Given that both Mg2' cations have all-oxygen coordination environments, unlike the situation with E. coli PCK, it is not surprising that Mg"+ binds at both sites. While it is clear that Mn2+is capable of binding at this site [1591, it is not clear which of M$+ or Mn2+ binds here with greater affinity. Both PCK and pyruvate kinase utilize the second divalent metal ion both as a bridge between the nucleotide and second sub.. strate molecule and as an. additional source of electron withdrawing capacity to increase the electrophilicity of the y-phosphoryl group, and therefore its susceptibility to nucleophilic attack. These metals, along with either K' or Na' that also coordinate to the terminal phosphoryl group, may help to screen the negative charges between the two substrates during catalysis.

A)

NA Polymerases A number of polymerases employ two metal ions in catalysis. Structural information is available for some of these, including bacteriophage T7 DNA polymerase bound to two Mg2+ ions, a primer template, and nucleoside triphosphate [87], As noted previously, several of the DNA polymerases belong to that category of enzyme that utilizes two metal ions in catalysis, usually with respect to their nuclease activity.

78

MATTE AND DEL

It is most likely that the two metal ions play a role in both substrate orientation and in charge neutralization of the transition state during catalysis. A metal ion located in the "A" position, most likely Mg2+,would serve to activate the S'-hydroxyl group of the primer strand. Taq DNA polymerase is found to possess three metal binding sites within the nuclease domain, two for Mn2' and one for Zn2' 1911. The distance of 5 between one of the two Mn2+ ions and the Zn2+ion suggests these are the important catalytic pair. In this case, two of the metal ions are thought to participate in a number of functions, including stabilization of a pentacovalent intermediate, generation of an attacking hydroxide ion, and departure of the 3' oxyanion. The exonuclease domain of E. coZi Klenow fragment contains two bound metal ions: a Zn2+ at site A and an Mg2+ at site B "ll. the Zn2+ and Mg2+ ions show the expected tetrahedral and octahedral coordination, respectively. The Zn2+ ion was found to bind in either the presence or absence of nucleotide, while Mg2' ion binding was nucleotide-dependent. Both metal ions are proposed to have distinct roles in catalysis, with the Zn2+ ion facilitating formation of an attacking hydroxide ion and the Mf;lL+ having a role in transition state stabilization and promotion of the leaving group. It is worth noting that both kinetic 11601 and structural evidence 1711 have recently demonstrated that adenylyl cyclase (E.C. 4.6.1.1) uses two divalent metal ions in catalysis. In the complex containing two M 2 + cations, one Mg2+cation (metal A) is tetrahedrally coordinated by the a-phosphoryl group of an ATP analogue, Asp396, Asp-440, and a water molecule. The second Mi2+ (metal €3) is octahedrally coordinated by the a-,0- and y-phosphoryl groups of an ATP analogue, Asp-396, .Asp-440, and the backbone carbonyl of Ile-397. With crystals soaked in combinations of Zn2* and Mn'+, Zn2+ is found to bind site A and Mn2+ at site B, with tetrahedral and octahedral coordination environments, respectively. Similarly,Mgz+occupies site A and Mn2+site B when crystals are soaked in Mg2+- and Mn2+-containingsolutions. Interestingly, adenylyl cyclase is structurally related to the "palm" domain of DNA polymerases and may operate with a similar dual divalent cation-based catalytic mechanism.

A

4.3. p21raS The p21 protein product of the ras gene is among the most thoroughly studied members of the G-protein family. This family consists of such members as small p21ras-likc proteins, the beterotrimeric G-proteins, the Rab proteins associated with vesicular transport, and the elongation and initiation factors associated with protein synthesis. The ~ 2 1 protein ' ~ functions in signal transduction through its activity as a molecular switch. In the presence of GTP, the protein is in the state; hydrolysis of GTP to GDP, a reaction dependent on the presence of +, results in the protein adopting ates is accompanied by conformastate. The conversion between on and tional changes in the protein that influence its ability to interact with various efyector proteins. ~mportantly,a number of human tumors have been found to contain ras

M A G N ~ S I U ~ - A ~ T I V A TNZYME SYSTEMS

79

oncogenes that contain mutations most commonly at either Gly-12 or Gln-61, resulting in the protein being locked in the state, i.e., the inability to convert bound GTP to GDP. A number of structures have been reported for p21ras,including its complexes with GppNp/Mg2'- [104,161], GDP, and GppCp [162], R and S diastereomers of caged GTP and dGppNp [163], as well as mutants of p21rasbound to substrate or substrate analogues [109,162]. As well, time-resolved Laue diffraction methods have been applied to study both the prehydrolysis GTP-bound structure as well as structural changes leading to the formation o f GDP [103,1641. The p21rasprotein possesses intrinsic GTPase activity that requires the presence of a divalent cation, either Mg2+ or Mn2+. The Mg2' cation is coordinated by the pand y-phosphoryl groups of GTP, two water molecules, Ser-170w,and Thr-350H[1041. It is clear that one divalent cation is sufficient for GTPase activity. While a variety of functions for the M g + cation have been suggested L1041, the current view is that the metal assists the process of substrate-assisted catalysis. In this mechanism, the yphosphoryl group acts as a general base, abstracting a proton from a nearby water molecule that in turn attacks the y-phosphoryl group via nucleophilic attack, resultwith ing in formation of GDP. Structural analysis of a complex of p21raS:p120GAP ~D~-AI~~-M suggests g 2 + that phosphohydrolysis proceeds through a pentacovalent arently no significant transition state that is associative in nature [loll. There is change in Mg2+ position or coordination in p21ra8::p120CAP P-AIF, complex compared with p21""' in the presence of GTP-analogue alone. Analysis of the crystal structures of p21ras(G12P)in the presence of either Mg2" n2+ and GppCp at 1.8-A resolution revealed very little difference in sLmcture [14lI--this despite significant differences in catalytic activity with either Mg2+ or Mn2' (ha&, = 2.1 and 13.5 with Mg2+ or Mn2", respectively), representing an overall acceleration in the presence of Mn2+of 4.4-fold. Comparison of the pKa values for the GTPase reaction with Mg2+ (2.9) vs. Mn2+ (3.4) suggests that Mn2' accelerates the reaction by shifting the pKa of the y-phosphoryl group by nearly 0.5 p C1411. Linearity of the Brdnsted plot demonstrates a direct relationship between the pK, of the y-phosphoryl group and the rate of the GTPase reaction. There is apparently no change in the reaction mechanism of GTP hydrolysis in the presence of Mg2+ vs. Mn".

Ribozymes are not proteins; rather, they are Mg"-dependent enzymes made of RNA. However, they catalyze hydrolysis and transesterification reactions of phosphate diesters or RNA and thus are included in this chapter. Ribozymes fall into different groups, including the group I self-splicing introns, hammerhead ribozymes, and hairpin ribozymes. In each case, divalent cations, especially Mg2+,have been shown t o play a functional role by contributing to the three-dimensional RNA fold, as well as also contributes to the kinetic and participating more directly in catalysis. I%$+

80

MATIE AND DELBAERE

thermodynamic folding pathway of ribozymes, as demonstrated with the ~ e t r a h y ~ group r ~ a I ribozyme ([1651 and references cited therein). Two fully hydrated Mg"+ ions have been identified structurally within the 160nucleotide P4-P6 domain of the Tetrahymena thermophilu group I intron [1371. The two Mg2+ binding sites are associated with G and U bases, although the metal interacts only through an outer sphere mechanism. These two sites are apparently of structural, as opposed to catalytic, significance. Studies on the hairpin ribozyme demonstrate that substitution-inert Co(NH3)F is able to replace Mg2+ in catalysis, although it is less e-ffective [166]. This result implies that catalysis operates using an outer sphere mechanism and that direct Mg2"-RNA interactions are not necessary for catalysis. The functional role of M 2 + in this case may be through electrostatic charge neutralization, or through correct orientation of the catalytic machinery via hydrogen bonding with water molecules in the Mg2+ hydration sphere.

Isocitrate dehydrogenase (E.C. 1.1.1.42)catalyzes the formation of a-ketoglutarate, PH, and CQ2 from isocitrate and NADP. The enzyme from E. coli is inactivated by phosphorylation of Ser-1J 3, which can be mimicked by the mutants Ser-113-Asp and Ser-113-Glu.The mutations inhibit the ability of isocitrate to bind to the enzyme. Crystal structures of the Ser-113-Asp and Ser-113-Glu mutants demonstrated minimal conformational changes within the protein, leading to the suggestion that electrostatic effects are primarily responsible for inactivation by phosphorylation [1301. The structure of the Ses-113-Glu isocitrate dehydrogenase mutant in complex with isocitrate and M$+ has been determined at 2.5-A resolution 11301. In this structure, the single Mg2+ ion is octahedrally coordinated by the a-carboxylate and 0 - 7 of isocitrate, Asp-283', Asp-307, and two ordered water molecules. The primary function of the Mg2+ cation in the catalytic mechanism has been suggested to be neutralization of the negative charge that develops on the hydroxyl group of isocitrate in the transition state. Structures have also been obtained with isocitrate dehydrogenase and nicotinamide hypoxanthine dinucleotide phosphate (NHDP), in place of NADP, as well as with Ca2' in place of MgZf [1311 (see Fig. 2). Binding of either NHDP or Ca2' results in small pertubations of the enzyme active site. Substitution of Ca2+ for Mg2' does not significantly influence the binding of substrates at the active site, although it reduces the overall enzymatic rate by 2.5 x m i l e both Ca2+ and Mg2+ both bind to the enzyme, Ca2+ has a tetragonal bipyramidal coordination environment with eight ligands, while Mgz+ binds within an octahedral environment [1311. Comparison of the Ca2+ and Mg2+ structures shows a displacement of the Ca2' by 1.4 A away from the Mg2+position. The net effect of Ca'+ coordination is to increase the hydride donor-acceptor distance by 0.55 A and at the same time alter the attacking and dihedral angles. Interestingly, Cd", which has an ionic radius similar to Ca2'

MAGNESlU~-ACTlVATED NZYME SYSTEMS

81

FIG. 2. Stereodiagram of the Mg2+ binding site in isocitrate dehydrogenase [131]. There is octahedral coordination of the Mg2+ ion by one of the a-carboxylate oqgen atoms and 0-7 of isocitrate, by one oxygen atom from each carboxylate of two aspartate side chains of the protein, and by two water molecules.

but prefers octahedral coordination like Mg2+,activates isocitrate dehydrogenase to a similar extent as Mg”. This result suggests that it is not the size o f the metal that is causing the structural perturbation and reduction in enzymatic activity, but rather the change in metal coordination environment.

4.6.

Xylose lsomerase

Xylose isomease (E.C. 5.3.1.5) catalyzes the interconversion of xylose and xylulose, and represents one step in the utilization of xylose as a carbon source by bacteria. Xylose isomerase also acts on glucose to yield fructose, an important process in the food industry. Unlike some isomerases, which utilize proton transfer mechanisms involving protein side chains, xylose isomerase utilizes two metal ions to promote a hydride shift from C-2 to (2-3 o f the substrate. In the structure of xylose isomerase from Streptomyces olivochromogenes with bound glucose or 3-O-methyl-n-glucose, two Mg2+ ions are bound on either side of the sugar and are separated by 1.8 [80]. In the native model magnesium 1 is coordinated by Glu-180, Glu-216, Asp244, and Asp-286 with a nonideal geometry. Magnesium 2 is octahedrdly coordinated by Glu-216, Asp-256, His-ZlSNE, the bidentate ligand Glu-254, and a putative hydroxide anion.

82

There are hundreds of known structures with Mg2+ as an essential component of the active sites of enzymes. In this chapter, we have included a representative subset of these proteins to show how this metal cation contributes to the various functions of these proteins. For the exact details of the interactions of Mg2+ with various proteins, the reader is referred to the following databases: Protein Data Bank-http:llw~.rcsb.or~lpdbl rosthetic Groups and Metal Ions in Protein Active Sites atabase (Promise)http:l~ioinf.leeds.ac.u~promise ~ e t ~ l o p r o t e i Database-http://met~lo.scripps.edu ns ~ a ~ e s i uismfound as an important component of a broad variety of enzymes, as compared to most other metals except calcium. The high concentration of magnesium in most biological systems likely has contributed to its selection in the function of many enzymes, where it can assume different roles, e.g., stabilizing high-energy substrate conformations, polarizing substrates for nucleophilic attack, orienting substrates for reaction, and so forth. It is also important to note that Mn2+ has often been used in place of Mg2+ for many in vitro enzymatic and structural studies, whereas in vivo Mg2 ' is often the biologically relevant metal cation; thus, one must be aware of the possible distinction between the in vitro and in vivo situations.

NTS This work was supported in part by Medical Research Council of Canada Operating T-10162 to LTJD.

A ADP AGS

ANIP P-PNP

AP5 ATF ATP

Azl BCL

adenosine 5 '-diphosphate phosphothiophosphoric acid-adenylate ester adenosine 5'-monophosphate 5'-adenylylimidotriphosphate bis(adenosine1 5'-pentaphosphate adenosine 5'4 p, y-difluororomethylene)triphosphate adenosine 5'-triphosphate 3 '-azido-3'-deoxythymidine 5 '-diphosphate bacteriochlorophy~a bacteriopheophytin cytidine 5'-monophosphate

MAGNESIUM-ACTIVATED ENZYME SYSTEMS

83

dia~nobenzophenone-phosphonoamidate-~r P 2 ’,3‘dideoxyadenosine 5 ’-triphosphate 2 ’,3’-dideoxycytidine 5‘4riphosphate 2 ’-3’-dideoxy guanosine 5’-triphosphate 2’-deoxythymidine 5’-diphosphate fructose-1,6-bisphosphate methylpiperazinoforskolin FECP fructose-6-phosphate F6P forskolin FOK guanosine 5‘-&phosphate GDP guanosine 5 ’-monophosphate GMP phosphoa~nophosphonicacid-guanylate ester GNP glycerol-3-phosphate G3P guanosine 5’-(p, y4mido)triphosphate GPPNP guanosine diphosphate monothiophosphate GSP guanosine 5 ’-triphosphate GTP guanosine 5’-0-3-thiotriphosphate GTPyS hadacidin A N-hydroxy-N-isopropyloxamic acid HO 2 ’(3’)-0-(N-methylanthraniloy1)-ADP “T-ADP nicotinamide adenine dinucleotide phosphate NADP dihydronicotinamide adenine dinucleotide phosphate NADPH NADP’ [2’-monophosphoadenosine5’-diphosphoribosel NAP nicotinamide-(6-deamino-6-hydroxyadenine)dinucleotide NDO phosphate nucleoside diphosphate NDP nicotinamide hypoxanthine dinucleotide phosphate NHDP 5-0x0-Z-norleucine ONL 1-cc-pyrophosphoryl-2rx,3a-dihydroxy-40-cylopentane PCP methanol-5-phosphate phosphoenolpyruvate carboxykinase CK PDB Protein Data Bank PEP phosphoenolpyruvate 3-phosphoglyceric acid 3PG Pi hydrogenphosphate ion PPi pyrophosphate PR-ANIP cyclohydrolase N1-(5’-phosphoribosyl)adenosine-5’-monophosphate cyclohydrolase PRP a-phosphoribosylpyrophosphoricacid

DABP-GTP DAD DCT DG3 dTDP FBP

RUB TPP TTP u-10 UMP

ribulose-1,5-bisphosphate thiamine pyrophosphate thymidine 5 ’-triphosphate ubiquinone-10 uridine 5 ’-monophosphate

84

MATTE AND DEL

P14 5 '-adenosyl)P' 45 '-uridy1)pentaphosphate xanthosine 5 '-monophosphate

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ivision of Physical Chemistry 11, Kemicentrum, Lund University, S-22100 Lund, Sweden

94

Aims and Scope Coordination Chemistry of Ca2' Bioinorganic Role of C g " Homeostasis and Metabolism Distribution of Ca"-Binding Proteins Introduction to Systems Chosen for Discussion

94 95 95

2. ~ N Z ~ ~ S ~ P ~ WIT O T E I ~ S 2.1. EF-Eland Proteins 2.1.1. Calmodulin 2.1.2. S l O O Proteins .1.2.1. Calbindin Dgk s.2.2. sloop 2.1.2.3. Calcydin 2.1.2.4. Psoriasin 2.2. Annexins 2.2.1. Annexin V and some General Structural Features 2.2.2. Annexin I and Annexin I1 2.2.3. Membrane Binding 2.2.4. Annexin III 2.2.5. Annexin 2.3, C2 ~ o m a i n s 2.3.1. S ~ ~ p t oI ~ ~ i n

100 100 102 109 110 110 114 114 115 117 117 119 120 121 121

1.1. 1.2. 1.3. 1.4. 1.5. 1.6.

95 97 99

"resent address: Department o f Biochemistry and Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, oxford OX1 3QT, United Kingdom.

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2.3.2. Phospholipase CF1 2.3.3. Cytosolic Phospholipase A2 2.3.4. Protein Kinase C 2.4. EGF-like Modules 2.5. Lectins 2.5.1. Mannose-Binding Protein 2.5.2. E-Selectin 2.5.3, Tetranectin

125 126 127 128 133 134 137 138

3. ENZYMESPROTEINS WITH UNKNOWPJ STRUCTURE

139

4. STRUCTURE-FUNCTIONRELATIONSHIPS

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5. P E ~ P ~ C T r ~ S

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6. Ca2+AND PROTEIN-RELATED INTERNET RESOURCES

141

ABBREVIATIONS AND DEFINITIONS

141

REFERENCES

142

1. INTRODUCTlON

1.1. Aims and Scope Our primary aim in this chapter is to present a summary of Ca2'-binding proteins from a structural perspective. Of course, any review that attempts to cover all Ca2' binding proteins will be a Sisyphan labor due to the enormous number of new Ca2+binding proteins that are continuously being identified. Even if we limit our discussion to those that have been characterized structurally, a search of the approximately 12,000 protein structures in the Protein Data Bank yields about 1700 hits for "calcium". Though it can be reduced further to those in which Ca2' is known to play an integral role, one is still faced with a daunting task. We have therefore chosen to concentrate this review on some recent studies of Ca2+-bindingproteins and discuss a limited number of representative systems. Our choice of systems to discuss has of course been influenced by our own research interests, but we hope that the broad ill give the reader a picture of the field as it is assortment presented in this chapter w progressing today. For more general reviews of Ca2*-bindingproteins, we can recommend two books. The first, Guidebook to Ca2'-Binding Proteins [I], is written from a biological perspective and gives brief introductions t o many Ca'" proteins. The other, Calciunz as a Cellular Regulator 121, gives more in-depth coverage of the major Ca2+ proteins and receptors from a biochemical perspective. Other reviews are available that cover various aspects of Ca2' proteins, including CaZf-binding[3], Ca2' -protein structures [4,51, and regulation of activity by Ca2' [61.

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1.2. Coordination Chemistry of Ca2'

Ca2+is preferentially coordinated by oxygens, usually from aspartic acid and glutamic acid side chains, as well as the carbonyl oxygens of the peptide backbone. The number of ligand atoms bound to Ca2+ can vary from five to eight, but the most common number is seven. In addition to oxygen ligands contributed from the protein, water molecules often function rn ligands. Though usually only one or two ligands are contributed by water, there are cases of Ca" being coordinated by three or even four water ligands, as is the case for proteinase K [71. Ca2' binding constants can vary up to seven orders of magnitude from weak binders such as certain EGF domains (logK, 3) to strong binders such as thermolysin or subtilisin (logK, 10). Surprisingly, however, in a recent survey of Ca2-'"-bindingproteins, absolutely no correlation was found between Ca2' affinity and many properties of the Ca2+coordination sphere, such as net ligand charge, number of water molecules in the coordination sphere, number of protein ligands, or number of backbone protein lgands [3] (Fig. 1).Instead, it was concluded that subtler forces determine Ca2' affinity. These included polypeptide strain, surface charges near the binding site, and the amount of conformational change induced upon Ca2' binding.

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ioinorganic Role of Ca2' In order to understand Ca2+-bindingproteins, it is useful to first have some information on Ca2+and its unique properties and roles in physiology. It is well known that in addition to carbon, nitrogen, hydrogen, phosphorus and sulfur, Ca2+is one of the major chemical components of living organisms. The most conspicuous and abundant form of Ca" is the biominerals of which our skeletons are composed, mostly in the form of crystalline hydroxyapatite (Calo(P04)6(OH),).Maintenance of this store of Ca2+ is important in hindering osteoporosis and other bone maladies and thus is important for the long-term well-being of the individual. However, the immediate well-being of the individual is more dependent on the availability of free Ca'" in the intra- and extracellular CaZf stores. While much smaller in total Ca2+ mass than biominerals, they are used to supply the Ca2' used in metabolic processes, predominantly as a signaling substance. 1.4. Homeostasis and The concentration of Ca2 ' is very strictly regulated in the intra- and extracellular Ca2' stores to the nanomolar concentration range intracellularly and dlimolar range extracellularly. These concentrations are maintained by a collection of Ca2' pumps that act both to move Cia' ions out of the cell and to sequester Ca2' in specialized organelles within the cell, such as mitochondria and endoplasmic reticu-

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FIG. 1. Comparisons of the correlation between Ca2+ affinity (logK ) and various properties of es in proteins. logK is plotted vs. A) The number o€ water molecules in the ) The net charge of all ligands in the sphere. C) The total number of protein ligands. D) The number of backbone ligands. (Reproduced by permission from ref. 131)

lum. These serve as stores for Ca2+ until it is needed. It is the large concentration en these stores and the c ~ o p l a that s ~ is e x p ~ o i t ein~ t r a n s ~ e ~ i n g proteins under~oc o n f o r ~ a t i o n ~ into the cell. Many ~a2+-binding , c ~ ~ o r ~ a t ichanges o ~ a l serve to activate re~ponset o Ca2+b ~ d i n gThese s by, for example, opening target binding sites. The Ca2

this range of critical GO g substance. If it wer ~ n c t i o n would s operate ~ ~ e ~ e fino orde ~e, ing ~ ~ o t e i nan s , equally complex ~

~A LCIUMAND ITS

7

For example, one well-understood system for Ca2’ release is that of the Ca2’ release channels (CRCs), which include the inositol 1,4,5-triphosphate receptors (InsP3Rs), ryanodine receptors nd spingolipid Ca” release-mediating protein from endoplasmic reticulu ER) [8,9l. InsP3Rs are activated by InsP3, which is a second-messenger molecule. RyRs are controlled by direct coupling to voltage-o~eratedCa” channels ( ~ O C C s )or binding of Ca” itself. They ryanodine, a neutral plant alkaloid used to characterize these receptors. SC less well characterized. Three different mechanisms are used to transfer signals from outside the cell to these receptors and trigger Ca2+ release: (1) triggering Ca2+ released from the ER/SR or from extracellular space via the CCs or other channels; (2) direct allosteric interactions between the receptors and smembrane proteiiis, such as, for example -proteins; and (3) stimulation of production of s i ~ a l i n gsubstances such as Ins by enzymes linked to extracellular signals. Each of these mechanisms has its own collect of regulating proteins that act to influence act will not delve further into the details of this both positively and negatively. other types of Ca” release mechanism but instead refer the reader to recent reviews s focused on CaZChomeostasis 1101. That this is only one example of the s y s t e ~ used to control Ca2’- concentration within cells gives one an idea of the intricacies of Ca2+induced signaling and the control exerted on it at a number of levels.

rot~ins Before we discuss specific examples of Ca2’-binding proteins, we shall st brief overview of the entire range of Ca2+-bin~ng proteins. It is a dif~culttask to ~ especially since the database of sequences is catalog all C a ’ + - b i n ~ nproteins, ing at a fierce pace. However, one can attempt to get a rough estimate using the resources currently available, mostly through Internet sites specializing in s e q u e n t i ~ and structural comparisons of proteins. As a first step toward exploring the prevamotifs, we can turn to the Pfam (protein family) database run lence o f CaZ+-bind~ng by the Sanger Centre “Ill. This resource uses idden Markov ~ ~ dto ~predict l s protein sequence simila~ity and thereby function using known seque “seeds” for the search. A keyword search with “calcium” returns 26, uted over 81 families of Ca2* bin mains. Of these 81 families, most c o n t less ~ than a few hundred members. r, certain motifs tend to dominate, notably EGF-&e domai ands (2159 hits) (for t hand”, see See. also some less obvious highly, such as collagen triple-helix repeat (2384 hits) and eukary domain (3479 hits). In the case of the former, the link is apparen zation. In the latter case, certain Jxinases that bind Ca2+, such have caused this very common motif to appear as a “hit”. This pitfalls in simplistic analyses of ~atabases:even one member that m cate the entire family (whether a few or all bind calcium). In the cas also a degree of overlap of which one must be aware, e.g., 8100 is 1

MURANYI AND FINN

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subfamily to EF hands but both appear as separate listings in the search for Ca2’. However, despite the limitations, one can see that there are a substantial number of Ca2’ proteins and that their sequences are spread over a significant number of sequential motifs. We can expect these figures t o grow as the number of sequenced proteins continues to increase. To get a feeling for the structural distribution of Ca2+-bindingproteins, there are several resources that one can use. For our purposes, we have chosen to examine their distribution in the SCOP (Structural Classification of Proteins) database [121. By running a keyword search of the database with “calcium+” (all terms beginning with calcium) we obtain approximately 1700 hits. Of these, we can remove those that we know to be irrelevant, such as nucleic acids, synthetic peptides, and other nonnatural or nonprotein substances. Also, any listing in which Ca2’ appears only once in multiple structures is ignored. For example, in DNA polymerase p, Ca2’ only appears in one structure out of a total of 90. This distillation leaves 1308 proteins, which SCOP divides into five classes: small proteins, all a-helical ( d - a ) ,all P-sheet (all$), “a and P” (a/p),“aplus p” (a+ p), and small proteins (those having too little secondary structure to be included in the other categories). The alp and a + P differ in that a / p has mixed a and p structures while in a p the a and P structures are segregated. In Fig. 2, we have analyzed the 1308 hits in SCOP for Ca2’ and divided them by structural classification. In Fig. 2A, the total number of structures is listed by structural type. This result gave the unexpected result that the all-a structural class was the least populated among the four major classes. This was a surprise given the predominant place that all-a EF-hand proteins have featured in discussions and reviews of Ca“ proteins, even our own [1,2,4,5]. We can refine this analysis by examining the Ca2+-binding protein structures by sequence and by fold. In Fig. 2B, the number of unique Ca2’binding sequences is shown by structural class. This differs from Fig. 2A in that there are often multiple structures determined for each unique sequence in various states (bound to various substrates, ligands, inhibitors, etc.). For example, for the all-a EFhand protein troponin C, there are a total of 24 structures determined for four unique sequences (fromfour different species): 17 for chicken, 2 for turkey, 4 for rabbit, and 1 for human. By filtering away the effects of multiple structures on the distribution of Ca2’ proteins, we find that the all-a proteins increase in relative distribution but are still overshadowed by the all+ class. We can take this analysis a step further if we wish t o examine how many “folds” are capable of binding Ca2’ as found in the structure database. In this case, fold is taken from the classification used in SCOP. For example, all EF-hand-like proteins are a single fold, a-amylase and related proteins are grouped together in the TIM-barrel fold, etc. The result of this grouping of Ca2’ proteins according to fold is that the distribution of folds capable of binding Ca2+ is remarkably evenly distributed between the protein classes, at least for the major classes (Fig. 2C). The small-proteinclass suffers from a negative bias due to the relatively small number of entries in that class. If we normalize the distribution by fold by dividing the Ca2+-bindingfolds by all known folds as defined in SCOP, we find a relatively even distribution over all classes, even the small-protein class. As shown

+

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E 3 $

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v1

200 100 0

i

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FIG. 2. Dar diagrams showing the distribution of Ca2+-bindingproleins by structural class as defined in the SCOP database [l21. See text for description of classes. A) Distribution of Lhe total number of structures by class. B) Distribution of unique protein sequences which bind Ca2' by class. C ) Distribution by unique fold which we able to bind Ca" . D) Distribution by fold normalized to the total number of known folds for each class.

in Fig. 2D, approximately 1545% of' all protein Iblds contain members capable of binding @a2'. This means that there is no intrinsic evolutionary preference of Ca2+ binding sites for a particular structural class.

1.6. ~ n t r o ~to~Systems ~ t i ~Chosen ~ for Discussion

As mentioned at the outset o f this chapter and in light of the statistics discussed in the previous section, any attempt to assay all Ca"-binding proteins, even those with known structure, is beyond the limitations of a single chapter. Therefore, we have chosen to select several proteins as examples and discuss these in some depth. This should give the reader a variety of examples of how Ca" is used in various proteins. Our selection criteria are based on a combination of the protein's physiological significance, the amount of structural data availahle, and its uniqueness. We hope that

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MURANYI AND FiNN

no one will feel slighted if their favorite protein has been omitted. However, in the interest of space, several highly significant proteins have been omitted when a similar protein has been discussed. For example, troponin G will not be discussed due to its similarity to calmodulin. We can instead refer readers to other reviews in these cases.

The EF-hand motif is one of the most widespread calcium-bindingstructures found in cdcium-binding proteins. Indeed, entire books have been devoted just to cataloging EF-hand proteins 1131. The most recent attempt to survey and compare all known EF-hand proteins was a series of articles published between 1990 and 1993 by inger and co-workers [14-171. Even during that short span of time the number -hand proteins grew from 153 in the first report t o 229 in the fourth one. In the h a 1 article of the series, 839 EF-hand domains had been identified and could be divided into 30 families. Subsequent overviews increased the number of families to 39 [ll. Today, according to the Pfam database, the number of calcium-binding proteins sequenced to date that exhibit this motif is over 2000 [ill. We will, therefore, limit our ambitions to covering only some of the more recent results from a handful of this family of &''-binding proteins. For those interested in a larger survey of EFhand proteins, we recommend reviews more specifically devoted to them, such as asaki and Kretsinger 1131 and Celio [ll. The EF-hand motif is composed of a helix-loop-helix that binds Ca2* in the loop between the two helices. The name EF-hand originates from the structure of parvalbumin, which contains such a motif between helices E and F and their connecting loop ClSl. An illustration of this arrangement is shown in Fig. 3. The canonical EFhand sequence is composed of 29 residues in which residues 1-10 and 19-29 make up the helices, and the Ca2+-bindingloop is made up of residues 10-21. As can be seen, hydrophobic and negatively charged residues dominate the sequence. The Ca" is bound in a pentagonal bipyramidal configuration. The negatively charged side chains w e of course used for chelation of the Ca" ion, specifically residues 10,12,14, and 21, the last of which contributes both oxygens as ligands. The remaining two ligands are the backbone carbonyl oxygen of residue 16 and a water molecule, which is in turn often hydrogen-bonded to residue 18. The hydrophobic residues point inward toward a hydrophobic core. This core is the result of the fact that EF hands usually (but not always) occur in pairs with a pseudo-twofold axis of symmetry between them. This arrangement forms a stable four-helix bundle, a common fold even among non-Ci"fbinding proteins. The number of EF hands in a single protein can vary from one to eight though four is most common. Their specific functions vary widely but can be divided into two classes: signaling and buffering. Their sensitivity to changes in Ca2' concentration is

C A L C I U ~AND ITS ENZY

101

FIG. 3. A ball and stick representation of a typical EF-hand and the cartoon drawing indicating the similarity to a hand with thumb and index finger extended. Liganding residues are indicated in gray and the oxygen atoms of the ligands in black. Hydrophobic residues which contribute to the EF-hand pair hydrophobic core are indicated by an "H". (Adapted from ref. [I411

used to regulate a large number of cellular processes. The signaling proteins propagate Ca2+ signals to their desired targets. This is usually accomplished by Ca2'induced activation of the EF-hand protein followed by binding to a particular target or targets. These processes are tightly controlled by the relationship between the Ca2+ concentration in the cell and the binding affinity of the EF-hand site. This concentration can range from lo-' M in a resting cell up to several orders of magnitude higher upon release. Since they occur in pairs, there i a good deal of interaction between the hands not only in the hydrophobic core ut also in the form of electrostatic and ot contributions. This gives rise to the cooperativity often observed in their Ca2+ ing. Cooperativity is a useful property because it allows the activation of a specific El?hand pair to occur over a much smaller Ca2' concentration ran This gives an extra -hand proteins and level of control over the Ca2+-inducedactivation of different ge of Ga2+ concenpresumably allows each to be active at a distinct and specific on ~ n d e ~ s tthe ~ ~ nature i n ~ tration. A great deal of time and effort has been s and causes of cooperativity in these proteins 131. there i s still much to learn about the ~ e c h a n i s mof this level. Indeed, even the methods for analyzing cooperativ

102

M U ~ A ~ AND Y I FINN

largely revolving around the misuse of ill coefficients when attempting to quantitatively determine cooperativity betwee a’’ binding sites 1191. In many cases, perhaps even up to one-third of them, the EF hand has lost its ability to bind Ca” [ZO]. In those cases where one o f a pair o f EF hands has lost the ability to bind Ca2’, the of a single Ca” to one of the pair i s presumably sufficient to maintain the activity. However, any advantage from cooperative binding is of course lost.

The hand ~ r o t e ~ are n s often collectively referred to as the c ~ m o d u ~s~perfami~y. in Therefore, it is not surprising that any discussion of EF-hand proteins will inevitably ~ n . is mostly because it is one of the earliest eharacterbe dominated by ~ d ~ m o d u lThis ized and best studied of its kind. However, there are also many unique and important aspects that make it a continuing subject of interest. It is ubiquitous, occurring in all tes. It acts on more than 100 target proteins, thus implicat~ngit in a correingly large number of cellular processes. These include signal transduction, thesis, cell division, motility, and secretion to name a few 113,211. he structure of calmodulin is composed of a single chain of 148 amino acids ighly conserved between species. Four EF hands are d~stributedpairwise in two domains that are connected by a flexible linker. The two domains act together to bind target sequences on proteins. As shown in Table 1,calmodulin is one ofthe most intensively studied proteins from a structural point of view. The first strucLure o f Ca2+-activatedcalmodulin was determined ray crystallography and later refined to high resolution in 1988 [37,431. was found t o possess an open hydrophobic binding patch which taken together would comprise the target binding site. This structure exhibited the classical “dumbbell” form of ealmodulin with an apparently rigid helical linker between the two domains. Further structures of calmodulin from other species [32,34,361 confirmed this view and the “central helix” became dogma in the field. This gave rise to the first great puzzle of calmodulin since most ofthe target peptides known to bind calmodulin were too small to span the gap between the two domains and bind to both simultaneously. In addition, X-ray and neutron scattering studlies indicated a more compact structure of the protein [44,451, implying that the two domains could come together to bind a target peptide. It was not until the determination of the high-resolution solution structure by NMR that this puzzle was resolved. In solution, the central linker was found to be flexible 146,471; thus, the “central helix” is most likely a crystallization artifact and is instead a “‘central tether” in solution (Fig. 4). Studies on calmodulin-target complexes confirm this view. In this case, X-ray crystallography and NMR studies are in agrecment that the two domains come together to bind the target peptide. The initial structures of calmodulin~peptidecomplexes all showed similar results, i.e., that calrnodulin’stwo domains acts t o surround and bind a helical peptide [23,2%,33].As shown in Fig. 5, the hydrophobic faces of the EF-hand pair domains interact with the hydrophobic side chains arrayed on the surface o f the peptide helix.

103


TABLE 1 Calmodulin High-Resolution Structures Found in the Protein Databank" Species Artificial consensus sequence (mutant; E84KI Bovine

Bovine (mutant A84) Bovine (TRIC domain) Bovine (TR& domain)

Ligand peptide Rs20

Ca",

Structure

lvRK [221

Ca", peptide from CmI(I1

ICDM r23i

Ca2+,peptide from CamKII ~ a " , peptide from CamKD Caz+, peptide from MLCK Ca", TFP (2:l complex) Cia+?TFP (4:l complex)

lCMl [241

ca2+

Chicken (mutant A79, A801 Drosophila

Ce'+ APO Ci"' Ca2 Ca2+ CiA+

Human

Ca2+

l C ~ 1.241 4 125i 1261

1LIN 1261 1 ~ [271 ~

G

1AK8 1CMF [a91

+

Cad+,peptide from MLCK Ca"', TFP

Paramecium

Rat Xenopus

(-y+ Ca2+ APO

APO Ca2+,inhibitor W-7 Ca2'-, peptide from Ca2+pump Ca", peptide from CdM

4CLN [321 2BRM [331 lCLL [341 lCTR [351 1CLM [36l 3CLN 1371

lCFF [411

'Protein source, ligands, and PDB code are indicated.

While these calmodulin-peptide complexes helped to explain the "dumbbell" puzzle, they also helped to address a second puzzle, namely, how c ~ r n can o ~ bind to a large number of target complexes with differing sequences and still with high aslinity. The explanation offered was twofold: first, that the two domains can maneuver relative to one another to adapt to many sequences; and second, that the predominance of large flexible hydrophobic side chains, such as methionino, provided for local adaptation in the binding surface to accommodate a number of sequences. However, the peptides used in these initial structural studies were a tiny subset of all calmodulin-binding sequences. When compared directly, they show a great deal of similarity in the distribution of hydrophobic and negatively charged side chains [48]

104

ANY1 AND FINN

FIG. 4. A ribbon diagram of Ca"-loaded vertebrate calmodulin (1CLL) with the secondary structure indicated. Ca2* ions are shown in dark gray and the flexible linker is indicated with an mow. The two domains, TRIG and TR&, are also indicated.

and therefore probably are not representative of the entire collection of ealmodulinbinding sequences. 'Phis suggests that additional levels of complexity in the calmodulin-target binding interactions are needed to acco~modateall c~modulin-binding sequences. A recent structure determined by NMR spectroscopy of calmodulin eomplexed with a peptide corresponding to the CaM binding domain of rat Ca2*/C is in protein kinase knase (CaMm) has shown that the all-helical ~ ~ m o d u ltarget in fact only a subset of the eonformations of c~modu~in-binding peptides [421. The peptide adopts a helix-hairpin loop conformation with both features important for calmodu~inbinding (Fig. 6). Not only does the conformation diEer from the originally c h ~ a c t e ~ z peptides, ed the orientation of the peptide is also opposite to that observed in the original complexes. The authors conclude that in addition to hydrophobic istributioii of electrostatic charges contribute to target specificity and addition, a more direct role of the central linker in binding specificity is The mechanism by which Ca2* activates c ~ ~ o d u l was i n also the subject of debate for some time since the first structures of Ca2'-loaded ealmodulin were presented. Tho first clues were provided by the structure of the closely related protein, troponin C, in a partially Ca2'-loaded form 149,501.This led to a model apocalmo~ulin from the &'+-free domain of troponin C 1511. o f calmodulin prevented a defrnitive answer t d e t e r m ~ n a t ~by o ~N R had matured ugh to allow protein s t ~ c t u r e sof intact d@terminedto high solution. TWON p u b l i s ~ es~in~taneously ~ with NMR structures of the isolated earboxy terminal domain in both the apo and CaZ'-activated forms [29,38,39].These structures showed ed model was largely correct in that the two hydro~hobi~ sed in the apo form. ese studies also addressed one of the ~nresolve ~ u e ~ t i owith n ~ respect to between the two domains ely, whether there is signific n~ention~ use y the term "signifitarget peptides or protein between the two domain^ covalent coupling via t h ver, whether there are additional ertain level of interacti


105

FIG. 5. Two views of the complex between calmodulin and the MLCK peptide UCLD, 1251). Calmodulin is shown in light gray and the peptide is shown in dark gray.

NYI

10

N

A

I

(

I

. 6 . Two examples of the importance of electrostatic interactions on protein”protein interactions in Ca2+-bindingproteins. ositive charges are indicated in red and negat indicated in blue. A) A three- dimension^ surface d i a ~ ofa the ~ calmodulin/C complex. The peptide is indicated as a ribbon. Critical interactions between pos residues on the peptide determine the orientation of the peptide in the negatively charged ) A representation of the electrostatic surface of the “electrostatic switch of 8yt I.” 8uccesi~ebinding of Ca2+ ions converts the protein fro having a highly positive to negative See Section 2.3.1 for discussion. (Fig. reproduced by permission from ref. surface 11571.) re 5.6 in the color insert.

direct contacts between the two domains suf~cientto exert a m e ~ s u ~ a beffect l e significant for function under physiolo~calconditions in vivo is the crux of the debate. art of the problem may be the result of misu erstanding of what is meant by e shown that for many of the ~~a~tonomous”. Previous studies functiona~aspects of c~modulin,such as Ga” ind ding, the intact rotei in acts simply as the sum of the two domains, i.e., that there is no apparent cooperativity between

CALCIUM AND IT§ E N Z Y ~ ~ §

107

the two domains [52]. The structural studies of the isolated carboxy terminal doinain showed that a single domain undergoes the same Ca2+-dependent conformational changes as the intact protein, indicating that these conformational changes also occur independently of domain-domain interaction [29], Studies of intact calmodulin indicate that while the rigid dumbbell model is incorrect, there is no single alternative structure that is correct. Rather, results from NMR relaxation [471, SAXS [45],and, most recently, paramagnetic NM r531 all indicate a flexible orientation between domains with a radius of gyration intermediate between the extended dumbbell and a compact domain-domain complex. Despite this flexibility, however, one cannot ignore the fact that the two domains are covalently linked by the central tether. There is no invisible barrier preventing forces such as long-range electrostatic interactions from occurring between the two domains. For example, a recent paper in which fluorescence energy transfer i s used to measure the apparent separation between the opposite ends of the linker connecting the two domains fkds that this distance decreases from 37 to 31 A upon calcium binding [541. A “direct structural coupling” is offered as explanation for this effect despite the fact that the apparent distance is still 31 A. A more likely explanation is that calcium binding reduces the eleGtrostatic repulsion between the two negatively charged domains so that it i s less energetically unfavorable for them to approach one another. Other evidence of domain-domain interaction has been presented in the form of mutagenesis, calorimetry, and proteolytic footprinting studies Ki5-591, However, these interactions me generally small, with energies on the order of 1 kcal/mol, and difficult to distinguish since the Ca2+ affinities of the respective domains lie too close to enable saturation of one domain without partially saturating the other 1571. Unfortunately, “structural interaction” is usually inferred from these effects though no direct structural evidence is presented. While structural interactions are an intuitively appealing explanation, several alternative explanations could be offered to explain the interactions. The most likely explanation for observed differences between individual domains and intact calmodulin is simply that they are due to the increase in the effective concentration of the two domains with respect to one another by linking them together, analogous to the “chelator effect” or “law of mass action” [48]. Two plausible models for effective concentration of the domains relative to one another are shown schematically in Fig. 7. In Fig. 7A a highly simplified model discussed in an earlier work 1481 is shown. In this case, one domain is considered fixed and the other occupies a spherical volume limited by the length of the linker. A simple calculation of the effective concentration (C,,) of the domains relative to one another is then given by:

where V is the volume, N,, is Avogadro’s number, and r is the distance between the domains. Using 30 for r, then C,f{ of the domains relative t o one another is 15 mM. A second, probably more realistic model for calmodulin is shown in Fig. 7B. In this

108

~ U ~ A N AND Y I FINN

B

FIG. 7. Two simple models for estimating the effective concentration of the two domains of calmodulin for one another. A) A model of two spheres coupled by a freely rotating linker. B) A model in which the orientation ofthe linker is fixed with respect to one of the domains such that the other is free to move within a conical segment of the sphere.

case the linker is considered to be fixed with respect to the first domain as is the case relative to the TRIC domain of calmodulin. The volume that the domain could occupy would then be limited to a conical segment of the sphere and its volume would be given by:

where 0 is the angle of the apex of the cone relative to the central axis. Evidence from the solution NMR structures of intact calmodulin indicate that the angle defining this cone could be quite large, perhaps up to 90" /391. owever, the evidence is imprecise and it is unlikely that all conformations are equal opulated. However, by taking 90" as the potential maximum angle for this model, the cone would resemble a half-sphere and the effective concentration would be twice that from the whole-sphere model, i.e., . This is then the minimum concentration of free domains one would need to use to co~pensatefor concentration effects when comparing results for free domains studies, where directly with those from intact calmodulin. However, except for millimolar concentrations are standard, all studies on free domains are carried out at concentrations several orders of magnitude lower. It has been demonstrated for calmodulin as well as cdbindin Dgk that simply increasing the protein concentration exerts a negative effect on Ca2* binding [601. This effect is attributed to electrostatic screening, which exerts effects over long ranges. We suggest that these concentration effects are not limited to Ca2+binding studies but could exert effects in many other studies that attempt to address the domain-domain interaction question. A similar ~~~

CALCIUM AND ITS E N Z Y ~ ~ S

109

effect has even been observed in vivo in which individual calmodulin domains were able to compensate for a calmodulin “knock-out” mutation in yeast but only when the domains were overexpressed six-fold over the normal level of calmodulin 1611. This directly shows the importance of the effective concentration of the two domains on nonadditive effects, even in a physiologically relevant environment. A resolution to this debate about the nature of interdomain interactions will have to await high-resolution structural evidence of an interdomain complex, if such exists. Indeed, it is unlikely that calrnodulin exists in a single conformation at equilibrium. NMR structures show that the flexibility of the tether between the d o ~ a i n s allows for conformational heterogeneity in the interdomain orientation [46,471. Even within individual domains, there is evidence from NMR relaxation and NOE measurements that indicates structural heterogeneity, especially in the less than fully calcium-loaded states 162,631. In summary, calmodulin continues to retain a prominent role in research on calcium-bindingproteins.New aspects of its structure and function are revealed almost on a daily basis. owever, each new insight yields additional questions, and despite intensive efforts many of these questions remain unanswered. In addit mentioned above, one could add less well understood questions such as: t u r d differences there are between the calmodulin-peptidecomplexes studied to date and the as yet unrealized goal of a structure of a complex of calmodulin and an intact target protein? Is the interaction of apocalmodulin with certain target peptides functionally significant or only an artifact? Is there a consensus sequence “code” among all of the hundred calmodulin-bindingsequences identified to date? The list goes on. 2.1.2. 8100 Proteins

The S l O O proteins are one of the major Subfamilies in the EF-hand superfamily. These proteins have several unique characteristics that distinguish them from other subfamilies of EF hands. They all possess two EF hands. The first EF hand contains an insertion of two residues, which causes a rearrangement of its conformation so that it is turned “inside-out” and the CL2+ ion is bound p r e d o ~ n a n t l yby backbone carbonyl oxygens rather than side chain carboxyl groups. This modified binding site is also called a “pseudo-EF hand”. Most members of the S l O O family form h e r s , whether hetero- or homodimers. The naming scheme for S l O O proteins has been particularly plagued by an overabundance of names, with some members having more than eight different names in the literature 1131. There have been attempts to reform the naming convention of the S l O O proteins from their present naming system ( S ~ O O N calcyclin, , Sloop, calbindin, D9k, etc.) to a new “genetic” system (S100A1, S100A6, SlOOB, SlOOD, etc.) 113,641. owever, this has not been universally accepted, perhaps because the new names offer no clear descriptive advantages over the old ones. It seems instead to have potentially increased the level of confusion, especially in cases where proteins have “switched” names. For example, the old SlOOD is now SlOOA5 whereas calbindin D,, is now S100D. The name SlOO was originally given to what is now Sloop and simply means that it is partially soluble in 100% saturated ammonium sulfate solution [65].

110

~ ~ R A N AND Y I FINN

Therefore, we will use those names most prevalent in the present literature. A summary of these is presented in Table 2. 2.1.2.1. Galbindin D Q k 9k is unique among the SlOO proteins in that it is monomeric. It is the most thoroughly studied of the S l O O family from a structural perspective. This is predominantly due to the fact that, like calmodulin, it has served as an excellent model system for biophysical studies of c~cium-bindingproteins. It has been especially popular for NMR studies due to its relatively small size and high stability. The structure of calbindin D g k has been determined both by X-ray crystallography [81,85,861and NMR spectroscopy in the Ga2+ form 1821 as well as in the apo 1781, (Cd"), and (Cd2'), forms 1791. ~nfortunately,while calbindin Dgk is extremely well studied structurally, little is known for sure about its physiological function. Unlike most other EF-hand proteins, even other S l O O proteins, it undergoes no significant conformational change upon binding to calcium. Thus, a role as a calcium second messenger i s highly unlikely. It is most likely that calbindin plays a buffering role in maintaining calcium homeostasis. owever, despite the fact that Ca2+appears to have little effect on the structure, ist physiologically relevant complexes that exhibit significant conformational changes. However, when comparing conformational changes between apo and Ca2'bound states, this is not necessarily the most relevant comparison from a physiological perspective. Given the high concentration of Mg" in vivo and the lower but still significant affinity for some Ca2+-bindingproteins for MgZ+,it may be more relevant to compare Mg2+ and Ca2+ structures. Recently determined structures of Mg2+ and &In2+ complexes of calbindin D9k show several interesting features [831. For example, at physiological concentrations of Mg2+ and Mn2+ only the second EF hand is occupied by an ion; the first site remains in the metal-free state. Also, the ion binding causes an alteration in the helix packing in the second EF hand, in stark contrast to the lack of effect observed for Ca" binding. Thus, calbindin may indeed have a significant conformational change that has been previously overlooked due to the fact that we have been looking at the wrong "C$+-free" state. In addition, ME;"+ and CaZ+exhibit negative allostery. like calbindin DSk, is a small acidic EF-hand pair with a molecular weight of' about 10,000 daltons. However, instead of being monomeric, it can exist in a number of dimeric forms, both heterodimeric and homodimeric. Thus, though calbindin Dgk is more thoroughly characterized than S l O O , the latter serves as a better paradigm for the S l O O family since most exist in dimeric form. For Sloop, these dimeric forms are termed SlOOa and SlOOb, respectively, thus adding another layer of confusion to the S l O O naming system. Sloop interacts with a number of target proteins such as cytoskeletal elements tau and MAp2, actin-binding proteins caldesmon and CAP-Z, and can inhibit phosphorylation of protein kinase C targets such as GM43, p53, and neurogranin [871.


111

TABLE 2 A Summary of the SlOO Protein Family. Earlier Naming Standards Are Compared to a Recently Proposed “Genetic” Naming Standard. Where High-Resolution Structures Have Been Determined, these Are Indicated along with the Ligands or Metal Ions Bound in the Structure

Name(s)

Ligand

Genetic Name

Structures

~-

s100, s10031 SlOOL, GaN19 SlOOE Calvaseulin, P9Ka, 18A2, pEL98,

SlOOAl SlOOA2 SlOOA3 SlOOA4

SlOOD Calqclin, 2A9, PRA, CaBP, 5B10

SlOOA5 SlOOA6

Psoriasin

SlOOA7

Calgranulin A, CFAg, MPRS, p8, MAC387, 60B8Ag, Lm,CP-10, MIF, NIF Calgranulin B, GI?&, MPR14, p14, WC387, 60BSAg, LlAg, CP-10, MIF, NIF Calpactin light chain, p l l , CLP11, p10, 426, Ca[ll SlOOC, calgizzarin Calgranulin C, p6, CAAF1, CGRP SlOQA13 Sloop, NEF, SlQQ

SlQOA8

APO

Ca2 Ho3 Ca2+ Ca2+ & Zn2’

‘

+

lCNP [661 2CNP I671 1A03 [681 lPSR I691 2PSR 1701 3PSR 1701

SlOOA9

SlQOAlO SlOOAll S100A12 SlQOA13 SlQOB

Annexin I1 peptide

1BT6 1711

APO

lCFP [72] lSYM [731 1B4C [741 l UW0 [751 lQLK [761 1 MHO [771 1CLB [781 lCDN [791 31CB [SO] 41CB 1811 2BCA [82] 51CB 1831 6ICB 1831

Ca2+

Calbindin Dgk, CaBPSK, calbindin 3, CALBS, IcaBP SlOOD

APO (Cd”),

tca2++)2 Mg2+ Mn2+

112

MURANYI AND FINN

'PABLE 2 (continued) Name(sj

Genetic Name

Calbindin D,k (mutant A15D, P20G, P43M) Calbindin Dgk (mutant A14 A, A15D, P20G, N21A, P43M)

CALB3, SlOOD CALB3, SlOQD SlOOP

s-loop

Ligand

Structures

(Ca2+)z

1BOC [841

(Ca2+),

lBOD 1841

Profillagrin Trychohylin Repetin The other major difference between calbindin D,,, and Sloop is that Ca2+bindk g causes a significant conformational change in the latter. This conformational

change produces an increase in the hydrophobic surface area, much like the case with chodulin. However, the details of the conformational change differ substantially from that of calmodulin. A number of high-resolution NMR structures are available for the apo forms from rat 173,741 and cow C7.21. This is also true for the Ca2+-loadedforms of the rat 1761 and human [751 proteins. In addition, an X-ray crystal structure of the Caz+-loadedbovine protein has also been determined E771. Taken together, these structures give a detailed view of the conformational change that Ca2' induces in Sloop. As shown in Fig. 8, the dimer interface in both the apo and Ca2" forms is an X-type four-helix bundle made up of the first and fourth helices from each monomer. The major conformational change upon Ca" binding involves a change in the loop. These conformational position of the third helix and the second (%?-binding changes open a hydrophobic binding site composed of residues from the hinge region, helices I11 and N,as well as the carboxy terminal extension at the end of helix N. The strudures confrmed previous predictions that the hinge region, the second Ca2'binding loop, the carboxy terminus 173,881 as well as helix 111 [89] are involved in the structural rearrangement. However, the structures were not identical to one another. The structures of rat and bovine apo-S100p differ substantially in several places. For example, the angle between helices I1 and III differs between the two structures by 75" and the angle between helices I11 and N differs by 53", despite precision ranges in the individual structures of only a few degrees. Since the two proteins only differ by four amino acid replacements, this leads to some questions about the accuracy of the stiuctures [67,89], A more highly refined structure of the rat protein using more powerfkl long-range dipolar coupling restraints has reduced the helix angle difference to 30" for the angle between helices I11 and IV [741. However, the remaining difference is still significant,suggestingthat a reexamination of the bovine structure might also shed light on these and other differences observed between the two structures.


113

FIG. 8. Ribbon diagrams of SlOOB illustrating the Ca2+-induced conformational change. A major part of conformational change involves movement of helix 3 (black) relative to the rest of the protein. A) Apo SlOOB 173 I (PBD code 1SYM) 13) Ca2+-loadedform [761 (PDB code 1QLK).

While the characterization of conformational changes in SlOOf! has been a very active area of investigation, studies of its hnction have been less so. Structural studies of SlOOf! in complex with target proteins have only just begun. However, preliminary NMR studies of the SlOOp in complex with a binding peptide from p53 have been successful in outlining the interaction surf'ace on Sloop. As had been predicted 173,88,891, the interaction surface is localized to the same area that undergoes the conformational change induced by CaZ+,namely, the hinge region, the 6terminal loop, and helix I11 [go].

114

~

U

~A N D ~ FINN ~ Y

2.1.2.3. Calqyclin Calcyclin (SlOOA6) is an SlOQprotein closely related to Sloop. It has been identified in a number of tissues, including lung, heart, platelets, and smooth muscle. It is overexpressed in some tumor cells such as those in leukemia, neuroblastoma, and melanoma. It has been shown to bind to several proteins in a Ca“-dependent manner, including anncxins 11, VI, and XI, glyceraldehyde-3-phosphatedehydrogenase, and caldesmon [ll. The first structure of the apo form of calcyclin was determined to low resolution by N in 1995 1661. It showed that calcyclin has a similar X-type four-helix bundle st e t o Sloop. However, just as with the two Sloop structures, there were differences, especially in the region around helix 111. This was less surprising due to the lower sequence homology calcyclin has to SlQOpproteins than they have to one another, but in light of the discrepancies in Sloop structures and the low resolution of the calcyclin structure, questions about its accuracy were also raised. More surprising was the slmcture of the Ca2+ form, also determined by NMR [681. Rather than showing a conformational change, as expected from similarity to Sloop, it showed little difference in structure from the apo form. This result is in apparent conflict with functional studies of calcyclin, which indicate that Ca2+binding induces exposure of a hydrophobic binding site [91,92], similar to the case for Sloop and calmodulin. Calcyclin’s behavior rather reflects that of calbindin DSk,which has no known signaling function. This, combined with the fact that calcyclin and other S l O O proteins have been shown in vitro to possess binding constants in the millimolar to micromolar range (i.e., below the range needed to respond to Ca2+fluctuations in vivo in the nanomolar range), raises doubt about their role as signaling proteins. just as was the case €or the apo form, the structure of the Ca” form of calcyclin was not determined to high resolution. A recently refined structure of apocalcyclin shows similar results to the original structure r671. Therefore, a final answer may have to await further refinement of the Ca2’ form. However, even if the refined Ca2+ structure of calcyclin also confirms the results of the original structure, there may be an alternative explanation for both the apparent lack of conformational change and low Ca2+-bindingconstants in vitro. As suggested by Chazin and co-workers [671, a “preassociation” of apocalcyclin with a target protein may serve to increase the Ca2+ affinity. Such an effect has been observed for SlQOpin complex with a peptide from p53, but in this case Ca2+ affinity increased only by a relatively modest factor of 3 [9Q].A preassociation would require that the apo form possess sufficient conformationd flexibility to at least partially adopt the conformation amenable to target binding. Support for this possibility can be drawn from recent studies of single domains of calmodulin, which possesses folds similar to those of the S l O O proteins. In studies of single mutants with reduced Ca2’ affinity, it was shown that the proteins existed in equilibrium between “open” and “closed” forms 162,631. However, no evidence of an equilibrium between multiple conformations in calcyclin has been reported. 2.1.2.4. Psoriasin Psoriasin (SlOOA7) is one of the most recently characterized members of the SlOQ family. As can be deduced from the name, the protein was found to be overproduced in

C A L C I AND ~ ~ ITS ENZYMES

115

humans suffering &om the skin disease psoriasis. However, little more is known about its potential function. It resembles the other homodimeric members of the SlOO family that have been characterized thus far, such as SlOOB and calcyclin. Sevcral rnetal-bound forms have been characterized: a Ca'+ form; a Ca2+ plus Zn2' form; and a Ho3+ form 169,701. It possesses several unique characteristics not the least of which is loss of the ability to bind Ca2* in the amino terminal EF hand due to the replacement of the bidentate glutamate ligand with serine as well as the loss of three amino acids, one of which was an aspartate Ca2' ligand. The Zn2+ binding activity of SlOO proteins as well as other members of the EFhand superfamily had been suggested some time ago. However, only Zn2' binding to SlOOB was believed to be physiologically relevant, raising the possibility that these structure proteins are also regulated by Zn2' as well as Ca2' 193,941.The Ca"-Zn" of psoriasin offers the first high-resolution structural data on the ligand sites. As shown in Fig. 9, the Zn2' binding sites are distinct from the Ca2' binding sites and are located in the interface between helix I of one monomer and helix IV of the other monomer, thus giving two sites per dimer. The ligands are three histidines plus a bidentate aspartate ligand. When comparing the Zn2'-free and Zn2+-boundstructures, Zn2' binding is shown to cause a change in the structure of the loop of the first EF hand. While Zn2+ and Ca2' do not compete for the same sites, Zn2+ binding causes a closure in the loop, which would inhibit Ca2+ binding. However, since this site lacks the bidentate glutamate ligand necessary for Ca2' binding, this function of Zn2+ binding can only be possible for other SlOO proteins that can bind Zn"'. Based on sequence homology, the most likely candidates for potential Zn2+-binding proteins are calgranulin B (SlOOA9) and SlOOA12, and perhaps even calgranulin A (S10OA8) and SlOOB [70].

The annexins are a family of mostly intracellular proteins found in a wide variety of species and cell types (see [95-991 for reviews). Based on a large number of in vitro studies, the annexins have been associated with exocytosis and membrane trafficking, ion movement across membranes, cell growth and proli€eration,cell adhesion, inflammation, anticoagulant activity, and interaction with cytoskeletal elements (reviewed in [95]). However, it is hard to determine the physiological relevance of most oE these properties; therefore, the true biological role of the annexins remains elusive. Usually the properties described above are related to Ca2+-dependent membrane binding, which is a feature common to all annexins. Binding to Ca2' and phospholipids is associated with a consensus sequence consisting of a G-X-G-T-X,,-D/E motif [1001021. The dissociation constant (Kd) for Ca2' binding is usually in the range 25-100 pM with the exception of annexin VI which binds Ca2' with a Kd of 1pM [95]. Up to a 100-fold increase in these Ca2+ binding affinities is seen when phospholipids are present. Members of the annexin family have different requirements for membrane

116

.-.&

FIG. 9. A ribbon diagram of psoriasin illustrating the Ca” (large gray ions) and zinc (small white ion) binding-sites (YDBcode BPSR) L701.

binding, but in general high-affinity binding requires that negatively charged phospholipids, such as phosphatidylserine, be present in the membranes [1031. The sequence is composed of four or eight (annexin VI) canonical domains of approximately 70 residues, which are sometimes called “annexin repeats”. This region has high h o m o l Qbetween ~ various members of the annexin family and is referred to as the “conserved core”. The consensus sequence mentioned above is repeated in every domain of the conserved core in most annexins, except in domain 111and in domain I of annexins I and II 195,1021. The amino terminal “tail region” exhibits much more sequence variation and has sites for phosphorylation and proteolysis. It is likely that this region is important for specific functions of the different annexins. Examples of interesting interactions initiated at the amino terminus include tyrosine phosphorylation of annexin I by EGF receptor kinase, which modulates Ca2* binding affinity [104-1071. Another event involving the variable amino terminal region is the binding to annexin If of p l l , a protein related to the SlO0 family of EF-hand Ca2’-binding proteins (see Section 2.1.2) 1108-1111. This binding event can be inhibited by phosphorylation of a serine in the binding region of annexin I1 by protein kinase G [112l.

CALCIUM AND IT§ ENZYMES

117

2.2.1. Annexin I 7and Some General Structural Features

At the time of writing this chapter, 30 structures of annexins I-VI and annexin XI1 can be found in the Protein Data Bank [113]. They represent different species and differences in conditions, e.g., in the Ca2+concentration, and in some cases structures of mutated proteins have been solved. e structures of annexin V are deposited t structures solved were those of human than of all other annexins combined. Th annexin V (lAVH, 1AYR) [102,114]. They show an a-helical protein where the four conserved regions identified on the sequence level correspond to structural domains (I-IV), each made up of five 01 helices (A-E) arranged in a right-handed superhelix as shown in Fig. 10 for rat annexin V. Helices A, B, D, and E are parallel and are connected by the AB and DE loops on the convex membrane-binding side of the protein, while the connecting helix C is perpendicular to the other helices at the concave side of the protein. Domains I and IV on one hand and TI and 111 on the other hand are tightly packed, and domain pairs I+IV and II+III are related by a pseudo twofold axis. In the center of the molecule, parallel to helices A, R, D, and E in all four domains, a hydrophilic pore is seen that is associated with the presence of a Ca"-specific channel [115-1201. Two types of Ca2' binding sit type IT and type 111 (where type I refers to the Ca2' binding sites found in the -hand proteins, discussed in Section 2.1) were inferred from structures of human annexin V crystallized in the presence of CaG1, and annexin V lanthanum derivates. The type I1 binding occurs to sites where residues in the first part of the consensus sequence donate backbone carbonyl ligands and the sequentially distant but spatially close aspartic or glutamic acid donates side chain carboxylate oxygens in a bidentate fashion. In addition to protein ligands, one or two solvent water molecules also act as ligands to the Ca" ion. A similar arrangement has been reported for the catalytic domain in phospholipase A2 /1211. These sites, Cal-Ca3, are located in the turns between helices A and B in domains I, 11, and IV and are often referred to as AB sites. The type 111 Ca" binding site is weaker and the ion is only coordinated by three or four sequentially adjacent protein ligands in the DE loop, water makng up the remaining ligands. Later structures of other members of the annexin family all show a lot of overall similarity to these structures of annexin V. Mqjor differences and new findings are discussed below. Electron microscopy studies of two-dimensional annexin V crystals on phospholipid monolayers showed that the protein binds as a trirner with the convex surface, which also harbors the Ca2+ binding sites, against the membrane, without penetrating the membrane very much [122,1231. 2.2.2. Annexin I and Annexin I1

The crystal structure of human annexin I shows that although the consensus sequence is not complete, a typical type I1 Ca2' binding site is formed in the third domain (LAIN) [1241. Comparison of the structures reveals that the 6" of K178 {this is residue X in the consensus sequence) is displaced by 10.0 A compared with the corresponding residue, W178, in the human annexin V structure. This arrangement

11

NYI

'

I"

,'#

'_ _

1

i

FIG. 10. Two views of rat annexin V (PDB code lA8A) rotated by 90 degrees. Ca2" ions are indicated in dark gray.

prevents the type I1 loop from forming in domain I in annexin V, while it of can form and has a bound Ca2' ion in the structure of annexin I. domain I the negatively charged residue ending the consensus sequence is which has prevented the formation of a type I1 site in the site is formed. Ca2+ binding to domains I and I11 in a analogously to annexin I [125].


119

There has been some interest of using annexins as model compound to study the folding mechanism of large multidomain proteins (126-1301. As part of that effort, Gao et al. have determined the NMR solution structure of' an isolated domain I from annexin I (1B09) [1311. Compared to the corresponding part of the crystal structure of annexin I there are few differences, and the most notable is located where the atypical type I11 Ca2+ binding site in the AB loop is found in the crystal structure. This can be explained by the fact that the NMR structure, in contrast to the X-ray structure, was solved in the absence of Ca". These studies show that while domain I is an independently folding unit with a structure very similar to the corresponding part in the intact protein, domain II does not fold independently as a result of missing hydrophobic interactions with domain IV [127,128,131].

2.2.3. Membrane Binding

Crystal structures of annexin V in complex with Ca2+ and phospholipid head group analogues, glycerophosphoserine (GPS) and glycerophosphoethanolamine (GPE), provide the structural basis for a model of the Ca2+-dependentannexin-membrane bind) [1321. The phosphoglycerol backbone of both head groups binds similarly along W185, a residue that had previously been shown by fhorescence experiments to interact with the phospholipid acyl chain 1133,1341. It had also been shown that this tryptophan becomes exposed to the solvent upon Ca" binding [135,1361. In the crystal structure two conserved residues (G186 and T187) in the Ca' ' binding loop make intimate contact with phospholipid head group analomes (Fig. 11). Furthermore, the Ca2+ion in the AB site is directly coordinated by phosphoryl oxygen in both complexes. In the GPS complex the serine carboxylate oxygen coordinates a second Ca2+ (type 111) in the same loop, denoted the AB' site, which is approximately 8.8 A away from the first Ca'' ion. This mechanism explains why most annexins preferentially bind to phosphatidylserine-containingmembranes. In contrast to the model of membrane binding proposed on the basis of monomeric structures, as discussed above, Luecke et al. proposed a completely different model based on a hexameric structure of' annexin XI1 (1AEI) 11371. In the crystal structure two trimers join with their convex faces toward one another. This diskshaped hexamer is stabilized by electrostatic interactions and ligation of six intermolecularly shared Ca2' ions. A n additional 18 Ca2+ions are bound on the perimeter of the disk. In the center of the disk a central pore lined with charged residues is found. It is proposed that the disk-shaped hexamer completely penetrates the phospholipid membrane, where it is stabilized by hydrophilic interactions mediated by the perimetral C,? ions to phospholipid head groups which are brought in place by rearrangement of the membrane in a local inicelle-like structure. In this arrangement the central pore could potentially exhibit ion channel activity.

120

MURANYI AND FINN

A

B I

I

j

23.20

0 I 2.34 I

3m ca2+

: 2.45 : HlQ

{ 2.54

[Aj FIG.11. A two-dimensional drawing of the phospholipid head groups and their interactions with annexin V. (Reproduced by permission from ref. r1321).

2.2.4. Annexin III

The crystal structure of annexin I11 revealed subtle but interesting differences when compared with the structures of annexin V (1AXN) [1381. Most notably, the tryptophan (W190) in the calcium-binding loop in domain I11 is exposed to solvent, just as in the Ca2'-loaded form of annexin V, despite the fact that the annexin I11 structure was determined at low Ca2' concentration. Fluorescence spectroscopy experiments confirmed that the tryptophan was much more exposed in the absence of Ca" than was


121

the case in annexin V. Furthermore, no significant perturbation of this tryptophan can be seen by fluorescence spectroscopy when Ca" is titrated in, which is in contrast to annexin V where large changes of the local structure around this tryptophan are observed by several spectroscopic methods [138,1391. Difference in the conformation of this tryptophan residue has previously been reported for annexin I when compared with annexin V [1331. Annexin I11 binds specifically to neutral phosphatidylethanolaine rather than negatively charged phosphatidylserine. This may be explained by a high density of negatively charged residues in the vicinity of the Ca2+ ions in domain I11 compared with annexins I and V 21401. In contrast, as described above, annexin V preferentially binds phosphatidylserine 11321. 2.2.5. Annexin VI

Annexin VI is the only member of the annexin family with eight instead of four domains. The crystal structure of the Caz+-free form has been solved and shows a structure consisting of two halves encompassing domains I-IV and domains V-VIII, respectively, each half closely resembling annexin I [1181. The two halves are connected by an a-helical segment and are arranged perpendicular to each other. As a result of this arrangement, the Ca2+ and membrane binding sites, predicted from other annexin structures, are not found on the same side of the structure. However, electron microscopy of two-dimensional crystals on monolayers indicates that a structural rearrangement takes place upon membrane binding, positioning the &"-binding faces of both halves coplanar with the membrane, as would be expected if each half behaves analogous to annexin V with respect to membrane binding. If the sequentially homologous core regions of the many annexin structures solved to date are superposed, large deviations are noted. This is, in general, an effect of differencesbetween the relative orientation of individual domains in different members of the annexin family and arises as a result of variability in the connecting regions. If' instead the individual domains are overlaid, a high degree of structural similarity matches the sequential homology. Interesting deviations are found at the Ca2'-binding loops on the convex side of the protein whFre key residues for phospholipid-membrane binding are located. Despite the wealth of information from these structural studies, many important questions remain. The most important issue might be the elucidation of the mechanisms responsible for ion channel activity. Furthermore, despite a widespread interest for this protein family, it is difficult to correlate the many properties devised for annexins in vitro with biological function in vivo.

2.3. C2 Domains C2 domains are found in intracellular multidomain proteins involved in signal transduction and membrane trafficking (see [141-1441 for reviews). C2 domain sequences comprise approximately 130 residues and the structures determined so far reveal a

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compact 0 sandwich composed of two lour-stranded 0 sheets. Due to the presence of four conserved 0 bulges one face of the structure is convex and the other concave. A subset of C2 domains bind Ca2+ and usually have a Ca2+-regulated phospholipid binding affinity. C2 domains have also been shown to interact with other proteins and in some cases the interaction is Ca'*-dependent. Ca" binds at the top of the domain where two and sometimes three loops from sequentially remote regions harbor the ligating residues. The best studied C2 domain are those of protein kinase C (PITIC), phosphoinositide-spec~~c phospholipase C 61 (PLCGl), cytosolie phospholipase A2 (cPLA2),and synaptotagmin I (SytI). 2.3.1. Synuptolagmin I

SytI is a synaptic vesicle membrane protein and a member of a large family of membrane proteins called the synaptotagmins (see C1451 for review). They share a common domain structure consisting of a short amino terminal intravesicle part, a single membrane-spanning region, and a cytoplasmic region consisting of two C2 domainsC2A and C2B [146,147]. SytI forms homomultimers via Ca2'-dependent interactions of the C2B donlains and binds cooperatively to negatively charged phospholipids in the presence o f Ca" [148-1501. SytI is involved in Ca2'-dependent fast exocytosis of synaptic vesicles, and it is likely that the first C2 domain, C2A, is primarily responsible for mediating this function [149,151,1521. It has been shown that isolated C2A exhibits similar Ca2' and phospholipid binding behavior as the intact protein, whereas isolated C2B seems inactive [153,1541. The first structure of a C2 domain was the crystal structure of C2A from SytI (1RSU) 11551. It showed an eight-stranded fi sandwich around a four-stranded motif which was designated the C2 key. Three loops at the top and four at the bottom of the molecule connect the 0 strands. The crystals were sensitive to Ca2+ concentrations higher than 0.1 mM but still one bound Ca" was found at the apex of the molecule close to the amino and carboxy termini in a bipartite structure formed by two of the apical loops. Only small structural differences were observed between the Ca'+-free form and the Ca" -bound form. Ca" titration experiments monitored by NMR indicated that three Ca2' ions bind at the top of C2A with dissociation constants of 75 pM, 500pM,and 1mM 1156,1571. Using NMR it was possible to determine the structure in the absence of Ca2" as well as in the presence of 30 mM Ca2'. These structures demonstrated that essentially no conformational change occurs as a result of Ca2' binding, with the exception o f a few side chains that rotate in order to bind Ca2+ (1BYN) [158]. In the Ca2+-bindingregion (CBR) the loops between strands 2 and 3, 4 and 5, and 6 and 7 are denoted loop 1,loop 2, and loop 3, respectively (Fig. 12). Three Ca2' ions denoted Cal, Ca2, and Ca3 were found in the NMR structure [157,158]. The Ca" ions are primarily ligated by aspartate side chains that serve as bidentate ligands for two or three Ca2+ions. All ligating residues are found in loops 1 or 3, and in total five aspartates are involved in Ca2+ligation (Fig. 13). These aspartate residues are conserved in most synaptotagmins and in C2 domains of many other proteins [143,1451.

-

123

FIG. 12. Structurcs of the C2-domainproteins. A) Ribbon diagram o f SytI. B) h ribbon diagram of PLCGI. C) Two-dimensional representation of the folding motilk of SytT and PLCGl. (Reproducedby permission from ref. [ 141I)

Sytl interacts in a Ca2'-dependent manner with syntaxin, a plasma membrane protein and an important part of the synaptic core complex that mediates intracellular membrane fusion [159,1601. 1H,15N-RSQCmonitored titrations of the cytoplasmic region of syntaxin and fragments thereof to SytI C2A in the presence of Ca2" revealed that the interaction surface on SytI C2A coincided with the CBR C1611. The CBR is characterized by five aspartate residues surrounded by several basic residues. Particularly large chemical shift changes were seen for the basic residues in loop 3 and for D238, which is a Ca2+ ligand. Upon binding of three Ca2' ions the zwitterionic CBR is converted to a strongly positive patch 1156,1581 (Fig. 6

MURANYI AND FINN

124

PLCS? FIG. 13. A schematic representation of the Ca2"-binding sites of SytI and PLC61. (Reproduced by permission from ref. [1411)

suggested that the interaction surfaces of the two proteins are complementary but as negative charges prevail on both proteins strong electrostatic repulsion prevents binding. Ca" binding to SytI C2A acts as an electrostatic switch converting the CBR to a positively charged region and thus allowing the two proteins to bind to each other. Mutagenesis of basic residues in loop 3 to acidic residues abolishes binding and thus supports this binding model. The large chemical shift change seen for D238, which ligates the weakly bound and incompletely coordinated Ca" in position Ca3 (Fig. 13), may imply a direct interaction of this Ca'" ion and syntaxin in such a way


125

that the coordination sphere is completed, making the ion an integral part of the interaction surface. Binding of SytI to membranes is probably also regulated by electrostatics as the protein preferentially binds to negatively charged phospholipids [150,153]. Comparison of NMR spectra of SytI C2A and Ca2+in the absence and presence of 6:O phosphatidylserine (below CMC) revealed significant chemical shift changes for H198, V205, and F206 in loop 2 and R233, F234, and H254 in loop 3 [1621. Tbis is in accordance with fluorescence experiments that point to loop 3 as being involved in membrane binding [1631. Thus the CBR is responsible for SytI CZA binding to both syntaxin and negatively charged phospholipids. For the former the interaction involves loops 1 and 3, while for the latter loops 2 and 3 are involved. The close involvement of the CBR in phospholipid binding may help to explain the strong positive cooperativity observed for Ca2+ and phospholipid binding to SytI 11531.

2.3.2. Phospholipase 661 PLCSl is composed of an N-terminal PH domain followed by an EF-hand domain, a catalytic TIM barrel domain, and a C-terminal C2 domain. It is involved in signal transduction. As a response to various extracellular signals, PLCG1 catalyzes the which results in two products, hydrolysis of phosphatidylinisotol-4,5-bisphosphate, u-myo-inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol,which act as second messengers ultimately regulating cellular responses such as cell owth, proliferation, contraction, excitation, and secretion. Several X-ray structures of a catalytically active deletion variant of rat PLCG1 in the absence (2ISD, 1QAS) as well as in the presence of Ca” (1DJI) or the Ca” analogues Ba2+ (lDJH), La” (lDJG), and Sm3+ (1QAT) have been determined [164-1661. Furthermore, a series of X-ray structures of ternary complexes of PLCG1, Ca2+,and inositol phosphates (bound in the active site of the TIM domain) have been determined (lDJX, lDJY, lDJZ, 1 D W [1671. The structure of the C2 domain from PLCFl is very similar to that of SytI C2A, with an overall RMS deviation of 1.4 A for 109 equivalent a-carbons [164]. the topology of the domain is strikingly different. The order of the p strand permuted, As a result, the amino terminal p strand 1 of C2 occupies the same position as the carboxy terminal p strand 8 of PLCG1 (Fig. 12). As a further result, the amino and carboxy termini that are located at the top and near the CBR of C2A in SytI are instead found at the bottom of the C2 domain of PLCG1 and are thus far from the CBR (Fig. 12). These two topologies, called topology 1 and topology I1 for the SytI and PLCG1, respectively, are expected to be found in other members of the C2 domain family. The mechanism of Ca2+-dependentphospholipid membrane binding o f PLCGl is unknown. Two different mechanisms are suggested [166]. Possibly the Ca’+ ions bridge the protein to the membrane by acting as a “glue” between the protein and phosphoryl head groups of the phospholipids in the membrane, as has earlier been demonstrated for annexin V [1321. One such model has been suggested supported by the observed conformational change seen for PLCF1 after Sm3+ has bound and the

126

R loop 1 has moved, leaving a cavity large enough to admit a phospholipid head group r1651. Alternatively, the Ca" ions may enable membrane binding in an indirect fashion by changing the domain structure or electrostatic characteristics. Such indirect influence of Ca2' on membrane binding via conformational change has been demonstrated for the Gla ~ ~ - c a r b o x y g l u tacid-containing) ~ic domain of blood coagulation factor X tl681.

2.3.3. Cytosolic P h ~ ~ ~ h o l iA2 p~se

contains an amino terminal C2 domain and a carboxy terminal catalytic domain. The C2 domain is responsible for Ca2'-dependent binding to intracellular membranes where the catalytic domain hydrolyzes arachidonic acid containing membrane phospholipids. The released arachidonic acid is used in the biosynthesis of l e u ~ o t ~ i e nand e s prostaglandins, which are inflammatory lipid mediators (see 11691 for review). LA, 6 2 domain in the presence of Ca" was solved by he structure of ray ~ ~ s t a l l o ~(1 a ~ h y and NMR (IBCI) [I 70,1711. Both structures contain two ding of two Ca2' ions agrees with Ca2+ ions in Ca" binding sites 1and 4 (Fig. 13) ric studies that show binding of results from equilibrium dialysis [1721 and c n an entropically driven process two Ca" ions with dissociation constants o f 1171I. The lack of additional C4' ions can be ed by a conservative substitution of D95N in loop 3 of the CBR. In SytI, PKCP, and PLCG1, this residue is ~ 2 3 2D248, , and D708, respectively, and ligates Ca2 and Ca3 in SytI and PKC and Ca2 in PLCG1 (Fig. 13).cPL& C2 has higher affinity for Ca2' in the absence of phospholipids than SytI 6 2 4 PKCP 62, and PLCG1 C2-an observation that can be explained by differences in the sequence in the CB . N65, which is a Ca2* ligand in cPLAz C2 corresponds to an arginine or $sine in SytI 6 2 that would repel CL". Furthermore, some of the hydrophobic residues in loop 3 in cPLA, C2 are basic in SytI. When these residues (R233, K236) were mutated to glutamine an increased affinity for Ca2* resulted 11611. On the basis of chemical shift changes observed between 'I_I,''Nof labcled Ca2'-bound cPLAz C2 in the presence and absence of micelles of dodecylphosphocholine, it was possible t o propose a model of membrane binding [1711. According to the ~~R data, the C2 domain interacts with the membrane at two sites. The first involves binding of the glycerophosphocholine head group by the CBR loops and their adjacent segments of attached strands. Such a conformation would restrict the entry and exit of Ca2' ions to the binding site, and this is in agreement with other studies showing that the on and off rates for Ca2' are reduced more than 10-fold in the presence of vesicles 11'721. The second site is composed of some residues on strands 2 and 3, and binding is conveyed by ionic interactions between basic side chains in this patch of cPLA2 C2 and the phospholipid head groups of the membrane.


127

2.3.4. Protein Kinase C

The C2 domain and its ability to bind Ca2+was first described for PKC, which belongs to a family of kinases that are regulated by diacylglycerol and other lipids [173,1741. Several isoforms exist whereof the so-called conventional (31, PI, BII, and y) isoforms contain a C2 domain and exhibit Caz+-dependent binding to acidic phospholipid membranes. The PKCP has an amino terminal regulatory region that contains an autoinhibitory pseudosubstrate, a cysteine-rich Zn2+-bindingC l domain, and one 6 2 domain. The carboxy terminal part of the protein constitutes the kinase region. NMRmonitored Ca" titrations showed that PKCP C2 binds at least two Ca2* ions 11561. In the crystal structure of the PKCP C2 (lA25)three Ca2' ions were seen in sites Cal, Ca2, and Ca3, i.e., at the same sites that are occupied in the SytI C2A solution structure (Fig. 13) L1751. All sites are hexacoordinated, or heptacoordinated with Ca2+ ligand coordination distances ranging from 2.4 to 2.6 A. Ca2'-coordinating water molecules are predicted but not seen in the structure. Two nearly identical molecules related by a dyad axis are found in the asymmetrical unit, and a glutamic acid side chain in one molecule supplements the Ca" coordination sphere of the second molecule. As few other contacts between the molecules in the asymmetrical unit are found, this is considered an effect of crystal constraints. However, based on this observation it is suggested that conserved aspartic acid residues in the C-terminal end of the C1 domain, which precedes C2 in intact PKCP, may contribute ligands to the Ca2' ion. The critical residue for coordination of a Ca2+ ion in site Ca4 (N65 in cPLAz 6 2 and N677 in PLCFl C2), which is a basic residue in SytI C2A, is a cysteine in PKCP 6 2 and is involved in formation of an intermolecular disulfide bridge to its dyad partner in the asymmetrical unit. PKCP C2 binds phosphatidylserine in a multivalent fashion that gives rise to an apparent cooperativity 11767. Ca2+ is not an absolute requirement for membrane binding or enzyme activity but increases the rate of phosphorylation significantly C176,1771. Although o-phospho-l-serine, a head group analogue of phosphatidylserine, was present in the crystallization buffer, no evidence of specific binding was found [1751. In a study on PKCor individual Ca2' ligands were mutated and it was possible to group the ligands according to their importance in membrane binding 11781. Proper ligation of Cal seems to be important for the initial phase where the protein binds to the membrane surface. Tn a second step where Ca3 plays a key role, a conformational change leading to activation and membrane penetration is implied. In line with this, four residues in loop 3 play a key role for the interaction with the membrane; R249 and R252 are responsible for electrostatic interactions with anionic membranes, and W245 and W247 penetrate into the membrane and make hydrophobic contacts. Further studies delineate the roles of the 61 and C2 domains in membrane binding and activation of PKCa 11791. In summary, when one combines the information that has become available from the structural studies described above a picture emerges of the C2 domain as a rigid scaffold with a variable CBR that is responsible for Ca2+-dependentbinding to membranes and other proteins. The variable reffion has evolved a structure fine-

128

MURANYI AND FINN

tuned for the task of binding proteins with quite different functions. SytI has a CBR that is adapted to bind many Ca2' ions at nerve terminals where Ca2+ channels provide high local concentrations of Ca" [180]. By binding three Ca2' ions the charge of the CBR is drastically changed from strongly negative to positive. This electrostatic switch allows binding to a negatively charged patch on syntaxin and negatively charged phospholipids. In contrast, cPLA, C2 must be responsive to the submicromolar Ca2' concentrations found in the cytosol of stimulated cells. Binding of only two Ca2' ions and a CBR where hydrophobic side chains are more prevalent than in SytI points to an interaction characterized by hydrophobic rather than electrostatic contacts. The question about the detailed role of the Ca2' in c P U 2 C2, then, remains to be answered. The reasoning above is based on data from the available structures and is in line with data from extensive sequence alignment of 6 2 domains that reveals significant homology in the positions implicated in Ca" coordination. However, a degree of variability remains, which may reflect specialization in terms of Ca2' affinity, cooperativity, conformational changes, as well as electrostatic and hydrophobic considerations adopted for the biological function of the particular C2 domain-containing protein L1431-

Modules homologous with epidermal growth factor (EGF) are ubiquitous in extracellular proteins. They are found in animal proteins exhibiting a wide variety of functions from cell development, connective tissue fibers, complement, and blood [181-183]. It is believed that EGF modules are present c o a ~ l a t ~ oand n ~br~nolysis in 1%of human proteins [184]. The EGF modules are independently folding domains that usually consist of 40-50 amino acids and three disulfide bridges in a characterattern 1-3, 2-4, and 5-6. The structure is characterized by an amino terminal part c o n t ~ n i n ga major two-stranded antiparallel p sheet in which the two strands are separated by a hairpin loop. S~metimesan additional third strand or a short helical segment is f h n d in the amino terminal end. The carboxy terminal part often contains a minor antiparallel p sheet. The main function of EGF modules is believed to be in pro~ein-proteininteraction, which can be Ca2'"dependent. A subset dules exhibit a consensus sequence th has been associated with C 8 X,-D" /N*-X,-Y/F (wher indicates any residue, rn and n cates possible ~-hydroxylation)D85-1871. Coupled to the sequence motif for Ca" binding is a sequence motif associated with hydroxylation of aspartic acid or asparagine residues to erythro-p-aspartic acid and erythro-&asparagine in a conserved position in many EGF modules of extracellular proteins ClSSl. implication of this hydroxylation is unknown. Since many of these GF modules also bind Ca2', there was speculation that there could be a correlation between hydroxylation and Ca2* binding. owever, c o m p a ~ ~ oofnthe Ca2' b i n ~ n g~ n i t between y hydroxylated and unhy~oxylatedforms of the amino

CALCIUM AND ITS ~

N

~

Y

~

~

§

129

terminal EGF module from clotting factors M (FIX) and X (FX) showed that hydroxylation did not affect Ca2+ &nity [1891. The first structures of calcium-binding EGF modules (cbEGF) were the NMR solution structures of the Ca2+-free&no terminal EGF (EGFl) modules from F and FX (IIXA, 1APO) [190,191]. Comparison of the structure of the Caz'-free form with the Ca2+-boundform t1CCF) of FX EGFl showed that binding of Ca2+ only led to local conformational changes close to the binding site, which is between residues in the m i n o terminal end of the sequence and residues in the major sheet just prior to the hairpin loop [1921. In the Ca2+-bound structurc the ligands for Ca2' could be deduced to be the backb e carbonyl oxygens of G47 and 6 6 4 as well as side chain 9, erythra-P-hydroxyasparticacid (Hya) 63, and D46. carboxylate oxygens of Analogous results were seen for the amino terminal cbEGF of clotting factors VII (1BF9, lDAN, 1F7E, 1F7M) and IX (1EDNI) [193-1961 (Fig. 14). The Ca2' affinity of isolated cbEGF modules has been measured in several cases and found t o vary from 0.2 to 10 mM [186,189,195,197-2011. Binding affinities are dependent on ionic strength and the highest affinity measured at physiological strength is 1.4 mM for FIX, which would imply that the Ca2' binding sites would not be saturated at physiological Ca2* concentrations (* 1.2 m in human plasma) [186] Nonetheless, reports have indicated that the Ca'+ binding is p h y s i o l o ~ c ~ l y important 11851. The explanation came when affinities for isolated modules were compared with affinities measured in module pairs where an additional module precedes the cbEGF that harbors the site of interest. In a fragment consisting of the two amino terminal modules of FX (Gla-~GFl), the Ca2' binding affinity of the cbEGF was 20-fold higher than in the isolated FX EGFl module, implying saturation at physiological Ca2+ concentration [2021. The affinity of cbEGFl3 of f i b f i n - l was

FIG.14. Diagram of Ca2+-ligationin an EGF-Gla module pair. Gla is a y-carboqglutamic acid containing nodule which also binds Ca2' and i s Found in a number of blood proteins.

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MURANYI AND FINN

70-fold higher in a cbEGF12-13 pair than in the isolated cbEGF13 module [201,203]. However, the addition of a transforming growth factor P-binding protein-like domain (TB) amino terminal of cbEGF32 from fibrillin-1 hardly affected the Ca2' binding affinity of cbEGF32 [204,2051. Protein S , which is a cofactor for activated protein C, has four EGF modules in tandem whereof the three carboxy terminal have a consensus sequence for Ca2' binding. A variety of Combinations of EGF modules have been produced and the Ca2+ affinity measured 12061. In an EGF3-4 pair the affinity of EGF3 was &most identical to that of isolated EGF3, while the affinity of E G was ~ approximately ~ 8600-fold higher in the pair 1200,2071. In each one of EGF1-4 and EGF2-4 three very high-affinity sites with Ic, from to M were found while Ca2+ affinities of EGF1-3 and EGF2-3 were two Lo four orders of magnitude lower 12081. These results imply that not only modules on the amino terminal side but also modules on the carboxy terminal side influence the Ca2+ binding affinities in the EGF modules of protein S. An investigation of the structure of these triplets or quadruplets will be necessary to fully understand the basis of this phenomenon. Although structures of triplets or quadruplets are not yet available, structures of module pairs have been determined and these have helped to elucidate the mechanism of increased Ca2"-binding affinity by an amino terminal module. Of particular importance has been the solution structure of the cbEGF32-33 module pair from fibrillin-1 (ZEMN, 1EMO) C2091. In this pair, the affinity of the Ca2 binding site in the carboxy terminal module is 25-fold higher than that o f the amino terminal module. Based on the discussion above, it is likely that the higher affinity of cbEGF33 is a result of the presence of cbEGF32. The structure reveals that the Ca2' ion in cbEGF33 is ligated mniquely by residues in that module, and it is suggested that the increased affinity arises as a result of a better defined binding site where the Ca" ion is shielded from solvent. In structures of isolated EGF modules it had been observed that the amino terminal end of the molecule was unstructured. This is not the case in the carboxy terminal module of the pair. One reason for the higher degree of order in this region compared with isolated modules is that a conserved aromatic residue in the loop between the last two cysteines in cbEGF32 is involved in hydrophobic interactions with an isoleucine and a glycine in the hairpin loop of the major p sheet in cbEGF33 (Fig. 15). Thus, it seems that the presence of a module amino terminal o f the Ca2' binding site is necessary to induce the rigidity that is a prerequisite for high-afbity Ca2 binding. These conclusions are supported by "N-NMR relaxation data C2091. Similar conclusions were also reached for the EF-hand Ca2* binding proteins I31 (Sect. 2.1). Thus, preformation of a rigid Ca2* binding site could be a general prerequisite for high-affinity Ca2+binding. In such a case, the negative effect of entropy loss upon ordering of the Ca'+ binding site upon binding is avoided. Fibrillin-l is the major structural component of microfibrils in the extracellular matrix, and mutations in the gene for fibrillin-1 lead to the Marfan syndrome. It has been shown that microfibrils from Marfan cell lines that contain mutations in Ca2+ligating residues lose their regular appearance [210 1. This is also observed for microfibrils composed of wild-type fibrillin-1 when Ca2" has been removed 12047. Based on

CALCIUM AND ITS

131

/

/

/!

\

c .

1

/I

l

i

FIG. 15. A ribbon diagram showing the interactions between sequential EGF modules in Fibrillin. The interacting residues are indicated with space-filling models.

the structure of the fibrillin-1cbEGF32-33 pair a model of‘how fibrillin monomers are arranged in microfibrils was proposed [209]. In this model the p-hydroxyl groups of the postranslationally modified aparagine/aspartic acid residues would be exposed and could participate in intermolecular hydrogen bonds. It has already been mentioned that modules other than EGF may modulate the CaZf-bindingaffinity in a succeeding cbEGF. In a combined WMR and SAXS study of the FX Gla-EGF1 module pair the role of the Ca2+ ion as a structural determinant was identified. On Ca2’ binding to the binding site in the EGF module the Gla and EGF modules fold toward each other 12111. The CaZf binding affinity in this site has been shown to be 20 times stronger in the Gla-EGF1 pair (Kd = 120 pM)than in the isolated EGF module CrC, = 2.2 mM) I2021. This is consistent with the hydrophobic interdomain interaction close to the Ca2+ion and the result of chemical shift comparisons between Ca2+-freeand loaded Gla-EGF, which revealed that not only residues in the EGF module but also several residues in the carboxy terminal end of the Gla module (the so-called hydrophobic stack helix) were affected by Ca2’ binding. Since the Ca2’ concentration in plasma is approximately 1.2 mM, it is likely that this site is saturated in vivo. It seems that Ca2+ binding to the first EGF module in factor X is essential in defining a biologically active domain orientation. Blood coagulation is initiated when factor VII (FVLI) binds to tissue factor (TF) and forms a complex that is essential for activation of FVII and proper inter~ction

132

ANY1 AND FINN

with substrates FIX and FX. All three coagulation factors VII, , and X have the same domain organization consisting of an amino terminal Gla module followed by tWQ sequenti~lEGF modules, where the first (EGFl) binds Ca2+.The carboxy terminal domain is a serine protease module. Mutational studies on FVII have demonstrated that mutation of Ca2+-ligatingresidues in EGFl leads to impaired TF binding 1212,2131.In the crystal structure of the FVII-TF complex (lDAN), the Ca2+binding site of EGFl faces away from the interaction surface with TF (1941. Further, comparison of the Cazc-free structure of FVII EGFl (1BF9) with the Ca"-bound EGFl in the structure of the complex reveals local structural changes in the Ca2+ binding site but no significant changes of residues that form the FYII-TF interaction surface L1931. Thcse observations are consistent with the idea that Ca2* binding to mJII EGFl i s important for rearranging and stabilizing the relative position of the amino terminal EGF module with respect to the preceding Gla module and thereby facilitating TF binding. In accordance with this view, molecular dynamic simulations of the light chain of FVII (Gla-EGF1-2)showed that the relative orientation between Gla and EGFl is significantly altered when the Ca2+ ion in EGFl is removed [2141. In summary, EGF modules play an important role in protein-protein interaction involving many extracellular mosaic proteins. The three &sulfide bridges define the fold. Few residues besides the cysteines are conserved among EGF modules Llt311. ~ o n s e ~ u e n t lthe y , large sequence variability allowed in this module allows adaptation to a number of protein-protein interactions and is probably part of the explanation for the reoccurrence of this building block in mosaic proteins with greatly divergent func~ions.One interesting example is the EGF module from the complement protease Clr, which exhibits a large loop between the first two cysteines [2151. This atypical feature may be a re ment for the specific protein~proteininteraction associated with Gar's function. es the structural integrity of the individual modules, interest has recently be sed on intramolecular mod~le-moduleinteraction. It has become evident that a preceding module may modulate the Ca2*binding asnity. This is probably not, in general, an effect of additional Ca2+ ligands donated from the preceding module; rather it is an effect of increased stability provided by the continuation of the polypeptide backbone and the presence of other contacts between modules, like the hydroph~bicinteractions seen in the FX Gla-EGFl module pair and the fibrillin-1 c b ~ ~ ~ 3 2 module - 3 3 pair [209,2111. Intemodule stability may also be imposed by other forces as dei~onstratedby the salt bridge stabilizing the EGFl and ~ G F in 2 FX, altho~gh,in this particular case, no Ca" ~ t e ~ a between ce ion binds in the vicinity o f the mQdUl r E2161. The effect of a preceding module on Ca2" afEnity varies greatly. The le only weakly affected Ca2+ binding to 1, while at the other extreme the prethe following cbEGF32 in fibrillinsence of E ~ F amino 3 terminally of EGF4 in protein S increased Ca'+ affhity to the latter 86OO-fold when compared with isolated ECF4 [200,2071. In the first case it is that the ~ ~ " c b E G ~ 3 2r is flexible and that pairwise interactions are relaxation data imply a rigid linker between 1. For the latter, "NGF3 and ~ ~ (A.FMuranyi, 4 J. Evenas, U. ~ t e n b e ~J. g ,Stenflo, and T. i o c ~ e ~ , iin~ tpress). ~ , Thus, it seems that the stFength of module-~oduleinterac-

133 tions and the flexibility of the linker separating the modules can vary greatly and may be modulated by Ca2+. It is also plausible that the Ca2" affinity correlates with the degree of rigidity at the intermodule interface. The importance of Ca'+ as a structural determinant in mosaic proteins is underlined by a number of point mutations identified in patients that affect Ca2+ binding to EGF modules and most likely disrupt supermolecular assemblies like formation of microfibrils in the case of fibrillin-1 and binding to substrate or cofactor in the case of blood coagulation factors, leading to the Marfan syndrome and hemophilia, respectively.

2.5.

Lectins

The lectins make up a structurally heterogeneous class of proteins that bind carbohydrates with high specificity (see [217-2191 for reviews). They are found in bacteria, viruses, plants, and animals and are responsible for cell recognition mediate^ by specific cell surface carbohydrates. They are important in a number of processes, including pathogen infection, host defense, leukocyte trafficking (i.e., migration to sites of in~ammationand homing of ~ymphocytesto specific lymphoid organs), endocytosis of serum glycoproteins in the liver?and metastasis. The carbohydrate binding site is usually in a shallow pocket or groove, and the dissociation constant for monosaccharide~i s in the millimolar range. Oligosaccharides usually bind with higher affinity and when several binding sites are combined the affinity for a multivalent ligand can be in the nanomolar range. Some subsets of the lectins require Ca2+ for carbohydrate binding and in some cases additional Mn2+is required along with Ca2+. Lectins that require both Ca2+and Mn2' are treated in Chapter 8 of this book. In the following section lectins that require exclusively Ca2' for binding are discussed. Lectins that require Ca2' for function are usually classifie as type c (for ~ a " dependent) animal lectins. They are a class of mosaic (multidomain) lectins consi of a carbohydrate recognition domain (CRD) and a variety of other modules. The contains approximately 120 amino acids, of which 14 are invariant and a further 18 are conserved. C-type animal lectins are grouped into three families, the endocytic lectins, collectins, and selectins. The endocytic lectins are receptors of hepatocytes and macrophages. These membrane proteins usually have a short amino terminal cytoplasmic domain brane-spanning region followed by a neck region, and a carboxy terminal C collectins are soluble proteins found in the extracellular matrix and in serum. They have an amino terminal cysteine-rich region ed by collagen-like repeats and an %-helicalneck region and a carboxy terminal The selectins are membrane proteins that mediate selective adhesion between cells. The three types of selectins are named after the types of cells where they are ~rimarilyexpressed: -type (endothelial cells), P-type (activated platelets and endothelial cells), and L-type (lymph node vessels). They all have one amino t e ~ n a l CRD followed by an EGF-like module, a variable number of' complement r e ~ l ~ t o protein repeats, a membrane-spanning region, and a short carboxy terminal &oplas-

134

MURANYI AND FINN

mic tail. The best studied C-type lectins, at least in a structural perspective, are the collectins; several structures of the mannose-binding protein have been reported, and some interesting mutants shed light on the determinants of ligand specificity. As an example of a selectin we will discuss E-selectin. Finally, we will discuss tetranectin, which belongs to a distinct group of C-type lectins. 2.5.1. ~ a ~ n o ~ e - 3 i r ~Protein ding

One of the best studied lectins is the mannose-binding protein (MBP), which is a collectin responsible for antibody-independent hosl defense against invading pathogens. It is usually found in oligomers of trimeric building blocks formed by interaction between the collagen-like repeats in coiled coils. In rat it is found in two forms: the serum type MBP-A in which the active oligomer consists of a hexamer of trimers (Mvv 650 kDa), and the liver type MBP-C which is a dimer of the trimeric building blocks 200 kDa). As MBP-C is found in the liver, it has been speculated that, in ion to its role in host defense, it may be involved in cell-cell interaction or glycoprotein trafficking. MBP-A and MBP-C bind carbohydrates that have hydroxyl groups in a similar position as the 3- and 4-hydroxyls of mannose, including N-acetylglucosamine, and fucose, whereas they do not bind galactosides and sialic acid. The first structure of a Ca'+-dependent lectin was the crystal structure of the CRD of rat MBP-A where the two required C&'+ ions had been replaced by holmium ions (€lo? (1MSB) [2203. It was soon followed by a structure of the complex between the CRD of rat MBP-A and an oligomannose asp~a~nyl-oligosaccharide (BMSB) 12211. In this complex only the terminal mannose interacts with the CRD. Three C&" ions are found per CRD but only the first and second are functionally relevant. Binding of the third Ca'" ion is an effect of the high Ca2+ concentration in the crystallization medium. The interaction surface is formed by direct ligation of the second Ca2' ion by the 3-hydroxyl and 4-hydroxyl groups of the terminal mannose to form an %coordinate Ca2' ion complex. The coordination complex is a pentagonal bipyramid in which the second apical position is bisected by the two hydroxyl groups of the sugar. In addition, four of the amino acid side chains of the CRD that are responsible for ligating the Ca'" (E185, N187, E193, and N205) also make hydrogen bonds to the 3-hydroxyl and 4-hydroxyl groups of the mannose, i.e., the same hydroxyl groups of the sugar that are responsible of ligating Ca2' (Fig. 16). Compared with the carbohydrate-free CRD-Ho3+complex, the protein structure is virtually the same but the No3 ' ion is 7-coordinate and a water molecule has replaced the position taken up by 4-hydroxyl in the tertiary complex. This arrangement leads to an intimately linked complex between the GRD, Ca2+, and sugar. This unusual conformation where the Ca'+ ion i s directly coordinated by the carbohydrate ligand contrasts the situation in the structures of legume lectins in which the Caz+ and Mn2+ions help to position the side chains, which are important for carbohydrate binding but do not interact directly with the sugar (see Chapter 8). of MBP-C has 58% sequence identity with the CRD of M important residues such as those involved in disulfide bonds, hydrophobic packing,

- -


135

Alp 206

FIG. 16. Ball and stick model of the intcraction sites of various lectins. A) Galactose binding in TC14.B) The galactose binding site in the QPDWG mutant of MRP-A. C)The mannose-binding site of MBP-A. All are shown in the Fame orientation. (Reproduced by permission from ref. [225 1)

136

~ U ~ A N AND Y FINN ~

and Ca2' ligation are identical. The structure of MBP-C has been solved in the absence of sugar as well as in complex with five different monosaccharides (lRD0, I, lRDJ, IRDK, 1RDL7 lRDM, 1RDN) 12221. As for the ~ B P ~structure A (BMSB), MBP-C exists as a dimer in the crystal. The structure of the individual GRD units of MBP-C are very similar to those of MBP-A whereas the dimer interface is different, resulting in dramatic differences if the entire dimers of MBP-C and MBPA are superpositioned. Mannose binds to MBP-C CRD in a way analogous to that of the MBP-A CRD described above, i.e., a tight complex in which the two hydroxyl groups of the sugar ring that ligate Ca"+ also form hydrogen bonds with oxygens on four of the amino acid side chains of the CRD that ligate Ca2*. Surprisingly, the sugar ring is turned 180" in comparison with the position in the MBP-A structure, and the positions of the 3- and 4-hydroxyls are interchanged. The other sugars are bound similarly to mannose. Galactose has a very low affinity for the MBP-C CRD carbohydrate binding site, and the structural basis for this is discussed in [2221. To function in the host defense it is essential that the MBPs recognize a broad range of monosaccharides. This is achieved by a shallow binding site and a specificity that is limited to a small subset of functional groups on the ligands, i.e., hydroxyl groups corresponding to the 3- and 4-hydroxyl groups of mannose. Significant affinity for the ligand is achieved by the intimate binding arrangement around the Caz+ ion. While the MBPs have specificity for mannose, N"acetylglucosamine7and fucose, other C-type animal lectins specifically bind galactose- or N-acetylgalactosamine-terminated oligosaccharides. It has been shown that the specificity for the latter sugars can be engineered into rat MBP-A CRD by changing three amino acids (E185Q, N187D, and H189W) and inserting a glycine-rich loop 12231. This mutant MBP-A and specificity comparable with the rat hepatic PDWG) has galactose WG has been solved in complex with @-methylThe crystal structure ne (1AFB) as well as galactoside (~-MeGal)(1AFAj and ~ - a c e t y l g a l a c t o s ~ i(GalNAc) in the absence of sugar (1AFD) L2241. These structures show that the same arrangement as in the wild-type MBP-A CRD-Ga2+-carbohydrateternary complex is responsible for binding of P-MeGal and GalNAc in the mutant complexes. consequence of the pentagonal bipyramidal Ca2+coordination geomet ring is found in a very different orientation in which it is flipped nearly 180"compared with the mannose in the wild-type complex. Furthermore, the apolar patch formed by carbons 3,4,5, and 6 of P-MeGal and GalNAc pack against the ~ ~ t o ~ side h achain n which has replaced the wild-type H189 (Fig. 16). This packing interaction is found in other galactose-specificlectins. The role of the inserted glycine-rich loop is to impose on W189 a conformation that is incompatible with the binding sugars, thereby making the mutant selective for galactose tion on regulation of the carbohydrate specificity among s t ~ c t u r oe f a tunicate C-type lectin (TCl.41, which cons 1 12251. It is the first example of a has natural galactose specificity (lTLG, 1 naturally dimeric 6-type lectin, and some o f the differences of this structure in comparison with other CRD structures are related to this fact. The spec~cityfor galactose is e ~ p l a i ~ ebyd two aspects of the structure of the interaction site. First, the


137

distribution of hydrogen bond donors and acceptors positions the 3- and 4-hydroxyls of the sugar ring in a position more similar t o that of mannose bound to MEW-A than to that of galactose bound to the QPDWG MRP-A. Second, the side chain of WlOQ, which is on the opposite side of the binding site than MBP-A W189 mentioned above, packs against the apolar side of the galactose molecule. A mannose ring positioned in this binding site would interfere sterically with W O O (Fig. 16). In an attempt to understand the role of the Ca2+ ion for the structure of the CRD of C-type lectins, Ng et al. solved the structures of two MBPs by X-ray crystdlography l-2261. The structure of rat MBP-A (CRD and trimerization domain) with a 03+ ion at site 1(1BUU) gave information about structural changes around site two when it was devoid of a bound ion. The ion-free structure of rat MBP-C CRD (1BV4)gave information on the conformational changes when both ions were missing. In both cases a large part of the CRD structure, including the hydrophobic core, waa unaffected while loops in the vicinity of the empty ion binding sites changed conformation. In addition, it was shown that Caz+ is necessary for maintaining a cis-peptide bond preceding a conserved proline residue in Ca2+-binding site 2, which in turn is determinant for the conformation of Ca2’ and carbohydrate-ligating residues. The apparent lack of structural rearrangement in the hydrophobic core of CRD upon loss of Ca2’ is puzzling as earlier studies had shown that mutagenesis of residues in the hydrophobic core reduced Ca2+binding affinity [2271. So f’ar structures of isolated CRD domains have been discussed. They are informative on the detailed Ca2+-mediated carbohydrate binding. However, the intact MBPs form trimers that further associate in a hexameric arrangement. To better understand the sole of these larger aggregates in the process of carbohydrate recognition, as a part of the innate immunity, larger fragments from the MBP have been studied. Structure determination has been performed on fragments from rat MBP-A and human MBP containing the a-helical neck region in addition to the CRD domain URTM, 1HUP) C228,2291. In both cases hexamers of trimers form, despite the absence of the collagen repeat region. The trimerization is mediated by the helices which form coiled coils and, in addition, hydrophobic packing of residues on the outside of the coil and residues of the neighboring CRD. The carbohydrate binding sites at Ca2’ binding site 2 are located 45-53 A apart. It is suggested that in solution the CRD “heads” are expected to have some freedom of orientation relative to one another in order to facilitate binding to arrays of branched oligosaccharides found on cell walls of pathogens, for which MBP has been shown to have affinity. 2.5.2. E-Selectin

Expression of the membrane glycoprotein E-selectin on endothelial cells is induced by cytokines. E-seledin mediates the specific adhesion of neutrophils. The smallest fragment that has neutrophil-binding capability consists of the two amino terminal domains: a 0, followed by an EGF-like domain. Graves et al. determined the crystal structure of this fragment (1ESL) [2301. The interaction between the two domains is somewhat limited although it was suggested that the relative domain

138

M U ~ A NAND ~ I FINN

orientation nevertheless is well defined. The overall fold of both the CRD and EGFlike domains were similar to their counterparts in other proteins. Although the CRD has only 30% sequence identity with rat MBP, the conserved disulfide bonds and several clusters of identical residues made it possible to superpose the two structures using 109C" pairs, which resulted in an RMS deviation of 1.94 Most of the differences between the structures were caused by insertions/deletions. However, the most interesting difference occurs in the Ca2'-binding loop where no insertion or deletion i s present. One of the two Cazf binding sites identified in MBP is missing. Only the one referred to as Ca2+ binding site 2 is present in the E-selectin CRD. Although all Ca'+ ligands in that site are conserved, the position and orientation of one of them, E88, is altered in such a way that it no longer ligates the ion. Instead, coordination has been replaced by a water molecule, which is also hydrogen-bonded to N83. Interestingly, the corresponding residue in MBP (D188) is a ligand for the other (site 1) Ca2+ ion. Mutations that impair E-selectin adhesion to neutrophils cluster around the Ca2+ binding site, a finding that is consistent with the observation that neutrophil binding is Ca2'-dependent 1230,231I.

A.

2.5.3. Tetranectin

Tetranectin (TN) is found in plasma and in a variety of cells as well as in the extracellular matrix of certain human carcinomas (see references in [2321). It binds the fourth kringle domain of plasminogen and i s thus implied in fibrinolysis. TN belongs to a distinct group of C-type lectins where one also finds a number of proteins that, in contrast to tetranectin, are composed exclusively of an isolated CRD module, e.g., pancreatic stone protein (lithostathine), sea raven antifreeze protein, and snake venom botrocetin. In TN the CRD is preceded by a 25-residues-long helix. Both in solution and in the crystal structure TN appears as a homotrimer (1EITN) [2321. The long N-terminal helix provides the trimerization domain by forming a coiled coil and the overall structure is similar to the trimers of rat MBP-A and human MBP discussed above. In addition to the structure of the intact protein, a structure of the isolated CRD domain has been reported (lTN3; Fig. 17) 12331. The isolated structure is a monomer in solution but in analogy with the MBPs it forms a dimer in the crystal. The combination of a plasminogen binding site, which is probably located in the @-helicalregion, and a Ca2+-dependent binding to sulfated polysaccharides and fibrin has led to suggestions that one function of the protein is to target plasminogen to specific carbohydrate ligands on cell surfaces in the extracellular matrix or to fibrin [2321. In summary, the CRD domain of all C-type animal lectins contains a unique Ca'+ binding site that is involved in a specific and intimate interaction with a variety of oligosaccharides. A rather shallow binding site and a centrally located Ca2+ ion in the interface between protein and its carbohydrate ligand is the explanation for sugar binding affinity. Amino acid side chains located close to the Ca2+binding site endow the protein with specificity for a certain type of sugar by sterically preventing undesired ligands from binding. The versatility of these carbohydrate binding sites is

139


FIG. 17. A ribbon diagram of the tetranectin triplex in two orientations rotated 90 degrees with respect to one another. (Reproduced by permission from ref. [2321)

clearly demonstrated by comparison of the two structures of galactose-bindingCRDs, the ~ P MBP-A ~ mutant, ~ and G the naturally galactose-bindingTC14, where specificity of the same ligand is organized in two different ways by rearrangement of hydrogen bond donorsiacceptors in the binding site and the position of critical amino acid side chains in the vicinity of the binding site. Trimerization mediated by the a-helical coiled-coil neck region yields a rnultivalent protein with a significant Aexibility of the individual CRDs adopted for avid binding to repetitive carbohydrate arrays on pathogen surfaces.

K~OWN STRU~TU

3.

As we stated at the beginning of this chapter, the size of the Cad+-bindingprotein field has forced us to make choices about which systems of known structure we could discuss in detail. We leave it to the reader t o continue his quest for other proteins among the books and literature references provided below. Having reached this point in the chapter, one might conclude that we know most of what can be known about the structure and fimction of Ca2' -binding proteins. Far from it! Despite years of intensive effort, we have only really come partway in our understanding of this huge and widespread family of proteins. Many fascinating systems have yet to be explored in detail, and many of the systems that have been examined have much more left to offer. An example of the former case is calbindin D28k, a protein that has been associated with neurodegenerative diseases such as Alzheimer's and Parkinson's disease [204]. Despite being first isolated over 30 years ago, no high-resolution structure has yet been determined. Even the question of how many of its six EF hands actively bind Ca2 is still a matter of debate.

'

140 In the latter case, even calmodulin, one of the most studied of all Ca2'-binding proteins, has many questions that are left unanswered. One of the major milestones yet to be achieved is a structure of calmodulin bound to an intact target protein. Structures of calmodulin bound t o peptide fragments have been numerous and offered some insights into the mechanism by which calmodulin functions as an activator of its many targets. However, the ultimate test of these models will be an intact c a l m o ~ u l i n - t ~pair. ~ e t Given the multitude of binding modes identified thus far, even the determination of one such pair will probably not suffice. These are but a few examples of the many challenges that remain in this exciting branch of the rnetalloprotein field.

ELATlONSkiI PS The wide range of structure and function of each type of Ca2+-bindingprotein makes the rendering of a single summary of these relationships difficult if not impossible. Each protein, from intracellular proteins such as most EF hands to extracellular proteins such as blood factors, has used available Ca2+in a unique way. One common thread among all is that their Ca2' binding behaviors have been finely coordinated with the local Ca2" concentration ranges available to them and under the specific conditions under which they must be activated and deactivated. Details of specific structure-function relationships can be found in their respective parts of Sect. 2.

5. We have attempted to present an overview of some recent studies of Ga2+ proteins, especially those for which structural data are available. If you have arrived here after reading the preceding sections, you will have already realized that we cannot do justice to Ca2'-binding proteins with a few sentences of summary. If you have skipped to here from the introduction: Shame on you! You missed the best parts! However, if there is one central theme that we wish to emphasize at this point, it is this: Perhaps more than any other type of metal-binding proteins covered in this book, calcium proteins are truly ubiquitous and possess an extraordinary range of structure and function. They occur in all types of organisms, tissues, cell types, and organelles. The range of functions in which they are involved is nearly all-encompassing; while often related to a signaling process, they can also involve structural, transport, and buffering functions. The sequences and motifs that can bind calcium are wide ranging, with no apparent basic preference for secondary structure or fold. Even the properties of Ca2* proteins that determine their Ca" affinity are manifold and at the same time subtle in their effects. We have tried to provide the reader with a glimpse into the field of Ca"-binding proteins as it is developing at the present time. Our highest ambition is simply to


141

spark a curiosity on the part of the reader in this collection of met~oproteins that we find so fascinating.

s

6. Ca2* A

ata Library (http://structbio.vanderbilt.eddcab Cellular ca2+Information Server (http://calcium.oci.utoron~o.c~) Pfam Protein Families Database ( h t t p : / / ~ . s a n g e r . a c . u ~ S o f t w a r e / P f ~ RCSB Protein Databank (http://www.rcsb.org./pdb/) Structural Classification of Proteins (SCQP) (http://scop.mre-lmb.c~,ac.uM scopi)

A B-MeCal CamKII CaNIKK cbEGF

CBR CMC CPLA,

CRC CRD EGF

ER GalNAc Gla

GPE GPS HSQC InsPs InsPsR lec MBP MLCK NMR NOE PH PKC PLCG1 RMS

p-methylgalactoside calm~dulin-dependentprotein b a s e I1 calmodulin-dependent protein b a s e kj.nase calcium-binding epidermal growth factor Ca” binding region critical micelle concentration cytosolic phospholipase A2 Ca2+ release channel carbohydrate recogniton domain epidermal growth factor endoplasmic reticulum. N-acetylgalactosamine y-carboxyglutamic acid glycerophosphoethanolamine glycerophosphoserine heteronuclear single-quantum coherence inositol 1,4,5-trisphosphate InsPs receptor lectin mannose-binding protein myosin light chain kinase nuclear magnetic resonance nuclear Overhauser effect plextrin homology protein kinase C phospholipase C61 root mean square

14

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. J. Rees, I. M. Jones, P. A. Handford, S. J. Walter, M. P. Esnouf, K. J. Smith, and 6 . 6. Brownlee, EMBO J., 7, 2053-2061 (1988). P. A. Handford, M. Mayhew, M. Baron, P. R. Winship, I. D. Campbell, and 6. G. Brownlee, Nature, 351, 164-167 (1991). M. Mayhew, P. Handford, M. Baron, A. Tse, I. Campbell, and 6 . Brownlee, Protein Eng., 5, 489494 (1992). P. Fernlund and J. Stenflo, J. Biol. Chem., 258, 12509-12512 (1983). M. Selander~sunncrhagen, E. Persson, I. Dahlqvist, T. Drakenberg, J. Stenflo, M. Mayhew, M. Robin, P. Handford, J. W. Tilley, I. D. Campbell, and G. G. Brownlee, J. B i d . Chem., 268, 23339-23344 (1993). M. Baron, D. 6. Norman, T. S. Harvey, P. S. Handford, M. Mayhew, A. G. . Tse, 6 . 6 . Brownlee, and I. D. Campbell, Protein Sci., 81-90 (1992). M. Ullner, M. Selander, E. Persson, J. StenAo, T. rakenberg, and 0. Teleman, Biochemistry, 3 1 , 59765983 (1992). M. Selander-Sunnerhagen, M. Ullner, E. Persson, 0. Teleman, J. Stenflo, and T. Drakenberg, J. Biol. Chem., 267, 19642-19649 (1992). A. Muranyi, B. E. Finn, G. P. Gippert, S. Forsen, J. Stenflo, and T. Drakenberg, Biochemistry, 37, 10605-10615 (1998). . W. BanneT, A. D'Arcy, C. Chkne, F. K. Winkler, A. Guha, onigsberg, Y. Nemerson, and D. Kirchhofer, Nature, 380, 41-46 (199 Y.-W, Kao, G. F. Lee, Y. Wang, M. A. Starovasnik, R. Kelley, M. W. Spellman, and L. h r n e r , Biochemistry, 38, 7097-7110 (199 Z. Rao, P. Handford, M. Mayhew, V. Knott, 6. G. Brownlee, and D. Stuart, Cell, 82, 131-141 (1995). E. Persson, M. Selander, S. Linse, T. Drakenberg, A.- . Ohlin, and J. Stenflo, J. Biol. Chem., 264, 16987-16904 (1989). P. A, Handford, M. Baron, M. Mayhew, A. Willis, T. Beesley, 6, 6. Brownlee, and I. D. Campbell, EMBO J., 9, 475-480 (1990). J.-F. Hernandez, €3. Bersch, Y. Petillot, J. Gagnon, and 6. J. Arlaud, J. Peptide Res., 49, 221-231 (1997). Y. Stenberg, K. Julenius, I. Dahlqvist, T. Drakenberg, and J. StenAo, Eur. J. Biochem., 248, 163-170 (1997). P. Whiteman, A. K. Downing, R. Smallridge, P. . Winship, and P. A. Handford, J. Biol.Chem., 273, 7807-7813 (1998). C. Valcare, M. Selander-Sunnerhagen, A,-M. Tamlitz, T. Drakenberg, I. Rjijrk, and J. Stenflo, J. Biol. Ch,em., 268, 26673-26678 (1993). R. S. Smallridge, P. Whiteman, K. Doering, P. A. andford, and A. K. Downing, J. Mol. Biol., 286, 661-668 (1999). P. Handford, A. K. Downing, 2. Rao, . R. Hewett, B. C. Sykes, and C. M. Kielty, J. Riol. Chem., 270, 6751-6756 (1995). S. Kettle, X. Yuan, 6. Grundy, V. Knott, A. Downing, and P. Handford, J. Mol. Biol., 285, 1277-1287 (1999).


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206. Y. Stenberg, T. Drakenberg, B. Dahlback, and J. Stenflo, Eur. J. Biochem., 251, 558-564 (1998). 207. Y. Stenberg, A. Muranyi, C . Steen, E. Thulin, T. Drakenberg, and J. StenAo, J. M o ~Biol., . 293, 653-665 (1999). 208. Y , Stenberg, S. Linse, T. Drakenberg, and J. Stenflo, J. BioZ. Chem., 272, 23255-23260 (1997). 209. A. K. Downing, V. Knott, J. M. Werner, C. M. Cardy, I. D. Campbell, and P. A. Handford, Cell, 85, 597-605 (1996). 210. C. M. Kielty and C. A. Shuttleworth, FEBS Left., 336, 323-326 (1993). . Sunnerhagen, G. A. Olah, J. Stenflo, S. Forsen, T. Drakenberg, and J. Trewhella, Biochemistry, ,?5, 11547-11559 (1996). 212. C. R. Kelly, C . D. Diekinson, and W. Ruf', J. Biol. Chem., 272, 17467-17472 (1997). 213. E. Persson, 0.H. Olsen, A. gstergaard, and L. S. Nielsen, J. Biol. C'hern., 272, 19919-19924 (1997). 214. L. Perera, T. A. Darden, and L. 6. Pedersen, Biophys. J., 77, 99-113 (1999). . Bersch, J. F. Hernandez, D. Marion, and G. J. Arlaud, Biochemistry, 37, 1204-1214 (1998). 216. 0.D. Christophe, P. J. Lenting, J. A. Kolkman, G. G. Bmwnlee, and K. Mertens, J. Bzol. Chem., 273, 222-227 (1998). 217. €€.Lis and N. Sharon, Chem. Rev., 98, 637-674 (1998). 218. W. Weis and K. Drickamer, Annu. Rev. Biochem., 65, 441-473 (1996). 219. J. Rini, Annu. Rev. Biophys. Biomol. Struct., 24, 551-577 (1995). 220. W. Weis, R. Kahn, R. Fourme, K. Drickamer, and W. Hendrickson, Science, 254, 1608-1615 (1991). 221. W. Weis, K. Drickamer, and W. Hendrickson, Nature, 360, 127-134 (1992). 222. K. K.-S. Ng, K. Drickamer, and W. Weis, J. Biol. Chem., 271, 663-674 (1996). 223. S. Iobst and K. Drickamer, J . Biol. Chern., 269, 15512-15519 (1994). 224. A. Kolatkar and W. Weis, J. Biol. Chem., 271, 6679-6685 (1996). 225. S. F. Poget, G. B. Legge, M. R. Proctor, P. J. G. Butler, M. Bycroft, and R. L. Williams, J. Mol. Biol., 290, 867-879 (1999). 226. K.-S. Ng, S. Park-Snyder, and W. Weis, Biochemistry, 37, 17965-17976 (1998). 227. M. Quesenberry and K. Drickamer, J. Biol. Chem., 267, 10831-10841 (1992). 228. W. 1. Weis and K. Drickamer, Structure, 2, 1227-1240 (1994). 229. S: Sheriff, C. Y. Y. Chang, and R. A. B. Ezekowitz, Nature Struct. Biol., 1, 789-794 (1994). . J. Graves, R. L. Growther, C. Chandran, J. M. Rumberger, S. Li, K.4. Huang, D. H. Presky, P. Familletti, B. A. Wolitzky, and D. K. Burns, Nature, 367, 532-538 (1994).

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~ U R ~ NAND Y I FINN

231. C. Hession, L. Osborn, D. Goff, 6. Chi-Rosso, C. Vassdlo, M. Pasek, C. Pittack, R. Tizard, S. Goelz, K. McCarkhy, S. Hopple, and R. Lobb, Proc. Natl. Acad. Sci. USA>87, 1673-1677 (1990). 232. B. B. Nielsen, J. S. Kastrup, H. Rasmussen, T. L. Holtet, J. Graversen, M. Etzerodt, H. C . Thqgersen, and I. K. Larsen, F ~ Lett., ~ S412, 388-396 (1997). 233. J. S. Kastrup, €3. B. Nielsen, H. Rasmussen, T. L. Holtet, J. Graversen, M. Etzerodt, H. C. Thogemen, and 1. K. Larsen, Aeta Cryslallogr. D Biol. Crystallogr., 54 ( Pt 51, 757-766 (1998). 234. C. W. Heizmann and K. Braun, Trends Neumsci. 15, 303-308 (1992).

rler, an

Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106-9510, USA

1. 1NTRODU~~’IQN 1.1. Coordination Chemistry of Vanadium 1.2. Bioinorganic Role of Vanadium

S KNOWN STRUCTURE: IUM E ~ Z ~ E TH A D I U ~~ O ~ E R O X I D A S E S . Vanadium Chloroperoxidase 2.1.1. Occurrence and Putative Biological Role 2.1.2. Molecular Structure: Overall Protein Structure and the Vanadium Site 2 -2. Vanadium Bromoperoxidase rrence and Putative Biological Role cular Structure: Overall Protein Structure and the Vanadium Site

154 154 155 155 157 157 158 165 165 166

3.

3.2. Structural Considerations and Reactivity

167 167 168 169 169

4.2. ~ o ~ ~ a ~ aAspects t i v eof the Vanadium Sites in V-BrPO and V-CIP 4.3. ~ e ~ h a n i s tConsiderations ic of the Catalytic Cycle 153

170 172

154

BUTLER, CARTER, AND SIMPSON

5. ~ERSPECTIVES

173

AC~NO~ED~MENTS

174

BREVIATIONS AND DEFINITIONS

174

~E~ERE~CES

175

emistry of Vanadium Vanadium compounds exist in oxidation states ranging from - 3, - 1and 0, to + 1 , f 2 , +3, +4, and +5.Only the +2 to +5 oxidation states have been identified in aqueous solution, and of these only V(III), V(IV), and VCV) are found in biological systems. Coordination complexes of these ions span a beautiful array of colors from brilliant blue to deep yellow. Interconversion between the V(III), V(IV), and V(V) oxidation states i s accessible at neutral pH, with reduction potentials of 0.99 V (vs, NHE) for vanadiurnCV) and 0.542 V for vanadium(IV) [I]. For additional information, the reader is referred to several recent books and reviews that cover the inorganic and bioinorganic chemistry of vanadium [2-6] . The oxyanion vanadate(V1 (IFVOg-/H,VO,; pK, 8.3) is the most prevalent and stable species in aqueous solution at neutral pH. Vanadate undergoes polymerization reactions depending on its concentration, pH, and the nature of other ions present in solution. For example, HzVzO$-, V40&, and decavanadate, H2Vl0O&, are formed at higher vanadate concentrations (e.g., 2 mM) and at lower pH values. Of relevance to the vanadium haloperoxidase enzymes is the propensity of peroxide to coordinate to vanadate. Under neutral and alkaline conditions, hydrogen peroxide coordinates to vanadate, producing oxoperoxovanadates, with one to four coordinated peroxide ligands and peroxodivanadates, depending on the reaction conditions 17-10]. The coordination geometry adopted by nearly all oxoperoxovanadium(V) and oxodiperoxovanadium(V~small-molecule complexes is pentagonal bipyramidal 1111. The most common coordination geometry of V(II) and V(I1I) complexes is octahedral, while V( ) and V(V) are much more flexible, adopting tetrahedral, trigonal bipyramidal, octahedral, and pentagonal b i p ~ a m i d ageometries. ~ The coordination chemistry of vanadium(IV) is usually considered to be dominated by the vanadyl, V=02+, group, while that of vanadium(V) is dominated by V03 and cisV02+ groups. Exceptions exist, such as “bare” (i.e., without 0x0 ligands) V W ) and V(V) compounds are known, even with biological ligands (such as siderophores and siderophore analogues), but these complexes are not very stable in aerobic, aqueous solution [12,131.

’

155

OTEINS AND ENZYMES

ole of Vanadium Vanadium is widely distributed in nature. In the Earth’s crust, vanadium is more abundant than many metal ions, at 100 ppm. In the ocean, vanadium is the second most abundant transition metal ion at 30-50 nM after molybdenum at 100 nM 1141. Vanadium shows a nutrient-like distribution profile with depletion in surface waters, where organisms grow, relative to deeper waters [ 141. Given the relative abundance of vanadium in seawater, as well as the abundance of halides (e.g., 0.5 M C1-, 1mM and pM I-) and hydrogen peroxide, which is present at concentrations as high as pM in daylight hours due to photochemical events, it is not surprising that marine organisms have adapted to their chemical environment by evolving haloperoxidase enzymes. Tunicates (order Ascidiacea),common sessile marine animals (also known as sea squirts), may have also adapted to their chemical environment by acquiring vanadium, yet the role of vanadium has eluded investigators since its discovery near the turn of the last century. The vanadium exists primarily in the trivalent oxidation state, although some tetravalent vanadium is also present. It is generally believed that tunicates concentrate vanadium from seawater through a reductive process. The reduction and possibly complexation is aided by an organic compound, Cunichrome (Fig. I),which is a modified tripeptide derived from three hydroxydopa residues [15191.

WN S T ~ U C T U ~

Biological systems have evolved haloperoxidase enzymes to catalyze the oxidation of chloride, bromide, and iodide by hydrogen peroxide. The majority of these enzymes in

H2Nq QH

An-2 R1= H,R2 = O H An-3 R, = R2 = H

FIG. 1. Structures of selected tunichromes. “An” refers to species of Ascidia nigra from which these tunichromes are isolated.

TER, AND S I ~ ~ S

156

terrestrial systems contain the Fe-heme moiety, including chloroperoxidase from the fungus Caldariamyces furnaga, and myeloperoxidase, eosinophil peroxidase, and lactoperoxidase from mammalian systems. Other haloperoxidases that do not contain metal ions are also known, such as the chloroperoxidase in Pseudomanas pyrrocina [201. In marine algd systems, the vanadium-containing halopei-oxidases predominate [21-241, although a few examples of Fe-heme haloperoxidases in marine algae and worms are known 125-271. Hydrogen peroxide does not have the driving force to oxidize fluoride; hence, there are no fiuoroperoxidase enzymes. Wistor~cally,haloperoxidases have been named based on the most electronegative halide that they can oxidize. Thus, chloroperoxidase catalyzes the oxidation of chloride, bromide, and iodide by hydrogen peroxide; bromoperoxidase catalyzes the oxidation of bromide and iodide; whereas iodoperoxidase can only catalyze the oxidation of iodide by hydrogen peroxide. Perhaps a better basis for the nomenclature of haloperoxidases would be a particular enzyme's physiological reactivity in viva However, in many cases the conditions under which the enzyme would function are not known, such as the halide content in a cell and the pW of the environment. The overall reaction carried out by the vanadium haloperoxidases is: +

---+R-X+2H$3

(1)

efficient halogenawhere X- is Cl-, Br-, or I- and R-W is an organic substrate. ogenating 1mol of tion, 1mol o f halide is oxidized by 1mol of hydrogen peroxide strate (e.g., R-H, in the equation above) 128,291. In this reaction, one is also consumed. In some h~operoxida~e-cata~yzed turnovers depending on the nature of the organic substrate, much more hydrogen peroxide is consumed than halo-organic compound produced. In this case, the oxidized halogen intermediate (i.e., X+ in Fig. 2, below) is reduced by a second equivalent of hydrogen peroxide, producing singlet, oxygen and the halide 1301. The standard assay for hdoperoxidase activity is the halogenation of monochlorodimedone (MCD; 2-chloro-5,5-~methyl-1,3-d~medone~ using hydro the oxidant of the halide 1311 (see Eq. (2)). The halogenation of spectrophotometrically at 290 nm, which monitors the loss of MCD in the enol form (c = 20,000 M-' em-'). Hdoperoxidase activity is expressed as micromoles brominated or chlorinated per minute per milligram of enzyme &e., units per m ~ ~ ~ aThe m )early . work on vanadium bromoperoxidase employed the oxidation of iodide by hydrogen peroxide [321, forming triiodide (13) which was followed spectrophotometrically at 353 nm (E = 26,400 M-' cm-I). However, this reaction is less V-HPO "X+-like" intermediate X- + H202 --.-.-+ (eg., HOX, X2, ,X , Enz-X, V O ,,X)

[Orgl X-Org

kZ[H2021 '02

+ X'

FIG. 2. Overall reaction scheme of vanadium haloperoxidmes.

RQTEINS AND ENZYMES

157

desirable for quantitation of haloperoxidase activity because of competing side reactions, such as the n o n e n z ~ a t i coxidation of iodide by hydrogen peroxide and reduction of triiodide by hydrogen peroxide.

X= Br', Cl* hmax= 290 nm E = 20,000 M-'cm''

X 290 nm E = 100 M*'crn-'

xtensive studies on the kinetics of halogenation by vanadium bromoperoxidase from several alga 33-35] and the fungal vanadium chloroperoxidase [36] have been carried out using as the organic substrate, as well as kinetic studies of dioxygen formation 134,371 (Fig. 2). It is clear from these studies that the rate of oxidation o f halide is rate limiting when MCD is the substrate, and that the KJMGD] and K 2 [ I j [ 2 0 2 ] pathways are competitive [28,34,381. It should be noted that MCD may not be the best substrate to quantitate haloperoxidase activity for dl enzymes. As we learn more ~ ~X-ray ~ ~stmctures, a ~ ~ we $ may &isabout the active site channels of h ~ o ~ e ifrom cover some haloperoxidases that catalyze halogenation reactions of other substrates more ~ ~ ~than i M~ G [39,40], n~ ~ or~ that y even fail to halogenate MCD a t all. A recent review focusing on mechanistic aspects of the haloperoxidase reactivity is available 161.

.1, Vanadiu~~ h l o r ~ p e r o x i ~ a s e 2.1.1. Occurrence and Putative Biological Role

The vanadium chloroperoxidases (V-ClPO, E.C. 1.11.1.10) have been isolated from the dematiaceous hyphomycete fungi Curvularza inaequalis 1411 and E ~ ~ e l ldidye s ~ mospora [42]. Other fungi in this class have also been shown t o possess c~oroperox" idase activity, in agreement with the initial screening reported by Hunter-Cevera and Sotos [431 Some of these enzymes also cross-react immunologically with a polyclonal antibody to the C. inaequalis V-ClPO, including Dreclzslera biseptata, D. s u ~ ~ a p e ~ r fii, E. d i ~ y ~ o s p o r and a , ~ l o c ~ a d chartarum, iu~ suggesting that these fungi also I

Halogenated natural products have not been isolated from these fungi (unlike from marine algae that contain V-BrPO; see Sect. 2.2); thus, the biological role of VClPO is s ~ c ~ a t i vThe e . fungi that produce the vanadium chloropero~daseare phytopathogens and must penetrate the cell wall o i the host plant. The proposed functio~ of the fungal V-CIP is in the degradation of the plant's cell wall through pro~uction of hypochlorous acid (WOCI), which is a strong oxidant. The apparent variation in the biological function of the fungal V-Cl~Oand the marine algal vanadium bromoper) in fact reflect reactivity differences between these two oxidase ( V - ~ r P ~may enzymes.

15%


2.1-2. Molecular Structure: Overall Protein Structure and the Vanadium Site

The X-ray structure of V-ClPO (C. inaequalis) shows that the protein is cylindrical with approximate dimensions of 55 in diameter by 80 in length [451. (See Table 1 for a summary of the X-ray structures of vanadium haloperoxidases (Le., vanadium chloroperoxidase and derivatives and vanadium bromoperoxidase) deposited in the Protein Data Bank). One molecule is present per asymmetrical unit. The amino acid sequence predicts a protein containing 609 amino acids with a MW of 67,488, which has been confirmed by SDS-PAGE. V-CIPO contains two cysteine residues that are present as free thiols and not disulfide bridges. The secondary structure i s dominated by a helices. Two 4-helix bundles comprise the main structural motif of the tertiary structure (Fig. 3) [45]. The vanadium(V) site resides at the top of one of the four-helix bundles in a broad channel that is lined on one half with prcdominantIy polar residues, including an ion pair between Arg-490 and Asp-292 and several backbone carbonyl oxygens (Fig. 4). The other half of the channel is hydrophobic, containing Pro-47, Pro-211, Tyr-350, Phe-393, Pro-395, Pro-396, and Phe-397.

A

A

TABLE 1 Summary of Vanadium Haloperoxidase Enzymes with X-ray Stnictures PDB code

Source

Enzyme form or mutation

Date deposited

Resolution Ref.

(A,

-

e EC: 1.11.1.10 (Native) 01-Sep-1995

1VNC 1VME

lVNF 1VN 1vN lvNI 1WS

inaequalis Recombinant Recombinant Recombinant Recombinant Rcxornbinant Recombinant

nodosum

D292A R360A H404A H496A Holo APO

20-Jan-1999 20-Jan-1999 20-Jan-1999 20-Jan-1999 20-Jan-1999 14-Jam1999

Native

11-May-1999

Native

14-Jun-1999

2.10

45

2.15 2.35 2.20 2.11 2.15 1.66

49 49 49 46 46

2.0

57

49

VANADI~ IN~PROTEINS AND ENZYMES

159

FIG. 3. Structure of vanadium chloropcroxidasc from Curnularia imequalis.

One of the more striking features of the vanadium site is its apparent simplicity, which resembles vanadate (€€VO;-) coordination to the protein by one histidine ligand, His-496, in a trigonal bipyramidal geometry [441. The first X-ray structure of V-C1PO to be solved was the azide-coordinated derivative, which was a result of crystallization of V-C1FO from azide-containing buffer. In this first structure, azide was found to be coordinated to the vanadium(V) ion in an axial position as shown in Figs. 4 and 5 . Three oxygen atoms are also coordinated in an equatorial plane and an apical oxygen atom of a proposed hydroxide ligand [461. The oxygen atoms are all hydrogen-bonded to amino acid side chains or the peptide backbone of the protein, reducing the negative charge around the vanadate center (see Fig. 4). The structure of the recombinant V-ClPO crystallized in the absence of azide [46] revealed an apical 0 atom in place of azide at 1.93 A, which was interpreted as being an OH ligand (Fig. 6). This apical oxygen ligand forms three hydrogen bonds of which two are to water molecules and one is t o His-404 (2.97 A). His-404 is an important residue because binding of peroxide is inhibited if this residue is doubly protonated. The third structure to be solved was the peroxide-bound derivative of V-CIPO. The X-ray structure reveals a distorted tetragonal bipyramidal vanadium site (Fig. 7) [46] in which peroxide is coordinated in a side-on bound manner in the equatorial plane, along with an oxygen atom and His-496. An axial oxide ligand completes the pyramidal coordination geometry. The oxide ligand is hydrogen-bonded to Arg-490;

160

BUTLER, CARTER,A N D S I ~ P S ~

FIG. 4, Active site channel of V-ClPO from Curvularia inaequalis (constructed from [451).

FIG. 5. The vanadium site of azide-bound V-C1PO from Curvularia inaequalis.

INS AND ENZYMES

161

t~is496)

FIG. 6. The vanadium site of‘native V-CIPO from Curvularia inmequalzs. (Adapted from 1461.)

the coordinated peroxide is hydrogen-bonded to a glycine amide backbone and Lys353; the remaining oxygen atom is hydrogen-bonded to Arg-360. -ray structure of the ago form of V-CIPO is superimposable on the native s t ~ c t u ~ . sug~esting e, that the protein matrix is rigid and provides a preformed metal binding site [46]. A. water molecule in apo-V-C1PO is bound in lace of the vanadate ion in the native form. This water molecule is hydrogen-bond amide nitrogen of Gly-403. The X-ray structure of apo-V-C1PO( expressed in yeast reveals a sulfate ion that is partially stabilized by electrostatic interactions and hydrogen bonds with positively charged side chain residues involved interactions in the native structure [ 471. ray structure of the tungst~te-substitutedV-GPO reveals rep1 vanadate by tungstate r471; however, is-496 is either not coordinated t weakly coordinated, since the distance between the Ne2atom i.e., 2.55 (Fig. 8). The tungstate oxygen atoms hydrogen-b and a main chain amide similar to native V-ClPO, including a long €3 bond between the Thus, coordination of vanadate and tungstate can be oxide and His-404 at 3.21 attributed to “rac~-inducedbonding” of these oxyanions to a rigid protein matrix 1481. The structures o f several single-site mutants of V-C1PO (C.~ ~ u ~ are ~ also u u ~ known 149,501.These mutants are the result of re ement of His-496, th h i s ~ ~ ~with i ~ ealanine , ( ~ 4 9 6 A )replacement ; of 404, the putative a 0, a residue involved in charge tidine, with alanine ( 04A); replacement of compensation of the vanadate site, with alan 60A); and replacement of Asp292, the residue involved in a salt bridge with Arg-490, with overall protein backbone structures of these mutants are the same as that of native V-ClPO, and virtually superimposable on one another except in certain regions of the vana~iumsite, as discussed below [49-511.

A

A.

FIG. 7. Thc vanadium site of peroxo-V-C1PO &om Curuularia inaequalis. (Adapted from 1461.)

162


FIG. 8. The vanadium site of tungsten-substituted-CIPO €rom Curvularia inaequalis. (Adapted from [47J.)

2.1.2.1. The H496A mutant 1491 The substitution of the ligand histidine His-496 by alanine does not prevent vanadate coordination; however, this mutant lacks haloperoxidase activity. Vanadate is now coordinated as a tetrahedral anion with three nonprotein oxygen ligands at 1.56 A and one nonprotein oxygen ligand at 1.67 A. All of the side chain residues that hydrogen-bond to the vanadium center in the native structure also form hydrogen bonds in this structure, including that between His-404 and the apical oxygen of the vanadate ion. The superposition of the vanadium sites H496A on the structure of the wild-type enzyme shows the differences in the coordination geometries of the vanadium (Fig. 9). The superposition also demonstrates the remarkable rigidity of the amino acids that frame the anion binding site (including His-404, Ser-402, Gly-403, Arg-360, hg-490, ASP 292).

2.1.2.2. The H404A mutant 1491 The most significant structural change in the structure of the mutant in which alanine replaces His-404 is the broken salt bridge between Arg-490 and Asp-292 (Fig. 10) r491. Arg-490 moves into the place of His-404, assuming hydrogen bonding interactions between other side chain residues in place of the vanadate oxygen atoms in the native structure. Vanadium still remains coordinated to His-496 (1.96 A). The apical 0 atom sits 2.0 A from the vanadium, corresponding to a mixture o f water or hydroxide ligation, and the equatorial 0 atoms remain at 1.60 A. The removal of His-404 completely abolishes chlorinating activity, which may be a direct result of the loss of the histidine residue or of the resulting rearrangements in the active site. 2.1.2.3. The D292A mutant (491 Asp-292, which forms a salt bridge with Arg-490, helps to orient the side chain of Arg490 so that it can also hydrogen-bond with two vanadate oxygen atoms at N" and N'. In the mutant in which alanine replaces Asp-292, there is very little effect on the structure (Fig. 11). His-496 remains ligated t o the vanadate ion, and vanadate remains in hydrogen-bonding contact with Lys-353, Ser-402, the amide backbone of 613.-403, and Arg-360 and Arg-490, each of which is hydrogen-bonded to two different oxygen atoms. Despite the similarity with native V-CP0, this mutant has only 2% o f the activity of the native enzyme.

V A N A D I ~IN~PROTEINS AND ENZYMES

163

H496A & wild type

FIG. 9, Overlay of X-ray coordinates of the vanadium site of wild-type V-C1PO and the H496A mutant. 2.1.2.4. The 13360A mutant 1491 Arg-360 is directly involved in charge neutralization oE the E-fvOi center through a single hydrogen bond to one of the vanadate oxygen atoms. In the mutant in which alanine is substituted for arginine, one might expect as a result of the change in charge compensation a significant reorientation about the vanadate to achieve charge neutralization. Somewhat surprisingly, however, the superposition of the vanadium site of R360A on native V-ClPO shows little change in the position of the residues other than the lack of Arg-360 (Fig. 12). The chloroperoxidase activity of this mutant is about 6% of that of native V-CIPO, illustrating the importance of the charge-neutralizing effect of Arg-360. In summary, it is surprising how similar the overall structures are OE native VCIPO, the apo and metal-substituted derivatives, and the mutant enzymes, yet seemingly small changes effect significant consequences in reactivity [49]. The protein forms a very rigid active site matrix which is framed to bind oxyanions through charge neutralization by hydrogen bonding to the side chain residues of Lys-353, Arg-490, and Arg-360. His-496 is essential to promote the trigonal bipyramidal geometry about the vanadium center. His-404 seems to play an important role in peroxide coordination. In addition, hydrogen bonds from Ser-402 and the amide backbone of Gly-403 may also be essential, although not tested at this point.

164

BUTLER, CARTER, AND SlMPSON H404A & wild type

FIG. 10. Overlay of X-ray coordinates of the vanadium site of wild-type V-GlPO and the H404A mutant. D292A & wild type

Arg360

FIG. 11. Overlay of X-ray coordinates of the vanadium site of wild-type V-ClPO and the D292A mutant.

V ~ N A D IN I ~PROTEINS ~ AND ENZYMES

165

R360A & wild type

FIG. 12. Overlay o f X-ray coordinates of the vanadium site of wild-type V-G1PO and the R360A mutant.

2.2. V a n a d i u ~~ r o ~ o p e r ~ x i d a s e 2.2.1. Occurrence and Putative Biological Role

Vanadium b r o m o p e r o ~ ~ (V-BrPO) ~se has been isolated from all the major classes of marine algae, including e ~ i l o ~ o p h(green ~ a algae), phaeophyta (brown algae), and i no ~ e r o x ~ d a $ e $ ~ h o d o (red ~ ~ algae). ~ a AB described above, F e - ~ e ~ ~ - c o n t abjr~o ~ have also been isolated from these classes of algae, as well as other or~anisrns(e,g., certain marine w o m s 126,271);however, the vanadium haloperoxidase seems to be more prevalent. In addition to haloperoxidase enzymes, the production of halogenated natural products is also widespread in marine organisms [52,581. These compounds s) range from halogenated indoles (Fig, 131, terpeiies (Fig. 13), ~ c e t o ~ e n i nphenols, cte., to volatile halogenated hydrocarbons (e.g., bromoform, chlorofofbrrn, brornomethane, etc.), which are produced on a very large scale 1,541. Often the h ~ o g e n a t e ~ compounds isolated from the marine organisms have important biological activities, such as antimicrobial properties, or feeding deterrent properties, suggesting that they play a defensive role in the marine organism. In many cases the halogenated compounds also are of pharmaco~ogicalinterest due to their ~ t i ~ i c r o ~ iaan lt i, ~ ~ n g a l , antiviral, and a ~ t i - i ~ ~activities. ~ ~ a t o ~

166

BUTLER, CARTER, AND SIMPSON H ' -

R ' /

N

\

B

B

R

'I

e

I

R = H, indiw'

a-Snvderol

R = Br, 6,6'-dibromoindigotin

-0

B

X

Prepacifenoi L. pacifics

Br

H L. brongniartii

H

Ruvularia fima Perfwene L suboppositol

violacene P. violaceurn

FIG. 13. Examples of halogenated indole and terpene marine natural products. (Adaptedfrom [52,53].)

2.2.2. Molecular Structure: Overall Proteein Structure and the Vanadium

Site The vanadium bromoperoxidases are all acidic proteins, with similar amino acid composition, molecular weight, charge (PI 4-5) and vanadium content. Analysis of amino acid sequence similarities of V-BrPO (AscophyZZum nodosum) shows a 89% similarity to V-BrPQ from the 'brown alga Fucus distichus [55], and a 40.9% and 42% homology to the two bromoperoxidases produced by the red alga C. pilulifera [561. The X-ray crystal structure of native V-BrPO from the brown alga A. nodosum has recently been reported at 2.0 fi resolution [571. The final model reports V-BrPO as a homodimeric protein with approximate dimensions of 90 A x 77 A x 75 A. The secondary structure of the monomer unit is predominately a-helical with a few short [3 strands. Each monomer has three intramolecular disulfide bridges, as well as two intermolecular disulfide bridges, resulting in the covalently linked homodimer. In addition to intermolecular disulfide connections, four salt bridges and numerous side-chaidside-chain, side-chaidmain-chain interactions stabilize the dimer interface. Each monomer of V-BrPO has been calculated to contribute more than 46% of the monomer surface to contacts in the dimer interface. The extensive surface contacts and the compact helical structure of the homodimer provides a structural explanation fur the observed thermostability and chemical stability of V-BrPO. The molecular weight of V-BrPO is 120,400 Da, which agrees well with the predicted monomer molecular weight of 65,000 Da from reducing SDS-PAGE gels [581. Previously the molecular weight of the homodimer predicted by SDS-PAGE was reported as 95,000-~00,000Da [59,603. The core of the V-BrPO homodimer is built up of two 4-helix bundles 1571. The active site of the enzyme is part of the core structure, with the vanadate cofactor bound at the end of a 15-A-deepsubstrate funnel. Both monomers of the homodimer contribute residues to the substrate funneI surface. The funnel entrance has a diameter of approximately 12 A and narrows to approximately 8 A near the anion binding site. The funnel surface is composed of both hydrophilic and hydrophobic residues,

V A N A D I U ~IN PROTEINS AND ENZYMES

167

with the hydrophobic residues dominating the surface region around the vanadate binding site. The vanadium atom at the active center of V-BrPO resides in a trigonal bipyramidal coordination geometry similar to the vanndium(V) site in V-ClPO [571. Vanadium(V) in V-BrPO is coordinated by four oxygen atoms and ML2 of Wis-486. The negatively charged cofactor is neutralized by several hydrogen bond interactions from side chain residues at the active site. The protein residues (Ser-416, My-417, His-418, Lys-341, Arg-349 N"', Nq2,Arg-480 N1") act as proton donors to the oxyanion and constitute the central part of the rigid vanadate binding site. The active site of V-BrPO coiitains an additional histidine residue not present in V-CIPO, His-411, which is not directly coordinated to the vanadate cofactor. His-411 is within hydrogen bonding distance to one of the vanadate oxygcn atoms and may participate as a proton donor/acceptor during the enzymatic reaction. The X-ray structure of the peroxo derivative of V-BrPO has not been reported yet. Curiously, when crystals of V-BrPO were soaked in millimolar concentrations of H202, Le., conditions similar to the preparation of the peroxo derivative of V-ClPO, the electron density did not change 1573.

IUM E N ~ Y OF ~ ~U S~ K ~ OS TW R U~C T U ~ V A ~ A D NITROGE~ASE I ~ ~ 3.1.

Occurrence and Biological Significance

The reduction of dinitrogen to ammonia is accomplished by the two-component nitrogenase enzyme system, consisting of a nitrogenase enzyme harboring one of the MoFe, V-Fe, or Fe-only iron-sulfur cofactors and a reductase enzyrne (also called the iron protein, or the dinitrogenase-reductase),which supplies electrons to the nitrogenase protein. Vanadium nitrogenase has been purified from several species within the Azotobacteriaceae family, including hotobacter uinelandii [61-631, A. chroococcum [64], and A. paspaEi 1651. A vanadium-dependent nitrogenase has also been found in the cyanobacterium Anabaena uariabilis [661. d e ~ t of the soil Reports of the ef€ectof vanadium on the ~ i ~ i t r o g e ~ - d e p e ngrowth bacterium A. uinelandii first appeared in the 1930s. Later reports suggested that a vanadium nitrogenase could be obtained by growth on vanadium salts in the absence of molybdenum [671; however, it was not until 1986, using the mutant strain of A. uinelandii lacking the structural genes for the Mo-nitrogenase, that molecular nuances of the alternative nitrogenase were realiaed I6SI. In 1987,vanadium was found to stimulate the growth of strains lacking structural genes encoding Mo-nitrogenase ( n i ~ D . K I64 ) I. Vanadinm nitrogenase was subsequently isolated from this mutant strain of A. chroococcum 1641 and the related strain of A. uinelandii 162,631.In 1988, the third nitrogenase that lacks molybdenum and vanadium was isolated (691. It is now known that each of these nitrogenases is genetically distinct. The alternative nitrogen fixation pathway allows each of these bacteria to survive in an environment

IM

1

in ~ o l ~ or ~v a n~a ~ei u In ~~. u ~ have hi~heractivit~at temper s at cold t e ~ p e r a t ~[70]. re~

tions

~

~in the n s t o~i c h i~o ~ eof st ~

ctivity

~ A N A D I UIN~P OTEINS AND ENZYMES s-cys

s - cys cys-

s - cys FIG. 14. Structures of the M and P clusters of nitrogenase. (Adapted from [90,911,)

the ~o-dinitrogenaseenzyme is ammonia, although hydrazine, NzH4,is observed in the V-nitrogenase system along with ammonia [79]. The proposed mechanism for Monitrogenase involves a series of enzyme-bound reduced &nitrogen species that are not released from the enzyme. All nitrogenases can also reduce acetylene to ethylene. The vanadium enzyme can also form ethane as a minor product of this reaction whilc the molybdenum enzyme cannot. Ethane production has been suggested as a test for nonmolybdenum nitrogenases [SO].

RELAT 1

0 ~IPS s~

4.1. Va~adium~ a l o p ~ ~ ~ x ixpression d a s ~ Systems In the last 5 years there has been a rapid increase in what is known about the structure and function of haloperoxidases. The wealth of information obtained from X-ray crystal structures and the applications toward mechanism and reactivity are the result of' successful cDNA sequence determinations and protein expression systems, The complete cDNA sequence and the derived amino acid sequence for vanadium chloroperoxidase has been determined [Sll. Native V-C1PO from C. inuequalis can be isolated from the growth media of this fungus, classifying the enzyme as a secretion protein. Howcver, when the cDNA clone for the enzyme was isolated no putative leader peptide could be assigned, which is normally observed in precursors of secreted proteins. The isolated cDNA of V-C1PO was initidly expressed in a bacterial expression system but afforded low yields for the enzyme. Recently, V-ClPO has been sucmssfully overexpressed in a yeast recombinant expression system [50]. The heterologous expression of V-ClPO in S ~ c c ~ a r ~ ~ y c cereuisiae afforded protein yields of LOO mgL o€ yeast culture for apo-rV-GlPO.


170

Experiments were performed to determine if rV-CiPO would be secreted into the growth media for yeast with or without being fused to the yeast mating type a factor, which directs secretion of proteins to the media. Surprisingly, no stable expression was observed when the a factor was fused in frame with the gene for V-ClPO. Furthermore, when the rx factor was omitted the protein was not secreted into the media but was found within the cytoplasm of the yeast in high yields. The isolation of large amounts of pure rV-C1PO obtained with the yeast expression system has enabled the creation of site-directed mutants of the enzyme and further structural characterization. In addition to cloning and expression of vanadium chloroperoxidase, vanadium bromoperoxidase from the red macroalga Corallina pilulifera has been cloned and expressed. Cloning of V-BrPO from C. pilulifera produced two separate clones; bpol and bpo2, where the two clones show 90% homology to one another 1561. The isolated cDNA for hpol was initially expressed in a bacterial expression system. The protein was successfully expressed in the bacterial system, but overexpression of the enzyme was not achieved. The recombinant bromoperoxidase (rV-BrPO1) that was isolated from the bacteria was in the apo form, and bromoperoxidase activity could be observed upon incubation with vanadium. In addition, N-terminal sequencing of rV-BrPOl was found to wholly agree with the sequence predicted by cDNA nucleotide sequence from bpol with the exception of the N-terminal methionine residue. The N-terminal amino acid or V-BrPO isolated from the alga was masked, but this was not found to be the case for the recombinant enzyme isolated from the bacteria. The isolation of cDNA clones and the overexpression of recombinant protein €rom various vanadium-dependent haloperoxidases have allowed the detailed structural characterization of native and mutant forms of the enzyme. In addition, the use of recombinant protein has aided the continued exploration of mechanisms of reactivity between vanadium-dependent chloroperoxidases and bromoperoxidases.

spects of the Vanadium Sites in Vv-CIPO The X-ray structures of V-BrPO €rom A. nodosum 1571 and V-ClPO from C. inaequalis 145,461, as well as apo-V-C1PO 1473, its metal-substituted derivatives 14'71, and the site-directed mutants of V-ClPO [491 are remarkably similar, but with certain notable differences. All o f the stiuctures reveal strong structural similarities at the vanadium site, which is not surprising given that all of the residues that participate in vanadate binding are conserved (Fig. 15). The protein provides a rigid oxyanion binding site, which is stabilized by hydrogen bonding interactions between these conserved residues and the oxyanion. In addition, both active sites for V-BrPQ and V-ClPO are found at the core of a four-helix bundle structural motif in the enzymes. However, the overall amino acid sequence similarity of the structurally aligned residues is surprisingly low, at only 21.5%. As described above, the quaternary structure of V-

VANA~IU IN~PROTEINS AND ENZYMES

171

FIG. 15. Overlay of the X-ray coordinates of V-C1PO (Curvulnriu inuequalis) and V-ErPO (Ascophyllum nodosum), (Reproduced with permission from l571.1 See Fiyrc 6.15 in the color inuert.

ClPO (C. inaepualis), a monomer, and V-BrPO (A. nodosum), a dimer, do differ, as well as their active site channels, undoubtedly as a result of differences in the primary stmcture. In both enzymes the vanadium binding site motif is P[S/AIvPS vanadate is diredly coordinated to a histidine in the axial position L45-491. The structures of V-BrPO and V-GlPO also render the proposed catalytic histidine as axially aligned with the vanadate cofactor, demonstrating again the highly conserved anion binding site, Equatorial ligands of the vanadate cofactor for Q-BrPO and VCLPO also appear to be conserved, although there are different residue-residue interactions and ligand stabilization that occur between the two haloperoxidases. h i n o acid alignments of V-GIPO and V-BrPQ for Ihe vanadate binding site show that QBrPO has an additional histidine residue that may be present in the vanadate binding site. Structural alibmment of V-BrPO and V-ClPO show the additional histidine aligns with a phenylalanine in V-ClPO. The crystal structure of V-BrPO indicates that the extra histidine has no direct interaction with vanadate oxygen. It has been proposed that the residue might possibly participate as a proton/donor acceptor during the enzyrnatic reaction. The role of the additional histidine found in V-BrPO will need to be explored further. In spite of the overall low sequence similarity between Q-BrPQ and V-CIPO, it appears that the residues involved in cofactor binding are highly conserved, which may indicate a common genetic origin.

~

172

U

~CARTER, L ~ AND ~ ,S I M ~ S O ~

echanistic Considerati~~s of the Cataiy~icCycle The first step in the catalytic cycle of V-BrPO and V-CIPO is coordination of hydrogen peroxide to v a n a d i u ~ (Fig. ( ~ 16, step 1). As discussed above, in the resting state, vana~um(V) is bound in a trigonal bipyramidal geometry ligated by a single protein side chain: His-496 in V-ClPO (C znaequalzs) or His-486 in V-BrPO (A. nodosum,). The three equatorial oxygen ligands and the axial hydroxide ligand are hydrogenbonded to multiple protein side chains or the protein backbone. The acidbase histidine (His-404 in V-CIPO (C. inaequnlis) or His-418 for V-BrPO (A. nodosum,)),which is present in the active site channel, must be deprotonated for H202 to bind 1823. Upon coordination of peroxide, the oxoperoxovanadium(V) intermediale is poised to oxidize the halide (Fig. 16, step 2). However, aqueous solutions of' oxoperoxovanadium(V)or oxodiperoxovanadium(V)do not oxidize bromide or chloride 171, Thus, it is significant that in the X-ray structure of V-CIPO Lys-353 was found to be in hydrogen-bonding contact with the bound peroxide, which could further activate it for oxidation of bromide or chloride. While the structure o f tlze peroxo intermediate of V-BrPO has not been reported, one might ~ t i c i p a t ethat Lys or His residues could participate in similar hydrogen-bonding interactions. Step 2 in Fig. 16 seems to depend on the type of haloperoxidase; thus, the nature o f the oxidized halogen intermediate (see Fig. 2) may vary between enzymes as well as the nature of the organic substrate to be halogenated in the case of &gal V-tr-BrPOs. In V-BrPO appropriate organic substrates can bind to the enzyme, blocking the release of an oxidized halogen intermediate [39,40J,whereas the function of V-GLPO (C. inaequalis) is proposed to be in production of HOCl in the absence of organic substrate. Thus, the oxidized halogen intermediates could differ between V-C1PO and V-B n either halogenate the organic subthe oxidized halogen intermedi , or oxidize a second equivalent

,.His 401

[

h(His)

FIG. 16. Proposed catal.ytic cycle of vanadium haloperoxidase.

V A N ~ IN~PROTEINS I ~ ~ AND ~ N Z Y M ~ S

173

What does the reactivity of the site-directed mutants tell us about the mechanism of V-ClPO? All of the changes (i.e., H404A, 292A H496A R360A, K353A, It49OA) eflectively abolish chloroperoxidase activity, although one mutant, retains 6% of the chlorinating activity of recombinant V-ClPO, which i s the highest activity of these mutants [49,50]. The lack of activity of H496A establishes the essentiality of the liganded histidine for reactivity [49l. Little has been reported about the activity of the H404A mutant except that it lacks chlorinating activity (and there was no mention of the brominating activity) [49]. Some of these mutants can, however, catalyze the oxidation of bromide by hydrogen peroxide, including K353A, ~ 3 6 0 Aand , 11490A [50].All o f these residues are involved in hydrogen-bonding interactions with the equatorial oxygen atoms ligated to vanadium(V) in the resting state. While the bromoperoxidase activity appears t o be substantial, the specificity constant, kcat/Km, is always one to three orders of magnitude less than rV-C1PO over the pH range investigated, pll 4.2-7.0 [sol. The kcat/&, value for the K353A mutant is the worst of the mutants, indicating the importance of this residue in the activation of vanadate-bound peroxide. Clearly, further studies of these mutants and others will be essential to our u~derstandingof the mechanism of the vanadium haloperoxidases.

5. The active sites o f V- rPO (A.nodosum) and V-C1PO (C. infaequalis)display sequence homology with a family of acid phosphatases [83-85]. This includes both soluble and membrane-bound isoforms of phosphatidic acid phosphatase (PAP), enzymes crucial in mammalian signal transduction, ~lucose-6-pho~phatase, which is the enzyme affected in von Gierke disease, also has a homologous domain. The PAP family all have a perfectly conserved motif ( is similar to the vanadate binding region of the haloperoxidases, The histidine covalently bound to the vanadate cofactor (residue 496 in V-ClPO) is conserved in each of the family of enzymes. In addition, the haloperoxidase residues that form hydrogen bonds to the equatorial oxygens of the vanadate are also well conserved among phosphatidic acid phosphatases. It is known that vanadale is a competitive inhibitor of'phosphatases. It has also been found that remetallation of apohaloperoxidases is inhibited by phosphate. This information suggests that acid phosphatases and vanadium haloperoxidases may have similar three-dimensional structures at the active site as well as similar amino acid sequences. The similarity was confirmed when apo-V-C1PO was shown to have phosphatase activity 1831. Recombinant apochloroperoxidase hydrolyzes ~-nitrophenyl phosphate, a commonly used phosphatase substrate. The Km was found to be 51 pM, which compares well with those for acid phosphatases (100-200 pM). However, the maximal turnover for apo-C1PO was only 1.7min-', which is much slower than the reported values of acid phosphatases (i.e., 102-103s-').

BUTLER, C A R T ~ RAND , SIMPS~~

174

In addition to a haloperoxidase exhibiting phosphatase activity, a vanadateincorporated phytase has been prepared that has peroxidase activity 186,871. Phytase isolated from the fungus Aspergillis ficuum is a phosphatase in its native state ke., without a bound metal ion). The vanadate-phytase derivative catalyzes the oxidation of sulfides by hydrogen peroxide. When the substrate is thioanisole, the ($3)sulfoxide enantiomer is the predominant product, formed with an enantiomeric excess (ee) of 56% and with a turnover frequency of 11min-l. Vanadium bromoperoxidases from A. nodosum L881 and C. piZuZiferu LS91, on the other hand, can also catalyze the enantioselective oxidation of sulfides t o sulfoxides. These reactions occur slowly, with turnover frequencies of 1min-'. V-BrPO from A. nodosum reacts with aromatic sulfides to form the (Rj-sulfoxide in about 80% ee I881.

Al3 gratefully acknowledges grants from the U.S. National Science Foundation (CHE 9529374) and the U.S. National Institutes of Health (GM38130). Partial support for our work on marine haloperoxidases is also sponsored by NOAA, U.S. Department of Commerce under grant NA66RG0447, project R/MP-76 through the California Sea Grant College System and in part by the California State Resources Agency. The views expressed herein are those of the author and do not necessarily reflect the views of NOAA. The U.S. Government is authorized to reproduce and distribute for governmental purposes.

A

V I AT10NS

enantiomeric excess extended X-ray absorption fine structure monochlorodimedone (= Z-chloro-5,5-~methyl-l,3-dimedone~ normal hydrogen electrode phosphatidic acid phosphatase PAP recombinant vanadium chloroperoxidase rV-C1PO SDS-PA G ~sodium dodecyl sulfate polyacrylamide gel electrophoresis vanadium bromoperoxidase V-BrPO vanadium chloroperoxidase v-ClPO vanadium haloperoxidase V-HPO

ee E M S MCD NHE

V A N A ~ ~IN~PROTEINS M AND ENZYMES

R

175

NC 1. A. Butler, in Vanadium in Biological Systems: Physiology and Biochemistry (W. I). Cliasteen, ed.), Kluwer Academic, Dordrecht, 1990, pp. 25-50. 2. H, Sigel and A. Sigel (eds.), Vanadium and Its Role i n Life. Metal Ions in Biological Systems, Vol. 31 , Marcel Dekker, New York, 1995. 3. N. D. Chasteen (ed.), Vanadium in Biological Systems: Physiology and Biochemistry, Kluwer Academic, Dordrecht (1990). 4. A. S. Tracey and D. C. Crans (eds.), Chemistry, Biochemistry and Insulin Mimetic Influences of Vanadium, ACS Symposium Series, 117 (1998). 5. D. C. Crans, Comments Inorg. Chem., 16, 1-33 (1994); D. C. Crans, Comments Inorg. Chem., 16, 35-76 (1994). 6. A, Butler, Coord. Chem, Rev., 287, 17-35 (1999); A. Butler, Struct. Bonding, 89, 109-132 (1997). 7. M. J. Clague and A. Butler, J. Am. Chem. Soc., 117, 3475-3484 (1995). 8. A. T. Harrison and 0. W. Howarth, J. Chem Soc. Dalton Trans., 1173-1177 (1985). 9. 0. W. Howarth and J. R, Hunt, J. Chem Soc. Dallon Trans., 1388-1391 (1979). 10. N. J. Campbell, A. C. Dengel, and W. P. Griffith, Polyhedron, 8, 1379-1386 (1989). 11. A. Butler, M. J. Clague, and G. Meister, Chem. Rev., 94 625-638 (1994). 12. P. Comba, L. M. Engelhardt, J. Harrowfield, 6. A. Lawrence, L. L. Martin, A. M. Sargeson, and A. €3. White, J . Chem Soc. Chem Commun., 174176 (1985). 13. T. B. Karpishin, T. M. Dewey, and K. N. Raymond, J. Am. Chem. Soe., 115, 1842-1851 (1993). 14. R. W. Collier, Nature, 309, 441-444 (1984). 15. R. C. Bruening, E. M. Oltz, J. Furukawa, K. Nakanishi, and K. Kustin, J. Am. Chem. Soc., 107, 5298 (1985). 16. R. C. Bruening, E. M. Oltz, J. Furukawa, K. Nakanishi, and K. Kustin, J. Nat. Prod., 49, 193 (1986). 17. E. M. Olta, R. C. Bruening, M. J. Smith, K. Kustin, and K. Nakanishi, J. Am. Chem. Soc., 110, 6162 (1988). 18. K. Kustin, W. E. Robinson, and M. J. Smith, Invert. Pepro. Dev. 17, 129-139 (1990). 19. M. J. Smith, D. J. Ryan, K. Nakanishi, P. Frank, and K. 0. Hodgson, Metal Ions Biol. Syst., 31, 423-490 (1995). 20. K.-H. van Pee, K. Hohaus, A. Altman, W. Burd, S Hammer, and J. Ligon, in Mechanisms of Dehulogenation, Proceedings of thc Royal Netherlands Acadeniy of Arts and Sciences, 43-54 (1997).

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22. 23.

24 25. 26. ~

27. 28, 29. 30. 31.

32. 33. 34.

utler and J. V. Walker, Chem, Rev., 93, 1937-1944 (1993). A. Butler, for Comprehensive Biological Catalysis (M. Sinnott, ed.), British Academic Press, 1997, pp. 427-437. R. Wever and B. E. Krenn, in Vanadium in Biological Systems: Physiology and ~ i o ~ ~ h e m(N. i s D. t ~ Chasteen, ed.), Muwer Academic, Dordrecht, 1990, pp. 81-98. . Vilter, Metul Ions Biol.Syst., 31, 325 (1995) J. A. Manthey and L. P. Hager, J. Biol. Chem., 260, 9 6 5 ~ 9 6 5 9(1985) $3, Franzen, M. P. Roach, Y. P. Chen, R. B. Dyer, W. H. Woodruff7and J. N. Dawson. J.Am. Chem. SOC.,120, 4658-4661 (1998). . P. Roach, Y. P. Chen, S,A.Woodin, D. E. Lincoln, C. R. Lovell, and J, H. awson, Biochemistry, 36, 2197-2202 (1997). . Everett and A. Butler, Inorg. Chem., 28, 393-395 (1989). nt Opin. Chem. Biol.,2, 279-285 (1998). R. Kanofsky, and A. Butler., J. BioL Chern., 26& 4908-4914 (1990). L. P. Hager, D. R. Morris, 3'. S.Brown, and H. Ebemein, J- Biol. Chem., 1, 1769-1777 (1966). Viltwtr,~ h y ~ f ~ ~ h 23, e ~ 1387-1390 i s ~ ~ , (1984). E. de Boer and R. Wever, J. Biol. Chem., 263, 12326 (1988) R. Everett, N. 8. Soedjak, and A. Butler, J. Bio2. @hem., 266 15671-

S. Soedjak and A. Butler, Biochemistry, 2Q, 7871-7981 (1990). W. P. M. Van Schijndel, P. Barnett, J. Roelse, E. G, M. Vollenbroek, and Wever, Eur. el. Biochem., 225, 151-157 (1994). 37. H. S. Soedjak and A. Butler, Biochim. Biophys. Acta, 1079, 1-7 (1991). . S. Soedjak, J. V. Walker, and A. Butler, Biochemistry, 34, 1 2 ~ $ 8 - 1 2 6 ~ ~ 38.

35.

39. 40.

41.

42. 43. 44.

. A. ~

s c ~ i ~ e tand - ~A.u Butler, ~ h J.Am. @hem. Soe., 226, 411-412 (1994). Butler, R. A. Tschirret-Guth, and M. T. Simpson, in Chemistry i o c h ~ ~ ~and ~ s ~Insulin r y Mimetic Influences of Vun'adium, ACS Sy~posium Series, (A. S. 'I'racey and D. C. Crans, eds.), 1998, pp. 202-215. J. W. P, M, Van Schijndel, E. C .& Vctllenbroek, I. and R. Wever, Biochirn,. iophys, Acta, 1161, 249-256 (1993). . Barnett, W, Hemrika, 1%.L. Dekker, A. 0. Muijsers, R. iol. Chem., 273, 23381-23387 (1998). -Cevera and L. Sotos, 211icmb. Ecol., 12, 121-127 (1986). E. 6 . M.~ollenbroek,L. H. Simons, J, W. 9. M. Van Schijnd M. Balzar, H. Dekker, C. Van Der Linden, and R. Wever, Trans., 23, 267-271 (1996). ,

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67. P. E. Bishop, ID. M. L. Jarlenski, and I).R. Heatherington, Proc. Natl. Acad. Sci, USA, 74, 7342 (1980). 68. For reviews see: R. R. Eady, Met. Ions Biol. Syst., 31, 363405 (1995); R. R. Eady, Chem. Rev.,96, 3013-3030 (1996). 69. J. R. Chisnell, R. Premakurnar, and P. E. Bishop, J. Bactpriol., 170, 27 (1988). 7'0. R. W. Miller and R. R. Eady, Biochem. J., 256, 429-432 (1988). . D. Joerger, T. M. Loveless, R. N. Pau, L. A. Mitchenall, B. H. Simon, and P. E. Bishop, J. Racteriol, 172, 3400-3408 (1990). 72. R. M. Allen, M. J. Homer, R. Chatteqjee, P. W. Ludden, 6. P. Roberts, and V. K. Shah, J. Bid. Chem., 268, 23670-23674 (1993) 73. R. Chatterjee, P. W. Ludden, and V. K. Shah, J. B i d . Chem., 272, 3758-65 (1997). 74. J. M. Arber, . R. Dobson, R. R. Eady, S. S. Hasnain, C. D. Garner, T, Matsushita, M. Nomura, and B. E. Smith, Biochem. J., 258, 733-738 (1989). 75. J. E. Morningstar, M. K. Johnson, E. E. Case, and B. J. Hales, Biochemistry, 26>1795-1800 (1987). 76. J. M. Arber, . R. Dobson, R. R. Eady, P. Stevens, S. S. Hassain, C. D. E. Smith, Nature, 325, 372-374 (1987). 77. J. Chen, J. Christiansen, R. C. Tittsworth, B. J. Hales, S. J. George, D. Coucouvanis, and S. P. Cramer, J. Am. Chern. Soc., 115, 5509-5515 (1993). 78. N.Ravi, V. Moore, S. Lloyd, B. J. Hales, and B. H. Huynh, J. Biol. Chem., 269, 20920-20924 (1994). 79. M. J. Dilworth and R. R. Eady, Biochem. J., 277, 465-468 (1991). 80. M.J. Dilworth and R. R. Eady, R. L. Robson, and R. W. Miller, Nature, 327, 167-168 (1987). 81. B. H. Sirnons P. Barnett, E. 6, M. Vollenbroek, H. L. Dekker, A. 0. esserschmidt, and R. Weaver, Eur. J. Biochem., 229, 566-4574 (1995). 82. J. W, P. M. Van Schijndel, E. 6. M. Vollenbroek, and R. Wever, Biochim. Biophys. Acta, 1161, 249-256 (1993). 83. W. Hemrika, R. Renirie, H. L. Dekker, P. Barnett, and R. Wever, Proc. NatZ Acad Aci. USA, 94, 2145-2149 (1997). 84. A. F. Neuwald, Protein Sci.,6, 1764-1767 (1997). 85. J. Stuckey and 6. M. Carmen, Protein Sci., 6, 469-472 (1997). 86. F. van de Velde, L. Konemann, F. van Rantvvijk, and R. A. Sheldon, J. Chem. Soc. Chem. Gomunun., 1891-1892 (1998). 87. F. van de Velde, L. Konemann, F. van Rantwijk, and R. A. Sheldon, Biotech. Sioeng., 67, 87-96 (2000). 88. H. Brink, A. Tuynman, H. L. Dekker, W. Hemrika, U. Izumi, T. Oshiro, H. E. Schoemaker, and R. Wever, Inorg. Chem., 37, 6780-6784 (1998).

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89. M. Andersson, A. Willetts, and S. Allenmark, J. Org. Chem., 62, 8455-8458 (1997). 90. M. K. Chan, J. S. Kim, and D. C. Rees, Science, 260, 792-794 (1993). 91. J. W. Peters, B. H. B. Stowell, S. M. Soltis, M. G. Finnegan, M. K. Johnson, and D. C. Rees, Biochemistry, 36, 1181-1187 (1997).


Chemistry Department, University of Virginia, Charlottesville, VA 22903, USA

181

1. I ~ T R ~ ~ U C T I O ~

ITH KNOWN ST 3. ~ N Z ~ ~ S ~ ~ WITH O T U E I N~ S ~ STRUCTURE O ~

385 186

187 188 188

RE~~RENCES

The role of chromium in living systems is controversial. For years it has been deemed an essential mineral in humans [11. On the other hand, some consider it merely beneficial, others a therapeutic reagent, and still others simply toxic. Chromium is clearly toxic in its Cr(VI) oxidation state, but there is growing evidence that Cr(II1)is also deleterious. The essentialists argue that Cr(III) is a crucial component. of the 80called glucose tolerance factor that potentiates insulin action, but in 40 years this factor has neither been isolated nor synthesized. Most recently, attention has been directed to a peptide that binds chromium, but neither the structure nor the function of this peptide is secure. Since these topics are interrelated, this chapter offers a brief overview before ending with a synopsis of reported peptide binding by chromium(II1). 181

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Two monographs on chromium chemistry emphasize environmental impacts but also cover biological aspects !2,31. A shorter review deals only with biological relevance 141. Owing to its low amount in living organisms and its ubiquity in the environment, reliable analysis for chromium has been an enormous problem in assessing its physiological importance and implications. The human body contains only about 1mg of total chromium. n the 20 years from 1960 to 1980 the “normal” blood serum concentration was reduced more than 1000-fold to ultratrace levels of about 0.1 ppb or 2 nmoW. At these levels, contamination by chromium in reagents and containers threatens reliability and even today makes accurate analysis a challenge. Caution is needed in citing all values obtained earlier than about 1980, and even later ones, unless stringent precautions have been taken. Chromium exists in several oxidation states, of which five may be significant for biological systems. Of the two most common, Cr(1II) is claimed t o be essential to mammals, while Cr(VI) is strongly oxidizing and toxic. The much less stable Cr(II), Cr(IV), and CrW) may be important as reactive intermediates. Cr(I1) is strongly reducing and slowly liberates Hz from water. About the only ore o f chromium is chromite, FeCr204, in which both metals appear in a low oxidation state, Fe(I1) and Cr(II1). In contrast, the main oxidation state of chromium in fresh water and seawater appears to be Cr(VI) [51,but it occurs only at nanomolar concentrations. In acidic solutions Cr(VI) exists as yellow hydrogen chromate, HCrO; , which undergoes proton loss with pKa 6.3 to yield tetrahedral chromate, CrOi-, the predominant intracellular form near pH 7. Not until 10 mM concentration in acidic solutions does red dichromate form in a reversible equilibrium with intermediate orange solutions: 2HCrOi + HzQ + CrzO;-. Chromate is a skin irritant and carcinogen [6-8]. It enters cells surreptitiously via anion transport systems. It is reduced in cells to Cr(II1)primarily by ascorbate and glutathione and in the blood serum by glutathione [91. It is important to recognize for Sect. 3 that intravenous injections of’ (&)chromate result in Cr(T1I) ekewhere in the animal. In the reduction process two species of intermediate oxidation states are produced, Cr(IV) and Gr(V), and these intermediate species are responsible for damage either by interacting directly with DNA or producing radicals that do D O 121. Cr(V) produced by inicrnsonial reduction of chromate is stable for 1h [MI. Cr(II1) produced by reduction provides cross-links between DNA and cysteine and glutathione /141, and proteins 1151. That up to 50% of the DNA-Cr3+adducts contained glutathione and lesser amounts with histidine and cysteine [16] belies the utility of designating Cr3+ as a “hard” metal ion 1171. Directly administered Cr(II1) complexes of glutathione, histidine, and cysteine are mutagenic in human fibroblasts [181. Although the contrast between the toxicity of Cr(V1) and the claimed essentiality of Cr(II1) has been described as a paradox 131, there are other cases of an element being essential in one oxidation state and toxic in another, e.g., Na’ and Na, 61- and Clz. In acidic solutions, with a nonbinding counterion, Cr(II1) exists as the violet octahedral hexahydrate, Cr(Hz0)36+.(When chloride ions displace water as ligands, the complex becomes green.) At about 25°C and 0.16 ionic strength, successive loss of

ARE THERE PROTEINS C O ~ T A I ~ CHROMIUM? IN~

183

protons from bound water molecules in the hexahydrate occurs with successive pK, values of 4.6, 6.1, 8.4, and 9.2 1191, making C r ( ~ ~ O j * (the O ~predominant )~ soluble species at pW 7 in dilute solutions. At greater than micromolar concentrations multimeric hydroxo-bridged complexes form slowly [19-221. In neutral solutions insoluble Cr(OH), precipitates; it is necessary to store CR,' solutions at pR < 3. Only about 1%of ingested Cr(II1) is absorbed by the body, and the insolubility of Cr(OHj, undoubtedly contributes to this low percentage. With phosphate Cr(I1I) very slowly forms several complexes and very insoluble precipitates [231, which may also contribute to its low absorption. If analogous to Al(III), mixed nonstoichiometric hydroxo and phosphate precipitates may also form [241. Also analogous to Al(III), absorption may occur from coordination with dietary citrate to yield a neutral complex that permeates membranes F251. Once in the blood plasma, Cr(I1Ij may well apportion similarly to Al(III), where about 90% is carried by transferrin (see Sect. 3) and 10%by citrate (0.1 mM in the plasma), both binding more strongly than plasma phosphate 124-261. Owing to the stability and inertness of Cr(II1) complexes, thousands have been characterized; almost all are hexacoordinate or octahedral. At 25°C the lifetime for exchange of a bound water in Cr(H20)i'is about 14 h (271. In sharp contrast, exchange of bound water in aqueous Cr(I1) is more than lOI4 times faster. If only a fraction of an inert Cr(I1I) complex is reduced momentarily to Cr(II), a pathway exists for ligand exchange. Sulfhydryl groups occurring in cysteine and glutathione in cells and serum may be able to effect this fractional reduction, providing a pathway for ligand exchange of otherwise highly inert Cr(II1j complexes through intervening Cr(1I) complexes with reoxidation. Cr(1I) has been used to affinity-labelthe electron transfer locus in plastocyanin; subsequent oxidation to Cr(II1) fixes the site 1281. Ligand exchange may be facilitated in Cr(I1I) complexes by enforcing irregular, not strictly octahedral geometries: tetragonally distorted Schiff base [29,30) and porphyrin complexes 1311, and quinquedentate EDTA 1321. The cited ligands themselves remain tightly bound; it is the remaining positions that undergo relatively rapid exchange. It is questionable, however, whether such distorted structures occur with the ligands that have been suggested for glucose tolerance factor below. Whenever considering Cr(III), rate effects rather than equilibria may indeed apply. Forty years ago chromium was advanced as an essential element in rats [33/. Eats on a low-chromium diet exhibited a reduced tolerance to intravenous injections of glucose that was alleviated by addition of several CrfIII) compounds. One of the coauthors of this report, Schwarz, subsequently proposed an essential role in rat growth for fluorine, tin, vanadium, silicon, and even lead [34,35]--elements not usually associated with essentiality in mammals. Fluorine, for example, may be beneficial for bones and teeth, but not essential. Meanwhile, Mertz has continued to advocate and has amplified an essential role for chromium in mammals, including humans 136-381. The best alleviating response to glucose intolerance was obtained with an insulin potentiating substance in brewer's yeast called glucose tolerance factor (GTF). In addition t o chromium its composition was suggested to be composed of two equiva-

184

MARTIN

lents of nicotinic acid (3-pyridinecarboxylic acid), glycine, glutamic acid, and cysteine [39,401. (With expected chelation by the last three amino acids there are too many donor atoms for the hexacoordinate Cr(III).) A disconcerting feature was that the most highly concentrated GTF fractions lost their activity upon storage even at 4°C [33,371, an odd result for a putative inert Cr(II1) complex. Noting that the last three amino acids are the components of glutathione (y-glut~ylcysteinylglycine),a synthetic mixture of this tripeptide (not found in yeast), Cr(III1, and nicotinic acid was tested and also found to exhibit GTF activity L41I. In later work on CTF, other investigators could not detect nicotinic acid, glycine, glutamic acid, or cysteine associated with the chromium 1421, or even other than adventitious chromium L43-451. Another rat study was unable to assign glucose intolerance solely to dietary Cr(III), suggesting a more complex relationship (461. Thus, more than 40 years after its proposal, the composition, let alone the structure, of the putative GTF from brewer’s yeast remains an open question. For positive reviews of GTFs see [361 and [471, and for a more measured evaluation, see [481. Three often quoted case studies, each with a single patient on long-term total parenteral nutrition, describe the need for chromium supplementation to counteract glucose intolerance [49-517. A seldom-cited follow-up to the first case resulted in doubt that added Cr(II1) is desirable for parenteral nutrition because more than sufficient Cr(I1I) impurities are introduced in solution components 1521. For all three cmes reported, “normal” serum values are up to 100 times greater than that currently accepted, and it is not certain that serum levels reflect those elsewhere in the body. These three cases stand in marked contrast to numerous others in which no glucose intolerance was observed in patients on parenteral nutrition [381. One should also ask about the speciation of Cr(I1I) that is added as CrC13 to a neutral parenteral solution in which insoluble Cr(OH), and CrP04would be expected to form unless the Cr3’ is complexed by more strongly binding ligands. The order of mixing and time of standing of parenteral solutions may be significant. Contrary to some studies, a carefully controlled double-blind investigation of elderly subjects with stable impaired glucose tolerance and low daily Cr(1II) intakes found no improvement from Cr(II1)-richyeast supplements 1531. Evidently the more controlled the study, the less effective to the vanishing point are Cr(l1I) supplements. Separate from other considerations in this chapter is the promotion by Gary Evans of chromium(II1)tris-picolinate (2-pyridinecarboxylate)as hoth a lean muscle builder and a fat loss agent [54]. The resulting publicity has gcnerated annual sales of $150 million of the complex to 10 million consumers, making this mineral supplement the second largest selling in the United States (after calcium) 1551. There is, however, a paucity of evidence to support such astonishing claims. In three carefully controlled studies, supplementation with chromium tris-picolinate to nonathletos or athletes during a period of strength training failed to increase lean body mass or strength, or reduce body fat [56-58). In all three studies, supplementation increased the urinary output of chromium. Chelation of three bidentate anionic picolinates with Cr3+ yields a neutral complex that may be better absorbed than net charged complexes. Greater absorption may be a disadvantage because the neutral complex causes chro-

ARE THERE PROTEIN5 CONTAINING ~

~ R O ~ I U ~ ?

185

mosome damage in Chinese hamster ovary cells and may accumulate in humans [591. Seriously impaired renal function arose from ingestion of large doses of c ~ o ~ u m tris-picolinate by a human patient [60] In addition, products generated by reduction to a Cr(I1) complex within cells cleave DNA [61]. The chromium(II1) tris-picolinate complex i s not related structurally to glucose tolerance factor and is not considered further in this chapter 1621. There are several reasons for the limited acceptance of chromium as an essential nutrient. Chromium occurs in only trace amounts in the body and there is no routinely reliable test for chromium status. It is difficult to induce chromium deficiency in s uncerexperimental animals. When offered in a diet, the bioavailability of Cr(I1I) i tain. Experiments with added chromium to diets of chromium-deficient animals often give weak eflects and contradictory results. Even after 40 years all attempts Lo isolate or synthesize GTF have failed, and the composition of' GTF from brewer's yeast remains unknown. Finally, there is no known mechanism for action by inert Cr(1II). What might Cr(II1)possibly be doing that other metid ions do not t o warrant the designation essential? Its inertness rules out ligand exchange at the active site of an enzyme. Remaining is a structural role. In this scenario, somehow ingested chromium manages to reach the relevant site, perhaps with its inertness as an aid. (Antitumor cisplatin, an inert lWl?complex, reaches the cell nucleus whereas the corresponding labile Pd(IX) complex does not 1631.) A n active CrfIII) compound then binds to appropriate ligands and forms a structure assisting the process by which glucose 1eveIs are modulated. The possibility of a structural role for Cr(I1I) directs our focus to the way its reacts with proteins.

S / ~ R ~ ~ WITH E I NKNOWN ~ STRUCTURE No proteins with bound chromium are listed in the Protein Data Bank. cr""is relatively small with an equivalent ionic radius in sixfold coordination of 62 pm (identical with Ga3"), greater than that of N3+at 54 pm, slightly less than that of high-spin Fe" at 65 pm, and less than IMg? at 72 prn 1641. Electron affinities, the primary determinant of metal ion binding strengkh, are nearly identical for Cr3', ' ' [171. Fe3+,and Ga3', with a somewhat smaller value for a Crystal structures reveal a variety of binding modes in always octahedral complexes of Cr(1II) with amino acids and peptides. Three glycine anions chelate in a highly insoluble mononuclear structure, Cr(gly), [651, and two glycinates are bound to each Cr(IT1) in a binuclear, dihydroxo-bridged complex, [Cr(gly)20H], [661. The latter complex typifies a tendency of Cr(IlI? to form oxygen-bridged multinuclear complexes. In a variety of mononuclear complexes, aspartate, histidine, cysteine 1671, and penicillmine 1681 serve as tridentate ligands either in pure 2:l complexes or in mixed complexes with two of the ligands. These structures demonstrate the ability of Cr(1II) to bond to carboxylate oxygen, amino nitrogen, imidazole nitrogen, and sulfhydryl donor atoms. Crystal structures of two dipeptide compXexes, i.e., gly-

MARTIN

186

cylglycine and prolylglycine, show that in a single complex each of two dipeptides serves as a tridentate ligand bound to Cr3’ through the amino nitrogen, the deprotonated amide nitrogen, and the carboxylate oxygen 1691. These peptide hydrogendeprotonated complexes were prepared by prolonged heating in excess base.

TEINS WIT^

~

N

K STRU~TURE ~ O ~ ~

By far the most common interaction of chromium with proteins is that in commercial leather tanning. Cr(II1) salts in acidic solutions cross-link triple-helical collagen molecules via their carboxylate side chains. The Cr(II1) content exceeds 2% of the protein by weight and therefore is of little guide in delineating the more subtle interactions of physiological interest. As for Fe(II1) and Al(II1) 1701, the main transporter of Cr(I1I) in the blood serum is transferrin. Typically transferrin is only 30% occupied with Fe(II1); about 50 pM of unoccupied binding sites are available for other metal ions, Electron spin resonance results obtained with Cr(III) have been used to show that the two metal ion binding sites of transferrin are distinct 171,721.Fe(lI1) binds many powers of 10 more strongly to transferrin than to albumin, and Cr(II1) is expected to do the same; at eq~librium albumin is not competitive with transferrin for the metal ions C701. Many studies report interactions of added Cr(II1) with a variety o f proteins, sometimes with activation and sometimes with inhibition. These reports have recently been inventoried 1731. Substitution of inert bidentate Cr(I1I)-ATP when used as an analogue for Mg(II)-Al’P provides information on the screw sense specificity o f several kinase enzymes C74,751. Since the GTF discussed in Sect. 1 potentiates insulin action, interactions of Cr(I1I) with insulin have been suggested as responsible for its action. Several Gr(1II) complexes capable of relatively rapid exchange when combined with insulin were found to relieve diabetic conditions in rats “761. However, since the CrGII) complexes are capable of rapid exchange, it does not appear ruled out that any relief is due to insulin itself. Zinc(I1) weakly stabilizes the insulin hexamer by interacting with imidazole side chains [771, and it is conceivablethat Cr(II1)might behave similarly. However, insulin circulates in the blood and interacts at its receptor as a monomer, so that it is difficult to see an advantage to any metal ion-induced multimer formation. With the failure t o describe the action of GTF in terms of small molecules, emphasis has shifted to low-molecular-weight chromium-binding substance Cr). Under challenge of intravenous dichromate injection, peptides of molecular weight 1500 were isolated in Japan from Livers of dogs “781, mice [791, and rabbits 180,811, and without challenge from cow colostrum [821. The peptides enhanced glucose oxidation and lipogenesis in rat adipocytes and lost activity upon removal of Cr(II1) by EDTA. As many as four Cr(Il1) were Found per peptide molecule. The proteins contains several glutamate, aspartate, and glycine residues and at least


187

one cysteine, but no nicotinic acid. These authors suggest that LMWCr serves as a Cr(I1I) carrier, including detoxification and excretion, and that it may also play an essential nutritional role. More recently, Vincent and co-workers have taken up the task of isolating L W C r from porcine kidney 1831 and bovine liver [841, and found similarities to the peptides reported in Japan. In particular they find the same molecular weight, similar amino acid composition, and four Cr(II1j per peptide molecule. They propose that the GTF described in Section 1is an artifact of acid hydrolysis of LMWCr [83,851. Since there are not enough peptide side chain donor atoms to bind to four Cr(III),they suggest a tetranucleax structure [841. This ratio of 4 metal ions to 11-12 amino acid residues in a peptide is more than twice as great as that found for other metal ions with metallothionein, Owing to the significantly weaker binding of the second Cr(I1Ij to transferrin, apoLMWCr slowly captured Cr(II1j from fully loaded transferrin, and the individual stability constants for Cr(II1)binding to each ligand have been reported [86]. However, it may be shown from the reported stability constants that at low total Cr(T1Ij concentrations transferrin allows the lower free metal ion concentration. The Vincent laboratory has also described new activities for LMWCr. Although it exhibits no intrinsic phosphatase activity, their peptide from bovine liver activates phosphotyrosine phosphatase activity in rat adipocyte membranes [871. They also discovered activation of insulin receptor tyrosine protein kinase activity in response to insulin and suggest that this may be the primary function of LMWCr [88]. Insulin was required, apoLMWCr waq ineffective, the maximum effect was at four Cr(II1j per peptide, and other transition metal ions were without effect. Regulation of both of these enzymes i s poorly understood, and the possibility that they are regulated by a peptide with four Gr(II1)is intriguing. A mechanism has been proposed for activation of insulin receptor kinase activity by L W C r in response to insulin [89,901. This activity was also mimicked by a well-characterized synthetic trinuclear Cr(1II) cation, [ C r ~ O ( O ~ C C H ~ C ~ ~ ) ~ where ( H ~ Oa) central ~ ] + , oxide ion binds to the three hexacoordinate Cr(II1) in a plane, and each of the six propionates bridgcs two Cr(II1j [91]. They further suggest that this inexpensive complex may serve as a new agent in the treatment of adult-onset diabetes and as a nutritional supplement. For a review of recent work from the Vincent laboratory, see [go].

The short answer to the title of this chapter “Are There Proteins Containing Chromium?” is no. This answer assumes that L W C r is not primarily a chromium protein or that an 11- to 12-amino-acid residue peptide is not long enough t o be considered a protein. Definitions aside, what is clearly needed is a crystal structure of LMWCr. With or without the crystal structure, there are also aspects of function that need to be delineated.

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188

It is puzzling that an 11- to 12-amino-acid peptide containing two Gysteine residues, and presumably elaborated to bind Cr(III), does not employ its sdfhydryl groups in metal ion binding. Demonstrated Cr(1II) binding to sulfhydryl groups is mentioned in Sects. 1 and 2. Perhaps the sulfhydryl groups are involved in some aspects of the peptide function. But since the peptide is always present even in the apo form, the activities uncovered so far may only be incidental to the as-yet-undiscovered function of the peptide.

VI AT1ON S

A ATP EDTA GTF r

adenosine 5 '-triphospbate e t h y l e n e d i ~ n e - NN, , N', N'-tetraacetate glucose tolerance factor low-molecular-weight chromium-binding substance

s

R

1. R. A. Anderson, Sci. Toi!ul Enuiron., 86,75-81 (1989). 2. 0. Nriagu and E, Nieboer teds.), Chromium in the Natural: and nuironments, John Wiky, New York, 1988. 3. S. A. Katz and H. Salem, The Biological and ~ n v i r o n m e n t uChemistry ~ of Chromium, VCH, Weinheim, 1994. 4. Vincent, in Encyclopedia of Inorgunic Chemistry (R. . King, ed.), John and Sons, Chichester, 1994, vol. 2, pp. 661-665. 5. R. Turner, M. Witfieid, and A. 6. Dickson, Geochim. Cosnzoeltirn. Ada,

6.

7.

8.

9. 10.

~ a ~ i l t and o n K. E. Wetterhahn, in Handbook on Toxicity of Inorganic ounds (H. 6. Seiler, H. Sigel, and A. Sigel, eds.), Marcel Dekker, New York, 1988, pp. 239-250. M, Costa, in Handbook of ~ ~ t a E - L i htteractiorzs ~an~ in iological Fluids-Bioinorganic Chemistry (G. Berthon, ed.), Marcel Dekker, New York, 1995, vol. 2, pp. 838-847. S. DeFlora, A. Camoirano, M. Bagnasco, and P. Zanacchi, in H u r ~ ~ b o oofk al-ligand Interactions in Biological ~ ~ u ~ ~ s - ~ ~ o i n o r~g u ne i c ~ (6. ~ thon, ed.), Marcel Dekker, New 'York, 1995, vol. 2, pp. ~ Q ~ ~ - 1 0 3 6 ~ tlerhahn, J. Am,. Chem. Soc., 107, 4282-4288 P. H. Connett and K. E. (1985). . Standeven and K. E. Wetterhahn, Chem. Res. ToxicoE., 4, 626-6215

(I991)

a

~

ARE THERE PR TElNS C O ~ ~ A I N I CHRO~IUM? N~

189

11. D. M. Stearns, L. J. Kennedy, K. D. Courtney, P. H. Giangrande, L. S. Phieffer, and K. E. Wetterhahn, Biochemistry, 34, 910-919 (1995). 12. A. Kortenkamp, M. Casadevall, S. P. Faux, A. Jenner, R. 0. J. Shayer, N. Woodbridge, and P. Q’Brien, Arch. Biochem,. Siophys., 329, 199-207 (1996). 13. K. W. Jennette, J. Am. Chem. SOC.,104, 874-875 (1982). . Borges and K. E. Wetterhahn, Carcinogenesis, 10, 2165-2168 (1989). 15. K. Salnikow, A. Zhitkovich, and M. Costa, Carcinogenesis, 13, 2341-2346 (1992). 16. A. Zhitkovich, V. Voitkun, and M. Costa, Carcinogenesis, 16, 907-913 (1995). Martin, Inorg. Chim. Acta, 283, 30-36 (1998). itkun, A. Zhitkovich, and M. Costa, Nucleic Acids Res., 26, 2024-2030 (1998). 19. 6 . F. Baes and R, E. Mesnier, The Hydrolysis of‘ Cations, Wi~ey-Inters~ien~e, New York, 1976. 20. H. Stunzi and W. Marty, Inorg. Ch,em., 22, 2145-2150 (1983). 21. F. P. Rotzinger, H. Stunzi, and W. Marty, Inorg. Chem., 25, 489-495 (1986). 22. L. Spiccia and W. Marty, Polyhedron, 10, 619-628 (1991). 23. R. F. Jameson and J. E. Salmon, J. Chem. Soc., 360-367 (1955). 24. L. Ohman and R. B. Martin, Clin. Chem., 40, 598-601 (1994). 25. R, B. Martin, Accts. Chem. Res., 27, 204-210 (1994). . Martin, in Aluminium in Biology and Medicine. Ciba Foundation Symposium No. 169, John Wiley and Sons, Chichester, 1992, pp. 5-25, 104108. 27. J. P. Hunt and H. L. Friedman, Prog. Inorg. Chem., 30, 359-387 (1983). 28. 0. Farver and I. Pecht, Proc. Natl. h a d . Sci. USA, 78,4190-4193 (1981). 29. r). R. Prasad, T. Ramasami, D. hmaswamy, and M. Santappa, Inorg. @hem., 21, 850-6364 (1982). . E. Gerdom and €3. M. Goff, Inorg. Chem., 21, 3847-3848 (1982). 31. I?. O’Brien and D. A. Sweigart, Inorg. Chem., 21, 2094-2095 (1982). 32. H. Ogino, M. Shimura, and N. Tanaka, Inmg. Chem., 18, 2497-2501 (1979). 33. K. Schwarz and W. Mertz, Arch. Biochem. Biophys., 85, 292-295 (1959). 34. 11. Schwarz, in Trace Elem,ent Metabolism in Animals, Vol. 2 (W. 6. Hoekstra, J. W. Suttie, H. E. Ganther, and W. Mertz, eds.), University Park Press, Baltimore, 1974, pp. 355-380. 35. K. Schwarz, Fed. Proc., 33, 1748-1757 (1974). ertz, Physiol. Rev., 49, 163-239 (1969). 37. W. Mertz, in Chromium in Nutrition and ~ e ~ a b o l i s(D. m Shapcott and J. Hubert, eds.), Elsevier, Amsterdam, 1979, pp. 1-14, 247-256. 38. W. Mertz, J . ~ u t ~ t i o 123, n , 626-633 (1993).

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39. W. Mertz, E. W. Toepfer, E. E. Roginski, and M. M. Polansky, Fed. Proc., 33 2275-2280 (1974). 40. E. W. Toepfer, W. Mertz, M. M. Polansky, E. E. Roginski, and W. R. Wolf, J. &Tic. Food Chem., 25, 162-166 (1977). 41. R. A. Anderson, J. H. Brantner, and M. M. Polansky, J. Agric. Food Chem., 26, 1219-1221 (1978). 42. E. Gonzalez-Vergara, J. Hegenauer, and P. Saltman, Fed. Proc., 41, 286 (1982). 43. S. J. Haylock, P. D. Buckley, and L. F. Blackwell, J. Inorg Biochern,., 29, 105-117 (1983). 44. D. D. Held, E. Gonzalez-Vergara, and H. M. Goff, Fed. Proc., 43,472 (1984). 45. P. R. Shepherd, C. Elwood, P. D. Buckley, and L. F. Blackwell, Biol. Trace Elem. Res., 32, 109-113 (1992). 46. D. L. Donaldson, D. E. Lee, C. C. Smith, and 0. M. Rennert, Metabolism, 34, 1086-1093 (1985). 47. J, Barrett, P. O’Brien, and J. P. I_). Jesus, Polyhedron, 4 , 1-14 (1985). 48. E. G. Offenbacher and F. X. Pi-Sunyer, Ann. Rev. Nutr., 8, 543-563 (1988). 49. K. N. Jeejeebhoy, R. C. Chu, E. B. Marliss, G. R. Greenberg, and A. BruceRobertson, Am. J. Clin. Nu&., 30, 531-538 (1977). 50. H. Freund, S. Atamian, and J. E. Fischer, J. Am. Med. Assoc., 241, 496-498 (1979). 51. R. 0. Brown, S. Forloines-Lynn, R. E. Cross, and W. D. Heizer, Dig. Dis. Sci., 31, 661-664 (1986). 52. W. SeeIing, F. Ahnefeld, A. Gi-unert, K. Kienle, and M. Swobodnik, in Chromium in Nutrition and Metabolism (D. Shapeott and J. Hubert, eds.), Elsevier, Amsterdam, 1979, pp. 95-104. 53. M. I. J. Uusitupa, L. Mykkanen, 0. Siitonen, M. Laakso, H. Sarlund, P. Kolehmainen, T. Rasanen, J. Kumpulainen, and K. Pyorala, Br. J. Nutr., 68, 20!2-216 (1992). 54. G. W. Evans and D. J, Pouchnik, J. Inorg. Biochem., 49, 177-187 (1993). 55. F. H. Nielsen, Nutr. Today, 31, 226-233 (1996). 56. S. P. Clancy, P. M. Clarkson, M. E. DeCheke, K. Nosaka, P. S. Freedson, J. J. Cunningham, and B. Valentine, Int. J. Sport Nulr., 4 , 142-153 (1994). 57. M. A. Hallmark, T. H. Reynolds, C . A. DeSouza, 6. 0. Dotson, R. A. Anderson, and M. A. Rogers, Med. Sei. Sports Exercise, 28, 139-144 (1996). , C. Lukaski, W. W. Bolonchuk, W. A. Siders, and D. B. Milne, Am. J . Ckin. 58. Nu&., 6t3, 954-965 (1996). 59. (a) D. M. Sterns, J. P. Wise, S. R. Patierno, and K. E. Wetterhahn, FASEB J., 9, 1643-1649 (1995). (b) D. M. Stearns, J. J. Belbruno, and K. E. Wetterhahn, FASEB J., 9, 16501657 (19951,


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60. J. Cerulli, D. W. Grabe, I. Gauthier, M. Malone, and M. D. McGoldrick, Ann. Pharmacotherupy, 32, 428-431 (1998). Speetjens, R. A. Collins, J. B. Vincent, and S. A. Woski, Chem. Res. 61. ol., 12, 483-487 (1999). 62. (a) M. K. Hellerstein, Nutr. Reu., 56, 302-306 (1998). (b) H, C. Lukaski, Annu. Rev. Nzitr,, 19, 279-302 (1999). 63. R. B. Martin, in Cisplatin-Chemistry and Biochemistry of a Leading Anticancer Drug (B. Lippert, ed.), Wiley-VCH, Weinheim, 1999, pp. 183-205. . Shannon, Actu Crystullogr., A32, 751-767 (1976). 64. . Bryan, P. T. Greene, P. F. Stokely, and E. W. Wilson, Inorg. Chem., 10, 65. 1468-1473 (1971). 66. J. T, Veal, W. E. Hatfield, D. Y. Jeter, J. C. Hempel, and D. J. Hodgson, Inorg. ChemT1., 12, 342-346 (1973). 67. K. Madafiglio, T. M. Manning, C. M. Murdoch, W. R. Tulip, M. K. Cooper, T. W. Hambley, and H. C. Freeman, Acta Crystallogr., C46, 554-561 (1990). 68. P. d. Meester and D. J, Hodgson, J. Chem. SOC.Dalton, 1604-1607 (1977). 69. 6. M. Murdoch, M. K. Cooper, T. W. Hambley, W. N. Hunter, and Freeman, J. Ch,em. SOC.Chwm. Commun., 1329-1331 (1986). . Martin, J. Savory, S. Brown, R. L. Bertholf, and M. Wills, Clin. Chem., 70. 33, 405407 (1987). 71. P. Aisen, R. Aasa, and A. G. Redfield, J. Biol. Chem., 244, 4628-4633 (1969). 72. D. C. Harris, Biochemistry, 16, 560-564 (1977). 73. A. Yamamoto and 0. Wada, in Handbook of Metal-Ligund In,teructions in BiologicuZ FZuids-Bioinorganic Chemistry (6.Berthon, ed.), Marcel Dekker, New York, 1995, vol. 1, pp. 248-253. 74. D. Dunaway-Mariano and W. W. Cleland, Biochemistry, 1.9, 1496-1505 (1980). 75. D. ~ u n a w a y - M a ~ a nand o W. W. Cleland, Biochemistry, 19, 1506-1515 (1980). 76. I(. Govindaraju, T. Ramasami, and D. Ramaswamy, J. Inorg. Biochem., 35, 137-147 (1989). 77. G. D. Smith, D. C. Swenson, E. J. Dodson, 6. G. Dodson, and C. D. Reynolds, Proc. Natl. Acad. Sci. USA, 81, 7093-7097 (1984). 78. 0. Wada, G. Y. Wu, A. Yamamoto, S. Manabe, and T . Ono, Enuiron. Res., 32, 228-239 (1983). 79. A. Yamamoto, 0. Wada, and T. Ono, J . Inorg. Biochem,., 22, 91-102 (1984). 80. A. Yamamoto, 0. Wada, and T. Ono, Eur. J. Biochem., 165, 627-631 (1987). 81. A. Yamamoto, 0. Wada, and S. Manabe, Biochem. Biophys. Res. Commun,, 163, 189-193 (1989). 82. A. Yamamoto, 0. Wada, and H. Suzuki, J . Nutr., 118, 3 9 4 5 (1988). 83. K. H. Sumrall and J. B. Vincent, Polyhedron, 16, 4171-4177 (1997).

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J. B. Vincent, Biochemistry, 36, 43824385 (1997). J. B. Vincent, J. Biol. Inorg. Chem., 2, 675-679 (1997). Am. Coll. Nutr., 18, 6-12 (1999). C . Royer, and J. B. Vincent, Znorg. Chem., 36, 5 3 1 ~ 5 3 2 0

avi School of Chemical and Physical Sciences, Victoria University of Wellin~on, P.O. Box 600, Wellington, New Zealand

1. I N ~ R O ~ U ~ T I 1.1. Coor~nationChemistry o 1.2. Manganese as an Oxidizin le of Manganese 1.4. Homeostasis and Metabolis~ 1.4.1. Manganese Uptake and Transport 1.4.1.1. Influx of Mn2+ into bacteria 1.4.1.2. Influx of Mn2+ into plants and yeast 1.4.1.3. Influx of 1.4.2. Manganese in t

OWN S T ~ ~ C T U ~ E 2.1.1. ~ehydrog~nases 2.1.1.1. Isocitrate d@hydrogenase(EC 1.1.1.41) 2.1.1.2. Isopr~pylmalatedehydrogenase (EC 1.1.1.85) 2.1.2. Oxidases 2.1.2.1. Arnine oxidase (EC 1.4.3.6) 2.1.2.2. Cytochrome c oxidase (EC 1.9.3.1) 2.1.3. Superoxide Dismutase (EC 1.15.1.1) 2.1.4. Manganese Catalase (EC 1.11.1.6) 2.1.5. Peroxidases 2.1.5.1. ~hloroperoxidase(EC 1.11.1.10) '1 93

196 196 197 198 198 199 199 199 199 200 201 201 201 201 201 202 202 202 202 206 207

194

2.2. Transferases 2.2.1.Kinases 2.2.1.1. Phosphorylase kinase (EC 2.7.1.38) 2.2.1.2. Pyruvate kinase (EC2.7.1.40) 2.2.2.Nucleases and DNA and RNA Polymerases 2.2.3.Carboxylases 2.2.3.1.Phosphoenolpyruvatecarboxylase (EC 4.1.1.31) 2.2.4.Sugar Transferases 2.2.4.1.Ribosyltransferases (EC 2.4.2-1 2.2.5.Adenosyltransferases 2.2.6.Arylalkyltransferases 2.3. Hydrolases 2.3.1.Peptidases 2.3.1.1. Aminopeptidases 2.3.1.2. Arninopeptidase P (EC 3.4.11.9) 2.3.1.3. Carboxypeptidases 2.3.2.Phosphatases 2.3.2.1. Protein phosphatases (EC3.1.3.16) 2.3.2.2. Inorganic pyrophosphatase (EC 3.6.1.1) 2.3.2.3. Inositol monophosphatase (EC3.1.3.25) 2.3.2.4. Fructose-1,6-bisphosphatase(EC 3.1.3.11) 2.3.3.Arginase (EC3.5.3.1) 2.4. Lyases Carboxykinases and Dehydratases 2.4.1. 2.4.1.1. Phosphoenolpyruvate carboxykinase (EC 4.1.1.32 and 4.1.1.49) 2.4.1.2. Enolase (EC4.2.1.11) 2.4.1.3. D-Glutarate dehydratase (EC 4.2.1.40) 2.5. Isomerases 2.5.1.Keto-Aldo Isom erases 2.5.1.1. Xylose isomerase (EC5.3.1.5) 2.5.1.2. L-F’ucoseisomerase (EC 5.3.1.3) 2.5.2.Cycloisomerases 2.5.2.1.Muconate cycloisomerase (EC 5.5.1.1) 2.5.2.2. Chloromuconate cycloisomerase (EC 551.7) 2.5.3.Racernases Mandelate racemase (EC 5.1.2.2) 2.5.3.1. 2.6. Ligases 2.6.1.Glutamine Synthetase (EC 6.3.1.2) 2.6.2.Carbamoyl Phosphate Synthetase (EC 6.3.4.16) 2.6.3.Aminoacyl-tRNA Synthetases (EC 6.1.1.-) 2.6.4.UDP-~-acetylmura~oyl~L-~anine:D-glut~ate Ligase (EC6.3.2.9) 2.6.5.Dethiobiotin Synthetase (EC 6.3.3.3)

207 208 208 208 210 210 210 210 210 212 212 212 212 212 213 213 213 214 214 215 216 217 217 217 217 218 219 219 219 220 222 223 223 223 224 224 224 224 225 226 226 226

195

MANGAN~SE-CONTAI~ING ENZYMES AND PROTEINS 2.7.

Proteins Containing Bound Manganese 2.7.1. Lectins 2.7.1.1. Concanavalin A 2.7.1.2. Other lectins 2.7.2. Integrins 2.7.3. Diphtheria Toxin Repressor 2.7.4. Integrases 2.7.5. Pneumococcal Surface Antigen Adhesin A 2.7.6. Mannose 6-Phosphate Receptor

(PsaA)

227 227 227 228 228 229 230 230 230

3. ~ G ~ E ENZYMES S E WITH UNKNOWN STRUCTURE 3.1. Oxidoreductases 3.1.1. Dehydrogenases 3.1.2. Manganese Dioxygenase (EC 1.13.11.15) 3.1.3. Lipoxygenase (EC 1.13.11.12) 3.1.4. Manganese Ribonucleotide Reductase (EC 1.17.4.1) 3.1.5. Oxygen Evolving Complex of Photosystem I1 3.1.6. Thiosulfate-Oxidizing Enzyme (EC 1.8.99.J 3.2. Transferases 3.2.1. Sugar Transferases 3.2.2. Sulfatases 3.3. Hydrolases 3.3.1. Prolinase (EC 3.4.13.8), Prolidase (EC 3.4.13.9) and

230 23 1 23 1 23 1 232 232 233 236 236 236 237 237

Other Aminopeptidases 3.3.2. Acid and Alkaline Phosphatases 3.3.2.1. Purple acid phosphatases (EC 3.1.3.2) 3.3.3. Dinitrogenase Reductase-ActivatingGlycohydrolase (EC 3.2.1.) 3.3.4. MutT Enzyme (EC 3.1.3.J 3.3.5. Enzymes Related to Arginase Lyases 3.4.1. Cyclases Isomerases 3.5.1. Keto-aldo Isomerases 3.5.2. Mutases 3.5.2.1. Phosphoglycerate mutase (EC 5.4.2.1) Ligases 3.6.1. Pyruvate Carbolrylase (EC 6.4.1.1) Proteins Containing Bound Manganese 3.7.1. Fur Repressor Protein

237 237 237

3.4. 3.5.

3.6. 3.7.

4.1.

C T ~ ~ ~ F U N C T I~OEN~ T I O N S H I P S Description of the Coordination Sphere of Manganese in Proteins

238 238 238 239 239 239 239 239 239 240 240 240 240 240 241

196

4.2. Description of Reaction Mechanisms 4.2.1. Superoxide Dismutase 4.2.2. Catalase 4.2.3. Hydrolases 4.2.4. Enolase Superfamily of Enzymes 4.2.5. Xylose Isomerase 4.2.6. The Oxygen-Evolving Complex

24 1 241 241 241 242 242 243 243 244

244 246

1. I

UCTlO

This chapter describes manganese-cont~ningand dependent proteins and enzymes. ere possible, references are selected from the literature since 1990. A number of books 11-31 and review articles r4-81 that cover the earlier literature are also available.

hernistry of Mang~nese Manganese in biological systems adopts a range of coordination environments and may have oxidation states of 4-2, +3, +4 and possibly +5. &inz+ in proteins has a high-spin d5 electron configuration, has no coordination geometry preferences, and forms relatively weak complexes with ligands, in general stronger than Caa+ and M$+ but weaker than the other transition metal ions found in biological systems. The ionic radius of Mn2+ lies between that of Mg2+ and Ca"+ and is about the same size as Zn2+and Fe2'. It has been found that MnZ+can substitute for these metals in proteins containing those metals. n(H20):' and Mn(HZO)g*and their complexes are labile. This lability of Mn2+ and the lack of thermodynamic stability of its complexes often results in the complete loss of Mn2+during the isolation procedure of an enzyme. Consequently, the enzyme must be reactivated by the addition of MnZ+once lated. Mn3+-containin~ proteins content is often substoichiowhen isolated usually contain some Mn3* but the metric. The coordination chemistry of Mn4+ is dominated by octahedral coordination and a tendency to form polynuclear mixed-valence (Mn3+iMn*+)complexes with p20x0 ligands. Although Mn4" with a d3 electron confi~rationwould be expected to be inert, the mixed-valence clusters are labile probably due to delocalization or electron exchange within the cluster.

'+

YMES AND PROTEINS

197

Ions such as Mn2+ bound to a metalloprotein are in dynamic equilibrium with the metal ions in their environment, and the identity of the metal ion bound to the protein will be dependent on the concentration of metal ions in the immediate environment of the protein and the relative affkiity of the protein for the ions. The total concentration of Mn2+in most cells under physiological conditions is not known but is usually considered to be in the micromolar range. Mn2+ is not likely to be present as the Mn(HZQ)2+6ion within the cell but will be bound to protein, ncentration is much smaller than the intracellular concentration of, for exmple, g2+ (0.3-3 mM), so that M g + has a concentration advantage that will usually outweigh the thermodynamic advantage of more stable Mn2+ complexes. Thus, it is more likely that a metal binding site in a protein will be occupied by M$+ than by &In2+,but in general a mixture of metal ions can be expected. Various compartments within a cell or different organs within a body may have much higher concentrations of manganese than average, so that it is possible for enzymes to be predominantly Mn2+-activatedin one part of an organism and I%$+-activated in other parts. Some organisms can accumulate high concentrations of manganese. For example, Lactobacillus plantarurn, which does not require iron, has an intracellular concentration of Mn2 ' of 0.025 M [91, and other species of lactic acid bacteria similar high concentration of Mn2+ [101. Micrococcus rudiodurans accu to an extent that there is one Mn2+ ion per 10 nucleotide bases present Mussels can have manganese concentrations that are much greater than the concentration in the sea or lake in which they live 112,131. Treponerna pallidurn, the causative agent of syphilis, also has few, if any, proteins that require iron and it may also use Mn as a replacement for iron 1141. Because Mn2+is EPR-active it has been used as a probe of the metal binding site %$'and ~a2'-containing enzymes, and this work has been re vie we^ in studies of I 1151. Substitution of M$' by Mn2+ can have a number of effects. Increases or decreases in reaction rates have been observed; the enzyme can become less specific in its reaction; and in some cases the stoichiometry of metal binding is different [16]. An example is exonuclease 111, which binds two &In2" ions in solution but on M&2+ or Ca2+ ion under the same conditions 1173. Mn2' can also replace Zn2+, NiA+and Co2' in the active sites of enzymes, and many of these substituted e n z p e s are functional with Mn2' in the active site. However, structural changes in the active site of the enzyme that result in inactivation have also been observed r o l l o ~ man~g ganese substitution [18].

xidizing/~educingAgent 20):+ ion i s a powerful oxidizing agent, Eo = +1.51V. The &In3 ox potential is highly dependent on the coordination sphere of the me nding nitrogen to Mn'+ lowers the reduction potential of the &In3+complex and carboxylate ligands have the same, though smaller, effect. Little information i s available on the oxidation state of Mn in cells, but it is clear Chat both the Mn(I1) and

WEATHERBURN

198

Mn(II1) oxidation states are common. In Staphylococcus aureus Mn2+ is oxidized to Mn3' in oxygenated cells and MnS+ is reduced to Mn2+ under anaerobic conditions C191. Manganese in enzymes such as catalase and superoxide dismutase that cycle between Mn2" and Mn3+ is bound to two or three histidine moieties. The Mn2+ion in manganese-dependent peroxidase, which is oxidized to Mn3+, has a coordination sphere comprising two water molecules, a propionate from the heme group, and three carboxylate residues [ZOI.

1.3. Bioinorganic ole of Manganese One of the most remarkable features of manganese-dependent enzymes is their very wide range of functionality. The enzymes discussed below include examples from every class of enzyme. Much attention has been focused on the manganese enzymes involved in redox processes, but manganese-dependent hydrolases (phosphatases, proline peptidases, and arginase), ligases, and transferases (particularly the sugar transferases) have important biological roles.

1.4.

Homeostasis and Metabolism

Humans consuming Western diets consume between 1 and 10 mg Mnlday. An estimated safe and adequate daily dietary intake of manganese for adults is 2-5 mg Mnl day. In food, the highest concentration of Mn is found in nuts, grains, cereals, and tea and coffee, with low levels in fish and meat. Absorption of Mn2+by adult humans and rats is quite low; only 3 4 %is absorbed, and absorption is influenced by the presence of Fe'". Uptake from the intestine is more efficient in young animals; infants fed human milk and infant formula retain 85-95% of the available Mn2+ 1211. The levels of manganese intake associated with adverse efl'ects (both deficient and toxic) are uncertain. The lowest observable adverse effect level for manganese in water is 4.2 mg Mnlday for a 70-kg individual [221. In animals, dietary manganese deficiency can result in impaired insulin production, epilepsy, arteriosclerosis, diabetes, altered lipoprotein metabolism, and impairments in the oxidant defense and cardiovascular systems. If the deficiency occurs during early development, there are pronounced skeletal abnormalities and an irreversible ataxia [23,241. Most animals show considerable resistance to dietary manganese toxicosis. Reports of toxicity in humans are restricted to cases of exposure to high levels of airborne manganese (manganese is unique in its capacity to be taken up by the brain via olfactory pathways), and t o cases when manganese excretory pathways are compromised [231. Exposure to manganese oxide dust produces a neurological syndrome similar to Parkinson's disease, characterized by muscle weakness, tremor, bent posture, whispered speech, excessive salivation, and behavioral symptoms that include nervousness, hallucinations, memory loss, cognitive problems, bizarre behaviors, flight of

MANGANESE-CONTAINING ENZYMES AND PROTEINS

199

ideas, and adverse moods [25-271. These effects are apparently irreversible and progress for up to 10 years after termination of exposure to manganese. 1.4.1. Manganese Uptake and Transport Considerable progress has been made in the last 5 years or so in the studies of the uptake of metal ions by prokaryotic and eukaryotic cells. Living cells have a wide variety of metal ion transporters in the various cellular membranes and organelles. Some transporters are driven by ATP and others are driven by an electrochemical gradient of protons or other ions [ZSl. The membrane-bound ATP-dependent transporters are believed to have similar structures to Na+/K+-ATPases and Ca2 ' -ATPases. ~ +bacteria 1.4.1.1. Influx of i ~ n into Bacteria have two or three different transporters for Mn" ions, usually one of high affinity and low capacity and the other of low affinity and high capacity. For example, with Lactobacillus plantarum and Synechocystis species PCC 6803, an Mn2+ transporter is induced under manganese starvation conditions. A second transporter system that is highly specific for Mn2+and is not inhibited by other metal ions is induced in the presence of micromolar Mn2+concentrations [29,301. On the basis of its amino acid sequence the L. plantarum transporter belongs to the family of P-type cationtranslocating ATPases. Similar observations have been made in the human pathogen Streptococcus gordonii except that there are two lower a f h i t y transporters that are inhibited by Zn2' and a proton motive force-dependent transporter that is active under Mn2+-rep1ete conditions 1311. In contrast, streptococcus sohriaus, Streptococcus cricetus, Streptococcus pneurnoniae, and Corynehacterium arnmoniagenes ATCC 6872 possess a single high-affinity transport system for Mn". Cd2+ was the only divalent ion to inhibit the uptake of Mn2' into the Slreptococcus species [32]. One Mn2+/Zn2+uptake protein, the pneumococcal surface antigen adhesin A, PsaA, from S. pneumoniae has been structurally characterized. There are two domains linked together by a single helix. The metal binding site is at the domain interface and is formed by the side chains of His-67, His-139, 61u-205, and Asp-280; however, in this structure the site contains Zn2+ 1331. 1.4.1.2. Influx of Mn2' into plants and yeast A review of the metal ion transport systems of Sacch,arom,yces cereuisiae has been published [341. There are at least two concentration-dependent transport systems for Mn2+.When Mn2+concentrations are less than 1pM, Mn2+uptake occurs using one system, whereas at concentrations greater than 5 pM, another system is active. Zn2+ and Co2+ inhibit Mn2+uptake in the high-affinity system; the lower affinity system is not as strongly inhibited by these metal ions. Mn2+ uptake is associated with a hydrolysis of low-molecular-weight polyphosphates and ATP and is strongly influenced by the intracellular M$+ as well as by the exit of K+ from cells [351. A gene for the high-affinity system, which codes for a hydrophobic protein located on the plasma membrane, has been identified [361. It shares homology with a mouse gene that may code for an Mn2+ andior a Zn2+ transporter 1371. Once inside the cell the

Mn2+ ion is immediately complexed or bound to the cell membrane [38]. A protein, Cdcl, which regulates intracellular, probably cytosolic, Mn2+ levels in S. cereuisiae has been identified r39,401. Another protein located in the membrane of a vesicsular compartment in yeast controls the homeostasis of Mn2+ ions 1411. Uptake of Mn2+ by plants and bacteria has been reviewed [42,43].Specific transport systems for Mn2+ have been identified in the filamentous fungus ~ s p e r ~ i l l uniger s 1441 and the green alga ~hlamydomonas species [45]. In ~ a b i ~ o p sthatiana is seedlings a mutation has been identified that causes the accumulation of Mn, Cu, Zn, and Mg in the leaves. Roots of these mutants also accumulated metals, but unlike the leaves they also accumulated Fe r461. A Ga2+pump (EC 3.6.~.38)may regulate Mn2+ homeostasis by pumping Mn2+ into endomembrane compa~mentsof the plant 1471. ~ a n g a n e s uptake e into wheat grains has been studied by Pearson et al. C481. In maize less than 5% of the Mn taken up by the maize root tissue is present in the vacuoles as soluble Mn2+.Vacuoles apparently act as a sink for Mn2+. Root tissues maintain a low concentration of free Mn'" in the cytoplasm during there is a nonequilibrium distribution of Mn2+ between the cytoplasm and the vacuole. The subcellular distribution of Mn2+ and Ca" is similar, suggesting that Mn2+,like Ca2", might have a control function in normal cells [49,501. 1.4.1.3. I ~ ~ ofu Mn'" x into animals Little is known about the uptake of ingested n by the intestine. After absorption roteins with albumin and a2-macrointo the bloodstream, Mn" is bound to seru being identified as the major ligands 1511, Mn2+is oxidized by ce~loplasmin binds to transferrin, which is a major serum transporting species 1521. The rate of oxidation o n2+ in the blood, uptake rates of protein-bound forms of Mn by the liver and the es, and neuronal transfer rates within the central nervous system deserve more study. Up to 25% of total human Mn is Iocated in the skeleton and may not be readily assessible. Storage sites for Mn have not been identified, although n does bind to the iron storage protein ferritin. The liver is the primary organ involved in manganese homeostasis; liver cells have three uptake systems, and release from these cells is an active controlled process (53,541. 2.4.2. ~ a n g a n e in s ~the Brain

a n g ~ e s eis essential for normal brain function. Enzymes present in mammalian brain are more likely to be ~n'+-dependent than similar enzymes in other organs from ~e the same animal. This has been suggested for glutmine s ~ t h e t 1551, ~ ~ n o p e p ~ i d[56], a s e ATPase [57], and adenylate cyclase E581. Kinetics of port across the blood-brain barrier, the role of transferrin in this process, the transport of Mn by astrocytes, and manganese homeostasis in the central nervous system have been reviewed 159,601. There are two systems for transport of n into the brain: a satura~lesystem For Mn2+ and an Mn3+~transferrinsystem. Uptake into the brain

§ E - ~ ~ N T A I~ N I ~Z GY ~ EAND § PROTEINS

201

during development and aging and the Mn concentration and localization in the brains of rats and mice has been studied [61-63]. In humans the highest brain manganese concentrations are in the pineal gland, the median eminence of the hypothalamus, the olfactory bulb, and the basal ganglia. In glial and astrocyte cells, &In2+i s distributed about 3040% in the cytoplasm and 60-70% in the mitochondria. Ca2+ions alter both the flux rates and distribution of &In2+ions in chick glia and rat astrocytes [641.

2.1.1. ~ e h y d r o ~ ~ n a s e s

ehydrogenases catalyze the reduction of a substrate molecule with NAD’ as the ing coenzyme. Most dehydrogenases share common structural features, their binding domains and their folding topology, but a family of dehydrogenases has been recognized that does not contain this folding topology. Three members of this family-tartrate dehydrogenase, 3-isopropylmalate dehydrogcnase (IPMD citrate dehydrogenase (ICDH), all require a divalent metal ion (Mg“?+ Manganese redox chemistry is not involved in the dehydrogenation and I metal ion serves to bind, orient, and activate the substrate. I ~ & I D H discussed below. Tartrate dehydrogenase has not been structurally characterized and i s discussed in Sect. 3.1.1. 2.1.1.1. lsocitrate dehydro~enaseIEC 1.1.1.41) ICDR catalyzes the decarboxylation of isocitrate to a-ketoglutarate and C02 via an oxalosuccinate intermediate I6fil. Mammalian tissues have two forms of EC 1.1.1.41, which occurs in the mitrochondria and requires Mn2+ o -t as cofactors, and EC 1.1.1.42 ich occurs in both the cytosol and the mitoria and requires Mg”?+and as cofactors. A crystal structure of the dependent enzyme from T. thermophilus with Mn2’ and isocitrate bound has been determined. Isocitrate and Mn2+bind in a pocket between two major domains in which both subunits of the dimer participate. Mn2+ is bound to Asp-283 and Asp-30’7, two water molecules, and the isocitrate via the carboxylatc and an OH group 1661. Structure determinations o€ mutant forms of the E. coli enzyme have been deposited in the Protein Data Bank [67]. 2.1.1.2. ~ ~ o p r o p y l m ~ ldehydrogenase ate (EC 1.1.1.85l I P ~ D Hcatalyzes the third step in the biosynthesis of the amino acid leucine in m i c r o o r ~ i s m and s plants I68 1. The reaction involves the conversion of 8-isopropyland the E. coli enzyme malate to 2-oxoisocaproate with the reduction of “I)+ requires Mn2” and K+ for optimum activity. Structures of the enzyme from E. coli, Salmonella t y ~ h i m u r i u mand , Thermus thermophilus have been determined. In the S. t y p h enzyme ~ ~ the~ asymmetric ~ ~ ~unit~ contains ~ a dimer with the two monomers in different, “open” and “closed” conformations. In the open conformation, Mn2’- is coordinated Lo three aspartate residues, two waters, and a sulfate ion. +

202

WEATH~RBURN

Sulfate is bound at a position thought to be occupied by the substrate, as in the homologous structure of ICDH the C1-carboxylateis bound in the equivalent position. In the closed conformation, Mn'+ is coordinated by the same three aspartate residues and a water molecule lS91. 2.1.2. Oxidases 2.1.2.1 Amine oxidase (EC 1.4.3.6) Amiiie oxidases oxidize mono-, di-, and polyainines to the aldehyde and ammonia. An amine oxidase from the liver of skipjack tuna is activated by Mn2' and inhibited by Cu2+ [701. Copper containing oxidases from fenugreek seedlings, pea, Lathyrus odoratus, L. sutiuus, mid humans but not from Hamenula polymovhu or E. coli contain an Mn2' ion "711. The role of the Mn2* is not known; it can be removed without affecting enzyme function and it is located 33A fkom the copper site. The Mn2+in the enzyme from pea seedlings is octahedrally coordinated to three aspartate cwboxylates (Asp-451, Asp-453, and Asp-5921, the amide oxygens of IIe-593 and Phe-452 and a water molecule F721. 2.1.2.2. Cytoclaroine G oxidase (EC 1.9.3.1) Cytochrome c oxidase (CcO) catalyzes the reduction of oxygen to water and pumps protons across the mitochondri~membrane, as discussed in Chapter 15 1731. Bacterial CcO from Paracoccus denitrificuns contains a number of redox active metals, a heme-a, a dinuclear iron-copper center (heme as-CuB), and two copper ions in a dinuclear CUAcenter. In addition to the redox active metals, CcO contains two non-redox active divalent metal centers. In mitochondrial CcQ these metals are ZnZCand M g + , but in bacterial CcOs at least 20% of the M$+ ions are repIaced by Mn2* and the Zn" is replaced by Ca". These nonredox ions may have a structural role, but the M a n site lies directly between the CLIAand heme-a3 sites and so may play some other role 174,751,In the enzyme from Paracoccus denitrificuns the Mn'"1 Mi2+ ion is bound to Glu-218 in subunit 11and Asp-404 and His-403 from subunit I, and at least one and possibly three water molec~les1761. Asp193 has been shown to have a role in binding Mn2+, although in the crystal it is not a ligand "771. 2.1.3. Superoxide Dismutase (EC 1.15.1.1)

Superoxide dismutase (SOD) catalyzes reaction (1): 20,

+ 2H'

-+ Hz02; I- 0

2

and so protects the cell from oxygen toxicity. Five different superoxide dismutases are known, three of which have been structurally characterized. These enzymes are a Gul ZnSOD, an MnSOD, an FeSOD, and an NiSOD. Strict anaerobes either lack a SOD or have FeSOD. NiSOD is discussed in Chapter 14 1781 and Cu/ZnSOD is described in Chapter 18. FeSODs and MnSODs are structurally related, but it has been demonstrated that in E. coEi these enzymes are not functionally equivalent, with MnSOD being more effective at preventing damage to DNA 1791, Inorganic complexes can act as functional mimics of SODS, and work on these complexes has been reviewed [801.

~ A N G A N E S ~ - C O ~ T A I N I ENZYMES NG AND PROTEINS

203

In general, if Fe is substituted into an MnSOD the enzyme is catalytically inactive. Cambialistic enzymes that are catalytically active with either Mn or Fe in the active site have been isolated from the anaerobes Propionibacterium shermanzi [Sl831, Bacteroides fragilis 1841, Bacteroides gingivalis [851, Bacteroides thetaiotamtcron 1861, Porphyromonas gingivalis la71 and Streptococcus mutans I881, and the aerobes Aeropyrum pernix t891, Mycobacterium smegmatis 1901, and Sinorhizobium melilolii [ X I . Cambialistic enzymes are often isolated that contain a mixture of Fe, Mn, and other metal ions, such as Zn. Only the iron- and manganese-containing forms of the enzymes are active. Primary structures of more than 120 iron and manganese SODS are known. Considerable homology, suggesting a common evolutionary origin, is evident in the sequences. Fourteen residues, including the four ligands to the metal ion, are invariant in MnSODs. Most of the other invariant residues are involved in subunitsubunit interactions. Primary structures of the FeSODs and MnSODs from E. coli, Bordetella pertussis, P. aeruginosa, and Nicotiana plumbagin~folia are known, and there is a low homology between the two forms of the enzymes, suggesting that the different forms diverged in evolutionary terms a considerable time ago 1921. Attempts at understanding the origin of the metal ion specificity of the enzymes in terms of the amino acid sequence data have not been successful. Two residues are regarded as distinguishing Mn from FeSQDs. Residue 77 (E. coli numbering) i s glycine in MnSODs and glutamine in FeSODs, and residue 146 is ahnine in all FeSQDs, and either glutamine or histidine in MnSODs. Enzymes from cambialistic enzymes that are active with either Mn or Fe usually have the Mn sequence at positions 77 and 146, but the enzymes from P. shermanii and Sinorhi~obium meliloti are exceptions 1911. Residues 77 and 146 are in close proximity in the active site, and the glutamine residues present in all structures hydrogen-bond to Tyr-34 that is a strictly conserved residue. The OH group of this tyrosine is 5 A from the metal ion. X-ray structure determinations are available for native and mutant MnSODs from five organisms ranging from bacteria to humans (cf. Table 1 for details). The metal ion is located deep within the protein structure in a hydrophobic pocket formed by two subunits. In structures without an inhibitor present the manganese coordination geometry is distorted trigonal bipyramidal with two histidine ligands and the aspartate donor in the equatorial plane. Another histidine and a water or OH- group are the axial ligands. A representation of the active site is shown in Fig. 1. FeSQDs have a very similar active site. Mn3+ in the structure of the enzyme from T.th,erm,ophiEus with N j bound has a distorted octahedral geometry r931. Variable temperature absorption spectroscopy of the E. coli enzyme and the N3 and F- bound forms of the enzyme have suggested that the geometry of the active site structure is temperaturedependent, increasing with decreasing temperature [%]. The ligand field spectrum of the enzyme with N3 bound is characteristic of five-coordinate Mn3+ at 295 K, but at temperatures below 200 K the spectra suggest a six-coordinatemetal ion. The ligand that is lost above 200 K is thought t o be either the water molecule or the carboxylate moiety.

w E A I H ERBuRN

T ~

O~dation statef Mmz6N

n3+

2.15 2.10 1.80 2.18 2.09

~ Bond Distances o ~ arounde

n2+ 2.14 2.12 1.84 2.21 2.23 ___

1

2.12 2.07 2.25 2.12 1.95 2.21

~

Mn2+

~

2.09 2.14 1.94 2.19 2.01

2.19 2.22 1.99 2.25 2.27

M

n

1

,Vote: residues are ~ u r n b e ~ eaccording d to the sequence in E. coli. 1931. Average values from two subunits [94]. [951. Average values from two subunjts [961. 1971. The oxidation state is uncertain in most cases as reduction in the X-ray beam may occur.

~

+

n3+ 2.13 2.17 1.98 2.2 2.31

Mn" 2.08 2.38 1.94 1.94

ENZYMES AND PR

205 ,Asn - 80

Tyr - 34

m

HIS- 26

FIG. 1. Active site in MnSOD showing the metal coordinationand hydrogen laonding network. Residue numbering i s from the E. coli enz'yme.

Structural studies of the Fe and Mn forms of the cambialistic enzyme from P. sherrnanii revealed structures that were almost identical [961. The tertiary structure of this enzyme hardly differs from the strictly Fe-dependent and strictly dent enzymes. The metal coordination sphere of the iron and manganese ions in the enzyme is trigonal bipyramidal, and it is evident that structural differences between the manganese and iron form cannot explain the cambialistic behavior. E suremcnts on the Fe form of this enzyme suggest that the coordinat changes to six with increasing pH [99]. inding of N; to the iron causes the iron to become six-coordinate over the entire p Determination of the crystal structure of the inactive Fe-substituted form of the MnSOD from E. coEi enzyme has shown that the protein structural framework is unchanged in this enzyme. There are two independent molecules in the unit cell. In one molecule the iron has a distorted square pyramidal geometry with the same five ligands t o the metal as in the native MnSOD. In the other molecule the iron is sixcoordinate; the additional ligand is thought to be a hydroxide ion. This geometry is similar to that in inactive forms of the enzyme with N3 bound 11011. The redox potential of the F'e-substituted MnSOD from E, coli (-240 mv) is almost 0.5V lower than ED o i the FeSOD from €2. coli [lo21 and thus Iies below the potential required t o oxidize 0, ion. This change in the redox potential i s probably associated with the change in the coordination sphere.

206

WEATHERBURN

2.1.4. Ma*nganeseCatalase (EC l.J.l.l.6) Catalase catalyzes the reaction: 2N2O2 --?.

0 2

4- 2HZO

Most catalases contain heme, but the caialases from the bacteria Lactobacillus plantarum DO31, T. thermophilus [104], Therrnoleophilum album 11051 and Therrnus species US 8-13 L106J contain a dinuclear manganese center. Reviews of these enzymes have appeared [4-6,107,1081. As isolated, the enzyme contains a mixture of oxidation states including an inactive "superoxidized" form of the enzyme that has an M n ( ~ ~ ~ ) / Mdinuclear n(~) center. The active form of the enzyme cycles between two diamagnetic ground-state ~ the n {M~n (~~ ~~ I ) / Mf o~m( ~[105,1101. ~~) The ~ n ( ~ ~ ) ~ forms: an ~ n ( I ~ ) and form of the enzyme has been isolated and characterized, but this form is not biologically active Lllll. Some properties of the enzymes from different organisms are summarized in Table 2. When isolated, the enzyme has a 16-line EPR spectrum together with a much broader signal that is consistent with both Mn3+/Mn4+and Mn2+/Mn2+ oxidation states in the active site. EXAF'S and other spectral studies have shown that the "superoxidized" enzyme has an Mn-Mn vector at 2.7 A that is consistent with an Mnz(kiz-0)2core in this inactive form of the enzyme 11121. EPR and microwave polarization studies of the Mnz+mn2+form of the enzyme from LactobacilZus plantarurn suggest that two high-spin weakly coupled manganese ions (J = 40cm-') are n ~ +and ~ present [1131. EPR characterizations of the Mn2+/Mn3', M n ~ + ~ 11091 ~ n 3 + m n 4forms + of the enzyme have been reported 11111. The metal binding site residues are conserved in the three sequences of these enzymes currently available. Anions such as F-, PO:-, and CN- bind t o the active site in all oxidation states and dramatically alter the nature of the active site 1113-1151. EXAFS determined that the Mn-Mn distance in the Mn2+/Mn2+,phosphate-bound form of the enzyme is 3.59 A E1161, in good agreement with the distance of 3.53 A derived from XAS and XANES spectra of the native Mn2+/NIn2+form of the protein 11171. Structures of the TABLE 2 Properties of Manganese Catalases Organism

Mol. weight/ subunit

Subunits

Lactobacillus plantarum

28500

6

Thermdeophilum album T. thermophilus 1: species strain YS 8-13

35250 35000 33303

4 6 6

j?l,(E)

Ref.

Mn3+ form 475 (330) 395 (310)

[1091

-

11051 Cl09l [lo61

492 (360)

~ A N G A ~ ~ ~ E ~ C ~ NENZYMES TAININ AND ~ PROTEINS

207

enzymes o f the Mn2'iMn2' and Mn3'/Mn3' forms of the enzyme from T. thermophilus and L. plantarum have been determined and the report of the structure of the T. thermophilus enzyme has appeared [log, 118,4881.The X-ray-determined Nn-Mn distances are 3.18 f in the Mn"'/Mn2' and 3.14 A in the Mn3+/M.n3+form 11181. A representation of the active site from the T. thermophilus enzyme is shown in Fig. 2. 2.1.5. Peroxidases

Manganese peroxidase, an enzyme involved in the degradation of wood, is discussed in Chapter 9 of this volume [1191. 2.1.5.1. Chloroperoxidase (EC 1.11. I.10) Chloroperoxidase catalyzes halogenation of the natural product ealdariomycin.

2RH + HzOz + 2C1- --+2R-C1+ 2HzQ

(3)

Chloroperoxidase contains a heme group and exhibits peroxidase, catalase, and cytochrome P-450-like activities. EPR, X-ray fluorescence studies, and the crystal structure of the enzyme from the fungus Caldariomyees fumago indicate the presence of a Mn'+ that is coordinated by a heme propionate, Glu-104, His-105, Ser-108, and a water molecule. The location of Mn2+ with respect to the heme is similar to that o f Mn2+ in manganese-dependent peroxidase (cf. Chapter 9)but the metal coordination sphere is different. The role of the Mn2' in chloroperoxidase is uncertain, and catalytic activity is not altered by the absence of Mn2+ [1201.

2.2. Transferases Mn2' is involved in many different types of transferase reactions. Particularly important reactions involve transfer of glycosyl groups, as well as transfer of phosphoruscontaining substrates (kinases), DNA and RNA polymerases, and sulfotransferases involved in the biosynthesis of sialic acid groups.

ASP- 70

FIG. 2. Schematic diagram of the &nuclear active site of manganese catalase.

20

Kinases transfer a phosphate group from ATP or GTP to a tyrosine, serine, threonine, or histidine residue of proteins or to the OH group of a sugar. The presence of the phosphate group serves a regulatory function. An M2'-nucleotide complex is usually required as the substrate and additional metal ions may be required. EPR spectra of Mn2+ bound to the active site of kinases have been used to characterize the active sites, and these studies have been reviewed 1151. There are reports of Mn-activated and M~+-inactivekinases [121-1261. Structurally characterized kinases probably activated by M g f in vivo but with Mn2+ in the active site in the crystal are listed in Table 3, ~ t r ~ ~ t ~characterized r a ~ l y kinases in which it is known that the Mn2+ participates in vivo are described below. Observations of and Mn2' interacting s ~ e r ~ s t i cin~kinases ly have been made with phosphati~y~inositol kinase C1381, pyruvate kinase (which also requires a monovalent cation) 11391, phosphoenolpyruvate kinase [1401, protein tyrosine kinase r141 I, and phosphorylase kinase 11421. With pyruvate kinase and phosphorylase kinase a dinuclear metal binding site has been demonstrated c r y s t a l l o ~ a p h i c ~ y . 2.2.1.1. ~ h o ~ p h o ~kinase l a ~ e(EC 2.7.1.38) ~ l i o s ~ ~ i o r y lkinase a s e is involved in glycogen degradation. Its main role is to regulate the activity of glycogen phosphorylase. A hexadecameric molecule with a subunit of (apyS)*,phosphorylase kinrrse is one of the largest and most complex stoichiomet~ of the protein kinases. The y subunit is the catalytic center. It has dual specificity; M$+ causes seryl phosphorylation but Mn2+ activates tyrosine phosphorylation. ~ ~ ~ ~studies c t ofuther catalytic ~ a r e of the y subunit of rabbit musclc p h o s ~ h o ~ ~ ~ a s kinase and the binary complex with Mn2+ and p, y-imidoadenosine 5'4riphosphate ) are available. Phosphorylase kinase has a dinuclear metal binding site; and y-phosphate oxygens of M ~ ~two~carboxylate P , oxygens er molecules, and the other Mn2+binds the a- and y-phosphate e bridging NH between the 8- and y ~ p ~ o ~uf~this h asubt ~ ~ gen of Asn-154, a carboxylate oxygen of Asp-167, and two water molecules 11431. This M P P N P binding mode is similar to the carbamoyl phosphate ~e in Section 2.6.2. synthesis domain in carbamoyl phosphate s y n ~ h e t discussed 2.2.1.2. pyruvate kinase (EC 2.7.1.40) Pymvate kinase catalyzes the final step in glycolysis: Phosphoenolpyruvate

+ MgADP + €3'

---f

pyruvate

+ MgATP

(4)

The reaction occurs in two steps. First the 0-phosphoryl oxygen of ~ g attacks ~ phosphoenolpyruvate, forming enolpyruvate and MgATP, and then the enolpyruvate is converted to pyruvate. The enzyme is allosterically activated by fmctose-l,6bisphQspha~e,and Mn2 ' mediates the allosteric communication between the phosphoenolp~uvateand ~~ctose~l,6-bisphosphate sites in an allosteric relay mechanism ic conformational change in going from the T (inactive) state to the R (active) state [145,146]. Pyruvate kinases require a divalent metal ion, a divalent metal-nucleotide complex, and usually also K+ for activity. Enzymes that do

P

Glycerol kinase ~ F - d e p e n ~ e p~otein nt kinase

2.7.1.30 2.7.1.37

Acetate kinase Phosphog~ycerat~ kinase

2.7.2.1 2.7.2.3

Adenylate kinase Nucleoside dip~osphatekinase Cyclin-dependent kinase

2.7.4.3 2.7.4.6 2.7.1.-

E. coli Mouse Pig ~ e ~ h a n o s u r c ~t hnear r n o ~ ~ i ~ a Pig muscle Yeast B. S t ~ ~ ~ o t ~ r m o p h ~ l L l s ococc~lsxanthus Homo supiens

1GL 1ATP lCDK

3PGK 1ZIP

lJST

210

WEA~HERBURN

not require K+ supply an internal monovalent cation in the form of a protonated lysine residue 11471. Crystal structures of the Mn"-substituted form of the enzyme, in the absence of nucleotide, from rabbit muscle shows that Mn2" is bound to Glu-271 and Asp-295 and the carboxylate oxygen and carbonyl oxygen of pyruvate. ENDOR spectra of the Mn-oxalate-ATP complex of pyruvate kinase had suggested that the oxalate is coordinated to the Mn2+ as a bidentate ligand; and it was suggested that a water molecule, a y-phosphate oxygen of ATP, and two protein residues completed the coordination sphere [148]. Mg2' binds to the protein through the same carbmylate side chains as Mn2+, and a crystallographic study of the ~ g ~ + ~ o x a l a t ~ -inA the TP rabbit muscle enzyme showed the M$* coordination sphere to be identical to that suggested by the ENDOR spectra 11491. The Mn2+ and K+ ions are 5.7 A apart. 2.2.2. Nucleases and DNA and RNA Polymeruses

Hydrolysis reactions of phosphate esters in DNA and RNA are promoted by Mg2+dependent endonucleases, polymerases, integrases, and ribozymes. These enzymes often contain more than one functional active size. DNA polymerase can act as a 3',5'-exonuclease, a 5',3'-DNA nuclease, and a DNA polymerase. In the DNA polymerase from 2'. aquaticus the 3',5'-exonuclease active site is separated from the polymerase active site by 33 A and from the 5',3'-nuclease active site by 70 A Il501. It is likely that two Mg2+ ions are required at each active site, but this view is controversial [151,152]. These enzymes are discussed in Chapter 4 11531. Structures of these enzymes with Mn2+ in the active site have been determined for the Klenow fragment of DNA polymerase I from E. coli Il541, rat DNA polymerase p 21551, bacteriophage T4 DNA polymerase l1561, human DNA polymerase [157,1581, for the exonucleases from 21 aquaticus il501 and E. coli il591, endonuclease FEN-1 11601, BamHI 11611, and EcoRV 11621, an avian sarcoma virus integrase C1631, and Maloney murine leukemia virus reverse transcriptase L1641. The role of &Inz" in the mechanism and specificityof endonucleaseshas been reviewed by Baldwin et al. 11651. 2.2.3. Carboxylases

2.2.3.1. Phosphoenolpyruvate carboxylase (EC 4.1.1.31) Phosphoenolp~vatecarboxylase (PEPC) catalyzes the earboxylation of phosphoenolpyruvate to produce oxaloacetate (Scheme 1)and requires Mn2+ or Mgz+ for its activity. Mn2' in E. coli PEPC is bound to Glu-506 and Asp-543, and so the bonding is similar to that of MnZ+in pyruvate kinase [166,1671. 2.2.4. Sugar Transferases 2.2.4.1. Ribosyltransferases (EC 2.4.2.) Structures of three sugar transferases containing Mn2+, hypoxanthineguanidine phosphoribosyltransferase (EC 2.4.2.8) from Trypanosoma cruzi 11681, quinolinic acid phosphoribosyltransferase (EC 2.4.2.19) from Mycobacterium tuberculosis II.691, and glutamine phosphoribosylpyrophosphate amidotransferase (PRTase) (EC


21 1

Scheme 1

2.4.2.14) from E. coli (1701 are available. The dinuclear Mn2' binding sites (illustrated in Fig. 3 ) in the first two of these enzymes are almost identical. One Mn2+ is coordinated by two ribose hydroxyl groups, two pyrophosphate oxygens, and two water molecules, whereas the other Mn" is bound t o four water molecules (or three waters and a carboxylate) and a-and @-pyrophosphateoxygens. The structure of PRTase contains two domains, one with a phosphoribosylpyrophosphate binding site, and the other with the active site for glutamine hydrolysis. These active sites are connected by a 20-A-long channel that is solvent-inaccessible. Mn2+ is octahedrally coordinated to two peptide C=O groups (Pro-302 and 61u-303), two phosphate oxygens, and two OH moieties of the substrate inhibitor la-pyrophospho~l-2a-3a~dihydro~-4@-cyclopentanemethanol 5-phosphate. This structure contains another Mn2+ located at a corner of the unit cell, this Mn2+ is bound to Glu-449, Asp-471 from a symmetry equivalent molecule, and four water molecules [1701.

Asp - 193

FIG. 3. The active site of hypoxanthine phosphoribosyltransferase with bound PRPP. The active site in quinolinic acid phosphoribosyltransferase is similar except that Asp-193 is replaced hy a water molecule.

212

2.2.5. Adenosyl Transferases

Eukaryotic mRNA is capped at its 5'-end by a 7-methyl-GMP moiety that probably cap F is catalyzed by protects the mRNA from digestion. Additioii of this 7 - m e t h y l - ~ ~ three enzymes: one to remove the 5'-termind phosphate, the second to form the guanylate derivative, and the third t o methylate this derivative. The guanylate derivative is formed from GTP in a reaction involving an enzyme-GMP adduct. The GMP moiety is then transferred to mRNA. Either M$' or Mn2+is required for the guanyl transfcr reaction. The structure of the capping enzyme (EC 2.7.7.50) from Chlorella virus PBCV-1 has been determined [1711, and the enzyme has two domains with a cleft between them. There are two conformations of the enzyme in the crystal with GTP bound, one with the cleft open and the other with it closed. In the crystal A h 2 + is bound in the closed conformer but not in the open form to the a-phosphate of the nucleotidc?and the a-phosphate was covdently bound to a lysine side chain,

Mn"-

or ~ ~ + ~ a c t ~ venzymes a t e d capable of transferring alkyl and aryl groups are known. The most important of these are the prenyltransferases, reviewed by Ogura m a C1721~Most structurally characterized prenyltransferases are M 8 + e n z ~ e that s contain three Nlg+ions in the active site 11731. There is one reported stnicture of an ~ n 2 + - ~ ~ o n t a i nenzyme, ing dihydropteroate synthase (EC 2.5.1.15), which catalyzes the reaction of p-aminobenzoic acid with 7,8-di~ydro-6~hydroxy" ~ ~ t ~ ~ ~p ~p ~t p~h ~ r si pnto h agive ~ e 7,8-dihydropteroate and pyrophosphate. Mn2+ is bound to the side chain of Asn-11, the P-phosphate oxygen of the substrate analogue h y d ~ # ~ ~ ~ t h ~ ~~olp ph ot ~e p~h ~ aand t ~e ,two water molccules [I741 I

2.3.1. Peptidus@s

2.3.1.2. Aminopeptid~es ~ j n o p e p t i d ~catalyze es the removal of the N-terminal amino acid from the end of a peptide chain. Leucine aminopeptidases (EC 3.4.11.1), which require MIS'+ and are inhibited by Zn2+,have been isolated € o m Salmonella ~ y ~ h ~ m11751, ~ r Fusciola i u ~ Izepatz'ca E1761, E. coli, pigs, and tomatoes 11771. Bovine lens leucine a m ~ n c ~ e p t i d ~ ~ e the most well-characterized aminopeptidase, has a dinuclear Zn2+ active site. Both of the metal binding sites must be occupied for activity; one site must be occupi~4by Zn2+,but if the other site is occupied by Mn'' instead of Zn2+ there is a significant increase in activity. The Mn'+-requiring enzymes have substantial sequence homology with the bovine lens enzyme, and the metal binding residues of the latter are conserved. Other structurally characterized aminopeptidases have dinuclear metal binding sites. The bovine lens leucine aminopeptidase [ 1781, glutamyl aminopeptidase e 3.4.11.-) from (EC 3.4.11.7) from E . coli [179], and an ~ n o ~ p t i d a s(EC Aeromonas proteolytica JlBO] have dinuclear Zn2" active sites. Methionine aminopep-

213

~ ~ O ~ T A I ENZYMES N I N ~ AND PROTEINS

tidase (EC 3.4.11.18) from E. coli US11 and Pyrococcus furiosus [1821 contain a dinuclear Goz+ active site. 2.3.1.2. A m i n o ~ ~ ~ t i d P a s(EC e 3.4.11.9) P r o l i n e ~ c o n t ~ ~pol~eptides ing are sequentially degraded by two aminopeptidases. Clostridial aminopeptidase (EC 3.4.11.-) cleaves off any N-terminal amino acid residue including proline from polypeptide chains but does not cleave the N-terminal peptide bonds involving a prolyl nitrogen 11831. Aminopeptidase P (EC 3.4.11.91 cleaves such prolyl nitrogen bonds. Aminopeptidase P from E. coli has a dinuclear Mn2' active site [184,1851 and has sequence homology with msthionine aminopeptidase, prolidase, and creatinase 11861. This tetrameric enzyme has been structurally characterized 11841 and studied using EPR, EXAFS, and ES 11851. The active site, in the C-terminal domain, contains a dinuclear manganese center (the Mn-Mn A representation o f the structure of the active site is shown in Fig. distance is 3.3 4. A bridging H20 or OH- apparently acts as the nucleophile in the attack on the scissile peptide bond of the substrate. In an inactive, low-pH form of the enzyme, the OH- group is replaced by a monodentate acetate. 2.3.1.3. ~ u r ~ o x y ~ e p t i ~ s e s Carbo~peptidases,which catalyze the removal of an amino acid from the C-terminal end of 8 peptide chain, are Zn2+-containing enzymes. Other metal ions, including Mn2+, can substitute for Zn2' and maintain activity; Mnz+ has been found bound to the procarbo~eptidasein rat pancreas. The structure of ~ n 2 ~ ~ s u b s t i t ucarted b o ~ p e p t i dA ~ e€rfrom os taurus has been determined I1871 but the results have not been reported in detail.

A).

2.3.2. Phosphatases

Phosphatases remove phosphate residues from phosphorylated proteins and sugars, thus mediating major signaling steps in biochemical pathways 1188,1891. ~ a n g a n e s e activated phosphatases include protein phosphatase (EC 3.1.3.16), alkaline phosphatase (EC 3.1.3.11, purple acid phosphatase (EC 3.1.3.21, inorganic pyrophosphatase (EC 3.6.1.1), phosphodiesteras~(EC 3.1.4.161, and phosphotriesterase (EC 3.1.8.1).

Asp - 260

Clu - 383

Glu - 406

FIG. 4. The dinuclear active site oC aminopeptidase P.

2.3.2.1. Protein phosphatases (EC 3.1.3.16) Protein phosphatases (PP) can be divided into two groups based on their substrate specificity, serinekhreonine phosphatases and tyrosine phosphatases. Some phosphatases efficiently hydrolyze both phosphotyrosiiie and phosphoserineithreonine. Although the reactions catalyzed by serinehhreonine and tyrosine phosphatases are similar, they have completely different structures and distinct catalytic mechanisms. Serinelthreonine phosphatases contain a dinuclear metal active site with 2Mn, 2Fe, and FelZn combinations found in different enzymes. Tyrosine phosphatases do not contain metal ions. Serinelthreonine phosphatases are subdivided into four groups (PP1, PP2A, PPZB, and PPZC) on the basis of differences in their biochemical properties. PP1, PPZA, and PPZB (also known as calcineurin) have homologous catalytic domains but differ in their substrate specificity. PPBC is unrelated to these enzymes by sequence but the active site is structurally similar. The structures of two PP1 enzymes have been determined. A human enzyme has a dinuclear active site containing Fe2" and &In2+, shown in Fig. 5a 11901, The rabbit muscle enzyme has a dinuclear Mn2+ active site and the metal ions are fivecoordinate; one is trigonal bipyramidal and the other is square pyramidal. The structure of the active site is identical to that in human PP2B (described below) except that the metal ions differ 11911. Human erythrocyte PP1 exists in two forms: Mn"-dependent and FeiZn-dependent. Thc Mn2+-dependent form has a higher activity [1921. Both Mn2' -dependent and Fe/Zn-dependent forms of PPSA have been identified in human erythrocytes, but a structure of this form of the enzyme has not been reported 11931. Human PP2B has a Zn2+/Fe2+-containingactive site shown in Fig. 5b [194,1951. Human PP2C binds two octahedral Mn'+ ions; the active site is shown in Fig. 5c. Protein architecture is similar to that of the PP1, PPZA, and PPZB enzymes despite the lack of sequence similarity [196]. Bacteriophage h protein phosphatase that is activated by Mn2' has been studied using EPR spectroscopy and a dinuclear active site is suggested 1197,1981. 2.3.2.2. Inorganic pyrophosphatase (EC 3.6.1.1) Inorganic pyrophosphatase (PP,ase), an enzyme found in almost all living cells, catalyzes the hydrolysis of PzQ$- to phosphate. All known PPiases require a divalent metal ion for catalysis; M 2 + usually has the highest activity but Co2' and Mn2+ are active. Up to four metal ions may be bound in the active site. The E. coli enzylne requires four metal ions for activity, whereas the yeast enzyme has highest activity with three M$+. There are significant sequence similarities between the enzymes from prokaryotes and eukaryotcs, although their subunit sizes differ (20,000 Da for the former and 28,000-35,000 Da for the latter). Crystal structures of the native enzymes from Sulfolobus acidocaldarius 11991, T. thermophilus I2001, S. cerevisiae, [201-2031, and E. coli [204-2061 have been determined. The enzymes from the latter organisms characterized with both M g + - and Mn2+-boundand active site mutants have aIso been studied. The structure of the active site of the enzyme from yemt with four Mn2' bound is shown in Fig. 6.

MANGANESE-CONTAIN ING ENZYMES AND PROTEINS

215

a)

173

pp-!

b) His -

0

FIG. 5. (a)The active site of PPI. The metal-metal distance is 3.3 A. (b) The site of PP1 and PP2B with PO:- bound. The met$ ions are Mn2' in the former and Fe2' and Zn2+in the latter. The metal-metal distance is 3.1 A. The amino acid nymbering refers to human PP2B. (c) The active site of PP2C. The metal-metal distance is 4.0 A.

PP,ase from B. subtiZis has an amino acid sequence with no similarity to other PPiases. This enzyme is activated by Mn2+but not by M$+, Ca2', Sr2+,Fez', Ni", or Cd2' [207-209]. Similar proteins may also occur in Methanococcus jannaschii, Archaeoglobus fulgidus, Streptococcus gordonii, and Streptococcus mutans (2071. 2.3.2.3. In,ositol m,onophosphatase (EC 3.1.3.25) Inositol rnonophosphatase is involved in the recycling of inositol from the inositol pyrophosphate second messengers. Crystal structures available include one containing three Mn2' ions in the active site [210,2111. It is believed that one of the &in2'

216

As;- 115 Asp - 120

FIG. 6. Active site in yeast inorganic pyrophosphatase.

ions (bound to Glu-70) is not involved in the mechanism of enzyme action as this Mn"' is readily displaced by the addition of PO:-. The other two Mn2+ions are 3.9 apart and are bridged by Asp-90. In addition, Mnl is also bound to Glu-70, Thr-95, the backbone oxygen of Ile-92, and a water molecule. Mn2 is four-coordinate and is bound to Asp-90, Asp-220, and Asp-93 and a el- or phosphate oxygen. Both metal ions are believed to bind the sugar phosphate 12121. 2.3.2.4. ~r~~tose-l,6-bisphosphatase IEC 3.1.3.11) Fructose-~,6-bisphosphataseconverts the a form of ~ - f ~ c t o s e - ~ , ~ - b i s p h o s ptoh oate f~ctose-6-phosphateand PO:-, and requires two divalent metal ions per subunit. There is evidence that the enzyme from rat brain is Mn"+ activated in vivo [213l. This enzyme is allosterically regulated and occurs in two forms (R and T forms). Structural changes in going from the R to the T form of the enzyme involve a 16" rotation of the upper dimer relative to the lower dimer. Mn"-containing forms of the enzymes from pig kidney [214], mutant forms of this enzyme [2151, and the spinach chloroplast enzyme [2P61 are structurally characterized. In the pig kidney Mn-FBP enzyme (T form) complexes with the a-analogue inhibitor 2,5-anhydro-n-glucitol-1,6-bisphosphate, AhC-l,&P,. The two 6-coordinate Mna+ are separated by 4.2 16 and are bridged by Glu-97 and Asp-118 and an 0 atom o f the phosphate of AhG-1,6-P:!, Mn1 is also bound to Asp-121and Glu-280. Mn2 is coordinated to the bridging groups and t o 61u-98 and the carbonyl 0 of Leu-120. In the absence o f metal ions the substrate binds in a similar way to the inhibitor AbG-1,6-P2so the inhibitor is thought to be a good model for the substrate binding. In the R form, Mnl is coordinated to the same residues as in the T form. Mn2 is not coordinated to the phosphate group of the substrate analogue. Changes in the coordination of Mn2 cause the metal ion to move about 1.6 A.

N T A I ~ I NENZYMES ~ AND PROTEINS

217

2.3.3. Arginase (EC 3.5.3.1)

Hydrolysis of L-arginine to urea and L-ornithine is catalyzed by arginase (Scheme 2). A number of reviews of the behavior and structure of arffinase have appeared [5,217,2181. Two forms of arginase are found in animals ranging from amphibians to mammals 12191. Structures of arginase from B. ealdovelox 12203 and both wild-type and mutant forms of the rat liver enzyme 1221,2221 have been determined. The bacterial enzyme is hexameric, the rat liver enzyme is trimeric, and both contain a dinuclear manganese active site. The manganese ions are located at the bottom of a 15-A-deep cleft in the monomer and are separated by 3.3 A, in reasonable agreement with the distances calculated from the EPR spectrum (3.4-3.6 A). An E trum of arginase did not reveal a peak attributable to Mn-Mn scattering [117]. Figure 7 shows the metal coordination sphere. The more deeply buried Mn i s bound to His101, Aqp-128, Asp-124 (bridging), Asp-232, and a bridging 0 - ion and a water molecule. The other Mn is bound to His-126, Asp-234 (bidentatel, and the bridging ligands Asp-124, Asp-232, and the OH- ion. His-141 functions as the base for deprotonation of the side chain amino group of L-lysine and the substrate guanidinium ( ~ and the ~ unprotonated ~ ~ side ) chain ~ of these amino acids is responsible for binding to the active site 12231.

Scheme 2

2.4. 2.4.1.

Lyases Carboxykinases and ~ehydratases

2.4.1.1. ~ h o s ~ h o e n o l p y r u v acarboxykinase ~e (EC 4.1.1.32 and 4.1.1.49)

hosp~oenolp~uvate carboxykinase (PCK) catalyzes reaction (51, which requires xaloacetate -I-AT /GTP

;It

phosphoenolp~vate+ C 0 2 +

(5)

either ATP (EC 4.1.1.32) or GTP (EC 4.1.1.49) as the phosphoryl donor and both Mn2+ and M p ' cations as cofactors. Crystal structures of the native enzyme from E. coli 12241 and with ATP, M[g2+, Mn2+, and pyruvate bound have been determined Mn binding site is at the base of a deep cleft between two domains. binds, PCR undergoes a domain closure with a 20" rotation of the

N EAT HER BUR^

21 8 His - 101

,Asp - 232

234

ASP- 128 \

Asp - 124 is - 126

FIG. 7. Schematic view of the binuclear manganese active site o f arginase.

two domains toward one another, trapping substrates and excluding solvent from the active site. M$+ and Mn2' are separated by 5.2 The M$+ is bound in a bidentate fashion by the p- and y-phosphoryl groups of ATP, three water molecules, and an oxygen of Thr-255. Mn2+ is bound to two water molecules, the y-phosphoryl group of ATP, Asp-269, Lys-213, and His-232. These metal-binding residues are strictly conserved in all ATP- and GTP-dependent PCKs. &If;"+ binding in the Mn2 ' binding site is not observed L2.261. The second substrate oxaloacetate is bound in the second coordination sphere of Mn2+ hydrogen-bonded to the coordinated water molecules. 2.4.1.2. Enolase (EC 4.2.1.11) Enolase catalyzes dehydration of 2-phospho-~-glycerateto yield phosphoenolpyruvate and requires a divalent metal ion for activity. Reaction occurs in two steps shown in Scheme 3. Deprotonation of the carbon acid, the first step, is a general feature of a number of other Mn2'-dependent enzymes, e.g., muconate cycloisomerase. M$+ is regarded as the natural cofactor of enolase, but a variety of divalent metal ions confer activity. There are three metal ion binding sites per subunit. Site I, the conformational site, is the tightest metal ion binding site. Binding at this site induces a conformational change that allows the binding of substrate. Following substrate binding a second metal ion binds at site 11,the catalytic site. At high metal ion concentrations, site 111, an inhibitory site, can be occupied. Crystal structures of enolase as a ternary complex of enolase-Mn2'-phosphoglycolate and a quaternary complex of enolase2Mn2~-phosphonoacetohydrox~ate ( P M ) have been determined L227-231 I. The site I metal has been well characterized in both Mn2'- and the M$ '-containing enzyme, but there is still uncertainty about the location of the site I1 metal, and the site I11 binding site has not yet been identified. Metal ions at sites I and I1 are separated by 4.2 in the M$+ structure but by 8 in the Mn2' structure. Mn2+ at site I binds to Asp-246, Asp-320, and Glu-295 beast numbering), and also to a water molecule. The carboxylate of the substrate/product i s bound in different structures as a bidentate ligand (M2+ is octahedral) or as a monodentate (M2-' is five-coordinate). Carboxylate and phosphate oxygens of the substrate/product are bound to the metal at site 11; the other ligands to this metal are two water molecules and the carbonyl and OH oxygens of Ser-39. A schematic diagram of the active site is shown in Fig. 8.

A.

A

A

~ A N ~ A N ~ S ~ - C Q N T A ENZYMES I N I ~ G AND PROTEINS

219

Scheme 3

EPR and XAS experiments using Mn2+have been used to characterize the metal binding sites [227,2321. These experiments indicate that early in the catalytic cycle two water molecules are bound to Mn2+ at site I in the e n o l a s e - M n 2 + - ~ ~ - M complex but that one of the water molecules is lost on conversion to the final enolaseMn2+-PhAW-M8+complex. Mn2+ at site I1 retains two water molecules throughout the cycle.

H21-

OH

FIG. 8. Schematic diagram of the active site of enolase.

2.4.1.3. 0-Gzutarate dehydralase (EC 4.2.1.401 D-Glutarate dehydratase catalyzes p elimination of water from n-glucarate to yield 5keto-4-deoxy-u-glucarate. Both the native M$+-containing P. putida enzyme and the Mn'+-substituted enzyme have been structurally characterized 12331, This molecule is homologous to both enolase and mandelate racemase and the metal ions are bound to Asp-241, Glu-261, and Asn-295. Other dehydratases activated by Mn2+and not by M$+ include imidazoleglycerol phosphate dehydratase from S. cerevisiae [234] and nglucosaminate dehydratase from P. fluorescens [235].

2.5.

Isomerases

2.5.1. Keto-Aldo Isomerases

Sugar isomerases interconvert keto sugars and aldo sugars, usually require a metal ion for activity, and use a variety of mechanisms to achieve the isomerization reaction. Two keto-aldo isomerases have been structurally characterized and are discussed below. There are a number of other sugar isomerases mentioned in Sec. 3.5.1.

220

2.5.1.1. Xylose isomeruse (EC 5.3.1.5) Scheme 4 shows the reaction catalyzed by xylose isomerase. Xylose isomerase also catalyzes the interconversion of n-glucose and u-fructose, and this activity is the basis of the commercial application of the enzyme. The enzyme requires metal ions to be present in a dinuclear site and Mg"', Co2', and Mn2+ activate the enzyme, isomerase is widely distributed in bacteria; it has been isolated from plants, and the catalytic and metal binding regions are conserved. Different metals ions have the highest activity in enzymes from different species, Mn2+ is superior to the other metal ions in Escherichiu, Bacillus, and Lactobacillus species. EPR studies of the Mn" form of the enzyme have been reviewed 12361. HzOH

H

H 01

- D -xylase

H a - D - xylulose

Scheme 4 Crystal structure determinations of xylose isomerases with Mn2+ in the active site are listed in Table 4. There are additional structure determinations with M 3 + and Coat in the active site that may be found in the cited references. Structures from M e r e n t bacterial species are similar; each monomer contains an eight-stranded a/@ barrel and a C-terminal domain that loops around the barrel of a neighboring molecule. The dinuclear metal center is located near the center of the barrel and the residues that ligate the metal ions are conserved in all species investigated. The metal ions are bridged by a glutamate residue and are 4.9 A apart in the substratefree form of the enzyme. A detailed discussion of all the crystal structures is beyond the scope of this chapter. The Mn2+ ions are octahedrally coordinated; the metal binding site 1 (or A site) has four carboxylate ligands and two water molecules in the native forms of the enzyme. Metal binding site 2 (or €3 site) has one histidine ligand, t h e e carboxylates (one bidentate), and water (or OH-) in the native enzyme (Fig. 9). inding of D-xylose and n-glucose has been studied in the crystal structures but the interpretation of the results is not straightforward. Both substrate and product are present in the active site and the active site is not fully occupied. There is good evidence that the site 2 metal moves upon substrate binding or during catalysis. An unexpected result of the structure determinations with n-xylose was that the sugar was bound in the open-chain conformation rather than the hemiacetal conformation normally found in sugars. Xylose is bound as a bidentate ligand via 0 - 2 and 0 - 4 to the metal at site 1.n-rrylitol, an inhibitor, binds in a similar fashion to xylose but there are two positions observed for the site 2 metal ion, one is identical to the site in the native structure, in the second position the Mn2' has moved 1.8 A toward the substrate, is bound to 0-1and 0-2 of the I->-xylitol (0-2 is also bound to the site 1metal); and the M n ~ distance ~ n is shortened to 3.5 A. Cyclic substrate analogues such as 5-thio-a-nglucose bind to the site 1 metal ion via the 0-3 and the 0-4 hydroxyl groups.

~ T A I N~I ~~ ~ Y M AND E PROTEINS S

221

TABLE 4 Crystal Structures of Xylose Isomerases

Qrgmism

Streptomyces olivochromogenes Str~ptomycesrubiginosus His-54-Ser mutant His-54-Asn mutant Phe-94-Ser mutanat Lys-183-Met mutant

Substrate

D-Xylose D-XYh3? Native pH 7.6 n-Xylose pH 9.0 r.-&corbate pH 7.4 1,5-anhydrosorbitol pH 7.4 D-G~UCO pH S ~8.0 n-Xylitol pH 7.4 D-Sorbitol pH 9.0

Arthrobacter

Asp-254-61u/Asp-256-G1u mutant Tyr-253-Cys mutant Actinoplanes missouriensis Glu-186-Gln mutant B. stearotl~ermt,;ph,iEus

Ref. 11.2371

~~~G

His-220-Ser mutant Es-220-Asn mutant His-220-6lu mutant

PD

9XIA

IXC

n-Threonate pH 9.0 n-Xylose D-Xylose n-Xylose u-Xylose Native u-Xylose 2,5-Dideoxy-.2,5-imino-~glucitol Native D-Xylose Native Gluconate 5-Thio-a-~-glucose Xylitol Native Xylitol Xylitol Xylitol Xylose

9x134 lAQD

W EAIHERE3URN

222

.Asp - 255

FIG. 9. Schematic view of the active site of the nabhe D-XylOse isomerase.

2.5.1.2. L-Fucose isomerase (EC 5.3.1.3)

r,-Fucose isomerase catalyzes the isomerization o f 1,-fucoset o t-fuculose (Scheme 5) and u-arabinose to n-ribulose, It has neither sequence nor structural similarity with other known aldose-ketose isomerases. A n X-ray crystal structure of t-fucose isomeraye from E. coli with an t-fucitol bound to the catalytic center shows that the active site is located between neighboring subunits within each trimer in a 20-A-deep pocket, terminating at a single Mn2+ ion. &In2+is bound to 0-1and 0-2 of L-fucitol, the side chains of Glu-337 and Asp-361 (bidentate with very long bonds to both oxygens), His-528, and a water molecule (illustrated in Fig. 10) [2501. It is thought that L-fucose is initially bound in its cyclic form but converts to the open-chain conformation before aldose-ketose interconversion occurs. After ring opening, rotation around C-2, with concurrent exchange of the 0-1 and 0-2 ligand positions at the metal ion, is required. During the isomerization two protons are transferred, one from 0-1to 0 - 2 and the other from C-2 t o C-1. Asp-361 and Glu-337 are positioned to assist in the transfer of the two protons via an em-diol intermediate. H CHO OH

HO

H

H 0

H

OH

H

OH

H

OH

H

OH

H

HO

HO (3-43 L - fucose

&I., L - fuculose

Scheme 5

M A N ~ A N ~ S ~ - C O N ~ A I NENZYMES ING AND PROTEINS

223

His - 528

H,C

A

OH

FIG. 10. Schematic view of the active site of L-fucose isomerase.

2.5.2. Cycloisomerases

2.5.2.1. Muconate cycloisomerase (EC 5.5.1.1) Muconate cycloisomerase catalyzes the isomerization of cis,cis-muconate to (4s)muconolactone (Scheme 6) although the substrate specificity is quite wide. This enzyme requires either Mn2+ or M2+with the former ion being preferred. Crystal structures of the enzyme from P. putida [2511, as well as mutant forms of this enzyme I2521 and of the apo enzyme from E. coli [253/,have been published. Protein folding is very similar to that of mandelate racemase and the metal binding site is in the same location. Mn2+ is octahedrally coordinated t o the protein side chains Asp-198, Glu224, Asp-249, and three water molecules.

CIS, C I S

- muconate

(4s) -muconolactone

Scheme 6 2.5.2.2. Chloromuconate qycloisomerase (EC 5.5.1.7) Chloromuconate cycloisomerase catalyzes the isomerization of mono- and disubstiLuted chloromuconates to chloromuconolactones with concomitant dehalogenation to form diene lactones (Scheme 7) [254]. Chloromuconate lactonizing enzyme from Alca,ligenes eu,trophus has 42% sequence identity with, and is structurally homologous to, muconate cycloisomerase from P. putida. Mn2+ is coordinated to Asp-194, Glu-220, and Asp-245 and a chloride ion 1255,2561. Dichloromuconate cycloisomerase (EC 5.5.1.11) from A. eutrophus JMP 134 is Mn"-dependent but has not been structurally characterized L2571.

H

o 2 - chloro -CIS,cis - muconate

(+) - 5 . chloromuconolactoiic

c

0

-00

CI (+) -2 chloromuconolactone

-0oc. 0 lrans -dienelactone

Scheme 7 2.5.3. Racemuses

ande elate racemase (EC 5.1.2.2) Mandelate racernase catalyzes the interconversion of the R and S enantiorners of mandelic acid (Scheme 8). It requires a divalent metal ion, being most active with M$', but is also activated by Mn2' l258l. Crystal structures of mandelate racemase from P. putida and of a number of mutant forms indicate that the enzyme is octameric, with each monomer comprising an N-terminal a i-domain, a central a/@barrel domain, and a C-terminal subdomain [259]. The active site is located between the two major domains at the C-terminal end of the strands. Mn2' and are inated to the side chains of Asp-195, Glu-221, and Glu-247, and either the inhib~tor(S~"a1trolact~te (bidentate) or a sulfate ion [2601.

2.5.3.1.

HO

Scheme 8

. ~ i ~ a s ~ ~ 2.6.1.

tamin^ in^ Synthetase (EC 6.3.1.2)

G l ~ t a ~ i synthetase ne regulates the metabolism o f cellular nitrogen and catalyzes the conversion of L-glutamate, ATP, and ammonia into L-glutamine, divalent metal ion is required for activity, and Mn" or Mi$' ar tors. X-ray crystal structures of glutamine synthetase from bot murium and E. co2i are very similar. There arc 12 identical subunits ~ r a ~ in~ two e d face-to-face s ~ e t r i c ahexamers, l with active sites located at the i ~ t e r f ~ cbetween es

MANGANESE-CONTAINI NG ENZYMES AND PROTElNS

225

adjacent subunits of the hexamer, in funnel-shaped open-ended cavities. These cavities are approximately 45 deep, 30 A wide at the top, and 10 wide at the bottom. About halfway down the active site cavity the two catalytically essential divalent metal ions and their ligands form a shelf. Structures of both enzymes and active site mutants were determined with two Mn2, ions hound in the active site; the metal-metal distance is 5.8 A. The more tightly bound Mn' ', in the 111 sitc, is coordinated to side chains of Glu-131, Glu-212, Glu-220, and two water molecules, one of which is shared by both metal ions. Glu-129, Glu-357, His-269, and two water molecules ligate the Mn2+ at the lower affinity or n2 site 1261,2621. The proposed mechanism involves the binding of ATP adjacent to the n2 Mn2+ and glutamate adjacent to the n l Mn2+ followed by gluta~nateattack on the y-phosphorus atom of A'I'P, producing the y-glutamyl phosphate intermediate and releasing ADP. Glutamine synthetasc then hinds ammonia, which attacks the y-glutamyl phosphate intermediate, forming an intermediate from which inorganic phosphate and glutamine are released 12611.

A

A

2.6.2. Carbarnoyl Phosphate Synthetase (EC 6.3.4.16)

Carbamoyl phosphate, a precursor for both arginine and pyrimidine biosynthesis, is produced from glutaminc, bicarhonate, water, and two ATPs via a mechanism that involves a number of consecutive reactions and three unstable intermediates. Intermediates are formed at three separate active sites connected by two molecular tunnels that run through the protein interior 12631. This enzyme is an (3. f3)4 tetramer, and the small p subunits are located at the ends of the molecule and catalyze the hydrolysis of glutamine. The J subunits have four distinct domains: an oligomerization domain, an allosteric domain, a carboxyphosphate synthetic domain, and a carbamoylphosphate synthetic domain. The three active sites contained within the ol, p heterodimer are separated by almost 100 A r264-2661. The active sites for both the carboxyphosphate and carbamoylphosphate domains contain bound MnADP and monovalent K+ ions that are octahedrally coordinated by three carbonyl oxygens and three side chain oxygens. The nucleotidc binding sites of these doniains are similar, hut they differ with different forms of the enzyme. In one structure determination there ws19 an inorganic phosphate and a second manganese ion in the carhoxyphosphate domain. The Mn"+ ions in this domain are bridged by the Glu-299 carboxylate side chain, inorganic phosphate, and a phosphate of ADP. Mnl is also bonded to a water molecule, a second phosphate oxygen of ADP, and the side oxygen of Gln-285. Mn2 is bound to Glu-299 (bidentate), the two bridging phosphates, a water molecule, and the backbone carbonyl of Asn-301. The single Mn2+ ion in the carbamoylphosphate domain is octahedrally coordinated to two phosphoryl oxygen atoms, a water molecule, and the side chains of Gln-829 and Glu-841 (bidentate). However, in a structure with AMPPNP present, the carboxyphosphate domain contained only a single Mn", bound t o Glu-299, Asn-301, two bridging phosphates, and a water, and the carbamoyl phosphate synthetic domain contained two Mn2' ions 12671. These ions are hridged by Glu-841, the Mn'+ in the previously identified binding site is

226

W E A T H ~ ~ ~ U ~

also bound to Glu-829, the cx- and y-phosphate oxygens and the bridging N o f ~ ~ P and the second Mn2+ is bound to the p- and y-phosphate oxygens of AMPPNP, Asn843, Glu-841 (bidentate), and a water molecule. 2.6.3. Aminoucyl-tRNA Synthetuses (EC 6.1.1.J

Protein biosynthesis depends on the binding of tRNAs to the correct amino acids, a reaction catalyzed by aminoacyl-tRNA synthetases. On the basis of their amino acid sequences mid the topology of their catalytic domains, tRNA synthetases can be divided in two classes: I and 11. Both classes are metal ion-dependent. Class I enzymes require one M$+ and class I1 enzymes require three &I$+, with the exception of histidine tRNA synthetase in which an asparagine residue takes the place o f one of the &I$+ ions. The structures of several different class I1 synthetases have been determined with Mn2+ ions present. Mn2' , which is catalytically active, occupies exactly the same coordination sites as M$+ ions in these crystals. Structures of T. thermophilus asparaginyl-tRNAsynthetase (EC 6.1.1.22 1 [2681, Pyrococcus kodukaruensis aspartyl-tRNA synthetase (EC 6.1.1.12) 12691, and T. thermophilus seryl-tRNA synthetase (EC 6.1.1.11) 12701 have been determined, and all contain an unusual bent conformation of the bound ATP. Three Mn2+ or M$+ ions are bound to the ATP. One bridges the u- and P-phosphates and is also bound to a Glu, a Ser, and two H2O molecules; the other two ions bridge the p- and y-phosphates and are located on opposite sides of the P-O-y phosphate linkage. One of these Mn2+ ions also has Glu345 as a ligand; the other metal ligands are water molecules. 2.6.4. U~P-N-ueetylmur.arnoyl-L-uZuninern-glutumate Liguse (EC 6.3.2.9)

uD~-~-acetyl~uramoyl-L-alanine:D-glutamate ligase (MurD) catalyzes the addition of u-glutamate to the nucleotide precursor UDP-N-acetylmuramoyl-L-alanine (UMA) to yield U~P-N-acetylmuramoyl-~-alanine-~~glutamate~ Crystal structures of the quaternary complex of the E. coli MurD, with the substrate UMA, the product ADP, and M$+or Mn2+ and the binary complex of MurD with the product UMAG have been determined. The metal ions are coordinated t o His-$3, the C = O of UMA, and four water molecules. A reaction mechanism proposed on the basis of these structures involves phosphorylation of the C-terminal carboxylate group of UMA by the y-phosphate group of ATP to form an acylphosphate intermediate, followed by the nucleophilic attack by the amino p o u p of u-glutamate to produce the product [2711. 2.6.5. ~ e t h i o ~ z o tSynthetase in (EC 6.3.3.3)

Dethiobiotin synthetase catalyzes the formation of the ureido ring of dethiobiotin acid, C02, and ATP (Scheme 9). The structure from (7R,$S)-7,8-diaminopelargonic of a subunit of the E. coli enzyme consists of a seven-stranded parallel p sheet, surrounded by cx helices, The mononucleotide binding part of the structure is very

M A N ~ A N ~ S E - ~ O N ~ AENZYMES I N I N ~ AND PROTEINS

227

similar to all GTP-binding proteins. A crystal structure of a complex of the enzyme with the ATP analogue adenylyl (&y-methylene)diphosphonate,7,8-diaminononanoic acid, and Mn2+showed the metal bound to P-and y-phosphate oxygen atoms and the side chains of Asp-54, Thr-16, and Glu-115 C2721. Mutations Lys-37-Glu and Lys-37Leu resulted in an inactive enzyme and shifting of the Mn2+ by 0.5 and the side chains of other active site residues also shifted 12731.

A,

Scheme 9

roteins Containing Bound Manganese 2.7.I . Lectins

Viruses, plant, and animal species contain carbohydrate-binding proteins, called lectins or agglutinins. Animal lectins contain Ca2+ as the metal cofactor, but plant lectins possess a dinuclear metal binding site that binds both Mn2+ and Ca2+. A number of reviews have been published C274-2771. Metal-free plant lectins have no carbohydrate binding properties. Binding of the Mn2+ to the protein induces a conformational change that forms the Ca” binding site. Binding of‘ Ca2’ to the protein forms the carbohydrate binding site. In the absence of Ca2+two Mn’+ can bind to the protein and form the carbohydrate binding site. 2.7.1.1. Concanavalin A The first lectin to be c ~ s t ~ l o ~ a p h i c acharacterized lly was concanavdn A from Cannvalia ensiformis (Jack bean) that specifically recognizes the trimannoside core of many complex glycans [278]. The dimanganese form of concanavalin A has been investigated using EPR spectroscopy and the spectrum is consistent with two octahedrally coordinated Mn2+ antiferromagnetically coupled I279 1. Concanavalin A has been the subject of many structural studies as follows: a triclinic form 12801, the apoprotein (2811, the orthorhornbic form at 0.94 resolution [282,2831, a neutron diffraction study [2841, forms with nonnative metals bound [285-2871, with 3,6-di-Omethyl-(a-D~mannopyranosyl)-a-D-mannopyranoside r2881, 4’-methylumbell~e~l-au-glucopyranoside I2891, methyl-cl-u-glucopyranoside [2901, methyl-a-u-arabinofuand 4’-nitrophenyl-cl-~-glucopyranoside [291j, 4’-nitrophenyl-a-~-mannopyranoside ranoside [292 I, methyl-3,6-di-O-(a-~-mannopyranosyl)-cl-~-mannop~anoside [293], P-GlcNAc- ( 1 42) -a-man-(1- 3)-[P-GlcNAc-(l+ 2J-ol-man-( 1 4611 -man 12941,

A

228

-

ASP 10 \

Asn - 14

A s p - 19

FIG. 11. Schematic view of the metal binding site in concanavdin A.

man-a-l,2-man-ol-OMe [295], methyl a-D-mannopyranoside 12961, and with trimannoside [2971 bound. A schematic view of the metal binding site in concanavalin A is shown in Fig. 11 and the metal-ligand distances to Mn2+ are given in Table 5. The saccharide binding site is close to the Ca2+ion, but there is no direct coordination of the sugar to the metal ions.

TABLE 5 ~anganese-LigandBond Distances Mn-~p”~9 ~n-~lu-8

2.222(6) 2.179(5) 2.170(5)

(A)in Concanavalin A

Mn-OH2(a) Mn-OH2(b) Mn-Asp-10

2.26~(6) 2.166(6) 2.146(5)

2.7.1.2. Other lectins

More than 70 lectin structures are available and studies have been initiated on many other lectins. Complete structures have been determined for lectins or agglutinins from Erythrina corullodendron [298,299], peanut 13001, two other leetins from Canaualia brasiliensis [301,302], Lathyrus ochrus [303,3041, garlic [305], G r ~ ~ o n i a s ~ ? ~ p l i c ~ f o[3061, l i a soybean (Glycine max) 1307,3081,pea (Pisum satzuurnj L309-3111, bean (~haseolusvulgaris) [312], snowdrop (G~lanthusn,ivalis) 1313,3141, daffodil (Narcissus pseudonarcissus) [315], mistletoe (Vzscurnalbum) [3161, lentil (Lens culinaris) [317], winged bean 13181, Vicia uillosa 13191, Dolichos biflorus [3121, Diocleu grandiflora [320], Canavalia brasiliensis [301], and gorse (Ulex europaeus) [3211. The metal binding sites are highly conserved and similar to the sites in concanavafin A structure, but the saccharide binding sites in these proteins difler considerably.

MA~GANE§~-CONTAI~I~N G

~ AND Y PROTEINS ~ ~ §

229

2.7.2. In,tegrins

Integrins are cell surface receptor proteins that mediate adhesion to other cells and to components of the extracellular matrix. Integrins consist of two ct subunits of 120-180 kDa, and a 90- to 110-kDa p subunit. Sixteen 41 subunits and 8 p subunits have been identified in humans, and these subunits can combine to form more than 20 cWerent heterodimers. Each subunit has a single membrane-spanning CI helix, a short Nterminal cytoplasmic domain, and a large extracellular domain [322-3241. The extracellular domains are implicated as being involved in cell-cell and cell-matrix interactions, and the cytoplasmictails interact with the cytoskeleton and regulatory proteins. Adhesiveness of these domains is controlled by binding events in the cytoplasmic tail. Interaction of integrins with ligands is dependent on the presence of a divalent metal ion [3251. Integrins CDllalCD18 (also known as LFA-1, or CLL&) and CDllbi CD18 (also known as C , aMPz,or Mac-1) have been the subject of a number of structural studies with protein and with Mn2’ and other metals bound to the protein. These proteins have a six-stranded p sheet and seven ct helices; a crevice at the C-terminal end of the p sheet contains the metal ion binding site and is the location of the ligand binding function. Two different forms of these proteins have been characterized, an “open” form in which two phenylalanines are solvent-exposed, and a “closed” form in which these phenylalanines are buried in the protein core. The spheres of the metal ions differ in the two forms. In the open form of M$+ cvld Mn2+ are coordinated to two water molecules, four amino acid residues, two serines, a threonine, and an aspartate (or glutamate) side chain from a neighboring molecule within the lattice 1326,3271. In the closed form, the metal ion moves about 3 A, and while the metal-serine and metal-water bonds remain intact the bonds to the threonine and glutamate are broken and replaced by bonds to an aspartate side chain and a water molecule (in CDllb) or a chloride ion (in CDlla) [325,326]. Changes to the metal coordination sphere are linked to a 10-A shift of the C-terminal helix of the protein and exposure of the buried phenylalanine residues. There has been considerable debate as to whether these conformations represent active or inactive structures, which form is active, and whether the open form is a structural artifact 1 3291. Surprisingly, given the similarities in the metal binding lb, the proteins have significantly different metal ion binding sites of CDlla and characteristics [3301. ~

2.7.3. Diphtheria Toxin Repressor

The transition metal ion dependent regulatory element that controls the expression of diphtheria toxin and several genes involved in the synthesis of siderophores in Corynebaeteriurn ~ i ~ ~ t ~ eisrthe i a diphtheria e toxin repressor, txR. Crystal structures of DtxR with bound Ni2+, Co2+, Cd2+,and Mn2’ have been d e t e r m i n ~[3313331 and have shown that the protein has three domains: a DNA binding domain, a metal binding domain, and a flexible domain. There are two metal binding sites. Site 1 i s occupied in all of the structures, and Co2+ and Mn2’ ions are bound tetrahedrally

WEATH~RBU~N

230

is-79, Glu-83, His-98, and an oxygen from a sulfate ion. Site 2 is only occupied by Mn2+ and CdZ+,the bonding is again tetrahedral, and the ligands we Glu-105, His106, the carbonyl oxygen of Cys-102, and a water molecule. The reasons for diflerential occupancy of this site in different structures are not understood 13321.

2.7.4. Integrases

Retroviral integrases catalyze the insertion of viral DNA into sites in the host DNA and contain two metal binding domains. The N-terminal domain includes a zinc finger motif, whereas the central catalytic domain includes the divalent metal cofactor required for enzymatic activity. The metal preference for in vitro activity of avian viral S integrase is Mn2+ > M 2 + and the coordination spheres of both metals are the same, i.e., two aspartate residues (Asp-64, Asp-121) and four water molecules [334].

2.7.5. Pneumococcal Surface Antigen Adhesin A (PsaAj PsaA is involved in the uptake of Mn2+ and Zn2+ in Streptococcus pneumoniae. The crystal structure has been determined and was described in the final paragraph of Sec. 1.4.1.1. 2.7.6. Mannose 6-Fhosphate Receptor

Mannose 6-phosphate receptor mediates the delivery of newly synthesized acid hydrolases to lysosomes by binding to mannose 6-phosphate residues on their N-linked oligosaccharides. This complex i s then transported from the Golgi to a compartment where the low pH induces dissociation of the complex. The released enzymes are then packaged into lysosomes and the receptors return to the Golgi or move to the plasma membrane. Two distinct types of receptor have been identified, and the cation-dependent form is activated by &in2+. A sti-ucture of the bovine receptor has revealed that Mn2+ is coordinated to side chains of Glu-101, Asp-103, a guanidino nitrogen of Arg111,and one of the phosphate oxygens of mannose 6-phosphate. There are numerous other residues forming hydrogen bonds to the mannose 6-phosphate 13351.

ENZYMES WIT^ U

~

K S ~T ~O~ C~ T ~~

There are many Mn-dependent enzyrnes that have not yet been characterized structurally or investigated using other biophysical techniques. A list of such enzymes has been published [336], and the following enzymes have also been identified as Mndependent: hydrogen sulfide oxidase (EC 1.8.J from Bacillus sp BN53-1 13371, mannose-l-phosphotransferase(EC 2.7.1.-> from Leishmania rnexzcana [3381, p-glucosidase (EC 3.2.1.21) from Aspergillus sojae [339], ~ D P - G ~ : ~ - D - ~ l c ~ A c - ~ - ~ n d o p e lyase (EC 4.2.2.2) from Erwinia chrysanthemi [340], and phenol carboxylase (EC

~ANGANES~-CONTAINING ENZYMES AND PROTEINS

231

4.1.1.-) from Pseudomonas strain, K172 [3411. A selection of these Mn-dependent enzymes is discussed in this section.

3.1.1. Dehydrogenases Dehydrogenases that are activated by Mn2+ and that have not been structurally characterized include NAD-malic enzyme from Ascaris suum [342l, D-arabitol dehydrogenase from Galdieria sulphuraria 13431, 6-phosphogluconate dehydrogenase from Schizosaccharomyces pombe [344], 2-oxoglutarate dehydrogenase from bison heart 13451, a bifunctional enzyme, I,-( +)-tartrate dehydrogenase-D-(+)-malate dehydrogenase (decarboxylating) (EC 1.1.1.93 and EC 1.1.1.39, respectively) from Rhodopseudornonas sphaeroides Y [346], tartrate dehydrogenase from Pseudomonas putida [347), histidinol dehydrogenase (EC 1.1.1.23) from cabbage, which is being structurally characterized [3481, threonine dehydrogenase from E. coli r3491, and malolactic enzyme from Lactococcus Zactis, the enzyme responsible for the conversion of malic acid to lactic acid in wine I350l. Human 17p-hydroxysteroid dehydrogenase, which is responsible for the synthesis of estrogens, has been crystallized with both M$+ and Mn2+ in the active site. Details of the structure are not yet available [3511.

3.1.2. Manganese Dioxygenase (EC 1.13.11.15)

Dioxygenases catalyze the incorporation of dioxygen into an aromatic substrate that contains a catechol cis-did structure 13521. Most known dioxygenases are iron enzymes; Fe3+ dioxygenases are generally intradiol dioxygenases that catalyze the cleavage of the C-C bond between the diol moieties. Fez+ and Mn2+dioxygenases are extradiol dioxygenases that cleave the aromatic ring to one side of the diol moiety. $An'+ dioxygenases have been isolated from Arthrobacter globiformis CM-2 [353,354] and B. breuis, and have been suggested as being present in B. steareothermophilus and B. macerans 13551. Chlorocatechol1,2-dioxygenasefrom Rhodococcus erythropoti may have both Mn and Fe in a homodimer enzyme 13561. Fe3+ and Fez+ dioxygenases have been structurally characterized (cf. Chapter 11). Fe2+ homoprotocatechuate 2,3-dioxygenase from Breuibacterium fuscum has 78.6% identity to the manganese-dependent homoprotocatechuate 2,3-dioxygenase from Arthrobacter globiformaisCM-2 [3571. A crystal structure of Z73-dihydroxybiphenyl 172-dioxygenasecontaining Fe2+ from P. cepacia has been determined and the iron atom has five ligands-Glu-260, His-146 (axial), His-210, and two water molecules in a square pyramidal geometry 13581. The ligands t o the Mn'+ center in the Mn-dioxygenases are likely to be the same as the iron ligands in this P. cepacia enzyme.

232

WEATHERBURN

3.1.3. Lipoxygenuse (EC 1.13.11.12) Lipoxygenase, which occurs in fung, plants, and animals, is a dioxygenase of polyunsaturated fatty acids. Mammalian lipoxygenases metabolize arachidonic acid whereas plant and fungal enzymes metabolize linoleic and linolenic acid, in both cases producing hydroperoxide metabolites. Active sites of most lipoxygenases contain a nonhemtt Fez+,which is coordinated t.0 three histidines, two other amino acid residues, and a water molecule. A linoleic acid lipoxygenase from the filamentous funbws Gnunmnnomyces graminis has been shown to form (1lbhydroperoxy(92,12Z)-octadecadieneoicacid (A) and ~13R)-hydroperoxy-(9Z,11E)-octadecadienoic acid (B) (Scheme 10) and it is activated by Mn2' 1359,3601.

A

R, = -(CHZ),COOH

B

R2 = -(CH*),CH3

A = (1 lS)-hydroperox~~(9Z,12Z)~~~dec~~enoic acid

B = (1 3R)-hydroperoxy(9Z,IlE)+ctadccsdienoic acid

Scheme 10 3.1.4. Manganese Ribonucleotide Reductase (EC 1.1 7.4.1) Ribonucleotide reductases convert ribonucleotides into deoxyribonucleotides 1361,3621. Three different classes of ribonucleotide reductases have been identified. These classes may be recognized by the metal cofactor and the chemical identity of the organic radical that initiates catalysis 13637. Class I enzymes contain a dinuclear iron center and a stable tyrosine radical; class I1 enzymes use adenosylcobalamin as a cofactor and use a 5'-deoxyadenosyl radical; and class I11 enzymes have an iron-sulfur cluster and a glycyl radical. The class I enzymes have bcen subdivided into class Ia and class Ib, which in Salmonella typhimurium and E. coli are encoded by different genes. The iron ribonucleotide reductase (Chapter 11)has been extensively studied and a Mn"-substituted version of this enzyme has becn structurally characterized L364j. Mn'+-dependent ribonucleotide reductases have been isolated from the bacteria B. suhtilis [36Ei], Corynebacterium (formerly Brevibacterrum) ammoniagenes (363,366,3671, Artltrvbacter species, Corynebcccterium glutamicum ATCC 13032 I368 I, and Prvpionibacferiurn freudenreichii 13691. These enzymes have bcen classified as class Ib enzymes on the basis of sequence comparisons (3631, but others have argued that these enzymes form a separate class, class IV 13681. Mn'+-dependent ribonucleotide reductases contain two different subunits and a dissociable cofactor. The reductasc contains substoichiometric amounts of Mn2+,and Fez is absent 13631. A stable protein-linked free radical has been detected in this enzyme but this radical has not bcen hlly characterized 1366,3701. A dinuclear metal

MAN~AN~S~~CO~~A ENZYMES I N I N G AND PROTEINS

233

binding site is suggested by the fact that the iron-binding residues in the class Ia and Ib enzymes are conserved in C o ~ n e b a c t e r i uammoniugenes ~ ribonucleotide reductase and the Q-band EPR spectra are indicative of such a site [363,3671. However, the Mn-dependent ribonucleotide reductase from B. subtilis may contain a mononuclear Mn site [3651. 3.1.5. Oxygen-Evolving Complex of Photosystem 17 The oxygen-evolving complex (OEC) of photosynthetic organisms, located in the chloroplast of plants and algae, contains a cluster of four manganese atoms at the site where water oxidation occurs. The reaction catalyzed at the manganese center is

2H20--+ O2 + 4E.I’

+ 4e-

(6)

The OEC and the role of the various polypeptides has been extensively reviewed, and in 1992 a very comprehensive review was published [3711. Since then many reviews on PSII have been published [372-3771. The development of inorganic and mechanistic models have been the subject of a number of recent publications [376,378-3801. The OEC contains at least 23 different proteins. Also present are about 400 chlorophyll molecules, which collect light; a “special pair’’ of chlorophyll molecules called Pfiao,carotenoids, which are also involved in light harvesting; and plastoquinones Qa and Qb. Experiments in which some of the proteins are removed suggest Dz, cyt b559, and the extrinthat the membrane-bound proteins called CP47,CP43,D1, sic proteins called 33 kD,24 kD,and 17 kD are necessary for 0 2 evolution. In addition to these proteins, the inorganic cofactors Mn’”, Fez+, Ca2-‘, 61-, and HCO; are required. The functions of these proteins are not fully understood. However, it is thought that the extrinsic proteins assist the binding of ?.!Inn+, Ca2’, and 61-, but they probably do not act as ligands t o these ions. Apart from providing the &,binding site, the function of Dz is also unclear. Both the D1 and D2 proteins contain tyrosines (called YZ and YT,, respectively) that are redox active. YZ is part of the electron transfer pathway in the water oxidation reaction; the function of YD is not known. YZ is believed to be about 5-8 A from the manganese cluster and about 10-15 A from Pfiao. The other tyrosine, Y,, is apparently 2 2 4 0 A from the manganese cluster and about 30-40 A from Yz 13811 and a similar distance from QA. Until recently, three-dimensional crystals of the OEC complex had not been prepared, but crystals of an 02-evolving PSII preparation from Synechococcus elongutus are now available r3821. The arrangement of the proteins of the oxygen-evolving core of the PSII complex from spinach and Murchuntia polymorpha has been studied using cryoelectron crystallography and scanning tunneling microscopy [383-3861. Crystals used in these studies contain a subset of the proteins of the OEC, the D1, Dz, CP47, CPd3 membrane proteins, the extrinsic 33-kDa protein, the M. and p subunits of cytochrome bsti9, and some low molecular weight subunits. The OEC is apparently dimeric. Within the monomeric unit the CP43and CP47proteins are located on upposite sides of the centrally located DI-D2 proteins. The region between the two monomeric units seems to contain low molecular weight polypeptides. The 23-kDa and 33-

234

WEATH~RBURN

kDa proteins have been located in a separate study [3871. None of these studies can identify the manganese binding site of the OEC. The function of the manganese cluster became apparent €rom experiments, in which dark adapted PSI1 preparations were subjected to intense short flashes of light* 0 2 evolution did not occur until the third flash and then peaks of oxygen evolution were observed every fourth flash. The accepted interpretation of these results is that dark adapted PSI1 preparations exist in a stable state called SI. Flashes 1, 2, and 3 each remove one electron from the manganese cluster to produce new states S2, S3, and S4 with increasing oxidizing ability. S4 oxidizes water to Q2, at, the same time gaining four electrons and forming the Sostate. Flash 4 returns the manganese center to the stable SI state and then the cycle is repeated. The over& process is illustrated in Fig. 12. Whether the manganese cluster is oxidized in each of the S-state advances is controversial. XANES experiments suggest that the Mn oxidation state increases on going from So to S1 and from Sl to S2 [388-3901. An oxidation state decrease is observed on going horn S3 to So (the S4 state has too short a lifetime to be observed). However, there i s no apparent change t o the W E S spectrum in going from S2 to S3, and so it has been proposed that another group may be oxidized. Histidine and tyrosine residues have been suggested as possibilities. The original interpretation of the ~~~S spectra has been challenged, however l3911. A model of the manganese binding site places it in an open, C-shaped structure modeled within a complex of D1/D2/cytochromeb559 proteins with Asp-170, Glu-189, Asp-342, and Ma-344 from the DI protein as putative Mn ligands 23921. Other models site 13931. His-337, His-332, Glu-333, have been produced that do not include the and possibly His-332 on D1 and Ris-339 on D2, the bicarbonatc ion, and 0x0 groups and water molecules have been identified as manganese ligands. There is disagreement about whether water is a ligand Lo the S2 state, with one group arguing that it is not a ligand [394] and another arguing that it could be a ligand [3951. Methanol and ammonia, which probably binds to Mn, can bind in the S2 state, so it would be remarkable if water could not bind 1396,3971. Water is bound to Mn in the S:+ state [398]. Asp-59 and Asp-61 of the D1 protein may be Ca2+ ligands [399i. Infrared studies suggest that a carboxylate acts as a bridging ligand between the Mn and the Ca2' ions 14001.

FIG. 12. The S-state diagram €or the oxygen-evolving complex.

MANGAN~§€-CON~AI~IN ENZYMES G AND PROTEINS

235

In the absence of suitable crystals for an X-ray structural study, a combination of XAS, EPR, ENDOR, XANES, FTIR, RR, and ESEEM has been used to characterize the manganese site in the OEC. Three major well-resolved peak3 are observed in the EXAFS spectrum ofthe S1 state of PSII. They are best fitted by approximately 2 0 or N atoms per Mn at about 1.82 A, by 2-4 0 or N atoms located 1.95-2.15 A from the Mn centers, by about 1.25 Mn atoms at 2.72 A and by 0.5 Mn or a Ga2’ at about 3.30 A [401,4021. The manganese cluster in the S2 state with the g 4 EPR signal (discussed in more detail below) is distinctly different from the Sz multiline EPR state or the S1 state. The second shell of back-scatters in the S2 state with g 4 is found to contain two Mn-Mn distances of 2.73 and 2.85 A probably due to two nonequivalent di-p-0x0-bridged Mn dinuclear structures. The shell at about 3.3 A also contains increased heterogeneity [4031. Four Ca2’ ions are bound with high affinity to PSII 14041. Results of independent EXAF’S investigations of the Ca2’ ions are apparently contradictory. One group suggested that the Ca” is about 3.3 A from the Mn cluster [4051, whereas another found no evidence for such an interaction 14061. Sr2+,which is presumably bound at the Ca” site, gives an EXAFS spectrum that is best simulated by two Mn neighbors at a distance of 3.5 A [407]. This close association is not supported by the observation that MI?’ substituted into the Ca2’ binding site does not apparently interact magnetically with the Mn cluster [4081. C1- is required only for the Sz + S3 and S4 --+ So transitions; however, it is not known whether the C1 acts as a ligand to Mn or Ca. These experiments and numerous studies of low-molecular-weight Mn complexes have led to the suggestion that the Mn cluster consists of a dimer of dimers with a structure similar to that shown in Fig. 13. EPR has played a pivotal role in understanding of the OEC!, and recently additional EPR signals have been observed. Three distinct EPR signals can arise from the SZ state of the OEC: a multiline signal at g = 2 (S = 1/2), a g = 4.1 state (S = 5/2), and a state with g = 4 t o g = 9 (S = 5 / 2 ; S > 5/2 is possible). Stability of these states differs between plants and cyanobacteria [409,4101. The multiline signal of the Sz state consists of at least 18 lines, separated by about 80 G, and it arises from the ground state ofthe Mn4 unit. A multiline EPR signal from the So state has been

-

-

FIG. 13. The “dirner of dirners” model of the active site in the oxygen-evolving complex.

236 observed that is wider than the Sz signal with structure over more than 2500 G, and at least 20 peaks on each side of g = 2. The Sostate has a ground S = $ state with no thermally accessible excited state [411,4121. Parallel polarization EPR revealed a spectrum from the S1 state of spinach OEC. The signal arises from an S = 1excited state and is centered on g = 4.9 with a width of 600 G [413,4141. In Synechocystis species PCC 6803 an 18-line signal is observed in the S1 state centered on g = 12 with a splitting of 32 G 14151. EPR signals from the S3 state are observed in a variety of PSI1 samples in which the OEC has been inhibited. These signals are thought to arise from interactions between the manganese cluster in an oxidation state equivalent to Szand an organic radical 1416,4111.Parallel polarization signals from the Mn cluster in the S3 state have been observed from an S = 1ground state 14181. The oxidation state of Mn in the manganese cluster continues to be controversial. The issue is complicated by the uncertainty about whether Mn is oxidized in the Sz-to-SB transition. Various proposals have been made that range from either ~ n ( ~ I I ) or~ ~~n (nI I~) &~ I n ) ( I I I ) ( ~ nin( ~So) ~to Mn(N), or ~ n ( I ~ I ) ( ~ n ( I V ) 3 in S3 [389,3~1,419,4201. 3.1.6. ~ h i o s u l f a ~ e - ~ x i d iEnzyme ~ing

~hiobaciZlusversutus uses a multienzyme complex that contains two colorless proteins and two c-type cytochromes to oxidize thiosulfate t o sulfate. Protein A binds thiosulfate and protein B contains a spin-coupled dinuclear &In'+ center f4211.

3.2. ~ r ~ n s f e r a s ~ s A glutathione transferase (EC 2.5.1.18)conferring resistance to the antibiotic fosfoacid, contains a mononuclear Mn" cenmycin, (l~,2S)-1,2-epox~ropylphosphonic ter. Sequence alignments and mechanistic considerations suggest that the active site is similar t o that in manganese extradiol dioxygenases discussed in Sec. 3.1.2. [4221. 3.2.1. Sugar Transferases

Sugar transferases catalyze the transfer of' a nucleotide sugar residue to a hydroxyl group on the target protein, lipid, or carbohydrate, although some use lipid-based dolichol phosphate sugars. Glysolated molecules of this type have a range of roles, including the determination of membrane, protein, and cell wall structure; protein folding; and acting as recognition elements in cell-cell interactions. Many different enzymes catalyze glysolation reactions. For example, 12 different groups and 5 families of galactosyltr~sferasesand 27 families of glycosyltransferases have been recognized on the basis of sequence comparisons 1423-4251. Many of these enzymes require divalent metal ions, frequently Mn2'-, and a list of the MnZt-activated enzymes has been published r3361. Little is known about the nature of the metal ion binding site in these enzymes with the exception of ribosyl-

M A N G A ~ ~ S ~ - ~ O N T A I NENZYM I N G S AND PROTEINS

237

transferases, which have dinuclear binding sites (Sec. 2.2.4.11.. The concentration of Mnz+required to give maximum activity with these enzymes is usually greater than 1 mh!13and such high concentrations would normally exclude them from consideration as Mnz+-activatedenzymes; however, Mn'" is often the only divalent metal ion tested that shows activity, 3.2.2. Sulfatases

Mammalian cytosolic sulfotransferases catalyze the transfer of a sulfonate group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) t o a hydroxyl an a sugar [4261, lipid [4271, amino acid side chain or amino group [428]. Three distinct forms of mammalian sulfotransferases have been identified and one of these and a bacterial form are ~n"-dependent [429,430]. Sulfotransferases contain a highly conserved region that has a similar sequence to the sequence of a loop found in many ATP- and GTPbinding proteins, and this region may be involved in the binding of PAPS 14291. Another conserved region is believed to be involved in substrate binding. Arylsulfatases are structurally similar to alkaline phosphatases, and alkaline phosphatases have weak sulfatase activity 1431,4321.Structurally characterized arylsulfaeases have only one metal ion (Ca" or M$') in the active site, whereas the alkaline phosphatase from E. coli has two Zn2+ and one M?' in close proximity to the phosphoryl group [433,4341.

3.3.1.

Prolinase (EC 3.4.13.8), Prolidase (EC 3.4.13.9), and Oth,er Aminopeptidases

Prolinase (EC 3.4.13.8) and prolidase (EC 3.4.13.9)cleave peptide bonds at the nitrogen atom of proline in dipeptides and tripeptides. Prolidase deficiency in humans results in deep skin ulcers, imidopeptiduria, and a unique facial appearance 14351. Both enzymes are activated by Mn", and prolidase, which shows significant sequence homology with aminopeptidase P (Sec. 2.3.1.2), probably contains a dinuclear Mn active site 11861. An aminopeptidase from Lactobacillus sake 14361, an aspartate aminopeptidase from rat brain [561, and di- and tripeptidases from Lactobacillus, are activated by Mn2' 1437,4381. 3.3.2. Acid and Alkaline Phosphatases

Most alkaline phosphatases are activated by Zn2', but the enzymes from Halobacterium halobium and Halobacterium cutirubrum are activated by Mn" 1439,4401. 3.3.2.1 Purple acid phosphatases (EC 3.1.3.2) Purple acid phosphatases are widely distributed nonspecific phosphomonoesterases. Mammalian purple acid phosphatases contain a dinuclear Fe3+/Fe2" active site and

the structures of the rat and pig enzymes have been determined 1441-4431. These enzymes have two stable redox states; the Fe"/Fe2+ state is the active form, and while the fully oxidized protein retains 5 1 0 % activity, this activity originates from an Fe"/Zn2+ "impurity7' [4441. Purple acid phosphatase from kidney beans has a dinuc/Zn2'-containing active site. The metal ions are 3.1 apart and bridged by - ion and Asp-164. Fe3+ is also coordinated by Tyr-167, His-325, and Asp-135 and the Zn2+ ion by His-286, His-323, and Asn-202 [4451. Despite only 18% overall sequence identity the mammalian enzyme has an overall structure similar to that of the plant enzyme. Metal coordination spheres and the geometry of the metal cluster are preserved in both enzymes; however, significant differences are found in the architecture of the substrate-binding pocket. There has been debate about the activating metal ions in purple acid phosphatases from plant sources. Early reports suggested that these enzymes contain only manganese, but later reports disputed this and suggested that the enzymes were activated by iron. It is becoming clear that both Fe- and Mn-activated enzymes and mixed-metal enzymes exist. Purple acid phosphatase of the duckweed Spirodela oligorrhiza contains both Fe and Mn [446]. Two purple acid phosphatases have been identified in sweet potato, one containing Fe and Mn [4471, and the other, like the enzyme from kidney beans and soybeans, containing Fe and Zn 14481. Both sweet potato enzymes have visible absorption maxima at 550-560 nm, similar to the maximum of the red kidney bean enzyme; however, substrate specificities are markedly different. Sweet potato and soybean enzymes show extensive sequence identity (> 66%) with the red kidney bean enzyme, and all of the metal ligands are conserved. 3.3.3. Dinitrogenase Reductase-Activating Glycohydrolase (EC 3.2.1.) Dinitrogenase reductase-activating glycohydrolase that is responsible for removing the ADP-ribose moiety from posttranslationally inactivated nitrogenase of Rhodospirillum, rubrum contains a dinuclear Mn2+ active site 14491.

utT Enzyme (EC 3.1.3.-) MutT enzyme catalyzes the hydrolysis of nucleoside triphosphates, by substitution at the rarely attacked p-phosphate, to yield NMP and pyrophosphate-a reaction that may be mechanistically related to the PPiase reaction. Two metal ions are required to achieve the hydrolysis, and &In2+ and M$+ both work. A cluster of five glutamate residues (41, 53, 56, 57, and 98) probably forms the metal binding site [450,4511.

3.3.5. Enzymes Related to Arginase Enzymes of EC class 3.5.3.- catalyze the hydrolysis of ~anidinocarboxy~ic acids to the co~espondingm-amino acid and urea. Enzymes that are activated by Mn2* and may have dinuclear active sites include arginine deiminase (EC 3.5.3.61, which

~ A N ~ A N ~ S ~ - ~ O ENZYMES N T A I ~AND I ~ PROTEINS ~

239

converts L-arginine to L-citrulline and NIS3 [452], and agmatinase (EC 3.5.3.111, which catalyzes the hydrolysis of agmatine to putrescine and urea [452,4531. Formiminoglutamase (EC 3.5.3.8), which hydrolyzes ~-formiminoglutamateto glutamate and formamide, has considerable sequence homology with arginase, and the metal ligands of arginase are conserved [4541. Other cnzymes in this class from Pseudonzonas species, e.g., guanidinoacetase (EC 3.5.3.2) 14551, ~anadinopropionase (EC 3.5.3.17) r4561, guanidinobutyrase (EC 3.5.3.7) [4571, and d i ~ a n i ~ n o b u t ~ a s e (EC 3.5.3.20) 1458,4593, are also activated by Mn2+, but structural and sequence data are not available.

3.4. Lyases Lyases that have been identified as Mn-dependent enzymes include ureidoglycollate lyase (EC 4.3.2.3) [4601 and a number of' cyclases that are discussed below.

3.4.1. Cyclases Mn2+-specificcyclases include adenylate cyclase (EC 4.6.1.1), guanylate cyclase (EC 4.6.1.2) 14611, cytidylate cyelase (EC 4.6.1.6) [4621, and sabinene hydrate cyclase (EC 4.6.1.9) 14631. A structure of adenylate cyclase, which converts ATP to , is available. It contains two internally homologous cytoplasmic domains ( C l and C2) that are inactivated when separated but are catalytically active when combined. Two metal ions are probably required for catalysis, but the structure contains only one gz* bound to the C1 domain 14641. The Mn2' ion binding site is contained within the C2 domain but its location is not known [4651.

3.5. Isomerases to-Ado Isomerases

A number of other sugar isomerases, apart from xylose isomerase and Id-fucoseisomerase, that were discussed in Sec. 2-51., e.g., L-arabinose isomerase (EC 5.3.1.4) 14661, D-mannose isomerase (EC 5.3.1.7) [467], 1,-rhamnoseisomerase (EC 5.3.1.14) 1468,4891, and D-ribose isomerase (EC 5.3.1.20) [4691, are all dependent on information on the nature of the metal binding sites is available. 3.5.2. Mutases

3.5.2.1. Phosphoglycerute mutase (EC 5.4.2.1) Phosphoglycerate mutase catalyzes the interconversion of 3-phosphoglycerate and 2phosphoglycerate. Two types of this enzyme are recognized depending on whether they need 2,3-diphosphoglycerate (DPG) as a cofactor. The DPG-dependent enzymes are found in vertebrates, most gram-negative bacteria and yeast, and the DPG-

WEATHERBURN

240

independent enzymes are found in plants and Bacillus species. Some organisms, e.g., E. coli, have both forms of the enzyme. The DPG-independent enzymes from E. coli and Bacillus species have an absolute and specific requirement for Mn2+, and two metal ions may be required 1470,4711. A crystal structure determination of the enzyme from B. stearothermophilus has been reported [4721. This form of the enzyme is a member of the alkaline phosphatase superfamily of enzymes identified by Galperin et al. [4731.

3.6.

Ligases

3.6.1. Pyruvate Carboxylase fEC 6.4.1.1) Pyruvate carboxylase catalyzes reaction (71, which is essential for carbohydrate metapyruvate t HCO,

+ ATP

--+

oxaloacetate + ADP + PO:-

(7) bolism. Pyruvate carboxylase, a homotetramer with a subunit M , of 120,000, contains one Mn2+ per subunit. Another divalent cation and a monovalent cation are also required for activity [4741. The function of Mn2+ is not understood. 3.7.

Proteins Containing Bound Manganese

3.7.1. Fur Repressor Protein The repressor protein Fur mediates iron uptake and regulation in E. coli. Mn2+ and Cu2+ ions have been shown to activate the protein in vitro. Fur binds one Mn2+ per monomer, and EPR evidence suggests that His-85, His-89, His-131, His-142, and His144 are ligands, along with a glutamate and aspartate in the C terminus 14751.

4. STRUCTUR E-FUNCTlON RELATlONSHIPS 4.1.

Description of the Coordination Sphere of Manganese in Proteins

In proteins Mn2+ is usually six-coordinate but four- and five-coordinate Mn2+ have been reported. Although some of these reports of lower coordination numbers may not be genuine, resulting instead from the resolution of the structural determination, genuine examples do exist. The coordination environment of Mn2+ and Mn3+ in all structurally characterized proteins contains oxygen donors, and frequently oxygen is the only donor to Mn2+.Some enzymes contain up to three imidazole nitrogens bound to Mn2+. Examples of Mn2+ bound to cysteine or methionine sulfur atoms are likely to be rare 14901. Mn2+ is often referred to as being thiophilic, and compared to M 2 + it is, but compared to, say, Fe2+ or Cu2+, Mn2+ has a low affinity for sulfur donors. There are no examples of Mn being bound to a heme or a corrin ring in any of the


24 1

proteins characterized to date, Mn3+ in all structurally characterized examples is bound to at least one histidine. 4.2.

Description of Reaction Mechanisms

With the very wide range of functionality displayed by Mn-dependent enzymes it is not possible to describe the mechanism of action proposed for all of them. In many cases the mechanisms that have been proposed are highly speculative but if they lead to predictions that can be experimentally tested they can be useful. Discussed below are some of the better characterized mechanisms, accompanied by some general remarks on enzymes that may use a similar mechanism. 4.2.I .

Superoxide Disrnutase

Extensive kinetic studies of the reactions catalyzed by MnSOD from T. therntophilus suggested that the reaction is a two-stage process in which 0, binds to the Mn(I1) center and oxidizes it to Mn(II1) [93, 476-4781. 0 2 is then released; 0, binds to the Mn(I1Ij center and is oxidized to water, and the Mn(II1) is reduced to Mn(I1). The overall reaction is extremely rapid and the rate-determining step may involve proton transfer. There is no direct evidence that 0; binds to the Mn ion but it is likely that it does so.

4.2.2. Catalase

A mechanism for the manganese catalase-promoted decomposition of HsOz that involves peroxide binding to the Mn(III)/Mn(III) form of the enzyme as a terminal ligand has been proposed rlO9l. Reduction of the &nuclear center in a two-electron step to produce weakly bound dioxygen and the Mn(II)/Mn(II) form of the enz.yme follows formation of this species. Subsequent binding of a second hydrogen peroxide to the Mn(II)/Mn(II) form of the enzyme, formation of p,-hydroperoxide bridge, loss of two waters, and formation of the Mn(III)/Mn(III) form of the enzyme completes the catalytic cycle. 4.2.3. Hydrolases

Hydrolysis of metal-NTP complexes to yield phosphate and the NDP or pyrophosphate and NMP is a common theme in Mg- and Mn-activated proteins. The hydrolysis reaction of NTP probably involves the conversion of a water molecule to a hydroxide group by the *:-phosphate of the protein-bound NTP. This hydroxide subsequently attacks the protonated y-phosphate, resulting in hydrolysis. It has been shown that the GTPase reaction rate is higher in the presence of Mn2' than M 2 + , although the metal ion coordination is identical. The increase in rate has been attributed to Mn" causing an increase in the pK,, of the y-phosphate group 1161.

In the structurally characterized proteins, coordination of Mn2' to the NTP moiety is variable. For example, three Mn2+ ions are bound in the tRNA synthetases and one and two Mn2+ ions are observed in carbamoylphosphate synthetase depending on crystallization conditions. ydrolysis of phosphate esters, arginine, and protein amide bonds is accomplished by enzymes (phosphatases, arginase, and aminopeptidases) which contain a p2-OH bimetallic active site. Metal ions involved in these reactions are Mn(lIj, Fe(I1j and Fe(III1, Co(II), and Zn(II), and presumably the function of the different metals is to orient and modulate the pKa and nucleophilicity of the OH- group. The protein environment undoubtedly plays a role, which is not clear at present, but stabilization of an intermediate, e.g., a trigonal bipyramidal phosphorus, is an attractive possibiifferences and similarities of the Zn and Mn hydrolases have been reviewed [2171. Most mechanisms for these reactions proposed to date have involved attack by the p2-0EI group on the substrate, which may or may not be bound to the metal centers. Nevertherless, an alternative mechanism that involves attack by a terminal M ( I I ) - O on ~ a substrate coordinated as a monodentate ligand to the other metal has received suppwt 24'19,4801. 4.2.4. Enolase Superfamily of Enzymes

Members of this family of at least nine different enzymes, including enolase, mandelate racemase, muconate cycloisomerase, and u-glucarate dehydratase, which were described above, catalyze very different overall reactions but are structurally similar. Each reaction is initiated by the abstraction of an x proton from a carboxylic acid; the fate of the intermediate is then determined by the structure of the active site [4811. A number of these enzymes require Mn2+for catalytic activity. Currently, the accepted mechanism involves the concerted enolization of a carboxylate group using both a general base to abstract the ix proton and a general acid to protonate the keto group as it tautomerizes r482-4841. Despite the large difference in the pKa values of the cx protons and the protein bases the reaction rates are rapid, indicating that the enolate intermediate is significantly stabilized relative to the substrate, enabling the activation energy for a-proton abstraction to be lowered [481,4821. 4.2.5. Xylose Isomerase

A hydride shift mechanism for the xylose isomerase reaction was first proposed on the basis of X-ray crystallographic studies and is now generally accepted. This mechanism involves the binding of the cyclic form of the sugar to the metal at site 1,which may or may not cause the metal at site 2 to shift. Ring opening occurs, which places the C-2 hydroxyl group of the sugar in a position to be deprotonated by the hydroxide bound to the metal at site 2 . The negative charge on 0 - 2 is stabilized by binding to both metal. ions. A hydride then shifts from 6-2 to 6-1and 0-1is protonated. Ring closure is the reverse of the ring opening step [242,244,485,48~].The other structurally

- C O N ~ A I ~ I NENZYMES G AND PROTEINS

243

characterized keto-aldo isomerase L-fixcose isomerase is believed to proceed via an ene-diol mechanism 12501. 4.2.6.

The Oxygen-EvolvingComplex

There have been many suggestions of mechanistic schemes for the QEC many of which have failed subsequent experimental tests. Two currently popular proposals, based on the “dimer of dimer” structural model, are due to Hoganson and Babcock [3791 and Yachandra, Sauer, and Klein 13721. These mechanisms are speculative but are in accord with most of the currently known structural details. The demonstration that a dinuclear Mn cluster can evolve O2 from HZ0 [487] will undoubtedly produce additional or modified proposals.

5. P

IV

Undoubtedly there are more Mn-activated enzymes and Mn-containing proteins awaiting discovery. It is known that manganese deficiency in animals can lead to abnormal fetal development, bone deformations, and impaired glucose tolerance. The proteins and enzymes inactivated by the deficiency have not yet been identified. The roles of manganese in brain chemistry, and its fixnction (if any) in bones remain to be established. Study of the uptake of manganese by bacteria and fungi, the regulation of this process in response to the external concentration of Mn, and the transport of Mn2+ within the cell are in their infancy. It is clear that, at least in some organisms, manganese-specific uptake and intracellular trafficking proteins exist, but biophysical characterization of these proteins is almost nonexistent, The mechanism of the specificity for manganese will be of considerable interest. In mammals, the currently available evidence suggests that manganese-specific uptake and transport proteins do not exist. Manganese appears to be opportunistic and binds to proteins, the main function of which is to bind other metal ions. For example, Mn” is transported in the bloodstream by transferrin, an iron-transporting protein. It would not be surprising if specific manganese uptake proteins were found in mammals in specialized cells or specific locations within the organism, e.g., the blood-brain barrier. The structural possibilities of mononuclear and dinuclear Mn2+-containing active sites of enzymes have probably been determined with the structures published to date and described above. Structural possibilities of Mn”-containing enzymes have not been explored in the same detail and higher nuclearity sites, in particular the OEC, are poorly characterized. Even when the structure of the enzyme is well established, e.g., SOD, there are many unanswered questions, such as what changes occur to the structure of the protein upon changes to the oxidation state of the metal? How do the protons necessary for the production of H202reach the active site?

244

WEATHE~BU~~

Enzymes that will undoubtedly be the subject of increasing attention include the Mn-dependent ribonucleotide reductases and the OEC. It will be of considerable interest to see how similar the Mn-dependent ribonucleotide reductases are t o the iron enzymes. Other enzymes worthy of more detailed study include the Mn-activated sugar transferases, aldo-keto isomerases, and the enzymes related to arginase that have not been the subject of biophysical studies. Major advances in our unde~standing of the Mn cluster in the OEC complex can be expected now that t h r e ~ ~ d i m e n s i o n ~ crystals are available E3821. A major challenge is to gain some understanding of the differences in the behavior of enzymes with difEerent metal ions in the active site catalyzing similar reactions, e.g., the protein phosphatases, SODS, and a m i n o p e p t i ~ a ~Why s . are there different forms of these enzymes‘?What properties of the metal ion or the protein account for the differences in reactivity? How i s the “right” metal ion selected by the apoenzyme? Investigations of MnSOD and FeSOD, the Fe and Mn forms of ribonucleotide reductases and purple acid phosphatases, and the Mg and Mn forms of endonucleases and exonucleases from a structural and mechanistic viewpoint will help provide some of the answers to these questions.

I thank Ms Teressa Gen who produced the figures. I also thank the following colleagues who provided manuscripts prior to publication: Professors Ted Baker, Larry Que, Jr., John Helliwell, David Christianson, and Jim Penner-Hahn. Professor Martin Schroder is thanked for his hospitality at Nottingham where this review was commenced and the Leave Committee of the Science Faculty at Victoria University for the opportunity of a period of research and study leave.

adenosine 5’-diphosphate AI)P l,6-bisphosphate AhG- 1,6-P~, 2,5-anhydro-~-glucitol5’-triphosphate ~ P P ~ P,y-imidoadenosine P AP4A AP5A ATP CAMP GCO CMP DFMG DPG DtxR

diadenosine tetraphosphate diadenosine pentaphosphate adenosine 5’4riphosphate adenosine 3,5-monophosphate cytochrome c oxidase cytidine 5’-monophosphate 3-deoxy-3-fluoromethylene glucose 2,3-diphosphoglycerate diphtheria toxin repressor

MANGAN~~~-CONTAINING ENZYMES AND PROTEINS

EDTA EN DO^

EPR ESEEM EXAFS FBP FTIR Gal GDP GlcNAc GMP GTP IGDH ~ P Man MAT MI3 mRNA MurD NAD NADP NDP NeuAc

NMP NTP OEC PAPS PCK PDB PEPC

P PP PP,ase PRPP PRTase PSI1 PSnA R1 R2

RR SerRS

SOD tRNA UDP

ethylenediarnine N,AT,N’iV”-tetraaceticacid electron nuclear double-resonance spectroscopy electron paramagnetic (or spin) resonance electron spin-echo envelope modulation extended X-ray absorption fine structure f~uctose-l,6-bisphosphat~e Fourier transform infrared spectroscopy galactose guanosine 5 ’-diphosphate N-acetylglucosarnine panosine 5’-monophosphate guanosine 5 ‘4riphosphate isocitrate dehydrogenase r Q p y ~dehydrogenase ma~ate ~ ~ sD~ ~ ~ ~ mannose ~ - a d e ~ o s y l ~ ~ e t h i osynthetase nine mandelate racemase messenger RNA ~P-N-acetylmuramoyl-~~-~anine:~-glutamate ligase nicotinarnide adenine dinucleotide nicotinamide adenine dinucleotide phosphate nucleotide diphosphate ~ - a c e t y l n e u r ~ i nacid, i c i.e., sialic acid nucleotide monophosphate nucleotide triphosphate oxygen-evolving complex 3 ’-phosphoadenosine 5 ’-phosphosulfate phosphoenolpyruvate carboxykinases Protein Data Base phosphoenolpyruvate carboxylase phosphonoacetohydrQxa~ate protein phosphatase inorganic pyrophosphatase phosphoribosylpyrophosphate glutamine phosphoribo~ylp~ophosp~~te amidotransferase photosystem I1 pneumococcal surface antigen adhesin A ribonucleotide reductase subunit 1 ribonucleotide reductase subunit 2 resonance h a n spectroscopy seryl-tRNA synthetase superoxide dismutase transfer ribonucleic acid uridine 5’-diphosphate

245

246

Urn

us

ES

EF

UDP-M-acetylmuramoyl-L-danine X-ray absorption spectroscopy X-ray absorption near-edge spcctroscopy

S

1. V. L. Pecoraro, ed., Manganese Re dm Enzymes, VCH, New York, 1992. m 2. V. L, Schrarnm and I?. C. Wedler (eds.), Manganese in ~ e t a b o ~ i sand Enzyme Function, Academic Press, New York, 1986. 3. A. Sigel and H. Sigel (eds.), Manganese and Its Role i n Biological Processes, Vol. 37, Metal Ions in Biological Systems, Marcel Dekker, New York, 2000. 4. D. W. Christianson, Prog. Biophys. Mol. Biol., 67, 217-252 (1997). 5. G. C. Disrnukes, Chern. Rev., 96, 2909-2926 (1996). 6. N. A. Law, M. T. Caudle, and V. L. Pecoraro, in Advances i n Inorganic Chemistry (A. G. Sykes, ed.), 1999, vol. 46, pp. 305-440. . C. Weatherburn, in Perspectives on Bioin,organic Chemistry (R. . Dilworth, and K. B. Nolan, eds.), JAI Press, Greenwich, CT, 1 pp. 1-113. kaira and S. M. Gorun, in Bioinorganic Catalysis (J. Reedijk and E. Bouwman, eds.), Marcel Dekker, New York, 1999, pp.355-422. 9. IF. Archibald, CRC Crit. Rev. Microb., 13, 63-109 (1986). . Walter, and M. 6. Montel, Meat Sci., 54, 41-47 (2000). 11. P. J. Liebowitz, L. S. Schwartzenberg, and A. K. Bruce, Photochem. Photobiol., 23, 45-50 (1976). Nlathew, J. Peterson, B. Degaulejac, N. Vicente, M. Denis, J. onaventura, and L, L. Pearce, Comp. Biochem. Physiol. B: Comp. Biochem., 113,525-532 (1996). 13. 6. P. Swann, T. Adewole, and J. IT. Waite, Comp. Biochem,. Physiol. B l., 119, 755-759 (1998). Wardharn, S . J. Norris, and F. 6. Gherardini, Proc. NatZ. 6, 10887-10892 (1999). . R. Poyner, in Metal Ions i n Biological Systems (A. Sigel and H. Sigel, eds.), Marcel Dekker, New York, 2000, vol. 37, pp. 183-207. 16. T, Schweins, K. Scheffzek, R. Assheuer, and A. Wittinghofer, J. Mid. Biol., 266, 847-856 (1997). 17. It. L. B. Casareno and J. A. Cowan, Chem. Commun., 1813-1814 (1996). . Holland, A. C. Hausrath, D. h e r s , and €3. W. Matthews, Protein Sci., 4, 1955-1965 (1995). 19. F. S,Ezra, D. S. Lucas, and A. F. Russell, Biochim. Biophys. Acta, 803, 9094 (1984).

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322 3. L. Guanylyl Cyclase 322 3.2. ~ y s t a t h i o n i ~0-Synthase e 323 3.3. ~ ~ ~ o l e~ ,a~ ~- ~i i ~ o xey ~ eand n a s~er ~ t o p 2,3-~~oxygena~e ~ a n 323

27 1

~ ~ ~ C RE ~ ~ ~ ~ ~ 4. ~ 4.I. Protein Expression 4.2. Detailed Structure- nction R ~ ~ ~ ~ t j o ~ s h ~ p s 4.2.1. ~ ~ o ~ h r o m e s 4.2.2. Glnbins 4.2.3. Heme-Based Biological Sensors 4.2.4. Gatalase 4.2.5. Peroxidases 4.2.6. Chloroperoxidase 4.2.7. ~ y ~ o c h r P450 o~~e itric Oxide ~ ~ t h a ~ e droxylamine Oxidoreductase 4.2,lQ.Nitrite Rediictases 4.2.10.1. ~ y t o c h r o mcdl ~ nitrite reductase 4.2.10.2. Cytochrome c nitrite reductase 4.2.3.1. Elerne Oxygenase 4.2.12 General ~ b s e ~ a t i o i ~ s 4.2.12.1. ~ e c o n d structure a~ 4.2.12.2. Proxinzal ligand stal interactions ~ o n € ~c ~~ royr ~ ~ n asphere t ~ o n effects 5.

E ~ T AND ~ ~W~~~~~ E ~ 5.1. Why Heme? Evolutionary AspecLs 5.2. An Outlook

~

N324 C 324 326 326 328 329 330 330 332 332 333 334 334 334 335 335 336 336 337 337 338 338 338 339 340 340

E~E~~NCES

1.

IN

342

CTI

Heme is one of the most versatile prosthetic groups in ~ e t a l l o p r o t e [1,21. ~ s The iron porphyrins present in heme proteins are heme a, heme h, heme c, heme d, heme d l , heme 0,~ h ~ ~ r oheme h e ~P460, e ~ and siroherne. Their stru€~ures are shown in Fig. 1. They share a common skeleton and differ in structural details due to substitutions at the various positions [2,3}. Although the extensive electronic delocalization of the t ~ heme , ~ s s e ~ a~ relatively e s high porphyrin imposes a s u ~ s t a i ~ t ipa l~ a n ~ ithe degree of p ~ ~ s t j ~and i t ys ~ ~ i~ e~v ic~ t ~ifrom ~ nt s planarity are usually e~countered in proteins.

TURANO AND Itl

272

Heme CI

Heme b

x

Heme c

Heme d

FIG. 1. Naturally occurring iron porphyrins.

The 6-type heme (or protoporph~inE l is the simplest representative. It has methyl groups at positions 1, 3, 5 , and 8. Two propionate groups are present at positions 6 and 7, and two vinyl groups at positions 2 and 4.Protons are present at o n the the so-cdled a, B, y, and 6-meso positions. In the free heme the c o ~ o r ~ a t i with vinyl groups coplzlnar with the heme is energetically favored, thanks to the interaction between n: orbitals of the vinyl group and the aromatie heme plane. However, in proteins, vinyl groups also orient thcmselves following the sterlc requirements pose^ by the protein matrix and the ~ y ~ r o p h o bi ni ~t e r ~ ~ ~ ~with o n sprotein amino acids. The c-type heme proteins have a heme covalently bound to the protein by two thioether bonds involving sulfhydryl groups of cysteine residues, A few c-type cytochromes lack the h t ~ o n ~ Cys e ~involved ~ d in heme linkage (see la&). ~~t~~~ proteins have been prepared in which one of the vinyl groups of a 6-type heme has

IRON IN HEME AND RELATED PROTEINS

Heme di

Chloroheme

FIG. 1. continued

273

274

been s ~ b s t i t ~ t with e d a thioether bond to a protein cysteine i41. The heme P460 in the N-terminal domain of hydroxylamine oxidoreductase is attached to the protein matrix through two thioether bonds analogom to those of c-type cytochromes~an additiorial covalent bond exists between the heme a-meso carbon and the Ct: of a Tyr of the adjacent subunit [5]. At variance with plants and fungal peroxidases, the ~ e r n e sof lactoperoxidase and myeloperoxidase contain a modified &type heme with CH3 at positions 1 and 5 replaced by RCH2, and R’CH2 groups (where R = Glu; ‘P’herefore, two covalent linkages exist through the terminal COO groups of the two amino acids 16,71. En ~YeIoperoxidase,an additional cov&lentbond has been p r o ~ o s ~ d ~er to exist at position 2 between the heme and the protein throi~gha ~ h i o ~ tbridge with a methionine residue 171. The other heme proteins contain non-covalently bound hemes. ~ h l ~ ~ r oish ae ~ e b-type heme with a formyl s o u p in position 2. Kemes a and o both carry a farnesylh y d r o ~ e t ~side y ~chain, but heme a diRers from heme o by an a d d ~ t i o n f~ lo ~ y group at position 8 of the tetrapyrrole ring. In heme d, the C ring of t e t r a ~ ~ r r is o~e s a t ~ r a t e d[Sl. Heme dl and siroherne have unconjugated A and B rings [9,lOJ. ~ i ~ e ~degrees e n t of conjugation in the various hemes impart different electronic t s ~ e s i ~ to e doptidelocali~ationon the heme plane, while different s u b s t i t ~ ~ nare eiprotein interactions. me iron in proteins can be five- or s~-coordinate,Axial ligation appears strongly related to protein function (Table 2 ) . In cytochromes that transfer electrons the coordinat~onsphere is saturated (Le., six- coordinate^ or blocked by hydrophobi€ a ~ that probably has g r ~ ~ ias p sin cytochrome c ’. Indeed, the only p e i ~ t a c o o r ~ nheme an electron transfer function is that of cytochrome c’, where the fifth ligand is a histidine residue. Having an open coordination site for exogenous ligands not only i s u n ~ e c e s s abut ~ could cause problems for the electron transfer (ET) function because of the cost of the p o t e n t ~ a ~larger ~ y r e o i ~ g ~ n i ~ aenergy t ~ o n at the heme center during ET. The ayial ligands of the heme iron are usually two histidines or one ~ histidine and one methionine. Cytochrome f has a histidine and a b a c ~ b o narnide nitrogen as Emidligands. In all of these cases, the donor atom for the histidine Iigand is the NEof the imidazole ring. A single exception has been reported np to now for the recently resolved structure of cytochrome c554twhere one of the four hemes has a N E His-NG Eis as the donor atoms for axial ligation. €’enta-coordination is usually found in globins and in heme enzymes. In globins the ~ ~ d & z oring l e of the “proximalssHis residue provides the fifth heme iron ~igand; the other axial heme iron position remains essentially free for 0 2 coordination. A similar situation is encountered in nitrophorin, the NO transport protein. En heme enzymes the sixth coordination position is available for substi*atebinding. The nature of the fifth (or proxinialf ligand modulates the redox potential of the heme iron. The distal site features are designed to facilitate the catalytic reaction. The nature of the axial ligands also influences the spin state of the heme iron 1211.~ e n t ~ ~ c o o r dori hexa~co~r~inatated ~at~ iron with a water molecule as the sixth ligand is high spin. Binding of 60, 02,NO, and CN- to the sixth axial position to low spin. ~ i s ~ ebis-His, t , or b ~ s -coordinati~~ ~ ~ t dwdys COP~ r o d ~ c ae change s

!WON lN HEME AND RELATED PROTEINS

~ i o ~ o ~~ c a ~nof

me type ~ rotei ins. t The heme ~ ~ and ax ~id ligation are also reporte and spin state for the heme

Function

I

as we11 as the resting state o

Axial ligation

states

Electron transfer ~ytochrQmes c: Class I Class XI (beside c’) Class II ~ ~ o c ~c’ r o ~ e Class III Class ~ ~ o c h r o mb: es ~ ~ o ~ ~b5,b2, o r bib65 n e Cytochxome b562 C ~ o e ~ rf o ~ e

ith the exception of eyt c’ that has Fe(I1) (S = 2 ) Fe(fII1 (8= 5/2)

His Nc/amide f\a of Tyr1

Globins: O2 storage t ~ a n s p ~ ~

b b or e h l o ~ o ~ e ~ ~ b

Fell11 (23 = 0)oxy

F'omal iron ~ ~ d a t ~ o ~ spin states

Fe(II1 (S = 2) FeCIII) ( S = 5/2)

Fe(II.1) (S = 5/21

His NE(+H20) CYS

Catalases P450 prateins

+

HzQ2 + HzO2 --+ 2 W 2 0 0 RH -iO2 + 2H" + 2e- -+ ROH + HZO

sv

Fe(II1) (S = 5/21 i

2

Oxidation of heme to ~ ~ v e ~ d i n ,

b b or OH-) I

277

Cytochrorne e oxidase (heme u3)

&

Q

is N&

c

heme 1

Fe(I1) (S = 0)

e(II1) (S = 1/2)

heme 3, heme 4

Hi? NE

heme 2 P460

His N E

I His NE or

His N E $- Tyr Uq C

Lys NC

cys s?,

~ e ( 1(S ~= ~ 5)/ 2 or S = 3/2)1 I ~ ~ i n ~ ~ otou ~ l e d heme 6

erne type

a1 ligation

Sulfite reduetases

Sir0heme

b Guanylyl eyelase

I

cys s

y

et S8,Met S6

~ o ~ v e ~ of s ~GTP o n to c

&

Fe(I1E) ( S = 1/2)

b

Fe(I1) (S b

is

=1:

0)

Fe(I1) (S = 2) deoxy I _

b

Fe(IIj (S = 2) deoxy

a€teferencesfor these systems are in the subsections dealing with the single proteins, with the exception of ~ t o ~ ~ r co oxidase, me cytochrome bo quinoX oxi&se, nitrite and sulfito oxidase, which are ~ ~ s c ~ in ~ Chapters bed 10 and 15 of this book. bAs ~ e ~ t ~inQthen text, e ~ the only e x c e p t ~ ios~~ o n s t ~ t u tby e d~ ~ c ~ o ~ e ~ and x i dm37elape~.oxi~~se, ase which contain ~ o d i f i hemes e ~ covale~t~y bound to protein amino acids.

responds to a low spin for the iron atom. The redox state of the heme iron in Ihe ~ ~ proteins is also reparted in Table 1,In heme e n ~ m e s , rest~n state ~ of n o n - heme iron redox states higher than + 3 are usually encountered during the catalytic cycle. ~ o w ~ d a the y s specific role of the heme substituents, axial ligands, and protein matrix in i ~ o d u ~ t i the n g biological function of heme proteins is unraveled thanks to our knowledge of high-resolution structures of many heme proteins with different ~ ~ o l o function, ~ c a ~ as summarized in Table 2,

i o s y n t ~ ~of~ Heme is

I n d e p e ~ ~ e n tby l y its ~ ~ t ~ther pe o,r p i ~ ~ rmacrocycle in binds iron with high a f f i ~ ~ t y and upon insertion in the protein matrix is able to fine-tune the metal ion redox p o t e n t i ~it l ~makes unusual iron oxid~tioiistates accessible that o t l i e r ~ ~are se d~f~cuIt to obtain in solution for simple iron complexes. A t the same time, heme biosynthesis is a multistep process that involves many enzymes [l%-151.Current experimental data for h e m e ~ ~ ~ n t h e s i zoi r~gi g~ i ~ m suggest s that all proceed via the same pathway once the first compound (5-aminolevulinate; -OOC - CN2 - CI32 - CO - CR, -NEE2) is formed. The fori~atio~i of this compound is known to occur via two distinct means. In plants and most bacteria there is the so-called five-carbon pathway whereby 5 - ~ i n o ~ e ~ i l iis~ formed a t e from glutamate, while in animals and certain bacteria this step involves the condensation of glycine with swccinyi ~ o e ~ A ~ y ~ (-0OC - CH2- CH2 - CQ -- CoA), and is calle e four-carbon pathway. h ~recursoi* to the formatio~o f p r o t o p o ~ p h ~ the reaction procecds t h i ~ o u ~the same i n t e ~ ~ e ~ i a talthough es, the enzyme d can vary from one organism to another. In particular, there are some steps in which the problem of aerobic vs. anaerobic conversion exists. Once the protoporph~inIX is obtained, ferrochelates are needed to catalyze the d ferroc~e~ates first bind ferrous ~nsertionof the metd ion. It has been p ~ o ~ o s ethat iron and then the porphyrin substrate. Metallation occurs when the macrocycle is bent, allowing metal insertion concomitant with proton release from the p o ~ h ~ i n . nce the heme is formed it becomes planar and is released as product (161. The resulting heme 6 not only is incorporated directly into 6-type heme proteins but s o rthe f o ~ a t i of o ~the hemes a, c, d, and 0,while heme also sei-ves as ~ ~ . e ~ u r for dl and siroheme are formed in pathways that branch off from the above-described pathway subsequent to the first three common steps following formation o f ii-aminolevul~na~, Non-covalent binding of the heme to the protein does not appear to require the a s ~ i s t a n co~f any other protein. On the c o n t ~ a a~key , step of c-type heme protein synthesis i s the covalent ligation of the heme to the protein matrix. From genetic and b ~ o c h e m i ~studies a~ it is clear that three distinct systems have evolved in nature to assemble these proteins [141. In gram-negative bacteria and p l ~ t ~ p r o t mitochono~o~ dria there is a system with eight genes (called ccm genes) involved in the cytochrome c ~ a t u r a t iIn ~ ~ .~ - ~ o ~ bacteria ~ t iand v chlorop~~sts e a simpler system exists invol-

280

Selected Home Protein Structures AvaiIable in the Protein Data Bank PDB Code

Protein

Class I C y ~ o c h ~ o ~c , e s Mitochondria1cytochrome c lcrc, lyeb, lyic, lyfc, lyea, lytc, 2ycc,lfib, lcrg, h i , Icty, lirv, lirw, lchh, lchi, lchj, lcsu, lcsv, lcsw, lcsx, lcrh, lcie, lcrj, lcif, Icig, lcih, Ictz, lycc, Bcyt, I.&, locd, lrap, Iraq, k y t , lcyc, Igiw, 2€rc5Zgiw, lhrc, lccr lcxa, Jcot, lc2n, 2c2c, 3c2c, lcxc, 2cxb, ~ y ~ ~ c h r oc2m e lc2r, Ihro, lcry letp Cytochrome c4 lcc5 ~ o ~ h r o cmg e 15Sc ~ y t ~ c h r c550 o~e 351c, lgks, 451c, 2pac, lcor, lcch Cytoehrome c551 lc52, layg, lc7m, la56, la8c Cytochrome c552 ldvh, 2dvfi, Sc53 C y t o c ~ r o ~c553 e 05Cl ~ y t o c ~ r ~c gm5 e~ Cytockrome cg lcyi, lcyj, la2s, Iced, lc6s, lctj lfcd Flavocytochrome c sulfide dehydrogenase 'Zrnta ~ e ~ h y l a m i n~ e e h ~ ~ o complex g o ~ ~ ~ e with a ~ c y a and ~ ~cytochrome n e m Yeast cytochrome c peroxidase (GCP) 2pcb complex with horse heart cytochrome c Yeast ~ o c ~c pre roo x ~~ ~ (CCP) ~a ~ e Zpcc complex with yeast iso-1 ~ o ~ c; ~ Saccharomyces certlvisiae

Class T I C ~ ~ ~e e ~ la%, lbbh, lcgn, lcgo, Scpq, lcpr, l e k , C y t o c h r o ~c'~ ljaf', lnbb, lrcp, Zccy

~

o

~

Clms I11 C y ~ ~ c c h ~ ~ ~ ~ s la%, lcth, lczj, fwnd, 2cdv, 2cy3, Zcym, Cytochrome c3 2cY& 3cyr Cytochrome c7 2new, lehj, llf22, h e w

lprc

Class IV C ~ ~ o c c h ~ ~ ~ e ~ Photosynthetic reaction center

~

s

281 Table 2 (Continued)

PDB

Protein _I_

C ~ ~ o ~b ~ r o ~ ~ s Cytochrome b5 laqa, law3, lmx, la-, lb5a, lb5b, l b h , lbfx, Xblv, lcyo, ld09, lehb, l e d , lfQ3,119)4, liet, lieu, lwdb, 2 m , 3bSc lcxy Cytochrome b558 ~ l a ~ o c y t o c h r obz m~ llco, lldc, lltd, lfcb lapc, lqpu, lqq3,256b Cytochrome b5G2 lbcc, lbe3, l b a , lqcr, 3bcc Cytochrome bcl

lctm, Shcz, 2pcf

Cytoehrorrtef ~ o c ~ o fm e

6106iTLS ~ ~ h r o c 111 ~ ~ o ~ n leca, leco, lecn, lecd lcqx Flavohemoglobin Zvhb He~oglobin( a ~ i ~ o m e t ~ Hemoglobin (deoxy) lcls, libe, 2hhe, lhdb, l d , lhga, lglj, llida, Zhbg, Bdhb, Ihbh, Zhhb, 3hhb, 4hhb (oxy) lhho Hemogl~b ~ lhgb, 2rnhb, 2pgh ~ ~ m o g ~ o(bai ~n L ~ o m ~ t ~ louu, 3sdh, lspg, lhbg, lhco, 2hc0, Spbx, Hemoglobin (carbon monoxy) lsct, Isdk, lsdl, lbbb, Is&, lsdl, Xcbm, 3hbi, Shbi, 'Ihbi Hemoglobin (complex with ethyl Zhbc, 2hbd, Zhbe, 2hbf isocyanide~ lith, 21hb Hemoglobin (cyanomet) Hemoglobin (deoxy) laOu, laOl, laOsv, laox, laOO, laOv, laOy, la&, ldsh, I&%,ldxu, ldxv, lhbb, 2hhd, lcbl, lout, 4sdh, lhbs, Bhbi, 4hbi, 6hbi Ihbi, la4f Hemoglobin (oxy) Other hemoglobin structures lash, lbuw, lhlm, lhba, laby, l&w, ffdh, lnih, lthb, Ihds, lcoh, lhgc, lhlb, lmoh, lap, lbab, lcmy, lvhb ~eg~e~oglob~n lfsl.,lgili, lgdj, lgdk, lgdl, llh2, 21h2, llhl, 21h1, llh6, 21h6, llh7, 21h7, llh3, 21h3, llh4, 21h4, 11h5, 2lh5, Bgdm, lbin Xbvc ~pomyoglobin

Table 2 (Continued) Protein Globins ( c o ~ t i ~ u e ~ ) Zfal yog glob in ( c y ~ o m ~ t j yog glob^^ (aquornet) Zmgb, lmnk, lmyi, lpmb, llhs, lmti, lmtk, zrngh, Zrngi, 2spm, Xspo, lmlh, lrnll, Imlo, Imfs, lvxb, lvxe, lvxh, lmyh, Imyj, liiiyg, 4nibn ~ y o g l ~ b(carbon in monoxy) lmbc, la6g, 2niga, Zmgc, lyca, lmyf, 2mb5, lajg, Zmgk, I m p , lmlu, 2mgf’, lmcy, Imoc, 2sp1, lmlf, lmlj, lmlm, lmiq, lspe, lvxc, l m f yog glob in (complex with azide) lazi, 5mba Myoglobin (complex with ethyl isocyanide 109m, 106111, Zmye, 2mya and sulfate) yog glob in ( c ~ m p l with e~ imida~~le~ 4mba yo glob^^ (complex with methyl f l O r n , Zmyh isocyanide and sulfatej ltes Myoglobin (complex with m e t ~ y ~ e t h y ~and ~ i nsulfate) e I l l m , 101m, 103m, 1071n,104m, Zmyc, Myo~lob~n (complex with n-butyl 105m, 112rn,2myd isocyanide and sulfate) lmgn, lvxa, lvxd, lvxg, lxch Myoglobin (complex with sulfate) llht, lemy, lymc, 2cmm, liop Myoglobin (cyanomet) lycb, lmbd, lyma, 2nig1, lmtj, Imob, ~ y ~ ~ g l(deoxy) o~in 2mgd, 2mgg, h o d , lmoa, lmlg, lrnlk, lmln, lmlr, 5mbn Ihjt, ljdo Myoglobirs (ferrous, ~ ~ - b o ~ n d ) 2 ~ b w lniba, , lmnh, lmni, lmiij, lmbs, yog glob in (met) lmyt, Xmge, Xmgj, lyrnb, 102m lltw, lmno, lmbo, 2mgm, Zspn Myoglobin (ow) llwy, lfes, Zmml, lyoh, lyog, lyoi, Other myoglobin structures 3mba, l h m , lajb, labs, lmbi, lmbn, lswm, %am, lirc

lnpl, Znpl, 3np1, 4apl lnp4

Nitrophorirts ~ i ~ ~ o p h o1r j n ~itrophorin4

283


Table 2 (~ontinued)

PDB

-~ Protein

~ l a r~~i ~~n,g aand l , ~ ~ ~ e r~ ie raolx ~ d ~ s e s lapx Ascorbate peroxidase; pea (Pisrim satiuum) C ~ o c c peroxidase; ~ o ~ ~~ a ~ ~ ~ i a r o ~ y c lccj, lryc, lbva, leyf, 2ccp, ticcp, 2cep, cereuisiae Gccp, 7ccp, ldcc, 3ccp, lcpd, Icpe, lcpf, lcpg, $ccp, lccp, lcca, lccc, lccb, lccg, lcce, QCCX, 4ccx, lcci, lcmp, lcmq, lcmt, lcmu, l&f, h a g , laa4, lac4, lac$, laeb, laed, laee, Iaef, laeg, taeh, laej, laek, laern, laen, Xaeo, laeq, laes, laet, laeu, law, 2cyp Lignin peroxidase; P ~ ~ ~ e ~ ~ c ~ a e llga, lflp, Iqpa, Ib80, lb232, lb85 clz~s~spor~um Manganese peroxidase; Phanerochaete lmnl, lmn2, lmnp clzrysosporium lsch Peroxidase; Arachis h y ~ ~ g a e a Peroxidase I; Hordeurn uulgare (barley) lbm gain latj Peroxidase C1A; horseradish Iarw, lam, Z w y , Igza, ~~~~~~e~ A r t ~ ~ o ~~y - &~ ~ so s u s Iayp, lam, 4 lgzb

Animal I”eroxidaseand a ~ ~ w - ~ o heme t e ~ (bL; t i ~656s or P1565; reduction pote~rtial-90 mV). Cyt b has eight t~ansmembranehelices (A-a-3) connected by F) and three short loops (BG, FG, GH) 1113-1171. The E loop is on the matrix side, while the r e ~ ~ three ~ nlong ~ loops ~ g are on the intermembrane space side. The and EF loops each contain one contains two short helices forming a hairpin structure. Both th of cyt b are located in the matrix, Tho two hemes are bound Four invtu-iant histid~nesare present in the sequences of@ b and be, the axial ligands of the two heme irons. In the cytachrome bel complex tho second redox subunit is constituted by cytochrome el. Cyt c1 is an alla p ~ o ~~~ os e~m~bclass l i ~T c~ y ~ c h r o E113-1171. ~~s It is anchored in the rne~ibraneby one mombrane-spannin~segment near the C terminus, Near the N terminus the sequence motif C-X-U-C-EX i s present, but Euglena grucilis c ~ o ~ h elr lacks ~ ~ the e first Cys. The c-type heme has a - 3 i s ~ axial e ~ ~ i g ~ tThe ~o~. second redox submit of the cytochrome bsf complex is the cytochrome f desc~bedin e da Fez S2 Sec. 2.1.3.The third redox subunit in both complexes i s c o n ~ t i t ~ tby center. 2.1.4.1.

Globins are heme proteins jnvolved in dioxygen binding and/or transport [1181. This collective name includes vertebrate hemoglobins, vertebrate myoglobins, invertebrate g~obins~ plant globins, and bacterial and fungal ~ a ~ o h e m ~ p r o t eIti ~has s . bemi proposed that all globins have evolved from a family of ancestral heme proteins ( 17,000 Da), which possessed the globin fold and functioned as redox proteins 11191. Once a t ~ i ~ s ~ h~eiro~~c ~ became g e n available, the acquisition of oxygen b ~ prop-d erties was initiated to finally reach the present function. All of them contain b - t ~ e heme, with the exception of c h l o ~ o c ~ o ~ iChlor~c~uorins ns. Irave a chloroheme, i.e., a me with a formyl group in ~osition2. yog~obinand he~oglobinare the first proteins for which an X-ray s t ~ ~ c t u r e has been determined [120,121]. They bind 0 2 in the reduced Fe(l1) state, and Q2 binding results in a transition from high-spin to low-spin state of the heme iron. ~ b o ~ The r e ~ ~ c ed ~ d o~x y ~ e n -form € r ~ is r e € e ~ e dto as deoxy, the d ~ o ~ ~ e n form ly oxidized states as met. as oxy,and the ~ u n c t i o n ~inactive,

-

298

FIG. 9. Overall (A) and active site (5)structures of the oxy sperm whale myoglohin (PDB code: fmbo [125]).

2.2.1. ~

~

~Occurrence, g ~ Biological ~ ~Bole,~and Motecular ~ s Structure :

~ y o g ~ o (Mb) ~ i n is the ovgen storage protein in vert~bratesf1221. protein of ellipsoidal shape (44 x 44 x 25 and molecular weight of Mk, exists as a monomer of 153 amino acids c o n s t ~ t ~ tby e d eight CI. helices (A-H). This represe~tsthe globin fold (Fig. 9A). The heme group is anchored in a cleft by an imidazole ligand from a histidine residue (the proximal histidine). The other side of the heme planc (the distal side) is ~ v a i l ~tob ac~ommod~te l~ a small sixtfi ligand, has of many hydrophobic amino an essentially hydrophobic character due to the pr 7His, or distal His) residues acid side chains, and contains conserved Val and ig. 93). An exception is represented by Aplysin plifb 1123,1241,which lacks the distal s whose role i s fulfilled by an Arg on the sane helix ElOArg. The structure has been determi~iedfor the three forms (deoxy, ow, and met) as well as in the presence of a variety of ligands [125-1311. In the deoxy form, the pentacoordinate iron atom lies 0.42 fi out of the heme plane, toward the proximal His imida~ole.Upon dioxygen binding, the Fe3+-€5 species is formed. aS a result, the metal ion radius decreases and the iron moves toward the heme plane. The displacement toward the proximal side of the heme iron is 0.18 & while the distance betwecn iron and the coordinated imidazole nitrogen decreases by only 0.15 fi in the oxy form. The latter figures indicate that the proximal amino acid is somehow following the iron ion in its ~ o y e ~ e nThe t . 0 2 molecule binds to iron in an end-on bent fashion with a

A)

IRON IN HEME AND RELATED ~ ~ Q T ~ I ~ §

299

Fe-Q-Q angle o f 115”.X-ray and neutron digraction studies indicate the formation of a hydrogen bond between the coordin~tedd i o ~ g e nmolecule and the distal histidine imidazole. It is notewo~hyto mention that from the analysis of the protein stmcture it is not possible to individuate a channel for the access of dioxygen to the distal cavity. ~ o l e c u l a dynamic r studies have demon st rat^, however, that Q2 can ~enetratethe protein thanks to local structural fluctuations E1321. 2.2-2. ~

e

~

oOccurrence, ~ ~ ~~ i o~l ~ ~~ iRole, c na l a.nd ~ Molecular : Structure

The dioxygen traiis~ortheme protein is ~ ~ e ~ o g l(Hb). ~ b i Hemoglobins n are found in plants, many invertebrates, crustaceans, mollusks, almost aU aniislid worms, and all vertebrates 1133,1341. The monomeric and oligorneric hemo globins all consist of a basically similar building block. of molecuiar weight of about 16 ma, with the typical globin fold eight conserved 05 helices constituting the scaffold for a well-defined heme-binding pocket, where the fifth ligand to the heme iron is constituted by a histidine residue [133,1351471. As in Mb, the distal histidine and valine are conserved, The vertebrate hemoglobins are tetrameric, usually of the type 052& where the 05 subunit lacks the due to deletion of five consecutive residues. In invertebrates, hemoglobins can be monomers, dimers, tetramers, or aggregates up to 200 subunits. Nigh molecular weight he~oglobinsare often referred to as erythrocruorins r1481. &itanyof them lack the distal His and Val that are always conserved in vertebrate hemoglobins. ~ o r n o d i m ~ rAscaris ic flb has an unusual structure 11891: each polypeptide consists of two tandem globin folds followed by a highly negatively charged trail (wfw 40,000 Da). Plant hemoglobins are monomers or homodimers I 1451. ~ e g h e ~ o ~ l o bfound in, in root nodules of leguminose plants, is structurally similar to H b and is involved in fixation of atmospheric nitrogen. The binding pocket o f leghemoglobiii appears more open, thus allowing water to enter the distal cavity and take part in the hydrogen bond with bound dioxygen togcther with the distal His 11491. An important feature of nonmonomeric hemoglobins is that the subunits can assume two d i f ~ e r e~~ot n f o r ~ a t i o ncalled s , T (tense) and R (relaxed) states [133]. The R state has dioxygen affinity similar to that of myoglobin and monomeric hemoglobins. The T state has mini shed affinity (P1,202about 60-100 higher than for the R state). The two conformations are in equilibrium with one another, correspondi~~ The T state is prevalent when the protein is in the fully deoxy form After about two equivalents (for tetranieric species) of oxygen have bound, the protein structure switches to the R state and the binding of 0 2 to the remaining two subunits is f ~ c ~ t a t e[1371. d While for the R stake s t ~ i c t u r adata l indicate similar b i ~ d i ~ disg tances for the iron and the proximal His in the oxy form, the iron-nitrogen distance in 1 ~ ~ ~ , ~ 4 ~ , It~ has 4 7 been , 1 ~ ~ the oxy T state is much longer, i.e., about 2.4 hypothesized that the source of this constrained conformation should be imposed by sLibuni~i n t e ~ a c ~ ~ oqnus :a t e r n ~ / t e r t ~structural a~ changes would be the origin

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300

of the reduced flexibility of the iron coordination sphere. However, despite many highresolution structures o€ different hemoglobin forms, the efl'ects responsible for this ~ o n s t r ca o~~~o r ~ a t of i othe ~ proximal histidine have not been c o ~ ~ l eunt ~ l ~ raveled.

Flavohemoproteins are fusion proteins consisting of a N-terminal globin-like domain and a C-terminal inding oxidored~ctasemodule, with a fold like that of ferredoxin r ~ u c t a s e ~ in fungi and bacteria 11511. The major deviation from the is a movement of the E helix that creates more space in s t a ~ globin ~ ~ structure d the distal site. The heme and the FAD molecule shortest distan s about 6.3 A, and the two cofactors have an i n t e ~ ~angle ~ a rof about 80", The ~ s ~ ~ring~ ~ and the heme propionate are bridged by highly conserved residues and a water molecule, which are thought to act as an electron conduit I1511. The proposed biological tio~ role is O8 sensing, scavenging or delivery [152,1531, and heme s ~ q u ~ s t ~ a11541. However, recently it has been de~onstrated that E. coli ~ a ~ o ~ e ~ o ~bas l oa brole i nin the catalysis of NO dioxygenation [1551.

2.3.1. Occurrence and Biological Role

~ ~ t ~ o p h o rare i n sheme proteins found in saliva of the b ~ o o d - ~insect e ~ ~~ i ~ ~o d ~ prolixus, which spreads Chagas' disease 1156,1571.The name nitr~phorinm-ises from the insect's ability to transport nitric oxide (bound to the heme iron) from i t s own ~~v~ to the host, where it ~nducesQ a ~ o d i l a ~ tand i o ~i n ~ ~ ipt ls a t ~ ~ ~ to this the host releases histamine, which can also bind ~ g ~ e g ~ t i In o nresponse . tightly to the heme iron. Nitrophorins were the first proteins shown to be capable of t r ~ n s p o ~NO i n ~from one tissue to another. ~~~~~

2.3.2, ~ u ~ r aArchitecture ll and Active Site Xlructure

prolixz~s~ 1 5 8 , 1 5The ~ ~ .overall structural ~ c h i t e c t u r eis distinct from globin folds and from those of all other known heme proteins. Instead it contains an eights t r a n ~ e dJ3 barrel (Fig. 1OA) that belongs to the lipocalin family, which ~ n c l ~ d e s ~ i n ~ insecticyan~n,and b ~ n " ~ ~ n d~i n~g o t e i such proteins as ~ t i n ~ l - b i n protein, L158,159]. In addition it has three CI helices and two disulfide bonds. The b-type heme i s enclosed in the barrel. The proximal ligand of tho heme iron is a His residue. The proximal h i ~ t interacts i ~ ~ with ~ the c ~ r b o x ~ ~ofa tan e Asp residue through a water~med~ated hydrogen bond (Fig. 10B). The same Asp also forms a hydrogen bond

301

FIG. 10. Overall (A1 mid active site fBf structures of ~ i t r o from ~ ~~ o~ o~ o~d - ~inscct ~ed~~ Rhodnilcs prolixus (PDB code: lnpl [1591).

with one of the two heme propionates, The sixth coordination position is occupied by nitric oxide or histamine. Cyanide and a m ~ o n i acan also bind the heme iron.

2.4.1. A x L

~ c c u r r e ~ and c e biologicat role FixL is a member of a b ow^^^ family of h e ~ e - ~ ~ bs ie od l ~ } sensor8 ~c~ [ ~ ~ ~ Specifically, FixL senses O2 and restricts the expression of specific genes to hypoxic o b ~~ zr ~~ ~~ ~ r ~ conditions. It is found in organisms such as ~ ~ i ~ melioti, japonicum m d ~ s c h e r ~ ccoli. ~ i aFixL contains two domains: a ~ e m e - b i ~ d sensory ~ng domain and a histidine kinase domain. The binding of 0 2 in the heme domain induces ~ nturns off Enase ~ ~ t i ~wh~ch t y , in turn chmges in the histidine kinase d ~ m that regulates gene expression in the system. 2.4.1.2. OueraZl architeelure and active site structure The X-rsy crystal s ~ ~ c t ~ of r emet s and c y a n o ~ e tforms of the FixL from ~ r a ~ y r h i 2 ~ bji au m p o ~ ~ (~ c u ~149,500) are known 11631. he structure i s dorniiiated by a ~ v e - s t r a ~ ad ~~dt i p ap ~bane3 ~ l ~and ~ belongs to class of sensory proteins called PAS (Fig- 1l.A) I164-1663. Therefore, the overall structure is different from ate~ globin folds and folds of other hernc proteins. The heme i s ~ e n t a ~ c o o ~ d i nthrough a p ~ o x h~~ s~ t ~ i d ~~(Fig. ~l e 11Bf. The heme pocket is p ~ ~ a h ~y d~ r lo y~ ~ with ~~~c 2.4.1.1.

^v

302

et192, ~ y r 2 and ~ 3 lile204 ~ on the proximal side and Ile215, Leu236, and. 118238 on the distal side. There i s 1x1polar residue, such as the distal histidine in Mb, to stabilize 02. 2.4.2. CoclA

Occurrence and biological role The ~ ~ - ~ etranscriptio~ n ~ ~ ~ activator i g CooA is found in the p h o t o ~ . ~ i t ~ ~bacteretic l ~ u ~ ~and i s the first metalloprotein known to have a clear ium ~ h o ~ o s p i r i rubrum biological role in C 0 recognition 11671.60 is believed to be a neurotransmitter in the m a x n ~ abrain. l ~ ~ The presence of CO causes CooA to bind DNA and turns on gene expression of a multicomponent CO oxidation system in ~ h o ~ s ~ i r rubrum. illu~

2.4.1.1.

Overall architecture and active site structure The CooA is a ~ o m 0 d iconsisting ~~r of a heme r e ~ ~domain t o and ~ a DNA ~ i ~ domain (Fig. 12A) [168]. The DNA binding domain contains a helix-turn- he^^ core and four antipsu-allel fi strands. The backbone of this domain is almost superimposaHA binding domains of other proteins in the catabolite activator protein (CAP) family. The heme regulatory domain, with eight antiparallel j3 strands and three helical segments, is also similar to the regulatory domain of that fact the effector in CooA is CQ-bound heme rather than c helix called the C helix dong the dixnei- interface is &own to be imp teric switching during the sensor regulation. The 6-type heme in the reduced CooA is six-coordinate (Fig. 12B) [1681. The heme in n ~ o n o ~A e ris ligated in the axial ~ositionby a h ~ s t i d i ~( e same monomer and the N-terminal nitrogen ofa proline (Pr02) from monomer B. The

2.4.1.2.

d

303

FIG. 12. Overall (A} and active site (B) structurc o f CooA from photosynthetic bactcriuni ~ ~ ~ d ~ ~rubrum ~ ~ (PUB r i lcode: ~ ulft9 m [ISS]).

latter finding is unprecedented and is unusual considering the relatively high pKa OC the Pro nitrogen ipKa = 10.4). Farther e x p e r i m e ~ t evidence ~l is needed to ~0~~ this finding from the X-ray structural study. The His77 is solvent-exposed and is next to a cysteine (Cys75).It is known that one of the axid Ligands must ~ i s s o c ~ ato t eallow CQ to bind. Furthermore, the oxidized CooA is shown to contain Cys75 as a ligand [l69,1701. Therefore, significant ligand and confornational changes may accompany CooA action L168f.

Gatalase is p r ~ ~ ine the ~ t p ~ ~ o x i s o of ~ ealmost s all aerobic cells. It catalyzes the o f hydrogen peroxide into molecular oxygen and water L1711: dispro~or~ionatio~ 2

~ --+ ~2HZQ Q

+ 02~

2.5.2. Querctll Ai.chitec~ure and Active Site Structure

The three-dimen~ionalstructures of several catalases have been reported [ 8 , ~ 7 ~ - 1 7 7 ~ . All of the enzymes are telramerie. Each monomer, constituted by about 500 amino ~ viL"n1e and ~ E. acids, consists of a iJ b m e l and an all-ol domain (Fig. 13Aj, ~

AND LU

FIG. 13. OvemXI (A) arid active site (B) structure ofbeef liver cataiase (PDB code: Teat f1721).

c d i hydropero~daseII have an additional domain with ~ucleot~de binding topology

~ l ~ ~~ ,o Iv ~~liver n~ e catalase i . does not c o ~ t this ~ ndomain but binds The parts of the protein with fewer secondary structure elements are ~esponsiblefor the quaternary structural interactions: several salt bridges at the interface between m o ~ o ~involve ~ e r ~k g , Asn, arid Glu residues. Most catalases have heme b as a prosthet~cgoup; however, a number of fungal and bacterial catalases contain heme d ~ 3 , I ~ 8 , ~In 7~ F 1~. ~ i ~ ivitak L l i and ~ ~E, coli hydrope~o~idase 11 the heme d is rotated 130" around the ct-y meso axis with respect to the heme b in other catalases. The fifth heme iron ligand is the phenolate of a sine residue (Fig. 13B). The heme group is deeply ~ ~ b e indeach ~ es~~~~~ ~ armd i s located between the p barrel and the cx helices (Fig. 13A). An access channel, about 30 A long with by h%c residues at the entrance and ~yd~~ophobic residues a8 the channel descends, access at, the distal site. Many residues of the distal cavity are c o n t ~ i ~ uby t ethe ~ barrel. A water molecule irJ found in the sixth coordina~ion ~ o s i t ~ o(Fig, n ISmS>.The aromatic ring of a Pke is shcked on the plane of one of the me pyrrole rings. Other conserved residues in the distal cavity are 811 Asn and a is, whose imidazole ring is almost parallel to the heme plane, This conformation is stabili~edby i n t e r a c t ~ owith ~ an Arg and a Thr.

ased on the known strL€ctural~eaturesand se~uencehomo lo^^ heme p ~ r o ~ ~ ~ a L130,IBll have been generally grouped into three superfamilies: plant (including fungal and some bacterial peroxidases), animal, and others that do not fit into the first two s u ~ e ~ ~ a[1~1,182~. ~ ~ j e sThe plant ~ u ~ e r f is~ the ~ i most ~ y studied family of

3Q5 ~ e r o x i d a ~and e s is further d i ~ into d three ~ classes [181,182]: ~ e r a x i d a ~with e s prokaryotic lineage, fungal peroxidases, and classical secretory plant peroxidases. Before 1992, yeast ~ o c ~ r o m c pee r o x i d ~ ~was e the only peroxidase with known structure t183,184]. Thanks mainly to the work of the Poulos group and their co~aborators, t r e m e n ~ o progress ~s has been made in the last 7 years so that at least one peroxidase from each rep~esentativegroup has a h o w n crystal structure. They are s u ~ m a r i ~ e d below, 2.6.1

~

~ u p c r f a r n ~of l y Plant, Fun,gal arid Bacterial Peroxidases

2.6.1.I, Class I: Ir~trucellularperoxidases

2.6.1.1.1. Yeast c ~ ~ o c h rc~~ ~@, er o ~ i ~ ~ s e 2.6.1.1.1.1. Occurrence and hiirlogical role. Yeast cytochrome c peroxidase the only known re~resentativeof m i t o c ~ ~ n ~d ~r ~ ~n o m e r ~ t is induced under aerobic growth conditions of bakerasyeast ~ a c c ~ a r o ~ cereuisiae. ~ces Its p ~ y s i o l orole ~ ~a ~~ p e to ~ be s the o x ~ ~ a t ~ofothe n ferrocytoehromec by reacting with H202. The enzyme may dso be present to remove Hz02 and prevent its oxidativ~~ a m a g ~ . 2.6.1.1.12. Overall a r ( ~ h ~ t e cand ~ ~ ractiue c site structure. The overall structural a r c ~ i t e c t is ~ ~generally e d e s ~ r ~ as b ecz-helical ~ prolate eflipsoid fold (Fig. 14) [183,1841. It is shared by all t h e e classes of plant peroxidases. Ten major a-helical s e ~ ~ span e nthe ~ whole ~ protein and account for about 50% of the protein. There is less than 12% j3 structure, with two antiparallel f-, pairs and one small f-, sheet in the promixal side of the heme. The heme is situated between the distal helix B and proximal helix F and is at the bottam of a distal heme access channel where s u b ~ t ~ a t e s can bind. CcP has a larger heme access channel than those of most other p e r o ~ d ~ e s and this can explain why the 6-meso position of heme in GcP can be readily modifi r1861. The eytochrome P450-Like 0x0 transfer reactions were also observed in Cc

~~~~

E: ~y~~~

FTG, 14. Overall structures of the t h e e classes of the superfmily of plant, fungalt and bacterid perouidases (PDB codes: cytochrome c peroxidase, Zcyp 11541; ~ a n ~ a n peroxidase, ~se lmnp [ZOO]; horseradish peroxidase, latj I1911,.

11871, but not in most other peroxidases. Unlike class II and 111 plant peroxidases, class I poroxidases such as CcP and ascorbate peroxidases contain no carbohydrate, no disulfide linkage, no calcium binding sites, and no signal peptide for secretion. Yeast cytochroi~ec peroxidase contains a b-type heme that is high-spin fivecoordinated by four nitrogens on the heme and one histidine (Fig. 15). The proximal iiiteraction from histidine carries some ~ i d a z o ~ acharacter te due a hydro~en~bondin~ the negative charge of the buried Asp235. The interaction is termed Asp-His-Fe triad, analogous to the catalytic triad of serine protease I 188I. This hydrog~n~bonding network o f triad is extended to proximal Trp191. On the distal side of the heme, three residues, Arg48, Trp5l and Flis52, form the ~ ~ ~ ~ pocket. - b i There ~ ~arei ordered n ~ water molecules in the distal pocket, one o f which is about 2.4 A above the heme iron. The NS1 atom of the His52 donates an H bond to the side chain oxygen of Asn82. Yeast CcP, Eke other class I peroxidases, contains Trp191 and Trp5l at the proximal and distal side, respectively, of the heme, whereas other classes of peroxidases contain Phe at the corresp~)nding~ o s ~ t i o n s ~ While CcP was the only peroxidase with known structure for more than 10 years since its first structure publication E1831, it is limited as a general model for other peroxidases because its substrate is cytochromc c, a protein, rather than small organic molecules. Therefore, the availability of other peroxidase structures such as that of ascorbate peroxidase is welcoming news. 2.6.1 1.2. Ascorbate peroxidase I

2.6.1. f "2.1. Occurrence and biological rob. Ascorbate peroxidase (APX? [ 1 ~ 1 , 1is$ found ~ ~ in plant cytosol, chloroplasts, root nodules with nitrogenase activity, and cyanubateria. It scavenges hydrogen peroxide by oxidizing ascorbate to monodehydroascorbate.

Hz&)z+ 2 ascorbate + RzO i-2 monodehydroascr,rbate The monodehydroasc~rbateis a much jess reactive radical than thosc involved in the oxidative damage of I1202. The monodehydroascorbate can be further oxidized to back to ascorbate. d e h y d r o ~ ~ c o ror ~ ~reduced te 2.&1.1.22. ~ ~ ~ ~~ r ac ~Z ~Z~ eartd c t ~actiue r e site s ~ r ~ & c The ~ ~ X-ray r ~ . crystal structure of pea cytosolic ascorbate peroxidase (W 57,500 per dimer) has been s , one heme in solvttd to 2.2 A resolution 11901. It consists of two h o m o ~ i ~ e rwith each of the four monomers. With 33% sequence homology, the pea cytosolic SYPX has the same overall strtictnral architecture as GcP (Fig. 14). The average root mean square (rms) difference for the 249 topologically equivalent a-carbon atoms between the four APX monomers and CcP is 1.3 A. The rms difference for the 137 helical Mcarbon atoms is even less, at 0.9 A APX contains less fewer p strands, with two of the three strands in CcP deleted in APX. The size and shape of the distal heme access channel is the same as that in CcP. AI?X has been shown to have the most average core fold of any peroxidases with known structure r1911 and therefore i s a good model peroxidase in terms o f overall structural fold,


307

The active site structure of the pea cytosolic APX is also the same as in CcP, including the hydrogen-bondinginteractions between the proximal histidine, a buried Asp, and a proximal Trp residue, on the proximal side and the distal His and Arg on the distal side (Fig. 15).The main difference between the two e n z ~ ei s the presence of a cation binding site (either Ki or Ca") in AI"X located about 8 from the acarbon atom of the proximal Trp179.

2.6.1 2. Cluss 11: ~ e € r e~ t ~ ~~peroxidases n ~

a

~

2.6.1.2.1. Ligrzirz peroxidase

2.6.1.2.1,l. Occurrence and biological role. Lignin peroxidase (LIP) is found in the white-rot b ~ s i d i ~ ~ y cfungi e t e t192,193J. Under ~trogen-limitingconditions, i u ~ ~at, least six the best-studie~white rot fungus, ~ ~ ~ n e ~ cc ~ ~~ sao es t~ eo rsecretes LiF isozymes and four manganese peroxidase isozymes (MnP, see next section). Together with an -generating system in the same organism, the extracellular LiP and MnP are responsible for biodegradation o f lignin, a complex p h e ~ y l ~ r l ~ p a n o ~ polymer compr~sin~ 3530% of w o o d ~ ~ p lcell a ~ twall materids. Lignin is the second most abundant biopolymer (after cellulose) on earth and is the most abundant renewable aromatic material. Therefore, controlled delignification is important not only for the global carbon cycle but for industrial applications such as wood and straw pulping, pulp b l ~ c ~ nand g , conversion of lignin to higher value products. 2.6.1.2.3.2 Overall architecture and active site structure. The structure of one of the major isozymes (LIP-2, FA&' 41,000) has been elucidated by X-ray crystall o ~ a ~ at h y2 A resolution by the Poulos group t194,1951, and then later at 1.7 A reso~utionby the Piontek group 11961. It is a glycoprotein. Despite of only 15%amino acid sequence homology, the overall fold of Lip is quite similar to that of the nonglycoprotein CcP, with an rms difference of 2.65 A for @-carbonsof the two enzymes (Fig. 141, C o ~ ~with a rCkP, ~ LiP has 49 extra residues at the C-terminal end, four d ~ s u l ~bonds d e and two calcium binding sites, one on each side of the heme. The distal calcium site is formed mainly from one continuous loop from residue 65 to residue 70. The ligands to the calcium are the side chain oxygens ofhp48, Asp68, Ser68, peptide ~ r b o n y of l GIy65, and two water molecules. ~ ~ m i lresidues ~ ~ y , 194-201 make the main contrib~~tions to formation of the proximal calcium site. The ligands are side chain oxygens of Ser177, Asp194, Thr196, Asp201, and peptide carbonyls of Pro196 0th calcium binding sites play important roles in maintaining the integrity (such as spin state) of the heme active site 1197,1981.The heme access channel in Lip is smaller than that of CcP. he heme active site is also quite similar to that of CcP and APX, such as the proximal His hydrogen-bonded to a buried Asp and distal His and Arg (Fig. 15). The major ~fferenceis that the proximal Trp191 and distat Trp51 in CcP are replaced by Phe in Lip. ~urthermore,the crystal structure at 1.7 resolution identified that a surface Trp17l residue is stereospecifically hydroxylated at the CB atom position. Evidence has been presented to support that a transient Trpl71 radical, generated from o ~ i d a ~ i oby n the heme center, may serve as substrate binding site for lignin degradation 11961.

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308

P Ph

FIG. 15. Active site structures of the three classes of tlte superfamily of plant, hngal, and bacterial peroxidases (PDB codes: cytochrome c peroxidase, 2cyp I184.1; ascorbate pernxidase, 1apx [1901; lignin peroxidase, lllp 1195,1963; manganese peroxidase, lmnp f2001; peanut peroxidase, lsch [204]; horseradish peroxidase, latj [191J).See Figure 9.15 in the color insert.

ON IN HEME AND RELAT

309

2.6.1.2.2. ~ a n ~ a ~~ e s e

~ ~ i d ~ ~ 2 . 6 . ~ ~ ~ *~~ , ~ 1 . ~ randr biological e ~ c role. ~ The o ~ u ~ r e nand c e biological role 46,000 Da) is quite similar to that ofLi o f manganese peroxidase ~ n P , (see Sec. 2.5.l.2lt1 for a detail 2.6.1.2.2.2. ~ ~ ~ rs ~ar lu lc ~ ~arch~tecture ral and active site structure. The crystal structL~~e o f Mn isozyrne 1from ~ ~ a ~ e ~c o~ c~ ~y ~~ e~was ~~ esolved ~ o ~at ~2.06 u r n r e s o ~ ~ ~ t[i 2o 0n~ , ~ 0 1With ] . a 43% amino acid sequence homology, MnP shares the e i ~ two same overall structural a r c ~ ~ c tas~ Lip ~ r (Fig. e 14). It is also a g l y c ~ p ~ o twith sites. MnP has five rather than four disulfide bonds. s t ~ i c t u r acalcium ~ The heme active site, consisting of a proximal His ligand ~ y d r o ~ e n - b ~ n to d e ad b ~ ~ r i Asp e d residue and a distal side ~ e ~ o x ~ d e - b ipocket n ~ i nwith ~ distal His and Arg, ~ Phe at the is almost identical to that of Lip and CcP (Fig. 15).Siniilar to Lip, M n has ~ r oand~distal i position ~ ~ corr~~ponding to TrplS1 and Trp51. A unique feature of MnP is that it contains an Mn2' binding site next to the henie ~ 2 0 ~ , 2 0The l l ~M i z + is in an o c t ~ e d r ageometry l and coordinated by carboxylate oqgens of GIu35, ~ l u 3 ~ , ~ p la heme ~ ~propionate , oxygen, and two water ~ x y g ~One n ~ of . the water ~ ~ ~ a is within bonding distance to the sccond heme propionate. A second c o ~ r ~ n ~ t e ~of the Mnzi Egands, Gfu35, is ~ r o p o ~ sphere c o ~ s i s t i of ~ gArgll? ~ - b o ~tod one to be important in orienting Glu35 and decreasing the excess negative charge around the &Inz+ ~ i n dsite. i ~ ~ 2.6.1 2.3. Coprinus cinereus peroxidase 2.6.1.2.3.2. ~ c c ~ r ~ e nand c e ~ i o l ~ ~ ' crole, a 1 C o ~ r cinereus ~ ~ ~ speruxidase (CiP) is found in the black inkcap mushroom, from the basidiomyeete family o f kngi [1811. Three p~rox~dases reported in khe l i t e r ~ ~ u r ~ ~ oc ~ ~ r i nn uperox~ ~ e idase, Coprinus rnacrorhizus peroxidase, and Arthrornyces rarnosus ~ e r o x i d a s ~ ~ e shown to be ~ ~ e n t ~and c a l appear to vary only in their degree of' glycosylation. Although CiF shares 4045% m i n o acid homology with Lip, it does not degrade lignin. Imtead, its enz.ynatic specificity resemble that of horseradish peroxidase. 2.6.1.2.3.2. ~ ~ e r aalrl~ ~ ~ i t e c tand i ~ r eactive site structure. The overall stmcturd architecture and active site structure o f CiP is almost identical to that of Lip, except CiJ? has a much larger heme access channel [2021.

-

2.6.1.3. Class 111: Classical secretory plani! peroxidases 2.6.1.3.1. Peanut ~ e r o x i ~ a s ~ 2.6.1.3.1. I . Occurrence and biological role. Peanut peroxidase (PNP) flt51,2031 i s found in cultured peanut cells. Its biological role remains L ~ ~ c e ~ t a i n due to the large number of reactions it can catalyze. 2.6.1.3.1.2. ~ i ) e r ~~~rl c ~ i t e and c ~ active ~ ~ ~ site e s t ~ u c t ~ ~The ~ e major , cationic isozyrne of PNP was the first class TI1 peroxidase whose X-ray structure was lurown 12041. The 10 main helices c o n s e ~ in e ~class 1 and II ~eroxidasesare also found in PNP. In addition, PNP contains three unique helices, two of which contribute to the with two cakcium binding sites, similar to heme access channel. It is a g~ycopro~ein that o f class 11peroxidases.

All the key features found in the heme active site of other peroxidases are preserved in PNP (Fig. 151, from the Asp-His-Fe triad on the proximal side to the catalytic His and Arg on the distal side. Like class I1 peroxidases, PNP contains Phe at both the p r o ~and ~ distal l side of the heme. 2.6.1.3.2. ~ o r s eperoxidase ~ ~ ~ s ~

2.6.1.3.2.1. Occurrence and biological role. Horseradish peroxidase (HRP, ~~~~0~ /181,2051 i s found in the root of the horseradish plant. Approximately 18%of its weight is due to the covalmtly bound carbohydrate moiety. There are many isozymes of HRP. The dominant form is the neutral NRP called RP is probably the most studied peroxidase because of its ready availability in the early stage of peroxidase research and wide use as a reporter enzyme in jmmun o d i a ~ o s t i ckits. Like PNP, ERP catalyzes a variety of reactions and its b i o l o ~ c ~ l role is not certain. 2.6.1.3.2.2. ~ v ~ r aar c~h l~ t e c t ~and ~ ~ uctiue e site structure. The overdl stmctural architecture of ERPC (Fig, 14) is quite similar to that of PNP ll9l1. The similarity includes the presence of 10 helices found in all class 1-111 peroxidases, and three unique helices present only in PNP. One of the three unique helices, an insertion between helices F and 6, i s variable between HRPC and PNP and is proposed do participat~in interaction dong the heme access channel. This region in HRPC is longer than that in PNP and contains a ring of three peripheral phenylalanines that may have an important role in influencing the interaction of HRPC with aromatic substrates. The heme active site structure of HRPC (Fig. 15) is very similar to that of PNP. ~~

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2.6.2, ~ u ~ e ~ aof iAnimal ? ~ ~Peroxidases l ~

2.6.2.1. ~ y e ~ o ~ ~ ~ o x i ~ u s e

2.6.2.1.1. Occurrence and biological role ~ y e l o ~ e r o x i d ~(MPO) ~ s e f l # l i is isolated from blood leukocytes, e ~ ~ e r i i rat ~ ~ n chloroma tumor tissue, bone marrow cells of the guinea pig, and pus o f infected i s d e s t r u ~ t ~ oofn dog uteri. The ~ r i m function ~ ~ r of the leukocyte is p h a g o ~ o ~and microorganisms. MPn may play a role in phagocytosis and therefore could be an antimicrobial agent.

2.6.2.1.2. ~ v e r a~~ cl h~i t e c tand ~ ~ iactive ~ ~ site s t ~ ~ c t u r e PO was the first MPO whose structure is known l206l. It is a dirneric enzyme with two identical monomers. The protein dimer is held together by a disulfide bond, Each monomer consists of a heavy chain with 466 residues, a light chain with 108 residucs, a covalently bound heme, a calcium-bindingsite, and at least thyee ed (Fig. 16). There are five d i ~ l ~~n d~ a~g e s sites for ~ s ~ a r ~ g i n e - l i n kg~~cosylation within each monomer. A single loop consisting of residues 168-174 provides most of the ligands to the calcium binding site, with ligands from side chains of Asp%, Thr168, Asp172, Ser174, and the peptide carbonyl uf AspllZ.

FIG. 16. Overall stm.tctures of chloroperoxidase and the superfamily of animal peroxidases (PDB codes: ~ y e I o ~ ~ r ~ x ilmhl d a ~[6 e J; , c h l o ~ o p e ~ o ~ ~1cpo a s e[2101). ,

The monomer is dominated by 19 helices ranging from 5 to 29 residues. The heme is located in a central core consisting o f four helices (H5, M6,118,and 1212) from the heavy chain and one helix (R2)from the light chain. The proximd His is ~ r o ~ d e d by Xis336 of helix H8. The is is H-bonded to Asn421 (Fig. 171, instead of an Asp residue found in the s u ~ ~ r f a m iofl yplant, fungal, and bacterial ~eroxida~es. The distal His95 and Arg239 are positioned in an almost identical manner as in CcP or Lip, and catalysis. The nature of covalent therefore may play a similar role in pernx~~ase attachment of amino acid side chains to the heme was not clew- until the structure of human MPO became available [6]. In c o ~ b i ~ awith t i ~s ~ e ~ t ~ ostudies s ~ o ~[?I,~ c the structural study indicates that the heme is a modified heme b with methyl groups at position 1 and 5 forming ester linkages with 6 1 ~ 2 4 2and Asp94, respectively. Furthermure, a thinether linkage between Met243 and position 2 of the heme vinyl group is also present.

FIG, 17. Active site structures of chloroperoxidase and of the superfamily of aniinal peroxidases (PUB codes: myeloperoxidase, lmhl 16I; ~ ~ o s t ~Hl synthaso, ~ n ~ nlprh E2081; chluroperoxiclase, lcpo [2101). See Figure 9.17 in the color insert.

31

2.622, ~ r o ~ t a ~ l aH~synthase '~in ~.~.2.2.1.~ c ~ u r r e and n ~ e~ i o ~ ~ rde g~~al synthase) /181,2071 has been isolated from both bovine and ovine seminal vesicles. Its role Is Lo convert ~ a c h i d o n acid ~ ~into prostaglandin 6 2 and EX2, ~ ~ ~ Overall ~ 2a r c~~ i ~~e c and . ~~ .site s t r u c ~ ~ ~ ~ ~ ~ u ractive The sti-ucture o f se isozyme 1from ram seminal vesicle wils solved 12081. The enzyme is a 70 0001 with two identical subunits. There are three d o i ~ ~ nThe s . first dom&n consists of 24. residues at the N t e r m i ~ u sand contains mainly two small p sheets. It s t ~ c t u r eis similar to that of the epidermal growth s first d o m ~ nc, o n ~ ~four ~ n sh y ~ ~ o ~ h factor. The second domain, which f o ~ o w the helices. The helices li the enzyme to the ~ e m b r a n esurface and provide a channel for substrate binding th the first and second domains w e unique to PGW synthase, 43. ow eve^, the hydroThe third and largest domain contains 19 heliccs, as in phobic channel from the second domain intrudes into the domain and makes the n t those o f other ~ ~ r o ~ ~ s t ~ c t u r of e the proximal side of heme quite d ~ ~ f e r efrom This structural a~.ran~ement resulted in the substr~tebinding site being on the proximal side of the hemc. ~ e s p these ~ ~ edifferences, the five helices that form the central core for the heme are present in PGH synthase, as are the proximal His (His388) and distal His his^^^) (Fig. 17). In addition, Gln203 is present in the distal side and may play a role in catalysis.

-

2.6.3.~ u ~ ~ r ~of aOther ~ i Peroxidases l y 2.6.3.1. ~ ~ l a r ~ ~ e r ~ x i ~ ~ ~ ~

2.6.3.1.1. Occurrence and hiological role ~ ~ ~ r o p e(CPO) ~ o r ~l ~i1 ~, ~ ~is 09 ae ~lycoprotein ~ isolated from mold ~ a l d ~ $ ~ o ~ ~ fumago. The primary biological function of CPO is chlor~nation.It is involved in the product~onof the natural product caldariomycin.

CPO i s one of the most versatile heme ~ n z ~ eIns additio~ . to chlor~nation,CPO can catalyze other oxidative reactions known for other peroxidases (dehydrogenation), ~ e ~ ~ ~ o s iand t i cytochrome o n ~ ~ P450 (0x0 transfer ~ e ~ c t such ~on~ catalases fE1202 as epo~dation). 2.6.3.1"2.ilverall archi~ec~u~.e and active site sttzccture Despite the functio~alsimilarities with other heme enzymes, the overall structural architecture of CPO r2101 does not resemble any of the known protein structures (Fig. 16). There are eight helical s e ~ e ~~ t sx p ~through ~ i nboth ~ sides ofthe heme. The proximal helix A that provides the proximal Ijgand is parallel to the heme rather

than p e r p e n d i ~ ~toa the ~ heme as observed in other peroxidaszscs and c~ochrome P450, Unlike other pero~idases,the heme edge is not c o ~ e c t e dto the ~ o ~ e c ever, there i s a small opening above the heme that could surface via a channel. allow substrate access e s it The most s t r ~ ~ dn igf ~ e r e n c~~e t w e e nCPO and other ~ r o ~ ~ ai ss that c o n t ~ ~ na sCys (CysZ~) as the proximal ligand rather than the norn~al (Fig. 17). In this regard, G P resembles cytochrome P450. The x just ~ ~the ~o ~ p ~ s itot ethat in globins an ning of the p ~ o helix, itive end of the helix dipole is proposed to stabili re importantly, the Gys sulfur atom forms bonds with the peptide amidee lfkr ligmd H bonds me residue 30 and 31, These p ~ p t i ~ proposed to be a characteristic and important Eeature for m e t ~ ~ l ~ p r o t ethat i n s utilize cysteines for iron c o o ~ d ~ n a t ~12101, on dases use distal H (HislO5j forms an 1~3.83 and may play a role in the correct orientation of the Glu.

~ 0 ) ~ontainsa b-type lieme and is n a ~ i e dfar the G ~ ~ ~ lP450 ~ r ~o ~~ 4e[21X,212] absorption peak of the GO-bound ferrous form of the enzyme. It is a ubi~uitou$ enzyme present in many orgmisms, including vertebrates (such as mammals, fish, t ~ ~ as insects and ~ o ~plants, s ~and, bacteria. It can be and ~~~~j~ i n v e ~ e b r a(such classified into two broad classes based on its redox partners that provide electrons for its Eunction. Class I P450s are reduced by ferredoxins, which are in turn reduced by c o ~ ~ n i ferredo~in ng r e d ~ ~ c Class ~ e . 11 ~ ~ are5 0 ~ ~ ~ ~ ~enzyme o ncalled t a~ ~ P ~ H -nc y ~ t o c~ h r o~m e 50s arc ~ o n ~ o x y ~ ~enzymes n a s e that catalyze a variety o f 0x0 t r a ~ ~*eact~ons ~ ~ c ~such as h y d r ~ ~ l a t epoxid~ti~n, ~~n, amine and s u ~ ~ d e oxidation^ and oxidative ~ealkylatio~. It is very i ~ p o ~in~the n t~ e t a ~ o ol f~xeno~m biotics and in the critical steps o f steroid hormone b i o $ ~ ~ h ~ s i s ~

450 are now a v a i ~ a ~ 123.3le ~ ~ ~ c tof~c~ochrome r e s share many s i m i l ~ t i e sin both the overall fold (Fig. 18A) and in the heme active site structure (Fig. 18Eb),despite low amino acid sequence homology. The overall fold of is ~ p p r o ~ ia~triangular ~ t ~ ~ prism, y dominated by M, helices. As e ~ ~ e cthe t ~ , main difference between different P450 structures is the s ~ ~ s t rb~i ~t dei n gsite, as ~ ~ e ~ P45Os e n t have ~ ~ ~ e substrate ~ e n t specificity. For example, in the ferric in the distal pocket by a single ~ ~ ~ 5 ~ c a m ~ c complex, ~ m p h othe r ca~iphoris stabil~~ed ~~~~

314

TURANO AND LU

A.

6.

ca FIG. 18. Overall (A) and active site ferric (B) and oxy (C) &ructuresof cyt P450cam (PDB codes €or ferric and oxy P450cam are: ldz6 and 1dz8,respectively 12181). See Figure 9.18 in the color insert.

W bond between its carbonyl oxygen and the side chain hydroxyl of Tyr 96. A different distal substrate binding site is present in other P450s. The proximal ligand to the heme iron is cysteine (Fig. 18B). The cysteine Iigmd loop, consisting of eight residues around the proximal Cys, is con~ervedin all P450s. Like CPO, the Cys in P450 is at the positive N-terminal end of the proximal helix and the Cys sulfur is H-bonded to three peptide amides. Qn the distal side, in addition to substrate binding site, there are two highly conserved residues, Asp251 and Thr252, and a water chain connecting Thr252 and another highly conserved 61~366. ~~terestingly, two new water molecules are present in the distal pockel, of the dioxygen complex of PCSOcam (Fig. 186) [2181 and they may play important roles in the enzyme catalysis (see See. 4.2.5) 1218,2221.

2.8.1. Occurrence and Biological Role

Nitric oxide synthase is an enzyme that produces NO by catalyzing a ~ v e - e l e ~ r o n o ~ d a t ~ of o na guanidino nitrogen of L-arginine (L-AT~)[223-2261, thereby c o n t r o l l i ~ ~ ~ NO distribution and concentrations in higher eukaryotes. Oxidation of r,-Arg to Lcitrulline occurs via two successive monooxygenation reactions:

~ ‘ n ~ ~ y d r o x y - ~-t-A Oa T+ g 1jZ N

~ ---f t-citrulline P ~ +NO

+ 1/2

~~~i

Three isoenzymes, encoded by different genes, have been found in mammals: iNQS and nNOS me soluble and main^^ located in the cytosof; eNOS is membra~e-associated. They difYer in size (130-160 kDa), amino acid sequence (50-60% identity botween any two isoenzymes), tissue distribution, and activation by intracellular calcium. However, they share a three-component construction: (1)an N-terminal ( N ~ that ~ binds * ~ a single ~ b-type heme, t e t r a h y ~ o b i 0 ~ catalytic o x y ~ ~ n adomain se ; a C-terminal reductase domain that binds terin (H4B>,and the substrate ~ A r g (21 ~ ~ FAD, N and? N ~ P(3)~an intervening ; calmodulin-bindingregion that regulates the electron flow from the reductase to Lhe oxygenase domain. Diinerization of NOS,,, is necessary for catdytic activity in vitro and may regulate activity in vivo. 2.8.2. Overall Architecture and Active Site Structure X-ray crystal structures have been reported for the oxygenase domain under a variety of‘ & o n d i t i ~ n [22~-2291. ~ N So, has an unirsud fold with a w i ~ g e dp sheet with projecting f3 hairpins and flanking a helices (Fig. 19AI. The overall shape is described as a “baseb;;bnllcatcher’s mitt with heme clasped in the palni” 12271. The heme iron is coordinated to a proximal 6y.s Xigmd (Fig. 19Etf. The distal heme pocket, constructed sensibly ~ r e sdiffers , from those of oxygenases, catalase, peroxidase~,and by p s t ~ ~ c ~ ~ oxidases, which are largely cx-helical. A proximal side Trp and a distal side Phe Lie

T

316

FIG. 19. Qverall(A) and active site (B) s t ~ ~of mfftlfje ~ r ~ ~ sa oxygenwe domain expressed in E. c d i iPDB code: lnos f2271).

e

U

~ AND A ~LU ~

~ nitric o ~oxide ~ synthase ~ ~ e

almost parallel to the heme plane. Many other conserved residues create a ~ ~ hydrophobic environment around the heme, with only the propionates protruding toward the solvent. It has been proposed that a c~nformationalswitching of heme ~ t yforrnation~ , ~ r o ~ ~ ~could ~ a bet ree *s p~o n s i ~for ~ ~c o ~ t r o ~heme l ~ n ~~ ~ a n ~dimer dlmer reveals a zinc ion and catalysis [227]. The e r y s t d structure of the tetrahedrally coordinated to pairs of symmetry-related Cys residues at the dimer interfax @29].

~

~~~~~~

~ y d r o ~ ~ oxidoreductase a m ~ ~ e (HAQ) catalyzes the four-electron ~ x ~ d a t i oofnhydroxylarnine to nitrite E2301. Each subunit of the homotrimeric enzyme contains eight c ~ v ~ e n tbound ly heme groups, d e s i ~ 1a-8~~ccoi*din~ ~ to the p o s ~ t ~ ofothe ~ hemein the sequence 151. These are the seven hemes c, with ~ i s - ~ ~ unusual heme P460 (heme 4) that i s ~ e ~ t a c o o r ~ nand ate probab~yrepresents the catalytic center (Fig, 20B). The eight hemes are ~ o in ~ clusters: group I ~ ~ r i p ~ e ~ hcluster} e r n e consists o f hemes P460, 6, and 7, stacked p l the ~d parallel to each other, Heme 6 appears to be e x c h ~ ~ ~ e - c o uto s3 Group 2 consists of hemes 3 and 5, and group 3 of hemes 1and 2. la ~ o u 2 ~and %hehemes are again parallel to each other. Heme 8 ~ ~ n s t ~the ~ ~~~g~~~~~~~ u t e ~ ~~u~ 4. The molecule exists as a trimer with the subunits arranged as cloves in a head of garlic, Each subunit is folded into two domains (the flexible ~ - t e r ~domain i ~ ~ i sl not c o ~ a short ~ ~~ w s o - ~ t ~ a fl ~ded r ~ s o l ~ e dThe ) * ~ - t e ~ i n a l (residues d o ~ ~1-269) ~ sheet and 14 01 helices (Fig. 20A1, with five heme c and heme P460, The central.

Arg 201

FIG. 20. Overall (A) and active site (B) structures of liydroxylamine oxidorductase from 7 ~ eumpaea ~ (PDB ~ code: ~ lfgi ~IS11. ~ ~ ~ ~ ~ f f ~

~

domain (residues 270-499) contains 10 c1 helices and the two remaining hemes. Striking similarities have been pointed out between the heme groups 3-6 of hydro~ l a o ~~ d o~r e e~ ~and c t the ~ et e t r ~ e care ~ e ~ o c h~554. r ~ ~ ~

2.20.2. Cytochrome cdl Nitrite Beductuse

~ ~ o c cdl~ nitrite o ~ reductase e is a b ~ ~ ~ i ~ tenzyme ~ o n athat l catalyzes both the one-electron reduction of nitrite to nitric oxide and the four-electron reduction o f oxygen to water r 2 ~ ~ - ~ 3 ~ 1 :

The first reaction is a key respiratory reaction in den it^^^ bacteria. The enzyme is a~oluh b o~ m ~ o d ~ ~located ~ e ~ in the p e ~ ~Each l ~~ Q~ n .o ~ofe ~r o c h ~ cdl o ~ e contains one heme c, bound to Ihe protein via two Gys residues, and a n o n " c o ~ ~ e ~ t 1 y b o u n ~heme dl. Heme d t appears to be the site of nitrite and oxygen ~ e d ~ c ~ i o where~sheme c accepts the electron from donors such as azurin, ~ s e u ~ ~ a and cytochrome ~ 5 5 1 . The ~ t ~of ~~ o ct h r~o cdl ~~e consists e of two structural d ~ m ~an~ Ns : terminal helical G domain and a C-termha1 dl domain k234-2 81. In spite of similar

31 8

TURANO AND

LU

~ c h i t e c t u r ethe ~ structure of heme binding sites differs s i ~ f i c a n t l yin the two en~ymesfor which the structure has been resolved. In the oxidized T. ~ a ~ t # t ~ o cyt cdl protein (Fig. 21) 1234236,2381,His17 and His69 provide the axial iron ligands for heme c; His200 is a fifth axial iron ligand for heme dl . An unusual feature of cyt cdl is that the main chain of‘ the c domain makes an excursion into the &-domain, ~ r ~ r 2 as ~5 the sixth v axial iron ~ ligand ~ for heme ~ dl. Upon ~ reduction, Tyr25 is released to allow substrate binding to the heme dl; c o ~ c o m i t ~ ta~“refolding” y, of the c domain takes place, resulting in a switch of one heme c iron ligand from Nisl7 to MetlO6. In the oxidized Ps. aeruginosa protein 12371, His51 and Met88 provide the axial iron ligands for heme c and His182 provides the fifth axial iron ligand for henze d l , The hydroxide ion, hydrogen-bo~idedto TyrlO, is a sixth axial iron ligand for heme

ti*. 2.10.2. Cytochrorne c Nitrite Reductuse

~ ~ o ~ c hnitrite ~ oreductase ~ e catalyzes the six-electron reduct~ono€ nitrite to ammonia in am-negative bacteria f2391. It is a pentaheme c-type enzyme of molecular weight 55-65 kDa. The physiologically active form of the protein probably consists of a dimer. A Ca2’ binding site appears to be an essential feature of the overall structural architecture.

IRON IN HEME A N D RELATED

~

~

0

~

~

1

~

s

31 9

The structure of cytochrome c nitrite reductase from Sulfurospirillum, deleyianarn has been reported recently (Fig. 22) [240]. Hemes 2-5 are conventional c-type hemes, with His as distal ligands. Heme 1 is five-coordinate with Lys as the axial ligand. A.U hemes are clustered on one side of the dimer, and hemes 2 and 5 have one solvent-exposed edge. Hemes 2 and 3, as well as hemes 4 and 5, are almost perpendicular to each other. This dihcme elbow motif is a feature of multiheme proteins s t ~ ~ from ~ n the g three-heme cyt c7, and is often a repeated motif in proteins with higher numbers of hemes (cyt c3, cyt c554, hydroxylarnine oxyreductase), but the functional reason for this arrangement is still unknown. Hemes 3 and 4 give rise to a parallel stacking of their porphyrin planes, with the shortest distance below 4 This is another motif often found in multiheme proteins. Analogously to hydroxylamine o x ~ e d ~ c t ~the s e active , heme is the only one that does not belong to any of these typical structural motifs. Reduction occurs at the positively charged distal site of heme 1, where relevant residues for catalysis am Arg, Tyr, and accessible through two different channels. ions enter via a funnel with significantly positive electrostatic surface potential. The product, constituted by an ammonium cation, leaves though an exit channel with predominantly negative electrostatic surface potential.

A.

FIG. 22. Overall (A) and active site (B) structures of cytochrome c nitrite rductase from ~ ~ ~ ~ ~ r ~ ~s p ~e r ~~ ~(PDB l ~u code: r ~n lqdb a [2~40]1.u ~

o ~ ~ o B1, ~ 6557, e s b567,5,and kSE9 are u ~ ~ protei~s s u ~that are also called [241-2431. They contain both II binuclear iron center and a lowbacteriof~r~iti~s spin s ~ ~ c o o ~lieme ~ n ~b,t with e bis-Met miail ligation (Fig, 23) [ ~ 4 ~ - 2 ~The ~1. Lthese ~~r eproteins ~ is very s i ~ i lto ~ that . of ~ ~ r ~ t e and ~~ u ~ ~~ t ~~t c ~eof~ form^^ 8 ~~~1~ spherical shell composed of 24 identical protein chains and 12 hemes, thus suggesting that their probable function i s iron uptake. The ~ ~ l d i n g blocks of bact~rio€err~tins are protein dimers constituted by two i d e ~ t ~~ c u~ b ~ g t ~ ~Each o s~~ ~ b ~@ n i ts bindi~ga single heme group through two ~ ~ r r e s p o ~ d~i n ~ is ~ o ~ s t ~byt four ~ t enearly ~ pmdlel ty: helices linked through the b i ~ ~ cmetal l e ~ b ~ n ~ site. n g Iron is stored as a hydrated ferric oxide minerd in the centrd part of the resulting shell.

n~ found in bacteria, ~yanobact~~ia, erne oxyg~naseis a ~ e m b r a n e ~ b o uprotein plants, humans and other mammals ~ ~ ~ ~It ~i s uniqu~ ~ 4 $among ] . the heme ~ ~ o t ~ in that it uses heme both a6 a prosthetic group and as a substrate. It catalyzes the

oxidation of heme to biiiverdin, CO, and free iron in a reaction that r e ~ u ~ r e s NADPH, and c~ochromeP450 red~ctase" ~ i ~ r t enzymatic he~ reduction of bilker results in the po . i s o ~ o ~ ~ s - H ~HO-2, - 1 , and ~ e s s e n g like e ~ N ~ 4 $ - 2 5 ~ ]Three id en ti fie^.

The X-ray crystd s t ~ c t u r e of s a soluble fomn o f human 12511 and rat [252] -1)are known. The overall structure is d o m ~ a t e dby cil helices (Fig' does not match any folds in the Protein Data Bank. ~ ~ s t ~ ~ l o ~ a p study on the human 0-1shows unusual h e ~ e r o g e ~ eof ~ tthe y protein m ~ ~ e inc ~ ~ a ti if ~f ne r between ~ ~ ~ e the two molecu~e~ in the ~~" the crystal, ~ ~ c ~ ~ u od~ i~ ~m ~ d a ~ i ~ h ~ t e ~ ~ e factor r a t u for r e certain regions of the ~olecule.The and the p ~ ~ p i o&re ~ aexposed t~ to the solvent, The heme pocket is ~ o ~ ~ ~~ es tby dl two y helices, one from xlmal side and another from the distal as the proximal ligand (Fig. 2 side. The heme is ~ n t a - c ~ o r ~ i ~wia t e d residues on the ~roximalside that are in contact with the heme inc culc), Ala28, and Phe207. 6 1 ~ 2 9 i s Within H-bon of the H-bond interaction between remin~~cent peroxidases, However, the t appear to form in s t the S 25 is neutral [ass]. pie study s ~ ~ ~ ethat 01m residue is p r ~ s e n on t the distal. side. ~ n t e r e S t ~ ~ gthe ly, distal helix c o n t ~ ~ nseveral i n ~ highly conserved, c o n f o ~ a t i o n ~flexible ly ~ l y c i n eis~ kinked by about 50", resulting in several residues in direct contact vith the heme,

incl~~ding the backbone atoms of two conserved glycines ( G ~ yand l ~ Gly143). ~ This close contact sterically restricts the access of heme ligands such as dioxygen to the f3-, y-, and &meso carbon, leaving only the cl-meso carbon available for attack. In ad&tion, despite the lack of polar residues in the distal pocket, the presence of backbone atoms makes heme oxygenase more polar than globins.

There are many heme enzymes and proteins that are ~nteresting,yet no t~iree~dimensional structures are available. Due to limited space we will cover only a few of these enzymes.

uanylyl Cyclase Guanylyl cyclases are a family of enzymes that catalyze the conversion of GTP to cGMP r25~,25~1. The family comprises both m e m b r ~ e - ~ ~ and u n dsoluble isofoms that are expressed in nearly all cell types. They are regulated by diverse extracellular agormists that include peptide hormones, bacterial toxins, and free radicals, as well as i n t r a c e l ~ umolecules, l~ such as calcium and adenine nucleotides. The soluble guanylyl cyclase (sGC) is expressed in the cytoplasm 01 almost all m ~ m a l i a ncells and mediates a wide range of important physiological functions, such as inhibition of platelet aggregation, relaxation of smooth muscle, vasod~~atation, neuronal signal transduction, and i m m ~ o m o d u l a t i o ~This . enzyme i s a heterodimeric protein consisting of cx and p subunits, and expression of both subunits is reqaired for catalytic activity. Each subunit has an N-terminal regulatory domain and a 6-terminal catalytic domain that shares sequence homology with the corresponding domains in particulate guaiiylyl and adenylyl cyclases. The heme binding domain is located at the N terminus of each subunit. The presence of the heme ~rostheticgroup is required for activation of sGC by NO. The unbound form is a ferrous, high-spin, five-coordinate heme iron. The fifth ligand is the imidazole ring of a His residue. It has been suggested thctt the heme in sGC may be bound to the protein matrix through a Cys residue in the f3 subunit. Iln sGC, each heterodime~con~ainsapproximately one heme with a high a f h i t y for NO. This is in contrast to heme in globins, which has a high afinity for oxygen and in an aerobic ~ n ~ o n ~binds e n toxygen prefe~ent~ally. Even in an aerobic environment, sGC prefers to bind NO rather than oxygen. NO binds to the sixth position of the heme ring, breaks the bond between the axial h i ~ t i d i and ~ e iron, and forms a bond with iron (Fig. 25). This results in a five-coordinate heme where NO is now in the fifth ~ndingof NO to sG6 appears to fit a model based on two po~ulationso f heme. The minor population, which contains 28% of the heme, initially forms a sixnitroc o o r ~ n a t ecomplex with NO and then is rapidly converted to a fiv~-coo~di~iate

323

N

His105

His105

FIG. 25. NO binding to the heme center of g?rmylyl cyclase.

syl complex. The second population, which contains 72% of the heme, also forms a sixcoordinate nitrosyl complex, but the conversion to a five-coordinate complex is much slower. The formation of a five-coordinate nitrosyl-heme complex creates a conformationat change in the structure of the protein that activates sGC. The dimerization state of sGC is not affected by binding of NO t o heme. Both the ~ ~ e ~ c o o r d i nferrou~ atc enzyme and the ~ve-coordinatenitrosyl form o f the cnzymc exhibited the same molecular mass of about 200 kDa.

y ~ t a t ~ i ~P-Synthase ~ine ~ y s t a t h i o n i n~~- s y n t h catalyzes ~~e the c o n d e n s a t i ~of~L-serine and ~ ~ h o ~ o ~ y ~ to give cystathionine and water, which represents the first step in catabolic removal of t-hetoxic homocysteine. It i s a cytasolic protein constituted by a h ~ m o t e t r a m eof~63 ~ a, with one pyridoxal5’-phosphate (PLP)and one heme per dimer, suggesting that the active site is constructed at the dimer interface 1256,2571.mile the role of PLP can be understood on the basis of other ~LP-dependentenzymes, the role of the heme is not yet clear. It has been proposed that the heme is a b-type heme in an unusual environment, with His and Cys ligands. The latter could be labile and removed by the substrate homocysteine. The heme cofactor is redox-active, the €emous form being two-fold less active than the ferric one. This redox sensitivity may be of s i ~ ~ i ~ cin~ n c the regulation of homocysteine flux in response to the ambient redox status o f the cell.

~ ~ ~ o l e2 ,~~ -i~ni ( e~ x y g e(ID81 n ~ s eand ~ ~ p t o p h 2,3-dio~genase an (TDO) are examples of heme dioxygenases [258-2601. Both enzymes have been isolated from mammals. TDO was also found in ~ ~~ e ~Both enzymes ~~ cakalyze ~the e ~incorporating ne both oxygen atoms into the oxidation o f L-Trp to ~ - f c ) ~ m y l ~ y n u rby ole ring at 2- and 3-positions. I D 0 i s a monomeric enzyme with a ,and TDO is a tetrameric enzyme with a Tvrw o f about 191 kDa. contains a predominantly six-coordinate, high-spin heme center, with a proximal His, and possibie another weaker nitrohen donor trans to the proximal His. The heme

324

T

~

~ AND A ~LU O

binding site in TDO is more common. As in ferric Mb, ferric TDO contains a high-spin heme coordinated by a proximal His and a water. ~ n t e r e s t ~ g l~y , ~ ~ ~ ci y ~o ~L l isou b ~~ believed to be evolved from a gene for in~olearnine2 , 3 - ~ o ~ g e n a sand e , not &om a to ~-formyl~ynurenine by TDQ globin gene 12611. The activity of oxidation of L-TI”F) makes it an i m ~ ~ amember n t of the Trp metabolic pathway, as the oxidation initiates c a l of is less the kynurenine pathway of L-Trp metabol~sm.The ~ h y s ~ o l o ~role certain. It may be involved in the antiviral and antiproliferative activities of interferon-?. It may also play a role in ~ e u r o p a t h o l oevents ~ c ~ ~or in ant~oxidantdefense. ~~~

Expression is a genetic term that defines the m ~ f e s t a t i o nof a heritable trait in an ~ ~ d i ~ ~ ~~ rdthe y~ principle a~ ~n ~gene or genes that d e ~ e r ~the e s trait. A simpler definition o f protein expression is the generation of a large amount of a protein of protein expreschoice from the gene coding for the protein in a selected host. nction relationt a n ~point for the elu~idationo f stmc &on i s an ~ ~ p o ~starting sing site-directed mutagenesis f262-2641. 0th homologous and heterologous expression systems have been used. In the ~ o ~ ~ l ~ eg~ op rue sss i ~system, n the protein is expressed in a host cell where the protein is isolated. Homologous expression allows the productioin of ~ecombinant proteins that are processed and modified in a manner that is the same as the wildtype e ~ ~ (is@.? y native ~ e enzyme isolated from its ~r~~~~ host ceEs1. This e x p ~ e s s i o ~ al system is particularly importa~tfor studying proteins where p o s t t r a ~ l a t i o ~modification is important for their structure and function. For example, expression of c~o~hrom o [265], e cytochrome c peroxidase C2661, lignin p e r o ~ C2671, ~ ~ ~mangae eroxidase [268,269], and chloroperoxidase 12701 has been carried out in a homologaus way. ~ l y c h ~ e ~as~ n ~ e n e r a l ~spesllring, y homolo~ousexpressio~is t ~ h ~ i i cmore care has to be taken to ensure that the endogenous gene expressing the d d - t y p e ~ o nexists ~ when the protein is either deleted or totally suppressed. An a ~ ~ t problem protein of choice is essential for cell. growth. For example, expressio~of ~ i o n ~ n ~ cerevisiae lacking the mutant proteins of iso-1-cytoehrornec in yeast Sac~haro~~yces c gene was not possible because the yeast celf~lacking the w i ~ ~ c ~~ ~t h~ r eo m e essential electron tran~ferprotein could not grow. A st rate^ was designed to overcome this problem by coexpressing both the nonfunctional cytochrome c mutant that e can s u p p o ~the cell growth at protein of choice and a ~ n c t i o n c~yl t o ~ h ~ ~c m the same time t2711. To separate the two cytochrome c proteins, a ~ e ~ ~ 5mutation 8 ~ i s is made onto the functional ~ o c ~ r o m c so e that a bis-His chelation is formed with 0 ~ in the protein. ~ The ~ f ~ ~i n ~ c~~1~o co~ nr o~~m e ce m then be easily the separated from the nonhnctional cytochrome c by a metal affinity column “2711. ~~~~~

Heterologous expression in a different host can avoid the potential problem of contaminat~onby the ~ l d - t proteins. ~ e The most c o ~ m o used ~ y host for heterologous expression is E. coli, as bacterial growth is significantly faster than the growth of eukaqotic cells and the expression efficiency is generally higher, In addition, facile isotopic enrichment of proteins can be achieved in E. coli. For protein expression in E. coZi, the choice of promoters before the DNA coding for the protein is critical. The promoter^ have been evolved from lac to tac and then to T7 pro~oters.The T7 promoter is the strongest and therefore is the oveiwhelming choice for the majority of protein expression nowadays, unless the expression level exceeds that of heme ~ i o s ~ t h e sand i s incorporat~oninto proteins. Even in this case, the heme-free apoprotein can be expressed and purified followed by heme incorpration in vitro [2722751, Anothe~advantage of using the T7 promoter i s that it often renders the rotei in expression efficiency less dependent on the choice of codon usage in E. coli 12763. For example, cytochrome c [2771, myoglobiizs [278,2791, hemoglobins 1276,~80-2861, cyto5 ] ~ pe~~oxidases f2871, m a n g ~ ~ peroxidases se chrome c p e r o ~ d ~ ~e s2 ~ 2 - ~ 7lignin [288,2891, and cyLochrome P450 [2901 have been exprcssed in E. coli. ~ o ~ p l ~ c a t sometimes ~ons arise during the heterologous expression in E. coli. One common problem is the formation of inclusion bodies during the ex~ression 1291,2921. Methods for sohbiliaation of the inclusion bodies and refolding of the ~ ] . com~on proteins have been developed and generally work well [ 2 ~ ~ , 2 ~other complication is khe additian of a Met residue at the beginning of the protein due to the r e ~ ~ e ~o ef the n tstart codon (ATG) that codes for Met in E. coli. For many heme 2 ~ 5 ~ proteins such as m~oglobin[278,2791 and cytochrome c peroxidase [ ~ ~ ~ - the additional Met residue has no observable effect on the structure or function properties of the proteins. On the other hand, for proteins such as hemoglobin ~ ~ ~ 6 , 2 $ 0 2861, the amino terminal residues of bath the a and fi chains play i i ~ ~ p o ~ troles a n t in modulation of the oxygen affinity of the protein by different allosteric effectors. must be deleted from the amino terminus of recombinant ~ e ~ ~ f othe r eextTa , hemoglobins. This task has been accomplished by coexpressing both the he genes and me~h~onine ~ i n o p e p t i d a s ethat will cleave the amino terminal dues after the hernoglobins are expressed 1285,2861. E. coli hosts rarely perform posttrans~ationalmodifications or proces the e ~ k ~ o tciocu n t e r ~ ~Int ~case . such ~ o ~ f i c ~and ~ oprocessings n s are needed for protein s t r ~ c t ~and r e function, new strategies have to be designed to overcome this problem if E. coli e x ~ ~ e s s is ~ otonbe used. For example, cytochrome c expression l ~ groups ~ tod the ~vinyll groups of the r ~ u i covalent ~ e ~ a~tachnientof two Cys ~ heme to form the a-thioether bonds. This step requires an enzyme called cytochrome c heme Iyase (see See. 1.2). ~ h e r e ~ o rthe e , E. colt e ~ r e s s i o nand p r o c e s s i ~of~cytochrome c was successfully carried out by coexpressing genes coding for both cytochrome c and cytochrome e heme lyase r2771. If other types of p o ~ t t ~ a n s ? a t ~ o nd~i ~ c a tor ~ processing o~s are ~ e q u i and ~d eoexpression of enzymes responsible for the modification and ~rocessingis not possible, other heterologou~e ~ p r e s ~ i osystems n may be considered. ~ o n those, g the s baculovims express~onsystem has proven to be an excellent way to e ~ r e s eukar-

yotic e n ~ ~because e s similar types of posttr~nslationalinodi~cationsand ~ r ~ c e s s i n ~ exist in this system C2951. MnP has becn expressed in this system [296].

The t h e ~ ~ o d y n ~ of m electron ic~ transfer reactions are based on the different reduction potentials between the partner molecules. Therefore, it becomes fundamentally o ii ~~~ tr ~ the h e factors ~d affecting the redox p ~ t e ~ iint proteins. i~~ The ~ ~ p o ~to tc ~ nature of' the metal ion ligands, geometrical constraints imposed by the protein, ~ y ~ bonding, r ~ solvent ~ n accessibility, the fractional protein charges, and the unit charges on the protein surface are all important factors in proteins [297-3041. As far as the licanes are concerned, the reduction potentials we tuned mainly by the number and nature of the axial ligmds and, to a minor extent, by the nature o f the heme itselfe The range o ~ r e d u c t i ~potentials n for the Fe3+/FeZCpair in cytochrornes is reported in Fig. 26. All other factors being equal, cytochromes that have one histidine and one

[FIG. 26. Ranges of redtiction potentials for the l?e3'/Fez' pair Covered by d i f f e r ~ cyto~t chromes. Values are compared with those of other electron transfer proteins as iron-sulfur proteins and copper proteins.


327

methionine as axial ligands are more reducible than cytochromes that have two histidines as axial ligands. This is expected on chemical grounds, as methionine is a softer ligand than histidine and favors the softer ferrous iron with respect to the harder ferric iron. In practice, the two negative charges borne by the propionates can be considered as protein charges, as these two heme substituents are usually largely so~vent~ex~osed in cytochromes. This has been. dcmon.strated for cyt b5 by using hemes where prop~onatesare substituted with methyl esters I30rSl. The reduction potential increases by about 60 mV, as expected from the deletion of two negative surface charges rclatively close to the metal. ons side ring the charge of the ~ r o p ~ o n ~ast easprotein charge, the overall charge of heme moieties is +1/0 for heme ferriciferrous redox couple. Thus, an increase in solvent exposure should cause a decrease in the heme iron reduction potential. Indeed, larger solvent exposure corresponds to a larger dielectric c(~nstant and therefore to a better stabilizat~onof the charged species. A typical example comes from comparison of the reduction potentials of cytochrome b5 with those of multiheme cytochrome c3 or c7. All of these proteins have bis-His axial ligation, arid the histidines presumably are all forming hydrogen bonds to backbone carbonyls. However, the solvent exposure is much larger in multiheme systems due to the high hemeiprotein residues ratio. These latter systems have reduction potentials about 300 m y lower than that of cyt ba. ~~ectrostatic effects also play an important role in the modulation of redox potentials. For example, replacement of a buried valine in Mb with a charged residue such as Glu caused up to 200 mV shift in the potential t3061. The effect o f surface charges has also been seen in cytochrornes. For instance, within the 3-heme containing cytochrome c7 and the 4-heme containing cytochrome cZ3,heme I and IV have similar solvent accessibility. However, heme IV has a higher reduction potentid than heme I because heme W is surrounded by several lysine residues on the surface [78,3071. In biological electron transfer, the kinetics is also of fundamental importance. According to the Markus theory [3081, the difference in redox potentials, the reorganization energy, and the nature of the intervening medium are all determining factors. The reorganization energy is related to the structural changes accompanying the change in redox state of the metal ion. Up to now the structural data available on both e cytochrom~c6 have redox forms of c~ochromeb5, ~ i t o c h ~ nc ~~ ro ic ~h r o c~ and pointed to small but meaningfiul changes involving the heme propionate-7 and surrounding residues 128,30,46,951. owle edge of the electron transfer pathway within proteins is also related to knowledge of the interaction surface with partner molecules. Many studies have been devoted to this aspect, but few examples are well characterized. For example, the Xray structure of the cytochrome cicytochrome c peroxidase complex is available f601. This structure shows how the van der Waals interactions are important for complex formation, although electrostatics is probably important in solution to allow the molecules to approach each other in a favorable orientation. Indeed, studies exist

328

that demonstra~ehow the ~ f f u s i o association n~ rate depends on the charge distri~ution on the protein surface [309J. A8 it appears from the s ~ r ~ c tdescription ~ r a ~ of cy~chromes ~ r o ~ d in e dSee. 2.1, heme is often involved in intramolecular electron transfer among the different redox n centers in the same protein or complex. It is noteworthy that the ~ x a ~ ~ n a tofi othe s t ~ c t u r e of s m u ~ t i h e mproteins ~ with completely different functions has resulted in the r e c o ~ ~ t i oofnseveral repeating structural motifs, such as the so-called me elbow motif and the parallel stacking o f heme planes with intraheme distances below 4 A, A c o m ~ r e ~ e n sof i othe ~ functional causes of these structural motifs represents a challe~~ fore a deeper u ~ i d e r s t a n ~ nofgthe electron transfer ~ a t , ~ win ~ py rsQ t e ~ ~ s . n s ~ mwhy~is heme ~ ,commonly involved in electron transfer? Apparently heme iron ~ e d ~ c t potentials ~on can be easily modulated by axial ligands. The 1-eorgani~ation~ ncan be ~ small r thanks ~ to the conformation^ freedom of the heme plane, heme s u ~ s t ~ t u e nand t ~ ,s u ~ o u n d residues. ~ n ~ Last but not feast, heme con~titutesan ~ i nelectron g e ~ t e n d n:e ~system, which could have the advantage of s h o r t ~ c i r ~ u ~the trans~er~athway.

Two mqjor a s p ~c~o ns s t i ~ uthe t ~ basis of ~ e ~ e - bdioxygen ~ e d t ~ a n and ~ ~ too ~r a ~ (1)selectivity toward dioxygen; (2) ability Lo bind or release dioxygen depending on the ~ i o ~ o ~dc~a ~l a n d . The a ~ n i t for y dioxygen depends on the distal site features of the globins, such as the p~eseneeof hydrop~obicresidues { d i n e , leacine, i s o i e ~ c i ~amrd e ~p ~ e n y l ~ a ~ nine) and the formation of a hydrogen bond between the distal histidine md the n t the picture that electron b ~ u n dd i o ~ g e n T . h i ~ -bond f o r m a t ~ ois~e o n ~ ~ s t ewith density accumulates on the dioxygen molecule upon coordination, resulting in a ~ ~ s s~ ~ion.~The ~ absence e y ~ of~H b ~ o n ~ en to g the distal imidaaoke of the coordinated CO ~noleculein CO addricts i s consistent with little a c e ~ ~ u l a tofi oelectron ~ ~ e n son ~ the t ~ carbonyl ligand. This interaction, together with other factors such as global steric inhib~tionof the bound €orm (1311, may explain globins's ability to e nunhin~ ~ ~ c r i ~the ~ toxic n a t CO, e which usually binds more tightly than d i o ~ ~ to dered iron p o r p h ~ complexes~ i~ The reduced ~ n i t for y dioxygen f o for Aplysia ~ ~ ~ b, whkh lacks the HisE7, is consistent with the ~ u n d ~ ~role e noft the ~ hydrogen nd with the distal histid~ne13101. As detailed in Sees. 2.2.1 and 2.23, binding of a sixth ligand produces changes in ~ Such c l ~ ~ n gcones the re~ativedistances between heme, iron ion, and p r ~ x i m aHis. ~ns stitute the core of cooperative dioxygen binding in hemoglob~n.Many h e ~ o ~ l o bare riot ~ ~ ~ e p e n~d o~ ~ n to ~with e ronly s one ~ ~ ~ xby~ ~n ed site ~n n but ~ rat he^ o l i ~ o m ~ ~ i e species with the protein composed of two or more similar subunits. The binding or o at one ~ site sensibly ~ ~affects nthe a f ~ n i and t ~ ~ n ~ t i of c s~~~a~~ release of ~ binding and release at a neighboring site. In this way a versatile dioxygen tran ~ a c ~ i isn eat^ e ~ that allows the protein to bind dioxyge~more t i ~at ~ t ~ ~ ~27, d ~ ~ ~ ~ e n t and r a ttoi release o~ it when needed. This concept is s ~ m m ~ini Fig.

where the percentage of saturation is reported as a Iunction of partial ~ ~ e s s of~ s e dioxygen. Dioxygen binding to an isolated site can be described by a h ~ e r b o l i csaturation cusve, whereas ~ o o ~ e r a t i interactions ve lead to a sigmoidai binding curve. From the comparison of the two curves it is clear how the fraction of oxygenated sites is lower at low ~ressuresfor the cooperative case but shows a larger change in the number of bound sites with small changes in pressure. ~heoi~etical models have been develope~to analyze cooperative figand binding, C model was developed, which relies on the existence of two In 1965, the so-called T (or tense state} and the basic ~ u a t e states--the ~ n ~ l o w - a ~ n conformation i~~ high-~finityconformation R (or relaxed state) I3111. Statistical therm~~ynamic nt ap~soach~ exist s that ex~licitlyaccount for dzerent affinities for ~ i ~ e r ebinding sites while stiU preserving the two basic quaternary states [312-3141. These l ~ a ~ p r o ~ c hgive e s improved fits to oxygen binding data. onet the less, the d e t ~comprehension of the structural origin of cooperativity still represents a challenge for structural b i o l ~ ~ s t s .

As in globins, the ~ s e f e ~ e n taffinity ~ a l for the signaling molecule in sensory prote~nsis governed by the nature of the amino acids present in the “distal” pocket. For example, in the case o f Goo& this pocket is constituted by hydrophobic residues as in globins, but it lacks the distal His that favors dioxygen binding in Mb and ct o f the protein axial ligands on function is more marked than in iron in these biological sensors can be five- (FixL, guanylyl cyclase) or s ~ - ~ o ~ r d i n(CooA). a t e The binding o f the signaling molecule causes bond bt-Faking with the sixth (Lea,the most labile) axial ligand in six-coordinatespecies, but may also induce bond breaking or Iigand exchange at the fifth coordination position. Such changes in the coordination sphere cause tertiary/qu.aternary structural changes

FIG. 27. Cooperative and noncooperative binding curves of &oxygen.

T

330

U

~

AND A ~Ltl ~

that activate the molecule. This latter effect is analogous to the canformational variations which are the basis of the cooperativity in Hb, but the detailed mechanism may be different. For example, in the movement of the proximal His plays a major role in the allosterie conformational change*However, the p r o x i ~ a His l in both the “on” and “off)’ forms of FixL is held rigidly [163], Instead, flattening of the hemo ~ and is believed to be the major determincauses a cascade of c o n f ~ r m a t i o nchanges ing factor in signal transduction.

The proposed catalytic cycle of catalase occurs in two steps:

1+ HzOz--+ P’-Fe(IV)=O

+

B‘-Fe(lV)=O -I-H 2 0 H202---j P-Fe(1II) HaO -t- O2

+

with P‘ being a porphyrin cation radical. The peroxide in the distal cavity interacts with His and Asn, which assist its heterolytic cleavage and produces compound I with Fe(TV)=O and porphyrin x-cation radical. Similar to those found in peroxidases, cytochrome P450, and NOS,, the t ~ o s i n a t eproximal ligand and nearby aromatic residues can stabilize the compound I-like form. 4.2.6. Peroxidases

The key structural elements for peroxidases (except chloroperoxidase) are the AspHis-Fe triad 11881 on the proximal side and the His-Asn hi bonding network on the distal side C315,316f. ‘The proximal triad imparts a greater imidazolate charaeter of the proximal histidine than that in globins and cytochromes, and it is important in defining the redox p r o p e ~ i of ~ sthe heme as well as the protein’s ability to activate H202r315,3171. The anionic character of proximal His can stabilize higher oxidation ials states of the heme and thus makes the heme possess lower ~eduction~ o ~ e n ~ than those of cytochromes and globins. The same anionic character can also stabilize the =O i n t e r ~ e d ~in t ecompound I and therefore Facilitate MzOz activation (see general mechanism shown in Scheme 2). The proximal triad may also help maintain the high-spin five-coordinate state of the heme center. In the case of Gc bonding network of the triad i s extended to the proximal Trpl91, and is believed to be i m p o ~ a n in t correctly orienting Trpl91 for efficieizt coupling of the free Trpl91 radical to the heme using a mechanism shown in Seheme 2 13181. Elydrogen-bonding networks are also present on the distal side of peroxidases 1335,3161. The donation of the N6f atom of the distal His to the side chain of a nearby Asn ensures that N E of~ the distal His is unprotonated and available to accept a proton from NzOz and donate the proton for ~ ” b o n ~ to ~ nthe g bound peroxide

IRON IN HEME AND RELATED ~ ~ ~ T ~ I N S His __ -:

His

331 His- -H.

Arg

:a

- *o n Y202

F

I

His

His

f

Fe -

I

His

Phe

Peroxidase A

Arg

Phe

ii

H20

Scheme 2 [318]. The Arg in the distal pocket can help polarize HsOz by stabilizing the developing negative charge on the peroxide OR- leaving group, and accelerate HZ02 activation by H-bonding to the ferry1 oxygen in compound I after 0-0 cleavage occurs. In CrtP, mu~ationof the distal His to Leu resulted in a decrease o f the 0-0cleavage rate by five orders of magnitude 13191, whereas mutation of the proximal His to Gln or Glu [ 2 ~ ~ , 3has 2 ~ no ] effect on the rate. Therefore, the distal pocket is much more important for controlling the 0-0 cleavage rates. In peroxidases the specificity for different substrates is provided by the protein matrix. Since the substrate o f CcP is a macromolecule, cyt c, long-range electron transfer is required through an internal amino acid tTrp191) radical 13211. Interestingly, another class I peroxidase, ascorbate peroxidase, also contains a Trp at the same location F1901. However, because the substrate of' APX is a small organic molecule (ascorbate), a normal Fe(I'V)=0 and porphyrin cation radical is required as the cornpound I intermediate rather than the Trp radical. To overcome this dilemma, MX contains a cation binding site next to the Trp residue, 80 that formation of a Trp radical is not favorable [3221. In all other peroddases, the Trp residue is replaced with a Phe that is much more resistant to oxidation. The extent of heme edge expo~ureis also different among different peroxidases and may play an important role in their sub~trateinter~ctions~ The substrate of I,iP and MnP is lignin, a biopolymer. The oxidation o f lignin is ~ ~ ~ o m ~ though ~ s h ea dmediator, veratryl alcohol for Lip [1~2,3231,and ~ n ~ I which is released from the Mn(1I) binding site in MnP [199,3231. In Lip a veratryl ) site in MnP alcohol ~~~n~ next to the heme is proposed [195], and an ~ n ( I Ibinding is clearly identified [200,2011. A surface Trp in Lip may also be involved in lignin d e ~ a d ~ t i o11961. n

332

T U ~ A N OAND LU

C h ~ o ~ o p e r o ~ d acatalyzes se the halogenation of a number of aliphatic substrates ~ c c o r d i nto~the following reaction:

is the substrate and X could be C1, Br, or I, but not F. though the primary biological function of CPO seems to be chlorination, the catalase, and monoo~genasea c t i ~ t j e sf2091. The en~ymedso exhibits perox~dase~ cycle is initiated by hydrogen peroxide or other oxygen atom donor binding to the high-spin, penta-coor~nate,ferric resting state Lo give compound I, a p o r p h ~ ~ i n cation radical containing FeIV. Then three p ~ t h w ~ are y s available d e ~ e n on d ~the ~ function. In the halogenat~onmode, it is proposed that compound I reacts with the e t I ~ Iadduct I ~ -termed ~ - ~ com~ halide to form a h ~ o t h e t i c a lferric h y ~ h a ~~ ~i ~ pound X, which then transfers a halogen atom to the substrate and regenerates the resting state. CPO shares the Cys thiolate proximal ligand with ~ ~ o c h r oP450 m ~but posseses a very polar distal environment, with a Glu residue in close proximity to the heme iron. In analogy to cyt P45Os, it has been proposed that the strong electron dona tin^ proximal ligand would have the role of € a ~ ~ l i t a tk~entge r o l ~ cleavage ~c of the 0-0bond and conferring low reduction potentials to the heme iron, The distal 61~183 may &so be ~ ~ p oIt ~has~been n p ~ o s. t u l a t ~that 61~183p r o ~ d ae d~d ~ t i o nelec~ trostatic destabi~i~ation to the oxoferryl cation intermediate, thus promoting the te o f oxidizing chlor~ o r ~ a t of ~ othe n highly reactive compound I i n t e ~ e ~ acapable ide at a low pH, It has also been proposed that Glu183 serves as a general acid-base catalyst and first reacts with the hydrogen peroxide-heme adduct as a general base to a b s t r a ~a proton from the adduct and g e ~ i e ~ aat chydroperoxo-iron complex. In the second step, the protonated form of’Glu183 would serve as a general acid to facilitate 0-0bond cleavage. An Mn2* ion is octahedr~lycoordina~edby the heme propionate, three m i n o acid residues, and two water molecules in an arrangement similar to that in MnP, However, the role of the in GPO remains unknown.

The most irnpoi-kantstructural feature of P450s is the proximal Cys ligand 1221,3151. Unlike peroxidases, P450s do not contain obvious acid-base groups in the distal group t k m His, is prop o c ~ ~Therefore, t‘ the Cys, being a better e1ectro~”~onating posed to help “push’ythe 0-0 bond cleavage. This functional significance may be the reason why the ligand loop containing the Cys i s highly conserved in P450s. Of equal importance may be the way P450s stabilize the anionic form of the Cys sulfur. Without the stabilization, as in the case of several heme proteins designed to mimic P450 1 ~ ~ ~ the 3 @ 2 37~ssulfixr 1 ~ dissociates easily from the heme iron when the proteins are reduced. The stabilization is accomplished by placement of the Cys at the

IRQN 1M HEME AND R LATED ~ R ~ T E I ~ S

333

positive N-terminal end of the proximal helix and by H-bonding interactions between the Cys and three peptide amides. The absence o f obvious acid-base groups in the distal pocket of mean that the distal pocket does not play a role in P450 catalysis ( shown in Fig. 18C, the highly conserved Asp251 and Thr252 form a ~ ~ b o n d ~ network with two new water molecules in the dioxygen complex of P45Ocam, but . of s i t e ~ ~ r e c m t ~udt a ~ e n ~and ~is not in other forms of P ~ 5 0 c A~ combination indicates that the conserved Asp251 is involved in proton c r y s t ~ o ~ a p [218,222] hy ivering protons to the iron-bound dioxygen through the Asp251p bond^^^ network shown in Fig. 18C. Thr252 is shorn to H-bond to the iron-bound dioxygen and to water molecules in the Asp25l-Thr252-water €3bonding network.

I s,

Scheme 3 4.2.8. Nitric Oxide ~

y

~

t

~

a

~

~

As discussed in See. 2.8, the five-electron oxidation of L-Arg to 1,-citrulline catalyzed by NOS oc~rarsvia two successive monoox~genationreactions:

PI3 --+ NW-hydroxy-L-Arg f NADPf ~ * ' ~ h y ~ r o x y -+LO2 - ~+-g1iZ

PH ---z r,-citrulline +NO + 3/22

In analogy to P450, NOS Iikely uses a P'-Fe(W)=O moiety (wilh P' being a porphyrin cation radical) in the first step of the reaction. However, in the second step a peroxo-iron species (P-Fe(III)=Oi-)is thought to operate. In P450 the coordinated thiolate and the presence of water in the distal site we thought to promote formation of 0x0-iron from peroxo-iron:

334

~

-

~

i ~213" ~4~ P'-Fe(fV)=O I ~ ~ -i- H

~ ~O o

~

-

NOS,, shares with P450 the common thiolate proximal ligand, Elowever, in its distal heme pocket no structured water molecules or protein residues are present that can donate protons to facilitate cleavage of the peroxo-iron Q-Q bond. The 0x0-iron formation needed for the first step is facj~tatedby two different effects: (1) a stacking with the heme with the aromatic rings of a Trp and Phe residues and (2) substratein GcP. The latter assisted activation. The former effect parallels the role of arises from the comparison with the role of the distal Arg for forming 0x0-iron in peroxidases. It is indeed proposed that L-Arg in NOS supplies the €€-bondinginteractions to help generate the 0x0-iron i n t e r m e ~ a t needed e for its own conversion E227l. The NOS,, catalytic center is at the proposed interface of tho dimer*This would allow the substrate H$3 and c ~ l ~ o d u l to i n modulate catalysis by influencing associat~on between domain and subunits. ~~~~~

(HAQ) catalzyes the four-electron o x ~ ~ a t i oofnhydroy ~ o ~ ~ a mo~doreductase i~ie xylamine to nitrite:

~ H 2 +-~ H20 H 4NO2 Jr 4e""-t 5H' The reaction could proceed in two two-electron steps 12301: ~

~~Q

H -+ E 2m0

~2e- f ~ 2H

'

+ H 2 8--+m02i 2e- + 2H'

The second step should closely follow to generate nitrite and ~ e c r e possible ~ ~ e side reactions from otherwise long-lived intermediates. It has been suggested that the Tyr residue attached to the P460 and the arrangement of heme ~ 4 ~ O ~ e6 m e exchange"co~~p1ed pair may allow HA0 to transiently accept two electrons at the game time. HA0 contains another pair of weakly interacting hemes with similar r e ~ u c t ~ potentials on near 0 mV (hemes 2 and 31, which may provide €€A0with a mean of transfer and storage of two electrons from the active site C3271. 4.2.10. Nitrite Reductases 4.2.10.1. Gytachrome cdI nitrite redicctase me c is the site of entry for electrons from donor proteins, while cll heme is the site

of nitrite and oxygen reduction. In the fully oxidized F. pantotrophus enzyme, the axial ligmds ofthe heme dl iron are His200 and Tyr25. In the reduced form, Tyr25 is no longer bound to the heme iron, enabling the substrate to bind in its place. Nitrite binds to the reduced dl heme iron: one of the oxygens of the bound nitrite forms Wbonds with His345 and Es388, which are p r o t o ~ in a ~the ~ uiilig~tedreduced form and are thought to participate in nitrite protonation. Following the transfer of two protons and one electron to the substrate, water and NO am produced. The molecule

of nitric oxide is in a bent c o n f o r ~ a t ~ o and n heme d l is probably in the oxidized form. The four pyrrole nitrogens of the heme dl have different electronic properties due to the disc~ntinuou~ conjugation of the p o ~ h ring. ~ n This, combined with probable steric, electrostatic, and hydrogcn bonding interactions between bound NO and distal ng pocket amino acids, aEects the Fe-N-O geometry and favors NO release. ~ e b i n d ~ of Tyr and return to the fully oxidized state may not be a mandatory part of the catalytic cycle 1236,3281

4.2.10.2. Cytochrome c nitrite reductase Nitrite is converted to a m m ~ n ~ inaa six-electron reaction by c~ochromec nitrite reductase [239,240]. Replacement of the usual His ligand by a Lys at the catalytic center is expected to raise the redox potential of the heme iron, generating an electron sirk in the system of redox-active cofactors. A minimal reaction scheme has been I1~ proposed that consists of the f o l l o ~ n gsteps. First nitrite binds to the F ~ ( ~with its nitrogen atom, while one oxygen atom is I-I-bonded to the distal His and Tyr, in cdle nitrite reductase where the interaction is analogy to that ~ e s c r i b for e~ c ~ o c h r ~ m with two His residues. A Fe(1I)-NO-boundspecies is then formed, followed by formation of a putative ~ e [ I I I ~ - b o ~hydroxylamine nd intermediate. ~ i n a ~ l ammonia y, is formed and released as an ammonium cation. 4.2.11.

Heme Oxygenase

The proposed catalytic cycle for the oxidation of heme to biliverdin, 60, and free iron is shown in Scheme 4 [24?1, It includes heme a-meso hydroxyla~ion,as well as conversion of ferric a-meso hydroxyheme to ferrous verdoherne, and then to biliverdin. The unusual h e t e r o g ~ ~ ~of~MO-1, i t y evident from the conformat~ona~ di~erence of the two €30-1molecules in the asymmetrical unit and high-temperature factors for certy tain regions of the probin, indicate that HO-1 is flexible f2EilI. This ~ e x i b ~may allow the easy entry o f the substrate and exit ofthe products. The presence of several conserved glycine residues, such as 6133139 and Gly143, in the distal helix provides this flexibility. In addition, they promote the approximately 50' kink in the distal nes, helix so that many residues, including the backbone atoms of the Iwo ~ l ~ ~ ~are in cloose contact with heme. This resulted in the more polar environment nccded for 0-0bond cleavage. Finally, steric restrjct~onof the access of heme ligands, such as dioxygen to the p-, y- and &meso carbons, makes the major contribution to the regioselective attack on the wrneso carbon.

4.2.12. Geizeral O b ~ ~ r u a ~ i o n s Upon inspection of dif'ferent heme proteins covered in this chapter, few people will not be amazed at how proteins can utilize the same type of heme (such as heme b) for such diverse functions as electron transfer, ligand transport, oxygen activation, and 0x0

6

Me

Me

transfer. Therefore, proteins have learned not only to adopt heme but also to control the heme for different functions, Following are several ways in which proteins can ~ n e - t the ~ ~heme e s t ~ ~ tand ~ rf uen ~ i o n .

~ e c o n strzcture d ~ ~ The heme proteins are clearly dominated by tx helices. In the majority of the (such as globins and P450), the heme active site i s usually defined by a sandwich, with helix ~ ofterr heme in the middle of a proximal helix and a distal helix The ~ r o x i m delivers the proximal ligand m d other residues that interact with the heme. The distal helix provides a distal ligand (in the case of six-coordinate hernes like cyto~ sn g~ chromes and Coo& or residues (such as the distal His in ~ l ~ c o~ ~ ~t ~ no the reactivity and selectivity of the protein. However, there are exceptions to the domination of helices at the heme binding site. As pointed out by Gong et al. r1631, the 0 2 sensing FixL utilizes a heme 5 ~ ~ ~ ed ~ w wae ~e~~ r ~~ xhelix ~ ~and ~a rll ~distal 8 strand (Fig. 28). It is even more interesting that the NO transporter nitrophorin contains a heme that is sandwiched between two p strands [1591. Despite this difference in ~ e c o R sdt ~ c t u r ael l e ~ e around n~ the heme, the ion^ of the p r o x ~ ~ His and ~f i ~ p o r t a ndistal t residues (such as Leu and Ile) are r e ~ ~close ~ among a ~ ~ CZ*ycera~ i ~ ~ u ~hemoglobin ~ h ~ u tr3291, a FixL [163], and nitrophorins [lS9]. Since the ~ found a three proteins are involved in ligand binding, s e n s i ~ gor~ t r a n s p o ~nature

4.2.12.1

~

337

FIG. 28. The heme-bindingpockets of' ITb from Glyeera ~ i ~ ~ n (PDB c h code: ~ a2hhg ~ ~f32911, FixL from Bradyrhizobium japonicurn iPDB code: ldrm t16311, and nitrophorin from ~ ~ o ~ n 1PDB code: Lnpl [159L),from left to right. (From " 3 1 , by permission).

~~~~~~

consistent way to control its binding properties fkom three evolutionar~~y diFfei~ent proteins with different s ~ o n structures. d ~ 4.2,12.2. ~ ~ ~ l x~ ~~ u ~~ a d l Histidine i s the overwhelming choice For heme proteins as their proximal ligand. It is

found in c ~ o c h r o m e sglobins, ~ n i ~ o p h o r ~ nheme s, sensor proteins such as FixL and GooA, ~eroxidases,and heme oxygenase. Gysteine is used as the proximal ligand in P450 and NOS, and Tyr is present in catalase. It is generally believed that histidine axial ligation is good enough for binding small diatomic ligands such as 0 2 in the other axial position, but not good enough to activate and split them. To accomplish the activation, cysteine has to be used., as in P450 [3301. "bile this observation is generally true, heme oxygenase is an exception. With a His ;~gits proximal ligand, ~ be n able to activate 0 2 and oxygenate the oe-meso heme heme o ~ g e n a s eis ~ h o to protein, Interestingly, chloroperox~dasealso uses cysteine as its proximal ligand, despite the fact that proximal histidine iwith the help of other residues from proximal anit distal sides; see next two sections) is known to be capable of splitting H202in dl other peroxidases.

4.2.12.3. ~ ~~ s ~~ u ~ ~ ~ r a ~ While the proximal ligands play important roles in heme protein reactivity, subtle diFFerences in the identity and position of distal residues can fine-tune the reactivity and control substrate specificity and selectivity. For example, the disgal histidines in both globins and pero~dasesare important in stabilizing O2 and E3cz02~, ~ e ~ p e ~ t ~ through H-bonding interactions. The exact position oE the distal histidine is critical in defining the activity of globins and peroxidases. The distal His in globins i s too close to ~ O ~ the heme center for the His to serve as a general acid for the activation o ~ ~ When the distal His in Mb is moved to the proper position through an L Z ~ H ~ double ~ 6 4 ~ mutation, the engineered Mb displays dramatically increased peroxidase activity f3311. In addition to the distal Eis, an arginine at the distal site of most peroxidases also plays a role in splitting HzOz. Water is present in the distal pockets of several heme proteins, including peroxidases and ~ ~ ~For0 a slong . time!,the role of these water molec~leswas not clearly

cent studies on P450 have demonstrated clearly that several water moleTherefore, attention cules are critical to the function of P450 [2~$,221,332,333~. should be paid not only to residues but to water molecules in the distal site. 4.2.12.4. Secondary coordination sphere efeets

It has been recognized that residues around the primmy coordinat~onsphere of metal ions and their ligands can have important roles in defining structure and function proper;ties o f the metal centers. This is particularly true for heme proteins [315~31~]. These secondary coordination sphere residues can exert their influence through Mbonding or through electrostatic and/or hydrophobic~ydrophilicinter example is the Asp-His-Fe triad in peroxidases such as CcP f315,3171.T interaction between the buried h p and the proximal His causes the irnidazole His to ~ o t ~ e r possess an i ~ d a z o l a t echaracter, thus making it possible to activate E202. example is the location of Cys at the positive N-terminal end of the helix aid the bonding to three a i d e groups in P450 and CPO that stabilizes the negative thiolate group 1221,3151.The hy~rophobicenvironrnent around the proximal cysteine residue in P450 is d s o important. For example, while mutation of the proximal His to Cys in 5 ~ , mutatioii Mb converted h4b to a P450-like resting state of enzyme r 3 ~ ~ ~ 3a2similar did not result in a P450-like protein 12741, Since the secondary coordination sphere around the cysteine Ligand in P450 is hydrophobic while the corresponding pasitions in G:P are hydrophilic and contain a buried negative charge from Asp that could destabilize the Fe-Cys bond, a fbrther mutation of Asp to Leu resulted in a protein that is very similar to P450 in its resting state as well as in its cyanide bond c~t form 13261. ThereFore, the secondary coordi~ationsphere makes s i g n ~ ~contributions to the coordination chemistry of metalloproteins. Other notable secondary coordination sphere effects in heme proteins include the distal W-bonding network involving distal His and Asxi, and the distal and proximal calcium binding sites in peroxidases. All of these are believed importaxit to the proper ~ o s i t ~ o ~ofi nthe g distal and proximal residues for their function. Steric hindrance of the distal site residues also affects reactivity. In peroxidases the substrate, even when constituted by a small aromatic molecule, cannot approach the Fe(lV)=O moiety, while in P450 it has been demonstrated that direct transfer of reacthe iron-bound oxygen to the substrate molecule occurs in the monoox~gena~e tion 11861. The latter feature of P450 reactivity requires the presence of a selective binding site for the different P450 substrates. Indeed, larger structural differences in the distal cavity are observed than within the family of ~eroxidases12211.

Iron is the most common metal ion found in metaIloproteins, This is not surprising because iron i s the most abundant tra~sitionmetal in the Earth’s crust and in the

IRON IN HEME AND RELATED PRQT

339

human body 13343. ~ r t l i e r r n o r e several ? different oxidation states o f the iron and extensive redox potential ranges of its redox couples are accessible at physiological conditions. The ready availability and high versatility &low proteins to possess a wide range of structures and functions. Nevertheless, the easy oxidalion of low oxidation states of iron in air and the insolubi~tyof complexes o f its high oxidation states (such as F‘es+)in water (as evidenced by rust formation) makes it difficult to transporr, md use iron by proteins. It is generally believed that in the early days of evolution Earth was under a reducing environment and complexes of the ferrous iron, the dominating iron under those conditions?were much more soluble in water than those of ferric iron, and thus posed little problem. However, Earth’s atmosphere became oxidizing due to photosynthesis. Organisms have learned either to keep the iron-containing proteins (such as iron-sulfur proteins; see Chapter 10) under anaerobic conditions, or to chelate iron so that its oxidation and solubility can be controlled. This latter adaptation may be one of the reasons that proteins use heme iron commonly, despite the complex and elaborate ways by which heme has to be s ~ t h e s i z e din b i o l o ~ c a ~ systems (see Sec. 1.2). Other unique features of heme, such as the extensive aromatic conjugation, also make heme more versatile,

The study of heme proteins rcpresents one of the earliest and most widely studied branches of biological inorganic chemistry, with the pioneering work on Mb, Hb, and cyt c. Nevedheless, the vein is far from being exhausted. Important s t ~ c t u r e - ~ ~ n c tion relationlihips in “historical” proteins such as the mechanism by which globins (now heme oxygenases) discriminate between CO and O2 still need to be fully understood. At the same time, the discovery of new complex heme-containing enzymes, as welt as the d i s c o ~ of e ~heme-based signal transductors anct sensors, poses new chafa l structural points of view. These tenges in the next few years from the ~ u ~ c t i o nand systems are the key mdecules for complex and fundamental biological processes, and their comprehension could represent a goal in research fields spanning from pharmacology to e ~ v i r o ~ m e n tchemistry. al Recent adva~cementsin overexp~essin~ c-type heme c ~ ~ ~ a i nprote~ns ing C2711 will allow isolation of isotopically enriched proteins and thus facilitate structural studies using NMR. From the structural point of view, new folds have been discovered, and the availability of structures of an increasing number of systems has allowed identification of typical structural motifs. The identificat~onof consensus se~uencesassociated with such motifs, as well as the comprehension of‘ their functional role, perfectly fits in the increasingly expanding field of structural genomics. From a more “inorganic” point of view, the fascinating discovery of new and unusual axial ligations for heme iron, such as the N-terminal nitrogen of proline in GooA [168j, promises new ways of m o d ~ ~ a t i nthe g heme protein structure and fknction and at the same time presents new challenges for bioinorganic chemists who study the enzymes by spectroscopy or modeling.

340

TURANO AND Lli

FIG. 29. S ~ ~ e ~ofthe o ~iron~ center t ~ in~nitrile n hydratase (thick Line, PDB code: 1ahj [3351) with a heme group (thin line), See Figure 9.29 in the color ineert.

The number of heme proteins discovered and characterized so far clearly rivals those of other metalloproteins. The domination may be due to both the versatility of heme and the ease in identification and purification of heme p~oteinsbecause of their strong color. Are there any other metalloproteins that may contain a metal binding site that resembles heme? The recent publication of a structure of nitrile hydratase may offer a clue. Nitrile hydratase contains a mononuclear, nonbeme iron chelated by three Cys residues and two main chain amide N atoms in an octahedron [335]. ~ n t e r e s ~ ~ nthe ~ l yplane , of the octahedron defined by an 11-atom protein chelate can be superimposed on the plane of the four pyrrale nitrogens and the iron of a heme group (Pig. 29). Although lacking the aromatic conjugation, this “pseudoheme”, or a “heme-want-to-be)’, could serve as an alternative to heme for certain types of reactions. It remains to be seen whether more of such types of p s e ~ d ~ h e m e e~~ with the stmcproteins are discovered in nature and how the ~ s e u d o h compares tural and functional versatility of heme.

We are grateful for the expert help of Mr. Thomas JI), Pfister and Steven K, Ma from at Urbana-Champaign in generating figures and schemes for the ~ n ~ v e ~ sofi Illinois ty ~iot~cnolo~e this chapter. P. T. thanks the Italian CNR (Progetto Fi~alizza~o 9 9 , ~ ~ ~ 0 Y.~ L. . ~ac~owledges F ~ ~ ~ . the financial support from the ~ a t i o n a ~ Science Foundation and the National Institutes o f Health, USA,

341

ascorbate p ~ r o ~ d ~ e adenosine triphosphate 3‘,5’-cyclic adenosine ~onophosphate cataloolite gene activator protein cytochrome c peroxidwe 3’,5’-cyclic guanosine monophosphate C o ~ ~cinereus i ~ ~ peroxidme s 60-sensing transcriptor activator c ~ l ooperoxid~se r cytochror~e electron transfer flavin adenine ~ n u c ~ e o t i d e a b ~ o l o ~ coxygen al sensor flavin mononucleotide guanosine trip~~o$phate tet~~~y~obiop~rin hy~oxylamineoxidoreductase he~og~~bi~ heme oxygenase horseradish peroxidw e i n d o l e ~ i 2~ ~e~ - d ~ o x y g e ~ a s e lignin peroxidase myoglobin ~ ~ ~p e r~o x ~ ~ ~ee s e myeloperoxidase molecular weight Monod, ~ y ~Change~x a ~ , n i ~ o ~ i n ~adenine i d e dinucleotide phosphate nitric oxide synthase an a c r o formed ~ ~ from the names of the first proteins r e c o ~ i ~ as ed sharing this sensory motif, Le., the eriod clock protein of Llrosophila, and ryt. ~ ~ d r o c a r b or n~ e p nzlclear ~ ~ r translocator of vertebrates, and the ~ i n g l e ~ ~ i protein ~ d e d of Drosophila. p ~ o s ~ ~ l a nH din p ~ i d o x a5’phosphate ~ peanut peroxidase photos~nt~etic reaction center root mean square soluble guanylyl cyclase tryptophan 2,3-dioxygenase

34.2

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242, 243.

244. 245. 246.

4 (2991j, ~ 8 ~ nd G. R. Moore, Struct, on^^^^, 88, 103-

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AND RELATED PROTEINS

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~ D e p ~ m eof n t~ ~ ~ s i11,o~niversity l o ~ of Tiibingen, Ob dem Himmelreich 7, ~ - 7 Tubingen, ~ ~ 7Germany ~ ~ D e p ~ t ~ nofe nchemist^, t ~ ~ ~ e r sofiCalabria, t y Via Pietro Bucci, C15, I-$7036 Rende (Cosenzal, Italy 'Magnetic Resonance Center ( C ~University ~ ~ o€ , Florence, Via L. Sacconi 6, 1-50019 Sesto Fiorentino { ~ l o r e n ~ eItaly ),

1. ~

~ ~ R ~ ~ ~ C ~ 1 ~ 359 Chemistry of Iron in Iron-Sulfur Proteins 359 1,l. ~oordinat~on 1.2. ~ v e of ~ Consensus e ~ Sequences and Structural ~ l ~ ~ ~ c a 362 t ~ o n 365 1.3, ~ i ~ i n o r ~Roles ~ n i of c Clusters

2. I ~ O ~ - S ~~ F ~~ R~ WITH ~ K.NOMN E I S T~R U SC T ~ ~ ~ 2 3 372 2.1. Rubredoxins and Other Proteins with Mononucle~I r o ~ - ~ ~ ~ u r Clusters 372 2.1.1. ~ c c u ~ e n cProperties, e, and Biological Role 372 2.1.2. M o l e ~ l a S r ~ ~ c t uofr eRubredoxin, D e s u ~ o r e d o ~and n, D e s ~f lo f e r r ~ d o ~ n 373 2.2. Rieske Proteins 375 2.2.1 Occurrence and Putative Biological Role 375 2.2.2. Molecular Structure of Rieske Centers in bel, b,f, and Dioxygenase C o ~ ~ l e x e s 376 2.3, 2Fe-2S Ferredoxins 379 2.3.1. Occurrence and Biological Role 379 2.3.2. Molecular Structure of Plant-Type Ferredoxin and Adrenodoxin 379 n s Fe3S4 and/or Fe& Clusters 381 2.4. F e ~ e d o ~ i with 2.4.1. Occurrence and Biological Role 381 2.4.2. ~ o l eStructure ~ l ~ of 3Fe-423, 4Fe-48, 7Fe-$S, 8Fe-$S Ferredoxins and Fe-Only ~ y ~ ~ g e ~ ~ s e ~ 382 357

BENTRQP, CAPOZZl, AND LUCHINAT

358

2.5.

igh-Potential Tron-Sulfur Proteins 2.5 1. Occurrence and Biological Role 2.5.2. Molecular S t ~ c t u r of e ~ i g h - P o t e ~ tIron-Sulfur ia~ Proteins Aconitase and Iron Regulatory Proteins 2.6.1. Occurrence and Biological Role 2.6.2. Molecular ~ t ~ c t uofr eAconitase Siroherne-ContainingProteins 2.7.L Occurrence and Biological Role 2.7.2. Molecular Structure of the Hemoprotein Subunit of Sulfite~itrite Reductase ~ ~ t r o g e n a Iron s e Protein 2.8.1. Occurrence and Biological Role 2.8.2. Molecular Structure o f Nitrogenrase Iron Protein Enzymes Fe4S4~ ~ u s t e ~ - C o n tDNA a i ~ Repair n~ 2.9.1. ~ c c ~ r r e aand c e Biological Role 2.9.2. Molecular Structure of Endonuclease 111 and MutY G l u t a ~ i n ePhosphoi.ibosyl~yropho~ph~~e Mdotransferase 2.10.1. ~ c ~ ~ r eand n c Biological e Role 2.10.2. Molecular ~ t ~ c tof~Glutamine r e Phosphoribosylpyro~hosph~te Amidotransferase ~ r i ~ e t h y l Dehydrogenase ~~ne 2.11.1. Occurrence and Biological Role 2.11.2. Molecular Structure of Trimethylamine ~ e h y d r o ~ e n a s e The “€iybrid’y or “Meatball” Cluster 2.12.1. Occurrence and Biological Role 2.12.2. Molecular Structure o f the Desulfouibrio sp. Pratein Fumarate Reductase and Succinate Dehydrogenase currence and Biological Role lecular Structure of Furnwate Reductase Pyruvate:Ferredoxin Oxidoreductase 2.14.1. Occurrence and Biological Role 2.14.2. Molecular Structure of D e s u l f ~ ~ iafricanus ~ri~ ~~vate:Ferred~ Oxido~ed~ctase x~n

387 387 387 389 389 389 39 1 391

~ U ~ T ~ R E ~ Ribonucle~tideReductase and Pyruvate Forrnate-Lyase Activase Biotin Synthase and Related Systems Ferredoxia:Thioredox~n Reductase The Regulator of Fumarate and Nitrate Reduction (F” The SoxR Protein

410 1810 412 413 414 415

I

2.6.

2.7.

2.8.

2.9.

2.10”

2.11.

2.12,

2.13.

2.118.

~ T 3.1. 3.2. 3.3. 3.4. 3.5.

4. ~ ~ ~ ~ C ~ ~~ E L ~ A T~ I O~~ ~ ”H I PFS ~ J ~ ~ 4.1. Role o f the Cluster and of the Protein Moieties in Electron Transfer by Iron-Sulfur Proteins

392 393 393 394 396 396 397 399 399 399 401 401 402 402 402 404 406 406 406 408 408 408

T 416 I 0 416

359

5. ~ 5.1,

~ ~ ~ ~ ~ ~ T Evolutionary Aspects 5.1.1. Sequence Alignment and Phylogenetie Trees 5.1.2, ~ m e r ~ ~n tg ~ c t u~roat ~ ifs Open Questions

5.2.

A ~ ~

42 1 424 425

Fe3S4/Fe4S4Interconversion Fe4S4/E’e2S4Conversions Fe-Only Hydrogenases: The H Cluster Role of the Cluster in ~ o l ~ i and n g Stabi~tyof Iron~Sul~ur Proteins

4.2, 4.3. 4.4. 4.5,

C

~

B ~

~

I

~

~

E AND ~ ~ T~

~ F

~

~

~

E

~

C

~

G

~

~

T

~

~ F

~~

~ ~ I S~

~

O

~

E

~

S

428

E

429S 429 430 43 1 442 445

~

S 446 447

The class of iron-sulfur proteins is perhaps the class of met~llopr~teins in which the same theme is played in the richest number of variations 111. Several factors contribute to this richness. Among them are (1) the intrinsically high affinity of iron ions for sulfur ligands; (2) the availability of sulfide ions, Fe2 ions, and organic thiols in the reducing atmosphere o f the planet Earth at the beginning of life and biological evolution (see Sec. 5.11; (3) the widely different redox properties of the various FeS aggregates that are t h e ~ o d ~ a m i accessible c~y (see Sec. 4.11, and (4)the evolutionary pressure to exploit all of“ them at best. ~ron-su~fur proteins contain two or more iron ions (either in the +2 or in the 4-3 oxidation state) coordinated by thiol ligands from cysteine residues provided by the protein and bridged by sulfide ions;, forming what is improperly termed an iron-sulfur “cluster”. It is customary to include in the class oC iron-sulfur proteins also mbredoxins, which contain a single iron ion coordinated only by four thiol ligands, without sulfide ions. The most common iron-sulfur centers are shown in Fig. 1. Each of them has been ~ ~ e ~ tand i ~ structurally e d characterized in several different proteins, and for each inorganic niodels are also available. In several instances, these model coniplexes suggesting that the resulting complexes indeed can be obtained by self-asse~b~y, represent thermodynamic “welXs9’121. As will be appreciated from See. 2, there are a variety of unrelated protein folds that allow the formation of iron-sulfur clusters in the presence of sulfide ions, provided a suitable number of cysteine ligands are available. It appears as if the clusters themselves are rather well defined in terms of

’

BENTROP,CAPOZZI,AND LUCHINAT

360

E

A

FIG. 1. Ligand geometries and arrangement o f iron and sulfide ions in (A) Fe(S-Cys)* cluster o f rubredoxins; (B) F%Szcluster: of plant-type ferrdoxins; (C) Fe& cfrusker:of Eeske proteiras; (Dt Fe3S4cluster of 3Fe-4s and 7Fe-8S ferredoxins; and fE) Fe4S4cluster of 4Fe-48, 7Fe-$8, 8Fe-83ferxedoxins and HiPIPs. This figure has been produced using the program ~ O L M O L as have all figures representing structures [299].

s t ~ c t u r a land metric features, and that there is enough ~ e ~ b i l i in t y~ o ~ ~ p chains of proteins to adapt to the “inorganic” re~uirementsof the clusters in several ways. Rubredox~nscontain the metal center shown in Fig. lA, This center is constituted by an iron ion, either in the +2 or in the +3 stato, coor~natedby four thiolate sulfurs from the protein in a roughly t~?trahedralg e o m e t ~ , el- iB , The bond distances and angles typical of both proteins and model complexes are summarized in Table 1. The expected decrease in bond lengths on passing from the reduced to the oxidized state can be a~preciatedonly for the model complexes, for wkch very h i ~ h - r ~ s o l u t ~s ot ~n c t u r e sare available. ~ t ~ i o rubredoxu ~ h ins are not iron-sulfur proteins, their structural and chemical properties are often discussed together with those of iron-sulfur proteins, to which they are ~ n c t i o n ~ y and evolutionarily related. Indeed, the properties of iron-sulfur clusters can be Better d ~ ~ c ~ with s e dreference to the ~ ~ o p e ~ofi ethe s simple ~ o n o ~ e iron ~ic tetrathiolate moiety. The simplest iron-sulfur cluster is constituted by two iron ions bridged by two sulfide ions, forming a so-called diamond (Fig. 1B). Each iron in the diamond is further coordinated by two thiolates from the protein. The resulting cluster is [ ~ ~ ~ ~ ~ ~ This ~ e ~ cluster ~ ~ is I 2found - ’ in ~ -~erredo~ins, . The idealized ~ ~ o of ~ this typc of cluster can be described as each iron being tetrahe~allyc o o r ~ a t e by d four sulfur donors (two thiolates and two sulfides). The two thiolate sulfur-iron planes are coincident, and perpendicular to t h diamond plane. The relewmt structural and

361

TABLE 1 S t r ~ c ~Features i r ~ of Iron-Sulfur clustersa Rubredoxins ~ Fe-SR distance RS-Fe-SR angles

Fe-SR distance Fe-S" distance S"-Fe-S*angles Fe-S*-Feangles

2.24-2.3 1 102.5-114.9

Model c;o ~ p o u ~ [212j ds ~ 2.252-2.278 (OX> 2.324-2.378 (red) 106.67-112.20 ( O X ) 103.5-114.9 (0x1

[Fe2Sd2* (1AWD)

Model compounds r.2131

2.27-2,36 2.16-2.23 101.4-104.8 76.3-77.2

2.303-2.306 2.185-2.232 104.7 75.3

[FesS4]'' Fe-SR distance Fe-S" distance Fe-S3 distance S*-Fe-S*angles Fe-S*-Sangles

11RB9> ~

(6FDR)b

2.26-2.30 2.23-2.33 2.20-2.32 100.2-113.9 69.4-75.6

Model compounds t214j 2.310-2.327 2.242-2.275 2.273-2.333 101.63-106.06; 111.5-113.8 70.0~72.70;71.71-74.42

[Fe4S4I2' ( F e r r e d o ~ ~ ~ l F CModel A ~ compounds L215,216f Fe-SR distance Fe-S* distance S*-Fe-S*angles Fe-S*-Fe arngles

2.29-2.32 2.29-2.34 101.3-206.7 71.3-72.7

2.291-2.299 2.337-2.368; 2.26~2.299 103.1-106.1 11.98-73.59

[Fe4S4I2"~ H i F ~ ~ s - l C ~Model ~ ) compounds 12171 Fe-SR distance Fe-S* distance S*-Fe-S"angles Fe-8"-Fe angles

2.25-2.30 2.23-2.33 103.5-105.7 72.1-73.7

2.291-2.299 2.337-2.368; 2.269-2'299 103,1-106.1 71.98-73.59

Fe4S413f ~ H ~ I ~ ~ - l I Model ~ ~ compounds ) Fe-SR distance Fe-S* &stance S*-Fe-S*angles Fe-S*-Fe angles

2.13-2.31 2.16-2.34 100.8-108.7 70.5-76.1

Distances in A. The structures of' the [Fe3S4J0and fFe3S4]+clusters in reduced and oxidized Azotobctctet. v ~ ferredoxin n $, respectively, ~ ~ are the ~ same within ~ the ~ expe~mental ~ error ~ of X-ray crystallography at 1.48 resolution [218].

362

BENTROP, CAPOZZI, AND L U ~ H I N A ~

metric features of this cluster in ferredoxins and model compounds are summarized in Table 1. It ctn be noted that in order to respect the pseudotetrahedral geometiy around the iron ions the S-Fe-S angles within the diamond are above lOO“, ie., close to the tetrahedral value, so that the Fe-S-Fe angles are below 80’. The same diamond structure is encountered in the so-called Rieske cluster (Fig. 16).At variance with the ferredoxin cluster, only one of the two iron ions is coordinated by thiolates, whereas the other is c o o ~ ~ i n a t ebyd two nitrogen donors &om histidines. Another very common cluster is the distorted cube called “cubane”. It is cons t h t e d by four iron ions bridged by four sulfide ions (Fig. IE). The eight atoms al~ernateat the vertices of the cubane, and each of the four iron ions is further coordinated by a thiolate &and from the protein. The resulting cluster is [(RS)4Fe4S41-’2 -/13- . Again, to respect the pseudotetrahedral ~ e o ~ i around e t ~ the iron ion, the S-Fe-S angles are close to the tetrahedral value, whereas the Fe-8-Fe angles are acute (Table I). It should be noted that, although each iron ion in both diamond and cubane clusters is surrounded by four sulfur donors, there are two thiolates and two suIfides coordinating each iron in diamond ferredoxins, whereas thcre arc one thiolate and three sulfides in cubane ferredoxins. It should also be noted that in all of the examples described above there are four cysteinyl ligands from the protein, In rubredoxins and in cubane femedoxins of idealized geometries the four tbio~atesulfurs occupy the vertices of a tetrahedron, the size of which is obviously larger in the latter case than in the former (Table 11, whereas in idealized diamond ferredoxins the four thiolate sdfurs all lie in the same plane. cluster, This cluster can Derived from the cubane cluster is the I(RS)3E’e3S4]Z-/3^be viewed as a cubane that is missing one iron and the corresponding thiol. The resulting stmcture (Fig. 1D) can still bind ;ifburth iron ion which, in the absence of a thiol donor, completes its coordination with one or more exogenous ligrtnds. This type of reactivity can be f~nctionallyrelevant (see Secs. 2.6 and 4.2). The metric features of this cluster are shown in Table 1. esides the main types of clusters described above, FeS moieties with mixed ligands and/or with mixed metals are also found in nature, as well as iron-sulfur clusters coupled to other me~al~contai~ing prosthetic groups. Some of these struce , some other structures, such tures will be described in Sec. 2 where a p p r ~ p ~ i a twhile as ir(~n-~no~ybdenum clusters, will be described in Chapter 22.

After having described the coordination chemistry requirements of iron-sulfur clusters, we present here the protein requirements to host them. Obviously, cysteines shoutd be present in the sequence, and in most cases four of them are needed to bold a cluster in place, Exceptions are Rieske clusters, which need two cysteines and two histidines, and Fe3S4clusters which only require three cysteines. There are interesting relations between the type of sequence that contains the ligands (consensus

sequence) and the cluster that is preferentially formed. These relations are discussed t u r ~ here and will be further elabor~tedon in Sec. 5 after presenting the ~ t ~ ~ cfeatures of many iron-sulfur proteins in Sec. 2. The typical consensus sequences of cluster-ligating amino acids are listed in Table 2. I ~ s ~ e c t i oofn the table shows that there is one or more typical consensus sequence for each type of cluster. A very recurrent feature is the CysXXCys sequence, but the CysXXXCys sequence is also often found. In several cases, there may be a variable number of residues (X,) between two cysteines. This occurs when the residues between two cysteines form a loose loop. However, with the exception of highpotential iron-sulfur proteins (HiPIPs), nitrogenase iron protein, the 6-terminal and of domain VI of pyruvate:ferredomain of glutamine PRPP amidotrans~~rase, doxin oxidoreductase, this variable spacer between two cysteines cannot occur more y s twice, than once in a particular protein. 2Fe-2S proteins contain the C y s ~ ~motif with a variable X , loop in between, while many 4Fe-4S proteins contain two adjacent CysXXCys motifs. These regularities are not unexpected. Protein residues that act as metal Iigands CL) in m~talloproteinsmust obey some obvious steric requirements in order to chelate a single metal ion or ligate two close-by metal ions in a cluster, such as being located on the same side of a p strand or of an a helix. In the former case, the “ c o n ~ e n s ~iss ~ ’ LXL, in the latter it is LXXL. Loops are more flexible, so that L Z , LXXL, L 9 and L L arrangements can all be favorable for metal chelation or cluster binding. After these considerations it is instructive to examine some statistics about the c~. occurrence of any two arnino acid pairs at a certain distance in a s e q ~ ~ nAmazingly, the probability of finding cysteine pairs spaced by one to four X is the highest among all possible pairs (Table 3). The probability is expressed as the ratio between the occurrences found and those statistically expected given the abundance of the amino acids involved. In other words, it appears that cysteines are the most “specialized” residues in proteins in terms of occurring close to one another in the sequence, obviously with the scope of being able to chelate metals, coordinate clusters, or form disulfide bridges. Before beginning a systematic illustration of the variety of iron-sulfur protein structures, we present a possible structural classification of all iron-sulfur proteins according to the accepted SCOP division of proteins in clmsm, folds, s ~ p e ~ a ~ i l ~ s , be somefamilies, and protein domains (Table 4) [31 . Like all c l a ~ s ~ ~ c a t i oitnmay what artificial, but it serves the scope of providing some kind of order. Together with Table 2, which relates the protein domains to the various consensus sequences and types of clusters, it provides a useful reference frame for the individual protein systems discussed in Sec. 2 where, however, the proteins are grouped according to the clusters they contain rather than the class in which they belong. A good example of I S (http:ll E hypertext management of different classifications is the ~ ~ O ~ site b r n b s ~ l l . l e e d s . a c . u ~ ~ ~ b ~ ~ / p The r o mstructure-function is~/~~ relationships that will be discussed in Sec. 4 will follow yet a different order, which will be more related to the function of the protein, as will be illustrated in See. 1.3.

364 TyIpe of cluster

C o ~ s e ~ s sequence us

[Cys4Fe]--I2-

Qs x, cys cys 6ys xfc cys x, cys Gys

I

~rote~ d on ~ a i n

xx

Cys X His X, Cys XX His Gys xx cys x, Gys x cys cys x, cys cys x, cys

Domain 2 of aldehyde oxidoreductase

xx

1 Cys XX Cys XX Cys X X X Cys X,-

~ r i ~ e ~ d e~~ y ~ ~ o g e~n a~s en e

1 Short-chain ferredoxin (2 clusters)

365 TABLE 3 Cases Where the Occurrence of Cysteine Pairs is Highest Among Amino Acid Pairs, and Corresponding Found/Statistically Expected hti.os CSS &!,CYS .............

Fou~d/statistic~ly expected ratios in ...

..

Loops

cys x Cys Cys X2C 6ys Cys xxx Cys cys xxxx Cys

.__.” ....

Edge f3 strands 3.1

2.1

3.8 4.4 6.5

...

rx Helices

2.6

Although the present chapter deals in the following sections with at least 20 different ~~r refiecting the chemical and s t r u c t u r ~~versityo f irontypes of ~ r o n - s u lproteins, sulfur prosthetic groups and the structural and functional versatility of the proteins harboring them, the list of cluster functions i s not as long, although it keeps growing. Excellent reviews on the b i o l o ~ uses c ~ for ~ron-sulfurclusters have been ~ u b l i s h e ~ t2,&7 I. This paragraph summarizes the bioinorganic roles of iron-sulfur clusters and gives a functional classification of iron-sulfur proteins with reference to Secs. 2,3, and 4 (see Table 5). The large majority of known iron-sulfur clusters in proteins is involved in electron transfer processes that exploit the chemical properties of these clusters for electron uptake, storage, donation, and exchange, both int*i*aand ~ t e r ~ o ~ eAll~the ~ a r . small iron-sulfur proteins described in Secs. 2.1 to 2.5 (also reviewed in 181 and 191) and most iron-sulfur clusters in redox enzymes such as tr~methyla~ine dehydrogenaBe, fumarate reductase, ~ ~ v ~ t e : ~ e ~ ~oxidoreductase edoxin and hydrogen as^ have such a function, The best studied example of a non-redox enzyme with an iron-sulfur n d ~ i ~which catalyzes the citratecluster at its catdytic site is ~ ~ t o c ~ oaconitase, isocitrate isomeri~ationin the triearboxylic acid cycle. Both Fe-only hydrogenase and sulfite reductase are redox enzymes in which a Fe4S4cubane is bridged to another b ~ o i n o r ~ ac~ ioc ~ ~ ~ofnthe ~ active n t site through a common cysteinyl sulfur figand. These special mfactors are involved in the biological production or consum~tio~ of hydrogen and the six-electron reductions of sulfite to sulfide and nitrite t o ammonia, respectively. The Fe4 “hybrid” cluster of Fepr (“putative prismane’,~protein is likely to be an active site as well; this is suggested by the interesting crystal structure of this protein whose function i s not known yet (Sec. 2.12). A number of Fe4S4 clusters stabilize protein structures that are ~ e ~ u i for r~d activity. The members of the endonuclease 111 family of DNA repair cnzymes contain a cubane cluster in a loop structure that helps to position basic residues for

BENTROP, CAPOZZI, AND LUCHINAT

366

Family

Class

Rubredoxin

MiPIP

HiPIP

Rieske iz*on-sulffur Rieske iron-sulfur W-P p ~~ Q water~ ~ i ~ , protein p ~ ~watert e ~ ~ soluble domain soluble domain

367

IRON-SUL~URPROTEINS

Rieske d o m of~ the ironsulfur subunit of ~ ~ o ~ g e ~ a s ~ s

6 [2481

omain 2 of aldehyde o~id~redu&t ase

Domain 2 of ~deh~de

Endonucle~e111 2Fe-ZS € e r ~ ~ o ~ ~

N-terminal domain N-terminal domain of aldehyde oxidored~&~ase of aldehyde ox~d5~e~uct~se C-terminal domain of C-terminal domain p ~ t ~ a l adioxygenase te of p h t h ~ a t e ~iQ~genase reductase reductase P~tidaredQx~n

Ql-cx proteins

r+P proteins


368

codes and r ~ f e r e n c e ~

Protein d o m ~ ~

IFe-8S ferredoxins

7Fe-8S ferredoxins

Archaeaf ferredoxins

kehaeal ferreduxins Single 4Fe-4S 4Fe-4S ferredoxins cluster f ~ ~ e d o ~ ~ ~ Domains 2 and 3 of Domains 2 and 3 Domains 2 and 3 of SiRW sulfite reductase ~ e ~ ~ ~ subunit r Q t ~ i ~

aldehyde ferredoxin Aeonitme, first 3 domains

UP proteins

ON-SULFUR ~ R O T ~ I N ~

lNIP, BNIP, 1N26, 16P2 [293-2951

369

N~trogenasgiron protein

~ i t ~ o g e niron as~ prote~-~~ke

g;lut&e PRPP ami~otr~sferase ~ - t e r ~ ndomain al of tri~eth~~a~ne dehydrogenase

Formate Formate d e h y ~ o g e ~ ~ e /~ e h y d r o g ~ n ~ ~ / DMSO reductase, DMSO redudrase, domains 2-3 domains 1-3 (el Protein engineered in expres&onsystem

BENTROQ, GAPOZZI, AND LUCHINAT

370

~~L~ ~

Electron transfer

i

5

~Functions l o of~1ron-Sulfur ~ ~ Clusters

Cluster

I Protein

j Section

[Cys4Fe]-/”-‘ 2Fe-2S ZFe-28 and/or 3Fe-4S ~ n 4Fe-4S ~ ~ a

~ ~ b r e ~d ae s~~ lnf o,r e d o ~ n Rieske proteins F ~ ~ e ~ o ~ ~ n s ~Iron-only hydrogenase

2.1 2.2 2.3; 2.4 2.4.2 2.13 f 2.5

4Fe-4S

~ i g h - ~ ~ ei nr ~t jn~- ~protein l ~ r

12.8

4Fe-4S onit it^^ Catalysis o f redox r e ~ ~ ~ o n s H cluster (4Fe-4S -t- 2Fe Fe-only hydrogenase

Sta~~~i~ o f~protein t i o ns for DNA repair

t

~

~

t

~

~

2.6 2.4.2;

IRON-SULFUR PROTEINS

371

Sensing and regulation: (1)oxygen " - sensors: loss of original cluster and of activity (2) sensor of 0, and NO: redoxregulated control of transcription (3) iron sensor: posttranscriotional remlation Redoxmediated generation of free radicals Stabilization of an intermediate in disuEde reduction

I 4Fe-4s

I Glutamine PRPP amidotransferase

I

4Fe-4S/2Fe-2S 4Fe-4SI3Fe-4S 2Fe-2S

FNR protein Aconitase SoxR protein

Apoprotein/4Fe-4S

I 4Fe-4S

4Fe-4S

12.10 3.4; 4.3 2.6; 4.2 3.5

Iron regulatory proteidaconitase

2.6

I

I

I formate-lyase activating enzyme

13.1

I Anaerobic ribonucleotide reductase, pymvate Biotin synthase Ferred0xin:thioredoxin reductase

3.2 3.3

substrate recognition at the positively charged D ~ A ~ b i n d i nsurface. g Such a passive structural role has been extended to an active one in proteins that employ iron. glutamine PRPP sulfur clusters as sensors of oxidants in regdatmy r n e c h ~ s m sIn a m i d o t r ~ s f e r ~the e cluster is destroy& by oxygen, leading to a destabilization of the protein structure and i n a c t i ~ a t i oof~ the enzyme. The dirneric t r ~ s c ~ p t i o n factor FNR loses its DNA binding capability by an oxygen-induced Fe4S4--+ FezSz cluster conversion that is accompanied by the ~ ~ s s ~ c i a t of ion the e~t was discovered for the two monomers. A purely redox s t a t e - d e ~ e ~ dregulation t r ~ s c r j p t ~ o nactivator al SoxR: only the redox state of its Feltsz cluster determines u l ~ is r the activity of SoxR. In all of these cases, the oxidation of the ~ ~ ~ - scluster the signal that triggers changes in the structure, c o ~ o ~ ~ or t i dynamics o ~ , of the protein, which then provoke the regulatory effect. Aconit~secould also be considered as an oxygen-sensing iron-sulfur protein since its cluster is readily converted to an inactive Fe3S4 cluster in the presence of oxidants. However, this cluster conversion is not accompanied by a change in the relevance is not clear. The apo € o m of cytoprotein structurc, and its physio~o~cal plasmic aconitase is identical to iron regulatory protein, which acts as an iron sensor. This protein regulates iron storage and uptake by ind din^ to the rnI3N.Aof ferritin (an iron storage protein) and transferrin receptor (which transfers iron into the cell); at high iron levels, it incorporates an iron-sulfur cluster and is thus transformed into a c o n i t ~which ~ , does not bind RNA anymore. An additional function of iron-sulf~rclusters in proteins is the generation of free e and biotin synthase, that radicals in enzymes, such as ~ b o n u c l ~ t i dreductase employ a radical mechanism to fulfill difficult synthetic tasks, In these cases the i s apparently linked to the f o r ~ a t i o nof a radical, eg., from ~-adenos~lm~thionine, previous r ~ u c t i o nof the iron-sulfur center. Chloropl~tferredoxin:t ~ o ~ e d o e reaction reductase is a disulfide reductase catalyzing a t ~ o l - d i s u l ~ dinterchan~e and accepting electrons from a 2Fe-2S ferredoxin. The active site of the enzyme contains two cysteines in close vicinity to a Fe4Sd cluster. The latter appears to stabilize a one-electron reduced intermediate in the two-electron reduction of thioredoxin by temporary binding of a cysteine to a sulfide ion in the cluster.

-

~ u b r e are ~ small o ~ bacterial ~ ~ electron transfer p ~ ~ t( e ~ ~6000, s ~ ~ i c a l52ly 55 arnino acids] with a mononuclear tetrahedral Fe(Cys), center. Little is known about their p h y ~ i o l o electron ~ c ~ donors and acceptors; however, they can replace ferredoxins as electron carrier in several cases. Their redox potential is about 0 mV fl0l. Recently, the rubredoxin from the green sulfur bacterium ~ ~~ e~ that ~

has an unusually low redox potential of -87 mV was identified as an electron acceptor il111. ~ e s u l f o r e d o ~isn a bacterial protein for p y ~ v a t e : f e r r e ~oxidoreductase o~ related to mzbredoxin and desulfoferrodoxin that was isolated from the sulfate reducer ~e.sulfovibr~o gigas. It is a homodi~~er, possibly also with an electron transfer function, Each monomer of 36 amino acids contains an Fe(Cys), center with distorted tetrahedral geometry. The distortion of the coordination sphere of the iron is caused by the sequence motif Cys-X-~-Cys-X,-Cys-Cysp r o ~ d i n gits four ligands. rans sf or mat ion of this sequence motif by site-directed mutagenesis to a typical rubredoxin~likesequence Cys-X-X-Cys-X~,-Cys-Pro~~-Cys (see Sec. 1.2) converts desulforedoxin into a protein from i sa hrubredoxin a ~ l ~ [121. Des~lfoferrodo~~n that is spectroscopically ~ n ~ s ~ i ~ ~ b re ~s u~ l f u ~ c ATCC ar~~ was isolated from the sulfate reducing bacteria ~ e s u ~ f o v i ~ 2'77'74 and D.uulgaris ~ildenborough~ and shown to be present also in other microc u ~~ ~ h ~ ~ gfulgil ~ b organisms such as ~ e ~ h a ~ o b a c t et hr ie ur ~~ o a u t o t r o p h ~ and dus. Its physiological function is unknown, but it shows superoxide dismutase (SOD) activity [131. ~ ~ s u l f o v ~~ ~~ sr u~ ~# ~ u rdesulfoferrodoxin lca~s is a twofold symmetrical dimer. Each nionomer of 125 amino acids contains two mononuclea~iron centers: center X with a redax potential o€ 1-4mV is an Fe(Cys), center with distorted tetrahedral coordination as in desulforedoxin, whereas the unusual center I1 ( + 2 M my) consists of a nonheme iron coordinated to one cysteine sulfur and four histidine nitrogen atoms in a square pyramidal geometry.

There are more than 20 rub redo^^ structures in the PDB, two o f them being WMR solution structures ( ~ ~ o s t rp ~a ~ ti ~u u~r i and a ~ Pyrococcus u~ furiosus mbredoxin, respectively) [14,151. Two of the X-ray stmctures have a resolution below 1 0.92 for D. vulgaris rubredoxin (lRB9) and 0.95 for P. fitriosus rubredoxin (IBRP, Fig. 2). The rubredoxin fold is characterized by 8 three-stranded antipardel fl sheet and two to three helical turizs. The p sheet i s formed by the N terminus p r o ~ d i n gthe first two strands and the S: terminus as the third strand, The iron ion is located close to the /3 sheet on the surface of the protein, with thc two Nterminal cysteines in the loop between the N-terminal p strands and the two Cterminal ligands in a hydrogen-bonded turn. The N-terminal ligands are arranged s - ~ l ybackbone conformation is very similar to that in a sequence ~ y s - ~ r o - X - ~ whose of the andogo~isresidues in the Zn binding domain of zinc finger proteins. Moreover, it is worth noting that the polypeptide fold around the mononuclear iron center in rubredoxin is similar to that around the FezSz cluster in Rieske proteins (see Sec. 2.2). A single point mutation in C. p a s ~ e u r ~ rubredoxin a~u~ iC42A) converts it to an Fe2Sz cluster-containing protein [llril. Desulforedoxin (1DXG) is a twofold symmetrical dimer (Fig. 3) with numerous h y d r o ~ ebonds ~ bet we^^ the monomer units. The monomer is stn*cturally similar to rubredoxin and contains five short /3 strands, four of which form an antiparallel fistructure that is extended to an incomplete f3 barrel by the dimer-

A

A

A:

374


FIG. 2. Three-dimensional structure of the rubredoxin from Pyrocaccus furiosus (PDB code IBRF) in a ribbon representation highlighting the elements of s e c o n d q structure. The iron ion and the four ligating cysteine residues are also depicted.

ization. The iron is situated at the surface of the molecule with an Fe-Fe distance of 16 A in the dimer. is arranged in two domains in the The desulfoferrodoxin ~ i o m o d i ~ e(1DFX) r same way as the monomer subunits. Each monomer domain contains one solventdomain exposed iron center (Fig. 4). The N-terminal domain I or des~~oredoxin"1ike has 34 residues and contains the Fe(Cys), center. Three antiparallel B strands from each monomer form two almost parallel 0 sheets in the dimer, resulting in an incomplete fJ b a r d with a hydrophobic core. The C-terminal domain TI (88 residues; homologous to neelaredoxin from D.gigas) has a ""34 sheet" structure like that in the fold of fibronectin 111; there is only one turn of x helix in the linker region to domain I. The four-stranded p sheets from each m o n o ~ e rare combined to an eight-stranded antiparallel sheet in the dimer with a lwgc twist between the first and the eighth es 68, 74, and 1181 strands, The iron center I1 is coordinated by four h i ~ t i d i ~(His-48, in the equatorial positions and Cys-115 in the axial position o f a squase pyramid. The sixth coordination site of the iron is solvent-accessible, A calcium ion, necessary for the c ~ y s t ~ l i z a t i owas n ~ assumed at the dimer interface and seems to contribute to diiner stabilization.

375

FIG. 3. Iltustration of the structure of' deeulforedoxin from Desulfovibria gigus (PDB code 1DXG). The symmetry-related subunits of the dimeric protein are shown in Light and dark gray, respectively.

2.2.1. Occurrence and Putative Biological Role

Rieske proteins occur in both prokaryotes and eukaryotes. They are commonly found in mitochondria1 bel complexes and in plastidial b& complexes as well as in bacterial bel complexes. Furthermore, bacterial Keske-type complexes are found in ring-hydroqlating dioxygenases, as well as in the electron transfer proteins often associated with the dioxygenase functions. The bcl and b6f complexes arc: multi-subunit membrane proteins containing four redox centers in three subunits: a cytochrome containing two heme b centers (see Chapter 9) in a transmembrane arrangement, a cytochrome c1 or f , and the Rieske iron-sulfur protein. These complexes oxidize hydroquinones and transfer electrons to their respective acceptors, i.e., cytochrorne c or p ~ ~ t o c y a n iIn n . dioxygenases, the Rieske center acts as an electron transfer center that transfers electrons to a catalytic nonheme non-FeS iron (see Chapter 11).The latter performs the aromatic ring hydroxylation reaction involving the attack of dioxygen to the aromatic ring producing a cis-arene diol. The electrons are provided by a reductase, that may also contain an iron-sulfur cluster, and are often transferred from the reductase to the dioxygenase by another soluble ferredoxin that i s often a Rieske protein.

376

BENTRQP, CAPOZZI, AND ~ U ~ ~ I N A

FIG. 4. Structnre of one monomer of the d e s ~ l € o ~ e ~from ~ o dDesulfouibrio o~~ ~ e s ~ l ~ ~ r ~ (PDB code IDFX). Both iron ians arc shown with their ligands: Fc(Cys), at the top and Fe(EW4Cys at the bottom. The putative Ca2+ ion at the dimer interface i s also shown.

Three X-ray structures of proteins containing Rieske clusters are availa~le~ two water-soluble fragments of the Rieske proteins from bovine heart bel complex and A lower resolution X-ray spinach b6f complex, and na~ththale~ie-l,2-dioxyg~nase. structure of the whole bovine heart bcl complex is also available (Fig. 5) E171. The Rieske fragment is anchored to the rest of the complex by a long t r ~ s m ~ ~ b helix acting as a membrane anchor. The bel dimer is arranged in a head-to-tail fashion and stretches across the membrane in such a way as to have the catalytic e of the other Rieske fragment of one monomer close to the t ~ ~ s m e m b r a nhelix monomer (see Sec. 4). All Rieske proteins for which the X-ray structure is known, al and presumably all other sequenced Rieske proteins, share an a r c h a e t ~ i ~struct u r d unit called the Rieske fold. The Rieske fold consists of three antiparallel @

377

FIG. 5, Structure ofthe whole bovine heart ~ ~ t o c h oh cnl ~ cmtnplex r ~ ~ (PDB code lBE3). The dark subunit represents the Rieske-iron-sulfur-protein(ISP) whose cluster binding domain is connected to its t r ~ n s ~ e helix ~ b by ~ a~Rexible e linker. In this perspective of the eltzster in the Rieske domain one iron (dark sphere) eclipses the other.

sheets (Fig 6) formed by the conserved j3 strands 1, 10, and 9; 2, 3, and 4;5-43 (bcl n ~ m b e r i n g )The ~ three sheets have been d e ~ n eas~f ~ r ~ i na g“double p sandwich”. The last j3 sheet and its loops form the cluster binding subdomain. The PqSz d i ~ ~ o is n dcoordinated by ligands located in loops fi4-PS and P6437. Each loop provides one cysteine and one histidine ligand. The inner iron is c o o r ~ ~ ~ a t ~ by the two cysteines, and the outcr iron is coordinated by the two histidines. The typical c o ~ s e ~ s u sequence s is:

Thus, the ~ ~ o ~ ~ ~ ~a tt t ieiQ sr 2+2, nn c o ~ to the ~ 3+l ~ pattern e ~ o b ~ e r in ~ the e~ ~our~cysteine-~oordinated Fe,S, diamonds (see Sec. 2.31, and is more closely related to that of ~ ~ ~ r e d (see o x See. ~ ~ 2.1). s The geometry of the Eeske cluster i s the same within the error in all three s t ~ c t u r e (Fig. s 6).

378

379

2.3,l. Occurreme and Biological Role

The first 2Fe-2S ferredoxins to be discovered were the so-called plant-type ferredoxins in spinach chloroplasts [lSl. They are found in many oxygenic p l i o t o s ~ t h ~organ~t~c isms, occur frequently in isoforms, and scrve as terminal electron acceptors to photosystem I (PSI). Their Fez& cluster (with a redox potential of about -400 rnV) is reduced by one electron from an Fe4S4 cluster of the PSI subunit PsaC and transferred to ferredoxin-NADP' reductase. Besides their role in photos.~thesis,they serve as electron carriers in a variety of metabolic pathways, such as nitrite reduction, nitrogen fixation, sulfite reduction, glutamate synthesis, thioredoxin oxidoreductio~i, and lipid desaturation. In bacterial ~ o ~ g e n asystems, se 2Fe-2S ferredoxins are the electron shuttle between reductase flavoproteins and oxygenase. Many redox active enzymes contain domains or subunits similar to 2Fe-2S ferredoxins, e.g., bacterial aromatic di- and m ~ ~ o o x y g ~ ~ afumarate s e s : reductase, eukaryotic succinate dehydrogenase, xmthine dehydrogenase, phenol h y ~ o ~ l aphthalate ~e, dioxygenase reductase, and cytoplasmic Ye-only hydrogenases. The soluble adrenodoxin-type 2Fe-2S ferredoxins (redox potential about -270 mV) occur in oxygenase systems and can be ~ o ~ a s ferredoxins, with divided into the v e ~ e b r a t ~ t y pand e ~ ~ e ~ d o putida-typc about 50% sequence similarity between these subgroups. Vertebrate ferredoxins are found in mitochondria1mo~ooxygenasesystems of different tissues where they transfer single electrons from ~ ~ ~ H : f ~ r ~ e reductase d o x i n to ~ e m ~ ~ a n e - b o ucytond chrome P450 enzymes involved in steroid hormone biosynthesis and other metabolic processes. The bacterial electron carriers putidaredoxin and t e r p ~ ~ e d o x i ~ to soluble cytotransfer electrons from NADH-dependcnt f e r r e d ~ ~reductases n i ~ the ~ chrome 1'450, e.g., P450,, (catalyzing the first step of camphor m e t a b ~ l i s in case of ~ u t i d a r e d o x ~ .

2.3.2, Molecular Structure of ~

l

~

~Perredoxin t - ~ and~ A ~d re ~ ~ ~ d o ~ i ~

There are more than 20 structures of 2Fe-2S ferredoxins in the PDB, most of them o f plant-type ferredoxins. The latter proteins (with a size of a p ~ ~ o x ~ ~ a96t eamino ly acids) adopt an a+p fold (Table 4)consisting of two ;x helices and a fou~-st~anded f3 sheet in which the first helix lies across the sheet. An additional short 310 helix is observed at the G terminus (Fig. 7A). The tertiary structure is stabilized by a consemed hydrophobic core; the Fe& diamond is near the surface of the molecule in a long extruded loop between the first cx helix and the third p strand and clearly determines the structure of this loop. The cysteine Iigands to the cluster are arranged in a conserved C y s X 4 ~ y sCys ~ sequence ~ s ~ ~ motif. ~ Halobacterial ferredoxins, as exemula (lf)c41),have a core plified by the 2Fe-2s ferredoxin from ~ a l ~ a r ~marismortui s t r u c ~ r esimilar to plant-type ferredoxins, but two additional amphipathic cl helices are inserted at the N terminw between the first and second strand of the fi sheet.

380

BENTROP, CAPOZZI, AND LUC~INAT

FIG. 7. (A) The plant-type ferredoxin fold

tls exemplified by the structure of the 2FeSS ferredoxin from Chdorella fusca (PDB code IAWD). (B) Structure o f the truncated bovine adrenodoxin (residues 4-108; PDB code 1 ° F ) shown in the same orientation as in (A)*Tho similarity of the folds i s clearly seen.

The crystal structure of a truncated bovine adrenodoxin (lAYF, Fig. 7B) reveals that vertebrate 2Fe-2S ferredoxins also have the compact a+ f3 fold typical of their plant-type analogues in spite of low sequence similarity. The secondary structure elements comprise three a helices, two 310 helices, and five p strands. The molecule consists of a core domain and a smaller interaction domain (35 residues) responsible for the binding to adrenodoxin reductase (1GJC 1191) and cytochrome P450. It exhibits a strikingly asymmetrical charge distribution 1201. Also, putidaredoxin (lPDX, lPU'I', 1GPX) and terpredoxin (1B9R) whose structures were determined by NMR

are structurally homologous to plant-type 2Fe-2S ferredoxins. However, similar to adrenodoxin, they contain more secondary structure elements. A new fold of an Fe2S2 c ~ u s t ~ ~ - c o nprotein ~ ~ i ~has l ~ nbeen ~ described for the second Fe2Xz cluster domain of the aldehyde oxidoreductase from D.gigas (lALO), a € a ~ t ~(see r member of the xanthine oxidase family containing a ~ o l ~ b ~ e ncuo m Chapter 22). Whereas the first Fe2S2cluster binding doniain has a plant-type ferredoxin fold, the second cluster is bound to a twofold symmetrical four-helixbundle and is found at the N terminus of two a helices (Fig, 8).

2.4. ~

~

~with ~ FesSa~and/or d Fe4S4 ~ Clusters ~ i

n

~

2.4.1. Occurrence and R l o l ~ g i ~Rote ul

F ~ r r e d o with ~ ~ scubane-like clusters are primarily found in bacteria, ranging from d~urn l2ll to photosynnonphotos~theticanaerobic species Eke ~ l ~ s ~ r ~pasteurianum thetic species and to ~ h ~ ~ " ~ t h e r m o p hbacteria. i 1 i c They have also been isolated from

FIG. 8. The two Fe& clustex+~ in the xanthine oxidase-related aldehyde oxidoreductase from e gigas are shown ~ within~ their respective ~ proteinf domains (PDB ~ code IAz1Q). ~ The first domain (lower left) hns a structure similar to plant-type femedoxins. The second E'e& cfuster binding domain is a four-helix bundle. The cluster is bound by two cysteines in the first and two cysteines in the third of the loops connecting the fOUT helices.

~

~

extremophilic archaea. The redox potential of the clusters in these proteins is within a range of -280 t o -650 mV, whereas the 3Fe-4S ferredoxins generally have a less negative redox potential (-100 to -150 mV) [lo]. The Fe3S4 cluster in 7Fe-8S ferredoxins has a reduction potential in the -130 to -450 m y range. Ferredoxins serve as low-potential electron transfer proteins in a wide range o f metabolic reacfactor in the tions, e.g. C.pasteurianurn SFe-8S ferredoxin is ail electron-tra~isfe~ing pyruvate o ~ d a t i o linked ~ to N2 fixation, the 7Fe-8S ferredoxin in Streptomyces griseus i s an electron carrier for the cytochr-omeP450 system in this organism, the zinccontaining 7Fe-8S ferredoxin from the archaeon Sulfolobus sp. i s an electron acceptor to a 2-oxoacid:ferredo~noxidorcductase. The FesS4 cluster in the ~ F e f~e r r~e dSo ~ n from the thermophilic hydrogen-oxidiz~n~ ~ a c ~ E schlegelii lu~ can mediate electron in (FPR). The 7Fe-8S fertransfer to cytochrome c from N ~ P H : f e ~ e d o x reduetase redoxin I from Azotobacler vineEaadii binds specifically to FPR, suggesting that tlie two proteins are redox partners in vivo; moreover, it has a regulatory fbnction and controls the expression of FPR via an oxidative stress response. Single-cluster ferredoxins have similar functions of electron transfer between redox active enzymes. A number of ox~do~eductases contain domains or subimnits that are h o ~ o l o g o to ~ sbact e r ~ ~ l ferredoxins, ~ t y ~ ~ e.g., Fe-only hydrogenase, fumarate reductase, succinate dehydrogenase, ~ ~ u v a ~ e : f l a v ~ d oreductme, xin formate dehydro~enas~. The PsaC subunit of photosyst~mI is a 9.3 kDa ferredoxin-~~ke 8Fe-$5 protein. It is necessary for the final electron transfer From the membrane phase to soluble 2Fe-2S ferredoxin or Atavodoxin in the cytoplasm.

~~us The 8Fe-8S ferredoxins from ~ e ~ t o ~ ~ r e ~ at ~ accoc c~ a~ r~o sl y ~ (formerly ~ e p ~ o c uerogerm, o c ~ ~ ~ ZFDX), C. pasteurianum (ICJLF, N~~ solution structure), ) approximately 55 amino acids can be considered as and C. acidi-urici ( 2 F D ~ with minimal f e ~ e d o x i ~and s prototypes of other dicluster ferredoxins. The molecules have an ellipsoidal shape and their structure is characterized by a ~se~do-twofold symmetrical a r r a n ~ m e onf~the Fe& clusters and the secondary structure elements (p strand, M helix9and strand) in a t / 3 ~ 4fold ) ~ (Fig. 9). The first short antiparalle~ sheet is formed by the N and C termini, and the second one consists of residues in a loop region between the two clusters. Short helical segments are formed by the sequential stretches between CysIII of one cluster and CysXV of the other cluster. s (see Sec. 1.2) are found The cysteine ligands in the typical sequence CysXXCy in two extended loops at the surface of the pxotein. ~ ~ d r o p h o bresidues ic protect the clusters from direct interactions with solvent molecules, The center-to-center distance of the two cubane clusters is always about 12 A. The 8Fe-$3 ferredoxins o f the ~ ~ r o r n ~ t i z ~vinosum-type rn, (approximately 82 amino acids) exhibit the same core fold as the clostridid ferredoxins, but have a six residue insertion between two cysteines of the second (C-te~minal)cluster and a C-terminal extension forming a 3.5-turn N, helix. This helix covers one side of the second cluster and has hydro~hobic

383

FIG. 9. The ( @ x P ) ~c o ~ fold e of femedoxins with cubane elusters. The structure of the We-89 femedoxin from Ctostr-idiun ~ c i ~ iPDB ~ - code u ~ZFDN) ~ ~ is shown in an orientation that emphasizes the pseudo-tworold symmetry of the molecule.

contacts to side chains of the six-residue insertion. The helix axis lies pe~pe~~dicular to the axis connecting the two clusters. ~ o t ~ ~ auineZundii-type ~ t e r ferredoxins with about 106 amino acids contain an (N-terminal) Fe3S4and an Fe4S4cluster in a core structure that is very similar to tlmt of clostridial ferredoxins. The more than 50 additional amino acids constitutc a long 6-terminal ex-kension that is mainly characterized by a four-turn helix from residue 62 to 75 followed by loops on the surface of the protein. In this case, the axis of the helix is also p e ~ e n d ~ c u l to a r the axis between the two clusters, but lies between them on the surface of the molecule and packs against the two @ sheets of the ferredoxin core fold (Fig. 10). Thermophilic bacteria like B. schZegeZii contain a 7Fe-8S ferredoxin of the A. ~ ~ J z ~ Ztype u n ~whose i ~ C-terminal extension is about 30 residues shorter, i.e.$their polypeptide chain ends with a four-turn helix in a position analogous to that; in A. vinelaadii-type ferredoxins. Otherwise, the structures are the same 122,231. g The recently solved crystal structure of a 103-residue ~ n - c o n t ~ n i n7Fe-8S ferredoxin from a t h e ~ ~ o a c i ~ o p harchaeon ilic (Sulfolnbus sp, strain 7; 1XER) reveals the special features of this new class offerredoxins (Fig. 11).Compared to clostridial ferredoxins, S u Z ~ o ~ ferredoxin ~us has an terminal extension of 36 amino acids and an insertion of 9 residues between the two cluster-binding sequence motifs.

384

BENTROP, CAPOZZI, A N D LUCHINAT

FIG, 10. Structure of ferredoxin 1 from Amtobarter vinelandii (PDB code 6FDl). The structural homology of the core of this 7Fe-8S ferredoxin to the smaller (2 acidi-urici ferredoxin (Pig. 9) is evident. The axis of the four-turn helix i s also shown.

~nterestin~ly, the photosystem 1 subunit PsaC has a homologous insertion in tho same sequence position. The N-terminal extension of SuZfolobcLs ferredoxin Eorms a a ~ lthat ~ lis ~ y ~ o g e n - b o ~to ded one-turn u helix and a t r i ~ l e ~ ~ t~r n~ td ie ~~ ~fl sheet the terminal f%sheet of the core fold part residues ~ ~ - 1 0result~ng 3 ~ ~ in a fivemain-chain folding of stranded f3 sheet. The core fold part has the typical (fl~$)~ clostridial f e r r ~ o ~ nThe s . single Zn binding site is located at the interface between core fold and N-terminal extension connecting the fl sheet in the e~ensionto the ~ othe n Zn centrd sheet in the core fold, This is achieved by t e t ~ ~ ec o~ oa~ l~ i n a t of e s the N-terminal extension mclone aspartate from the core ion by three h i ~ t i d i ~from fold. (For a seminal review on Fe3S4clusters in proteins, see [24:1.) The PDB contains three-dimensional structures of ferredoxins with only one cubane c b t e r from four digerent prohryotes. Generally, they are a ~ p r o x ~ a t e l y ~ ~ - r e s i dproteins u~ with a h~drophobiccore and the same n u ~ of~ ~ e c ~o n d a ~ structure elements as clostridial dieluster ferredoxins (i.e.*two a helices and two ta the latter, single-cluster f e ~ r e ~contain o~~s short ~ t i p ~fl sheets). ~ ~ e Compared l t ~ d ~The only the N-terminal cluster in a highly similar €old of the p o l ~ ~backbone.

385

FIG. 11. Structure of the zinc-containing fexdoxin &om Sulfolobus sp. strain 7 (PDB code 1XER). The coordination o f the zinc ion by three histidines and one aspartate at the interface between the ferredoxin cure fold and an N-terminal extension is shown (lowerright), The zinc ion connects the fl sheet in the extension and the core fold. Although the femedoxinfrom ~ ~ & ~ f o ~ o sp. strain 7 is a 7Fe-88 ferredoxin, the structural model contains two I?e8S4cluslexs. This i s due to the loss of one iron ion from the original Fe& cluster during aerobic purification [1431.

second cluster i s missing but substituted by a longer ct helix to stabilize the structure (Fig. 12). 8everal single-cluster ferredoxins contain a disuEde bridge in a position that is equivalent to the C-terminal cluster in dieluster ferredoxins. ~ r d i ~bridge ~ l ~ n~e ~~ e s for s ~a stable fold. ~ ~ ~~ i ~ ~ e ~ r u~ so4Fe45 femedoxin (2 with 81 amino acids has some insertions and a l ~ - r ~ s ~ Cdue terminal extension in comparison with the other single-cluster ferredoxins~but still shares the same folding pattern. ly that are represenCurrently~crystal s t r ~ c t u r eof~ two ~ e ~ o n hydrogenases are known (for N i ~ o n t ~ hydro~~ng tatives of the two groups o f F~~hydrogenases 14), The c ~ o p l a s m ~monomeric c F e - h y d r ~ ~ e nfrom ~e C ~ e ~ a see s e Chapter ~ ~ ~ a s ~( 1 e ~~1C4A, ~ ~ ~lC46) ~, ~is a~574residue ~ , ~protein with five distinct iron-sulfur clusters in four domains. The three smaller domains form the stem of a mushroo~-sha~ed molecule and are classified according. to the cluster(s) that they contain: FS2 contains an Fe2!2S2 cluster in a plant-type f e r r e d a ~ ~fold n at the N t e r ~ n ua f~the protein; F ~ - contains ~ Stwo ~ Fe,& ~clusters in a clastridial f e ~ e d o x ~ n fold ~ ~ iand ~ e is next to the large active site domain; FS4C ~ o ~ t a i an sFe4S4 cluster that is c ~ o ~ d i ~ iby a t three ~ d cysteines and one hi mated in a loop hetwe two a helices. The sequence M S4C domain links the FS2 and the Fe2S2 cluster and the histidine-caord~naed~e~~~cluster

386

FIG. 12. The structure of tho 4Fe-4s forredoxin from Themaotoga mnritirna (FDB code 1VJW) is stabilized by a longer CL lxelix with respect to the &cluster ferredoxins (cf. Fig. 9 the orientation of the molecules is similar).

are close to the protein surface and are possible sites for intermolecular electron transfer with physiological redox partners. The active site domain comprises resi7 4caps the protein molecule. It contains the c a t ~ ~“H i cc h ~ t e r , ~ ’ dues 2 ~ ~ - ~and Lee, a cluster of six Fe atoms consisting of a Fe4S4 cubane subcluster covalently bridged by a cysteine sulfur to an Fez subcluster with octahedral coordination of both Fe ions (see See. 4.4 for a detailed description). The H cluster is located at the i ~ t e ~ ~ of a ctwo e equivalent lobes constituting the active site domain and has cluster is an edge-to-edge distance of about 9 A from the FS4A cluster. The about 10 A from the FS4A cluster and seems to serve as a junction in the presumed electron transfer pathway to the H cluster because it is in a triangular a r r a n g e ~ e n with t the remaining two eluslers. It could accept electrons either from the FS2 cluster (at about 11 or from the FS4C cluster (about 8 A), The F’S2FS4C i n ~ ~ r ~distance ~ u ~ ~iseabout r 17 The periplasmic heterodimeric ~ ~ h y ~ r ~ ~ from e n a~s ee s u ~ ~ do~ ~s u~ ~~~ ru r~ cans ( 1is composed ~ of~a large ~(421 residues) ~ and a small subunit (123 residues), It has a close e v o l u t i ~ n arelationship ~ with the monomeric C. pasteurianurri enzyme [as]. The m i n o acid sequence of its large subunit is highly homologous to the FS4A FS4B and active site domains of C. ~ ~ s ~ Fe-hydrogenase. ~ u r ~The ~large~subunit u ~ contains the six-iron H cluster and two Fe4S, clusters. The latter ones are located in a clostridial ferredoxin-like domain at the N terminus and correspond to the FS4A and ~n~?n The active site domains of both clusters of C. ~ a s t e u ~ ~ Fe-hydrogenase. proteins are essentially superimposable, but the FS2 and FS4C domains of the C. ~~~~

A.

pasiteurinnum enzyme are missing in D. desulfiricans Fe-hydrogenase. The small subunit o f D. desulfuricans Fe-hydrogenase exhibits a peculiar fold: it sur-rounds ~ the large s u b ~ ~ nlike i t a ring or belt that consists of four a helices a l t e r n a t i ~with stretches of extended polypeptide chain. ‘There is some sequence homology between the C terminus of the large subunit and the C-terminal approximately 50 amino acids of C. ~ a s t e u r ~ a Fe~hydrogenase nu~ that fold into a number of helices parikdly embracing the active site domain.

2.5.

~ i g ~ - ~ a Iran-Sulfur t e ~ ~ i ~Proteins l

2.5.1. Occurrence arid Biological Role

~ i g h - ~ ~ ~ e inr ot ina- sl u l ~ proteins r t ~ i P I are ~ ssi~gle-cluster ~ 4Fe-48 ~ r o t e i with ~s a positive redox potential in the +90 to 1-500 mV range 1101. They vary in size from 6 to 10 kDa (53-85 amino acids) and were mostly isolated from purple ~ h o ~ o s ~ n t h bacteria et~c such as ~ ~ r o ~ a ~t h~i ouc a?~ ~s~a~c~ t o t h ~ o r ~ ~ o ~ Rhodocyclus, and other members of Rhodospirillaceae. Also the denitrifying bacterium Paracoccus h a ~ o d ~ ~ ~ ~ the t ~ iaerobic c a ~ ~ sulfur s, bacterium ~ ~ ~ fer- ~ r ~ o and ~ the ~ ~aerobic ~ ts h e~ ~ o ~ ~ o ~n ohn ip h~o~t ocs ~ ~ ~ h ~bt ~ i cc t e ~ ~ ~ h , Q ~ o t niarinus ~ ~ r ~ contain ~ s EIiPIP. In phototrophic bacteria, H ~ ~ are P s found in the periplasm and serve as soluble electron shuttles in the cyclic electron flow between two t r ~ s m e m b r a n ecomplexes: the ~hotosynthetic~ e a c t ~ ocenter n (RC)and the cytochrorne bcl complex. Reduced MiPIP i s the immediate electron donor to an bound c-type tc-?traliemiccytochrome 126-281. There is also evi~ s an important role in bacterial respiratory electron transfer dence that ~ i P I play 1291. Chromatiurn vin,osurri HiPIP was reported to have an additional fmction as electron acceptor for a thiosulfate-ox~di~i~g enzyme in that bacterium 1301, In I? ~ i ~ l o ~ n i ~ r~i ~ a~niss Fassumed ~ Pto be an electron donor to a nitrate reductase r311, whereas the homomultimeric 53-residue HiPIP of T. ferrooxidans is supposed to function as a iron(T1) oxidase [321. The recently discovered ~ ~ i from P 1 R. ~ ~ a r ~ is n umntral ~ to the electron transfer chain of this o r g a ~ i ~ and m s~~~ttles electrons between its cytochrome bc complex and a caag terminal oxidase. u n u s u a ~ l ~this , H ~ is ~ membrane”bound I ~ in vivo; however, it is w a t e ~ ~ - s o l u b ~ after ~ ~ i r ~ f i c afrom t , ~ othe ~ m e ~ ~ r [33,34If ~ e s 2.5.2. Molecular Structure of lifgh-Potential Iron-Sulfur Proteins

~ h r e e - d i m e ~ s istructures o~~l of ~ ~ Pfrom I ~fives different bacteria are deposited in the PDR, Solution NMR structures of C. uinosum HiPIP in the reduced and oxidized state show that there are no significant structural dirferences between the two oxidation states. The sequence homology between I-fiPIPs is relative^^ low, w also evidenced by the variations in the cluster binding sequence motif n ~ea tyrosine ~ ~ - ~ ~ ~(see~Sec.s 2.1; ~ Z ~is a~ t ~-t ~ ~p h ~a or Z ~ in y R.s tenuis

>.Nevertheless, the structural similarity of different HiPIPs is high, especially around the cluster. The structure of all HiPIPs i s characterized by the absence o f extended secondary structure elements. Their fold can be described as a series of turns and loops that bury the Fe.& cluster in the interior of the protein so that it is inaccessible to solvent (Fig, 13). Aromatic side chains from the ~ ~ t e ~ m ihalf n a l of al the po~ypeptidechain and a strictly conserved tyrosine from the ~ " t e r ~ nhalf ( T ~ - l in 9 the C. uinosum n L i ~ b e ~ system) in~ form a hydrophobic pocket for the cluster that is essential to protecl it in the oxidized state from hydrolytic degrada~ P s tion [35,361. The only conserved secondary structure element in all ~ i ~ except that fiom E. ~ ~ a isZa short o ~ CI helix ~ ~ in ~the ~N~terminalregion, before the ~ o n s e ~ etyrosine d residue. This CI helix is part of a dimerimtion interface that was observed in crystals of several HiPIPs. HiPI s tend to dimerize about a consewed hydrophobi~surface patch consisting o f three to four d ~ s c o n t ~ n ~~ ~eu s~ e ~ of the ~ o ~ y p e pchain t ~ ~ e137-391; however, the orientation of the two monomers at the dimer ~ t e r f a c eis different in the various NiPIPs. The physiological relevance of the ~ e r ~ a t i observed on in crystals is not yet clear.

FIG, 13, ~ i ~ h - p oiron-sulfur ~ ~ ~ t proteins i ~ (HiPfPsl fold around the cluster as ~ x e ~ p ~ f i e d by the crystal structure of Chrornatiunz uinosum HiPIP (PDB code IClkT[J).

389

Aconitase (aconitate hydratase or citrate hydro-lyase; EG 4.2.1.3) is an F’e4S4 clustercontaining enzyme of the tricarboxylic acid (Krebs) cycle in eukaqrotes and pro&yoLes. It catalyzes the stereospecific isomerization of citrate ta isocitrate via cis-aconitate by an elimination (dehydration~-addition ~ ~ i y d r a ~mechanism on) in which the iron-sulfur cluster is directly involved, Two forms of aconitase with difyerent relative distr~butionsin tissues and organs are known: a mitochondria1 (m-) and a cytoplasmic (c-) enzyme. The m- and c-aconitases have 3096 sequence identity, but c-aconitase is 135 amino acids longer than the 754-residuem - a c o n ~ ~and s e more stable to dative loss of ac~iv~ty. c-Aconitase is not only an enzyme but also plays a key role in iron e to the iron regulatory protein honieostasis. The apo form a€ c ~ a c ~ n ~ ti as sidentical or IRP1, formerly called ferritiii repressor protein or IRE-BP (iron responsive element binding prote~n)].This protein controls iron uptake and storage at the level of translation by binding to IRES, i.e., conserved stem-loop structures in the untranslated regions of mRNAs, when the iron content of the cell is low. When the iron level is high, the IIWP becomes a hQ~~protein with an iron-sulfur cluster and with aconitase a ~ i v ~ t The y . iron-d€ur form of the protein does not bind RNA and thus cannot interact with IRES. This means that the function of I~F/c-aconitaseis regulated by the assembly/~sassemblyof the iron-sulfur cluster (see 140 and [411 for recent reviews). IRPS is a protein that has 57% sequence identity to I P; there is, however, a 73-residue insertion near the N terminus, and no stable iron-suffur cluster has been it hcontains the same cysteine residues that ligate thc observed in IRPZ, ~ t h o ~ g cfttster in aconitases. PRF2 is regulated by proteolytic d ~ ~ a d a t i and o n the insertion serves as iron~dependentdegradation motif [42,431. 2.6.2. ~ o l ~ ~~ ut lr ~ u croft Aconitase ~ ~

There are 15 X-ray structures of aconitxase (wild type and two mutants) in the PD All of them were solved with crystals of m-aconitase, either from pig heart or from beef heart. The m-aconitase molecule comprises four domains (domain I: residues 1200,II: 201-319,111: 320-512, “hinge-linker”: 513-536, PV: 537-754). ~ o ~P-111 a ~ are tightly associated a d contain a structural motif similar to the so-called n~cleotide binding domain with a central parallel p sheet of 5, 4, and 5 strands, respectively. ~ d d ~ tp istrands ~ n ~and cx helices in each domain cont~ibuteto their association. The iron-sulfur cluster is coord~natedby three cysteines (Cys-358,421,424) in and is close to the center of the protein molecule (Fig. 14). Domain IV is linked to the first three through an exxtcnded piece of p o l ~ e p t i d echain (hinge-linker) and has a shape Chat fits onto the surface formed by domains 1-111. Domain Tv has an unprecedented topology with a central five-stranded parallel p sheet that is e ~ t e by~ a~ e three-stranded @ meander. A f o ~ r ~ s t r a ~ antiparallel ded p sheet is fomd on one side of the central p sheet; on the other side there are cx helices. The interface between

BENPROP, CAPOZZI, AND LUCHINAT

390

FIG. 14. Structure of bovine m i t o c ~ o n daconitase r~~ (PDB code 1ANLJ) with suifate as bound species. The Fe4SQcluster is close to the center of the molecule between &he6-terminal domain IV (upper part of the mulecide) and the three N-terminal doniains (lower part ofthe molecub), The central sheets of the latter are clearly seen. The active site deft between domain IV and domains 1-111 is open to the left in this perspective.

domain IV and domains 1-111 is occupied with water mo~ecu~es or made up of polar amino acids and forms a cleft leading to the iron-sulfur cluster in the active site of the enzyme. This supports the idea of hinge motion between domain IV and the rest of the molecule that would allow opening and closing of the cleft during turnover, '~hus, substrata inolecules could diffuse into or out of the active site. The active site of aconitase is formed by the side chains of 23 amino acids from all four domains. It is a large polar cavity with a net positive electrostatic field to active protein has a diamagnetic bind anionic substrates. The native, en~ymatical~y Fe4S4cluster in the active site. Only three of its iron ions are ligated by the abovementioned cysteines in a ~ y s ~ ~ ~sequence y s ~motif. ~ yThe s fourth (labile) iron ion (Fe41 points to the cleft and has, in the absence of substra~e,a tetrahedral c o o r ~ i ~ ~ twith i o n a hydroxide ion as fourLh ligand. In the p ~ e s e n ~ofe substrate, Fe4 is sixfold coordinated by three sulfur and three oxygen ligands in a slightly distorted octahedral geolzletry. The three oxygen ligands are a water molecde, a ~arboxylate,and the hydroxyl group of citrate or isocitrate. The position of Fe4 is about 0.2 away from the Fe3S4moiety compared to its position in a s.ymmetrically

A

ligated Fe4S4 cluster. Loss of the labile iron from the cluster (e.g., during purificaLion) leads to the formati(~nof an inactive form of aconitase with an Fe3S4 cluster and no changes in the protein structure (PDB code 5ACN). Under reducing conditions, aconitase with this cluster can easily incorporate a fourth iron from solution and regain activity.

isean iron t e t r ~ ~ ~ o p o r p h yofr ithe n iso~acteriochlorinclass (with eight Siro~ei~ ~ ~ ~ ~ - c ap er r~~ pi hne rsubstituents) g~ found in sulfite reductases (Si related nitrite reductases (NiRs). These enzymes are found in arcliaea, bacteria, and eirkaryotes and catalyze the six-electronreductions of sulfite to sulfide and nitrite to ammonia during the biological assimilation and d i s s i ~ a t i o iof i sulfur and nitrogen compounds. In their unique active site a siroheme i s bridged to an Fe4S4cluster via a shared cysteine sulfur ligand. Based on biological function, SiRs and related NiRs r ydissimilato~.~ s ~ m i l a t o sulfite ry reductases are classified as either ~ s i m ~ a t o or (aSiRs) occur in bacteria, fungi, and plants; they generate sulfide for inco~~oration into methionine, cysteine, and sulfur-containing enzyme cofactors and use S;r or reduced ~ e ~ ~ d o as x i electron n donor. Dissimilatory sulfite reductases EC 1.8.99.3) are found in s u ~ f a t e - r e d ~ cbacteria i~g and some t ~ e r ~ ~ o p h ar ilic they reduce sulfite as a terminal eIcctron acceptor in anaerobic respir~tion. are multimesic enzymes with cna& or a2fJzy2structure that often contain additional Fe4S4 clusters not bound ta siroheme. They rclease primarily ~ n c o ~ ~ l ereduced te~y sulfur in the form of trith~ona~e <S&-t and thiosulfate (SzOi-). oreo over^ the ~ e can forin NH;. Several types of dSi reduce nitrite, NO, and ~ y d r o x y l a m ~and were isolated from sulfate reducers, namely, d e s u l f o ~ ~ d idne, s u ~ f o ~ ~ bdesulfoi~n, fuscidin, and P582, all of which accept electrons from cytocbrome c3. A d S ~ R - l ~ ~ e ic ‘‘reverse’’sulfite reductase is involved in sulfur oxidation in tlnc ~ h o t o t r o ~ hsulfur bacterium C h , r o ~ , a ~ ~ uvinosum m and the sulfur-oxidi~in~c h e ~ o l i t h ~ t r o ~ h ~ h , i ~ b dcnitrificans. ~ ~ ~ ~ u Yet s another type of SiR was identified in s ~ l f a t e ~ r e d u c ~ n ~ bacteria (e.g., ~ ~ s ~ ~ ~ aetoxidans, f u r ~ ~ ~ oe ~s i, ~~~ sfvulgaris): ~ v ~ ~ these r ~ oare assiinilatory SiRs of low molecular mass (-- 25 D a ) containing one siroheme and one Fe4S4 cluster per polypeptide chain. Assimilatory nitrite reductases (aNiRs) in bacteria, fungi, and plants reduce nitrite directly to ammonia and participate thus after the initial reduction of nitrate to nitrite in nitrate assimilation ~denitrificat~on~, Bacterial and fungal aNiRs accept electrons from N ~ t and P are ~ homodimers, ~ whereas higher-plant aNiRs (e.g., from spinach) are monomeric and accept electrons from plant-type 2Fe-2S ferredaxins. Sequences and properties of laniirS are similar to those of aSi&. Bacterial dissimilatory nitrite reductases tdNiRs) form a diverse e ~~ ~~ r co h~ e ~~ ~ a e - c oreductaae, n t ~ ~ i nbut ~ group of enzymes, i~cludingan ~ s c ~ ~ coli also cytochromes and Cu-containing NiRs.

The E. coli assi~ilatorys a i t e reductase (EC 1.8.1.2) is a component of the cysteine regalon that €unctions to reduce sulfite to sulfide for cysteine biosynthesis. It cornsubunits prises four catalytic, 64-kf)a hernoprotein 66 protein subunits. In vivo, the latter transfer electrons from to vitro, isolated SiRHP reduces sulfite to HS- and nitrite as well as hy~~oxylamine to NHi w ~ ~ h # ureleasing t ~ n t e r r n e ~ a t ewhen s suitable electron donors are present. S~~~ is reduced by one electron at the siroherne with a redox potential of -340mV and at the Fe4S4 cluster with a ~ t ~ n t iofa l-405 rnV. This reduction of the cofactors activates by a X06-fold ~ ~ ~ cof substrate e ~ ~binding n tand ~ng ~ s s o ~ i a t i orates n 1441, E. coli SiRHP is currently the only s ~ r o h e m e - c o n ~ i pro. than 10 crystal str~ctureswith ~ h o s ~ h substr~te, a~e~ tein of known s t ~ c t u r eMore in the active site of the enzyme are deposited inhibitors, ~ n ~ ~ e d i aand t e products s~ in the PDB. The crystallized species was always a klly active 497-residue protein ~rodueedby prdeolytic removal of the ~ - ~ e r r n ~ n aamino l 7 3 acids from native SiRHP. ~i~~~ is a trilobed molecule; the cofactors are bound in a pocket at the interface of n s 15). The central j3 sheet of each domain ~ a ~ ~ ~ cin i ~ three or-+@ d o ~ n ~ (Fig. ~~~~~

FIG. 15. Structure ofthe hemo~roteinsubunit ~ € ~ cofi ~ sulfite ~ ~re duct^^ , e (PDB ~ code ~ c ZGEP). The three a+J3 domains and the cofactor (siroherne+Fe4S, cluster) are clearly men (dornak 1, upper right; domain 2, upper left; domain 3, lower). The binding' o f sullite through the sulfur atom to the siroherne iron is also visible.

inter do ma^ contacts and in binding the prosthetic groups, whereas solvent-exposed helices form the periphery o f the enzyme. The Fe4SIcluster is buried from solvent on the proximal face of the siroheme, where it shares a cysteine s a u r ligand with the siroheme iron, so that the two metal centers are covalently linked and exchangecoupled. The siroheme itsex and fi sheets fhrn domain 1 and 2 form the walls of the highly adapted active center cavity close to the molecular surface. Thus, the substrate binding site i s on the distal face of the siroherne where positively charged Lys-215, Lys-217) assist in directing substrate anions to residues ( ~ g - 8 3Arg-153, , the remaining axial coordinatio~site of the siroherne by electrostatic and hydrogen b o n ~~ n t ~~~ a~c t i oSulfate n s , binds the siroherne iron o f SiRHP through sulfur. S i domain ~ 1 ~resembles ~ a parachute and consists of two subdoma~ns(1: residues 81-145, and 1’: residues 347421), each of which contains two a ~ ~ ~ ~ a four-stranded fi sheets and two p e r i p h e r ~3: helices. The two subdornains are relaled s ~ m eaxis t ~that also relates domain 2 (residues 146-346) to by a ps~udo-twofo~d 7 0 ~indicates . a gene duplication that has led lo two domain 3 (residues 4 ~ ~ - ~ This pseudo symmetric^ halves o f the protein, termed “sulfite or nitrite reductase repeats” ~ ~ s ~ ~ b d o ~1aand i n domain 2 (residues errninal S N i encompasses of s u b ~ o m ~ i1’ n and domain 3 ~ “ t e r i repeat ~ ~ ~i sa composed ~ (residues 347-570). An 18~re~jdue linker (residues 329-3463) connects the two SNiRRs and acts as structural mimic for substrate-bound siroherne in a residual i n v ~ ~ n a t i othat n is symmetry-related to the active site cavity. The homologous domains 2 and 3 are composed of mixed five-stranded fi sheets and flanked by several helices. Domain 2 contributes to ~ i ~ d i onf gthe siroheme and forms part of the active ~ d center on the distal side of the saddle-shaped siroheme, while domain 3 ~ ~ otwo highly conserved pairs of cysteine ligands for the proximal Fe4S4cluster (Cys-434/ Cys-440 and ~ s ~ 4 ~ ~ / C y s - 4The $ 3 )sulfur . atom of Cys-483 ligates both the cluster and the s i r o h e ~ eiron. Conserved residues in the s y ~ m e t ~ ~ r e l ahalves t e d of Si and other SiRs and NiRs have been identified as key determinants of s t ~ c t u r eand function of these anion redox enzymes, although the proteins show considerable eric state. The patterns of conservation are c o n c e ~ t ~ a t e d variations in size and o H5 (from N to C terminus). Regions Hl-H4 correspond in five h o ~ o l regio o~ ~whereas i H5 is~ the sev~n-r~sidL~e ~ , segto the structural dete~minant~ of one ~ ment at the C-terminal end of the linker that structurally mimics siroheme. (See [45l for a detailed review on the structural imp~cationsof the consei~edS i homoIogy ~ ~ regions.)

i t r o g Iron ~ ~ ~ ~ ~ 2.8.1. Occurrence and Biological Role

~ i o l o fixation ~ c ~ of ~ atmospheric nitrogen is achieved solely by bacteria and ~ e ~ u i r e s the nitrogenase enzyme system (EC 1.18.6.1) which converts Ne to ammonia in a sixclectron reduction that is accornpanied by the hydrolysis of MgATP (see 1461 for a

394

~ E N T ~ Q CAPOZZl, P, AND L U C ~ I ~ A T

recent review). ~ i t r o ~ e ~ consists ase of two met~oproteinsd e s ~ ~ the ~ airon t ~ protein (Fe protein, component 1, NifIH) and the molybdenum iron protein (NloFe protein, component 2j. The latter is a h e t e r o t e t r ~ e rof appro~mately230 kDa mid contains two different types of metallosulfur clusters: an unusual iron-sulfur cluster ~ ~ )is assumed to be the active site and an iron-molybdenum cofactor { F e ~ o cthat for nitrogen reduction (see Chapter 22). AZI Fe proteins from nitrogen-fixingbacteria (including eubacteria, archaebacteyia, and cyanobacteria) are 60 to 70 kJDa homodimers with sequence identities 2 48% and contain an o ~ ~ e n " s e n s i t iFe4S4 v~ cluster that is coordinated by invariant cysteine residues, Upon treatment with an iron-~he~ating reagent this cluster undergoes conversion to a single ferredoxinlike FezS, cluster /47,481; however, this cluster conversion does not seem to be of ~ h y s i o l o relevance. ~c~ In the process of nitrogen fixation the Fe protein accepts electrons fi-om the oneelectron donors Aavodoxk or ferredoxin and donates them to the c a t ~ ~ i ~active lly MoFe protein after formation of a protein-protein complex by the two nitrogenase components. There is no known subst~tuteof Fe protein in this electron transfer r e ~ c t i o nDue ~ to the recent discovery of a two-electron reduced, ali-ferrous state of the Fe4S4cluster with a physiologically relevant redox pot en ti^ of -460 mV, it is not clear ~ h e t tho ~ ~Ferprotein acts as a specific one- or two-electron donor to MoFe protein ([49,60] and references therein), The nitrogenase Fe protein has one Binding g ~ and binds ~ Ftwo nucleotides with positive cooperasite for M ~ T ~ per~subunit y affinity than tivity, with the oxidized Fc4S4cluster state having a s i ~ i f i c a n t ~higher the one-electron reduced cluster state. The exact role of MgATP hydrolysis in nitrogenase function is still undefined, but it requires both the Fe and the MoFe protein and is apparently associated with electron transfer in the nitrogenase complex. F u ~ t ~ e i - ~the i o rFe ~ ~protein participates in the biosynthesis of FeMoco and i s required for the insertion of this eofactor into MoFe protein polypeptides during ~ i o s ~ t h ofe MoFe ~ ~ s protein. However, the iron-sulfur cluster of Fe protein is not involved in these activities. Fe protein may also have a regulatory function in the nitrogenases. b i o s y n t ~ e sof~ alternative ~

There are currently four structures of nitrogenase Fe protein in the PDB: one is the Fe prote~nfrom C. pasteurianum (lCP2; 1.93A resolution two structures are of the (1NIF ~ d and ~ ~ 2NIP with 2.9 and 2.2 resolution, free Fe protein from A. u i n e ~ ~ res~ect~vely~, and one is a structure of the A. uinelandi~~iitrogenasecomplex (1N26; 3.0 resolution). The Fe protein (Fig, MA) is composed of two equal subunits that a single, solvent-exposed Fe4S4cubane cluster at one end of s ~ ~ e t r i c a lcoordina~e ly the dirner interface through two cysteines from each subunit (Cys-97 and 132 in A. uinelandii Fe protein). Each subunit is folded as a single a/P-type domain with a three-layer c t / f i / ~ sandwich architecture: a central eight-stranded 0 sheet is flanked by numerous c1 hefices, This polypeptide fold is similar to other nuc1eotide"bj~ding ding p r o t e ~ ~such s , as G-proteins, and contains a higlily conserved p h o ~ p ~ a t e ~ ~ i nloop

A

A

395

FIG. 16. (A) N ~ ~ r o ~ iron e n ~protein e is a honiodimer with a Fe&, cluster at the diincr interface. The crystal structure of the iiitrogena~eiron protein from ~~~g~~~~~~ (289 residues) is shown as a ribbon r ~ p ~ s e n t a t i oinn a view along the dinier interface (PDB code 2NIP). {B) The structure of the nitrogenase iron protein from A. u~~~~~~~~ in the nitrogenase complex (PDB code 1N2C). The protein is shown in the samc orientation as the uncomphxed form shown in (A). It is clearly seen that the dimer interface i s much tighter and the quaternary structure more compact than in the free iron protein. The 15 C-terminal residues of the iron protein j275-289) are disordered in tho nitrogenase complex and are not included in the cry&illograpkic model. ~~~~~~~~

~~~~~~~S {P- loop^ close to the N term~nus(residues 9-16 in A. u ~ Fe protein). Within the structure of the Fe protein, the nucleotide binding sites arc close to the subunit interface. Binding of M g A T ~or MglllsP induces large conformationd changes in the Fe protein that are necessary for effective complex formation with MoFe protein, and leads to a lowering of the redox potential of the Fe4S4 cluster e the A. u i ~ n i te ~ ~ ~e n a ~s e by approximately 100 mV. In the crystal s t ~ c t u r of P-loaded Fe protein docks with the surface, which includw the Fe4S4 cluster, to MoFe protein resulting in comp~eteburying of the cluster in the Fe protein/MoFe protein interaction area. The conformational changes in Fe protein upon ~ ~an e da p p ~ o ~ ~ ~ a13" t erotation ly of c o ~ p l ~ xo r ~ ~(Fig. t ~ 16B) o n can be r a t j o ~ a ~ as each subunit towards the subunit interface, resulting in a more compact quaternary structure of Fe protein. This motion shifts the Fe4S4cluster by about 4 A towmd the Fe prote~nsurface in the nitrogenase complex, oreo over, rotation of the subunits leads to a new dimer interface with interactions between highly conserved side chains of the monomers. Most of the structural changes in complexed Fe protein involve the CI helices and IOOPYsurrounding the structurally conserved central 0 sheet. The two mo~eculesbound per Fe protein dimer are oriented roughly parallel to the dimer interface, Each nucleotide is mainly associated with one monomer; the phosphate g ~ o u pi n ~ e r ~with c t ~ the P-loop and the ~ ~ ~ ~ c l e o s i d e away from the MoFe protein. F o r ~ a t i o nof the nitrogenase complex from Fe and MoFe protein seems to be a & c o ~ p a ~by ~ eshifts d in redox potentids of the i r ~ n - s clusters. ~ ~ ~ rIn a nondissociating complex of MoFe protein with a Leu-127 deletion mutant of Fe protein, a redox p o t ~ ofn -620 ~ ~ mV ~ was observed for the IFe4S4J2+'+couple of the Fe protein d [Sll. variant, wberea its potential was -420 m'v in the u n c o ~ p l e ~ estate ~~~

2.9.1, Occurrence and Biological Role

DNA repair is of fundamental importance to protect the genomic information in all cells. Enzymes of the helix~hairpin-hel~ (HhH) superfamily of DNA glycosylases initiate the base excision repair pathway by r e c o ~ i ~ i nand g removing damaged or ~ i s ~ a t c h ebases d from DNA. Endonuclease IJI (EG 4.2.99.18; D N A - ~ a p u ~ n ~ c or a p ~ ~ m i site) d ~ ~ lyase; c nTH gene product) from E. coli is such a DNA repair enzyme that contains one Fe4S4cluster with a redox potential of less than --600 mV in a 2l~-residue~ o ~ ~ ~ pchain. t i d It e has both a DNA ~ - g l ~ c o ~ y l activity ase to remove damaged pyrimidines from DNA and an a p u ~ ~ n ~ c / a p ~ i m ~lynse d i n iactivity c ~ ~ nofi cthe dam~ged that cleaves the C-0-1' bond 3' to the a p ~ r i n i c / ~ p y r i ~site strand via a fi elimination mechanism, thus leaving a 3'-terminal unsaturated sugar and a product with a terminal phosphate. A recent study on o~~onucleotides that contain a single modified pyrimidine has shown that endonuclease 111 cleaves the DNA backbone mainly through hydrolysis, and no 6 elimination product 1x1 does not directly interact was detected f521. The Fe4S4 cluster of endon~ic~ease

with the substrate or contribute to the catalytic activity, but has a s t ~ c t role ~ r ~ (see below). Its four cysteine ligands are arranged in a unique C y s ~ C y s ~ ~ C y s ~ sequence motif that Is conserved in a number of DNA repair enzymes t ~ o u g h o u t phylogeny, e.g,, endonuclease XII-type proteins were identified in archaea, yeast, mouse, md human. According to a recent classification 1531, the endonuclease 111 family of DNA repair enzymes has five members: (1)endonuclease 111; (2) MutY (EC 3.2.2.";~ ~ s p e cadenine i ~ c glycosylase), which excises adenine from ~ ~ i s with p ~ s ~~ i c~auDNA ~n guanine and El-oxoguanine;(3) ~ e t ~ a n ot ~~~ r~~ o~ a tu t~o frr o~~Mig, N-glycosylase that removes uracil or thymine mismatched with guanine; (4) endonuclease from ~ i c r a ~ o luteus, ~ u s which cleaves the N-glycosidic bond of the 5 ' ~ p ~ ~ i in~ ai np ~e i m i d i n edimer; (5) MpgXI, a ~ o n o f ~ n c t i o nmethy~~urine al ys~~~ DNA glycosylase found in bacteria and archaea with a ~ y s ~ ~ C y s ~ 2 Cironsulfur cluster loop, Some members of the MpgII group have lost this sequence motif, o ~ y ccontains e ~ Euncthus lacking the Fe4S4 cluster. Similarly, $ ~ ~ ~ ~ r cerevisiae tional h o ~ o ~ o g u eofs E. coli endonuclease 111 with and without the Fe4S4 cluster t54,551.

The crystal structure of E. coli endonuclease I11 at 1.85 A resolution ( only structure of this enzyme in the PDB. ~ n d o ~ u c l eI11 as~ is a monomeric basic protein of elongated shape with primarily a-helical secondary structure (Fig. 17A1, The malecufe consists of two s i ~ ~ l a rsized l y globular domains that are separated by a deep cleft on one side o f the molecule. Residues 22-132 form a so-called six-helix barrel domain of antiparallel helices, whereas residues 1-21 and residues 133-2111 constitute the Fe4S4cluster domain. The latter domain comprises a ~ - t e r ~and n ~ l three ~ ~ t ~helices ~ with i ~ thea topology l of a Greek-key helix bundle and the Fe4S4 cluster loop (FCL) with the cysteine ligands to the cluster (Cys-187, 194, 197, 203). Both domains have extensive hy~rophobiccores and charged s u ~ a c e they s ~ are connected by two hinge loops. The FCL folds as a right-handed spiral around the cubane cluster and packs against the four helices of the domain core, resulting in s h i e l ~ n gof the Fe4S4 center from solvent. The cluster of the native enzyme is resistant to O2 oxidation, but ferricyanide treatment partially converts it to an Fe3S4 cluster (less III is inactive. than 25%). Apo~nd~nuc~ease The proposed DNA-binding motifs in endonuclease XI1 are (1)the Hh the C-terminal end of the six-helix d o m (residues ~ 108-130) and (2) the FCLdmotif [561. They are at opposite ends of the interdomain cleft and lie within a positively charged and sequence-conserved surface region that can bind DNA in a sequencei n d e ~ ~ n ~manner. ent Specific recognition of damaged pyrimidines is provided by an ~ s the formation of active site pocket in the interdomain cleft. The FCZ c o n t r ~ h u t to the ~ N A - b i ~ d i nsurface g mainly by positioning conserved basic residues for interaction with the DNA phosphate backbone. ~ d u~lycosylase e from E, coli whose sequence is conserved MutY is a ~ ~ 0 - r e ~DNA from bacteria to humans. Compared to endonuclease 111 it has a 140-residue exten-

39

PIG. 17. The structures of the DNA repair enzyme endonuclease III (A, PDB code BARK) and of the catalytic core of the DNA glycosylase MutY (B; c ~ u PDB t ~code ~ l m ) are shown in the same orientation, revealing the siructw-al homology of the two enzymes. The proteins consist of two domains: a six-helix barrel domain (lower part) and a non-sequencecontinuous Fe4S4 clwtex domain (upper part). Both N and C termini of the proteins are located at the top of tho figures. In both proteins, DNA binding occurs at the intei"~omain cleft (occupied by imidazole in the structure of cMutY) which runs &om the Fe4S4 cluster to the h e ~ ~ - h a i r ~ ~ ~ nmotif, - h e lwhich ~ x has the short helix at the lower left of the figures as its first h e k

sion of unknown function at thc G terminus, whereas the N-terminal part i s sequentially and structurally homologous to endonuelease 111. The crystal structure of this hlly active c~talyticcore of MutY ~ ~ ~ uresidues t Y , 1-225) has recently been solved to a resolution of 1.4 (IMUY; Fig. 17B). cMutV has the same bilobal structure as endonuclease III, with a six-helix barrel domain (residues 22- 130) containing the HhW moiif, and the Fe4S4duster domain (residues 1-18 and 135-2251 c ~ r n p ~ i s i ~ g five a helices and the C-terminal FCL ~ C y s l ~ ~ X ~ C y s 1 ~ 9 ~ ~ C y s 2 0 2The X~Cys c~ly buried Fe4S4 cluster arranges loops and 3~ helices at the e ~ ~ ~ r o s t a t ipositive DNA binding surface and positions key catalytic residues such as Asp-138 npar the interdomain clef%.This cleft is about 5 A narrower than in the case of endomelease 111, but it still harbors the a d e n i ~ e - s p epocket, ~ i ~ ~ i.e., the active site of c ~The ~ substrate recognition role of the Fe4S4 cluster of MutY was also investigated by folding studies [571 and site-directed mutagenesis of the cysteine ligands [581.

I R ~ N ” § U L ~ UPROTEI R NS

399

2.10. ~ l ~ tPhosphoribosyfpyrophosphate a ~ j ~ ~ A ~ i ~ o ~ ~ a ~ s f ~ r a 2.10.1. ~ c c u r r ~ n and c e ~iologicalRole Glutamine phosphoribosylp~ophosphateamidotransferase (GPATase; EC 2.4.2.14; an~dophos~horibosy~ti.ansferase~ catalyzes the first reaction in de novo biosy~thes~s + E l 2 0 -+ of purine nucleotides: L-glutamine 5-pliospho-~-~-ribose-l-diphosp~te L-glutamate + 5-phospho-P-o-ribosyl-1-amine +- diphosphate, i.e., it transfers the glutamine amide nitrogen to 5”phospho~ibo~eGPATase is also the key ~*e~;yuiatory enzyme of this pathway because it is inhibited by adenine and guanine nucleotide end products of purine biosyntbesis, which bind to an allosteric A site and a catalytic C site, respectively. The synergistic inhibition by certain nucleotide pairs has been studied in detail 1591, Numerous GPA’l’ase sequences from eukaryotes, prokaryotes, and archaea are known and show pairwise sequence identities of the order of 40%. Only the enzymes from BaciEEus sublilis and E. coli are structurally characteri~edby LGPN. and lECB, LECC, LECF, 1ECG, IECJ, respecX-ray c r y s t a l l o ~ ~ p h(1A80, y tively), and both of them are homotetramers of similar overall structure. However, they represent two classes of GPATases: enzymes of the B. subtitis class (e.g,, in ~ t a ~ contain a Fe4S4cluster that is ligated humans, rat, chicken, ~ r o s o p ~ soybean) by four conserved cysteines, and are usually synthesized with an N-terminal propeptide, whereas the E. coli-type CPATases (e.g., in yeast, Haemophilus influemane, ~ ~ t ~ a ~ o c oj cac un s ~ ~ are ~ metal-free h ~ ~ } and lack the p r o p e ~ t i ~ Similar e. to the cluster in endonuclease 111, the Fe4S4 cluster of B, subtilis GPATase has a redox potential of less than -600 m y and does not participate directly in catalysis. It has a structural and a ~ e ~ lfunction ~ t o(see~below).

+

210.2. Molecular Structure of Clutamine Ph~ospharibosytpyrophospha~e

Amidotransfwase Each of the four identical subunits of B. subtilis GPATase consists of a single polypeptide chain of 465 aniino acids that is folded into two domains of similar size, with the Fe4S4cluster lying at the domain interface m d shielded from bulk solvent (Fig. 18).The GPATase tetramer is a doughnut-shaped molecule (Fig. 19) in which the subunits are related by three mutually perpendicular twofold symmetry axes. Xn m the t.etrameric and a funct~onaldimeric solution, there is art ~ u i l ~ b r i u between state. The N-terminal domain of‘ each subunit (residues 1-230) (Fig. 18) functions as “glutamine’~domain for glutamine binding and utilization. It contains a highly conserved cysteine at position 1 whose sulfur atom acts as the nucleophil~attacking the carboxamide of glutamine. The structure of the N-terminal domain resembles a four-layer sandwich with two antiparallel P sheets of seven and six strands, respectively, between two exterior layers of three R helices each. The C-terminal or “transferase” domain (residues 231-465) contains the substrate (PRPP) binding site for NH~-dcpendentsynthesis (catalytic C site) and also provides all amino acid residues d e ~ fi for the allosteric A site. This domain has a core consisting of a ~ v e ~ s t r a n parallel

400

FIG. 18. ~ t ~ ~of one t ~monomer r e of the homotetrameric glxdemine PRPP midotransferase from Bacillus sitblilis (PDB code lAoO). The N-terminal domain is at the upper right part of the molecule.

FIG. 19. Structure o f the Bacillm suhtilis ghtarnine PRPP a ~ i d o t r a ~ ~ f eletramer r a s ~ showing the symmetry and the ~ o ushape ~ of~the ~molecule t IPDR code MOO). The ~ o n oi s~ ~ shown in Fig. 18.

sheet packed between a helices in a topology similar to that of the six~stran~ed nudeotide binding fold. The C site is found in this core of the transferase domain e ~ (residues 341-3531. and is structurally defined as a conserved s t r a n d - l o o ~ - h motif ~~, Moreover, the t r ~ s f e r a s edomain contains a loop structure (residues 3 0 2 - 3 ~ calle the “Bag,” which forms a short double-stranded antiparallel p sheet and a short a helix, This “flag” is in contact with the glutamine domain of the n e i ~ h b o ~ subunit ng in the GPATase tetramer and is, as an important part of the A site between two subunits, involved in the end-product regulation of enzyme activity by bind~ngof feedback inhibitors. There are a total offour A sites between subunits of the tetramer along one of the twofold axes. Each of the four Fed& clusters of GPATase is coordinated by cysteines from one transferase domain: Gys-236, 382, 437, and 440. Although the B. subtilis ~ ~ A Tsequence ~ e contains the ]Fe,&& femedoxin-like sequence motif C y s 4 3 ~ X ~ C y s 4 3 7 ~ ~ C(Sec. y s 4 ~1.21, 0 Cys-434 is not a ligand to the duster but resides se in a loop on the outside of the molecule. Both oxidized and reduced G ~ A ~ a are e n z ~ a t i c ~active l y T60l. Due to the location of the cluster between the two d o ~ a ~ n s of a subunit and the position of Cys-236 in the peptide linker of the domains, the s t ~integrity ~ tof the ~ protein ~ critically ~ depends on the cluster. The Fe4S4cluster ~on p r e ~ ~ aacts b l as ~ an oxygen sensor in a mechanism to detect nutr~entl i n ~ i ~ a t via the increased O2 concentration that is a consequence of low levels of res~iration.Loss of cysteine ligation upon oxidative decomposition of the cluster destabilizes the tertiavy sLructure of GPATase, leading to denaturation of the enzyme followed by proteolytic degradation. Thus, the o~ygen-d~pendent inactivation of B. suhfilis ~ ~ ~ ~ appears to regulate, at least in part, de novo purine biosynthesis in this organ~sm. The c r y s t ~ l ~ ~ a pstudies h i c on GPATase have been reviewed [61,62]. be noted that the peptides su~oundingthe iron-sulfur cluster in B, subtilis and their homologues in the metal-free E, Cali enzyme adopt very similar conformations, suggesting a common cluster~cont~ining ancestor for the two classes of ~ P A T ~ [631. es

l ? r i m e t , h y l ~ id~eeh y ~ o g e n ~ ~T~~ e C 1.5.99.1) is an iron-sulfur flavoprotein ~ i l (bacterium u ~ that was isolated €?om ~ e t ~ ~ ~m o ~ trophus ~ ~It catalyzes ~ ~ the oxidative d e ~ e t h y l ~ t i o ofnt r ~ ~ t h y l a m i n (or e some ~yl-substituted S> to ~ m e t h y l a m ~ nand e formaldehyde. The physiological electron acceptor €3 is an electro~-tra~sferring flavoprotein (ETF). TIViA.DE is a homodimerr;each subunit has a molecular mass of about 83 kDa (729 amino acids) and contains one Fe4S4 by four cyshines, one cofactor that is covalently cluster that is coord~~ated bound to the protein (6-cysteinyl FMN), and one molecule of ADP. The detailed enzymatic mechanism of t r i m e t h y l ~ n eoxidation by TMADW and the intramolecu~~~

lar electron transfer are still a matter o f intense research E64-6131(see 169I for a recent review). Protonated trimethylmine binds to the enzyme and is oxidized by the FMN cofactor; then, fully reduced FMN passes one electron to the Fe4Sq cluster (redox potential of about -1-100 mV [70I), giving rise to a spin-spin interaction between the flavine semiquinone and the reduced cluster under high pH conditions f7lJ. I n t e r ~ o ~electron e ~ l ~transfer to the acceptor protein ETF occurs solely &om the one-electron reduced FeqS4cluster of Ti.MADI3C and is not the rate-limiting step during steady-state turnover [721. 2.1 1.2. i ~ o ~ e c u l Structure ar of T r i i ? i e t ~ i y l ~ m Dehydro~enase ~n~

The crystal structure of ( 2 T ~ Fig. ~ ; 2OA) shows that the ~ o ~ ~ unit m e r (Fig. ZOBf is folded into three domains: N-terminal domain I comprises more than half of the polypeptide chain (residues 1-384) and contains the Fe4S4cluster and the FMN with a distance of 4 A between the closest atoms o f the two cofactclrs. The FMN is bound to ~ y s - ~ whereas O, the Fe4S4cluster is coordinat~dby Cys-345,348,351, and 364 in a ferredoxin-type sequence motif (Sec. 1.2).The substrate trimeth~lammonium ion is bound through organic cation 71 bonding by three aromatic residues (Tyr-60, Trp-264, Trp-355) in the active site. The protein fold of domain I can be described as an eig~it~stranded pnrallel alp barrel. Domain I1 is not sequence-contin~ous(residues 385491, 644-704) and binds an ADP molecule of unknown function. Its fold resembles the FAD and Ni$.IlPN binding domains of g l u t a t ~ o n ereductasc! and can be ~ Domain 111 comprises residues 492-643 described as a three-layer a / f l / sandwich. and has a folding pattern of helices and fi sheets similar to that o f domain 11. ~~~

The ~ ‘ ~ y b r i dor’ ’ “ ~ ~ a ~ b aCluster ll’’ 2.12.1 Occurrence an,d Biological Role

In 1989, a so-callcd putative prismane iron-sulfur protein was discovered by Hagen and coworkers in the sulfate-reducing bacterium Desulfovibrio vulgaris (strain s t ~ c t uorf ~ this protein with very unusual spectro~ ~ l ~ e ~ br731. o r The o ~crystal ~ h ~ scopic properties 1741 was solved to a resolution o f 1.7 A in 1997 1751 and showed the presence of two unique Fe4 clusters (see below). The Eunctian of this protein (now termed Fepr or fuscoredoxin) is not known yet, but 011the basis of the X-ray structure it is generally assumed to have an enzymatic activity involving one of the Fe4 clusters. Another Fepr protein with 66% sequence identity to the D. vulgaris protein was isolated from D. desulfuricans 1761. The Fepr amino acid sequence (553 residues) us does not show homology with other known proteins; however, the N t e ~ * ~ i nis homologous to part of the sequence of CO dehydrogenase from ~ l o § t r ~ therrnod~u~ aceticum, Methanotrix soehngenii, Methanosarcina frisia, and Rhodospt’rillum r ~ as well ~ as torthe anaerobic ~ ~ ribonucleotide r e d u ~ t ~ from s e ELcoli, Sequences coding for Fepr-type proteins were identified in the genomes of a number of‘prokar-

IRON-SULFUR PROTEINS

403

FIG. 20. (A) Structuye of the iron-sulfur fiavoprotein trimethybamine dehydrogenase from ~ ~ ~~ ~. ~ ~e t~ ~(PDB oy ~code ~ o 2TMD). ~ t ~ ~~ Its ~i monomer ~~ ~ ~s (El s comprises three domains: the N-ternljnal domain (upper) i s considerably bigger than the other two and binds both the ftavin cofactor and the Fe4S4duster with electron tramfer function. Domain I1 (lowor left) binds an ADP molecule. ~

yotes and archaea: E. coli, T h i o ~ a c ~ ~ferrooxidans, l~s ~ o r ~ a ~ e morganii, llu Thermotoga rnarilima, Pyrococcus abyssi, Methanococcus jann,aschii, and ~ e t ~ a ~ o dtLenrroautotrophicum. ~ a ~ l ~ r ~ ~One~ of the most unusual properties of Fepr is that one of its Fe4 clusters, termed the ‘%ybrid’’ cluster, has four stable o x ~ d ~ tstates i o ~ with redox ~ o t e~ ~e t~ ~ i+300 e ~~ ~nand -200 mV, The four iron ions of this cluster are exchange coupled.

lecular Structure of thw ~esulfovibr~o sp. Frotein Fepr is an approximately 6Q-kDaprotein that consists of three domains. The Fe4 ‘ ‘ h ~ b r cluster i ~ ~ is found close to the interface of the three domains and at 12-13 A distance from a regular Fe4S4cubane cluster in the N-terminal domain (residues 1221, Figure 211, The latter cluster is close to the surface of the protein and is cool-&nated by four cysteine residues (Cys-3, 6, 15, 21) that are arranged in a unique sequence motif ~ y s X ~ C y s ~The ~ ~N ~terminus ~ ~ y ofs Fepr . is wrapped around cluster in a series of turns and loops before it forms the hs.l long hefix of the Fe4Sza the protein (residues 24-50). The second (“hybrid”) cluster o f Fepr has a total of seven protein ligands from domains 2 and 3. It is a unique Fe4 cluster with two p2-oxo and two p2-sulfido bridges and an additional unidentified p2 bridge X with partial occupantry in the crystal. X is possibly a small substrate molecul~tor ion) or a solventexchangeable oxygen. The protein ligands to the “hybrid” cluster are cysteines, a p ~ r s ~ f i d o c y ~ t e iglutarnates, ne, and a histidi~e{Fig. 22). The cluster is open and asymmetrical; only two iron ions (Fe5 and Fe6) and two bridging sulfur ions form a Fe2Szunit reminiscent o f one face of a cubane cluster. Fe5 is t e t ~ a ~ e ~coordir~ly

FIG. 21. The Fopr (“prismane”) protein from ~ e ~ u l vulgaris ~ o vcontains ~ ~ ~an Fe4S4 ~ ~ cluster and an ~ i ~ p ~ e c ~ d eE’ee, n t e‘‘hybrid” d cluster in s three-domain protein structure in which the hybrid cluster (cf. Fig. 22) is Imated centrally at the domain interface 1751.

nated by Cys-434, two bridging sulfurs, and X; in the absence of X its c o o r ~ a t i o n geometry is trigonal. Fe6 is bound t o Cys-312, the same two bridging sulfurs as Fe5 and to one bridging oxygen in a tetrahedral geonietry. Fe7 is probably the most by is-244, Gluu n ~ iron s ~ion ~of the “hybrid” cluster since it is ~oord~nated 2 ~ ~ ~~ ,s ~ a4bridgmg ~ 9 , oxygen, and X, re~ultingin a trigonal b i p y ~ a ~ in ~ dthe ; absence of X, the coordination of Fe7 can be described as square pyramidal. Fe8 is bound to Glu-494, to a persulfide group involving Cys-406, to two bridging oxygens, to one bridging sulf~irof the Fe5-3Fe6 couple, and p~ssiblyto X, resulting in an octahedral g e o m ~ twithout ~; X the geometry of Fe8 is a ~ ~ s t o r t tripnal ed bip~ra~~~. The N-lerminal domain of Fepr is mainly a-helical. Six of the helices form two antiparallel three-helix bundles, each consisting of one short and two long helices. It is an unusual feature of the structure that the two bundles are almost p e r p e n d ~ c uto l~ 2 2 2 - 3 ~and ~ ~3 (376-553) both comprise central 8 each other. ~ ~ m ~2 i(residues n s sheets s ~ r o u n d e dby helices in a three-layer [3-a-[3,doubly wound fold. A new 1.6 A resolution cyrstal structure of the “hybrid” cluster protein from D.udgaris is now available in the protein data bank (PD

FIG. 22. A closer look at the open structure of the Fed ‘%ybrid” cluster in the Fepr (“prismane”) protein fkom ~ ~ s u l f o ~vulgaris ~ ~ r z o(see text and Fig. 21 E757).Fe ions arc in dark gray, 3.i atoms of cysteines and the coordinating n~trogenatom of His-244 are in ,gray (thin spheres), bridging sulfides and the terminal wlfixr atom of the Cys-406 persulfide g o u p are represented by gray thick spheres, pz-oxa bridges and the unknown ligand X are evidenced as thick white spheres, whereas the carboxyl oxygen atoms of the coordinating Glu-2438 and 494 are depicted as thin white spheres. For clarity, the two Cys that ligate Fe5 and Fe6, i.e., the two iron ions forming an Fc2S2unil, are not shown (Cys-434 and Cys-312, respectively),

Fumarate reductase (EC 1.3.99.1) is an integral membrane protein containing FAD, three different iron-sulfur clusters, and at least two quinones l771. It belongs to a family of membrane-bound enzymes that function in central carbon metabolism and energy prod~ctionin cells. Bacteria and eukaryotic cells that can grow under anaerobic conditions often use endogenous fumarate as terminal electron acceptor in a respiratory process in the absence of oxygen. Fumarate reductase is the enzyme catalyzing the final reaction in this anerobic pathway; it reduces fumarate to succinate. Usually, the l o w ~ ~ o t e nelectron ti~ donor for this reaction in prokaryotee is the whereas eukarreduced n a p h t h o ~ u ~ ~ o~ nee ~ a ~ u i (or ~ ode~ethy~menaquinone~, ne yotic fumarate reductases were shown to interact with the benzoquinone rhodoquinone. As a respiratory enzyme, fumarate reductase is located in the cytoplasmic membrane of bacteria or in the mitochondrid inner membrane of eukaryotic cells. Succinate deh~drogenaseis a homologou~enzyme that catalyzes the reverse reaction of fumarate reductase, the oxidation of succinate to fumarate. It both is an enzyme of the Krebs cycle and constitutes complex I1 of the mitochondria1 aerobic respiratory chain. Fumarate reductase and succinate dehydrogenase are similar in primary struclly each other ture, cofactor composition, and mechanism, and can ~ u n c ~ i ~ n areplace under certain c o n ~ t i o n s[181. But in viva catalysis occurs only in one direction, and the cnzylnes are expressed differentially depending on the external conditions [791. 213.2. ~ ~ ~ ~ cStructure u l a r of Furnarate Reductase

The crystal structure of fumarate reductase from E. coli was recently ~ e t e i . ~ ~ at n ead resolution of 3-3 (1FUM). The molecule has a molecular mass of 121 kDa and c0nsist.s of four subunits in a modular q-shaped arrangement (Fig. 23A). The highly conserved flavoprotein (FrdA, 601 residues) and iron-sulfur protein (FrdB, 243 residues) subunits are water-soluble and located in the cytoplasm (equivalent to the matrix in mitochondria for succinate de~yd~ogenase~. They fomi the upper part of the “q” while the tail. of the “q” is formed by two hydrophobic ~ e m b r a n eanchor subunits (FrdC, 130 residues, and FrdD, 1J&residues). The FrdA subunit harbors the active site for fumarate reduction (succinate oxidation) at a covdently linked FAD. Except for the core FAD binding domain, which has a typical nucleotide binding fold, this subunit does not show structural simila~itiesto known folds in the PI2 the membrane anchor subunits FrdC and FrdD contains three t r ~ s ~ e ~ b helices connected by extramembrane loops. The membrane anchor binds two menaquinone molecules that are found at a distance of 27 on opposite sides of the ~ e m b ~ ~ e - s p a n nregion. ing The FrdB subunit of fumarate re duct as^ i s attached to the m e ~ b r a n eanchor and serves as linker to the catalytic subunit FrdA. FrdB is a unique iron-sulfur protein containing three distinct clusters in two domains (Fig. 23B). The N-terminal

A

A

I ~ ~ ~ - S U RL PROTEINS FU

407

FIG. 23. (A) The molecule of fumarate reductase (Frd) from Esckerichia coti (PDB code 1FUMi resembles the letter “g”. (B) The 27 kDa subunit €3 of Frd, evidenced in dark gray in {A), contains three iron-sulfur clusters (FezS2,Fe&, Fe&) in an almost linem array. The subunitrconstitutes a central part of the molecule with electron transfer function between the more external cofactors (FAD and m e n a ~ u ~ ~ o ~ i e ~ .

domain (residues 1-91) is structurally very similar to plant-type ferredoxins (see Sec. 2.3) and binds a Fe& cluster through a typical C37~57XqC;vs62~~6ys65X,~ys77 sequence motif (see Sec. 1.2). The C-terminal domain resembles bacterial dicluster ferredoxins (see See. 2.4). It contains an Fe4S4cluster that is coordinated by Cys-148, 151, 154, and 214, and an Fe3S4cluster that is coordinated by Cys-158,204, and 210. Tbis means that the cluster arrangement in FrdB i s reversed with respect to bacterial 7%-8S ferredoxins in which the Fe3S4 cluster ligands are always found at the N terminus of the protein. Moreover, there are substantial structural differences of ~ core structure: the short the all-helical 6-terminal domain to the ( f 3 ~ @ ) ferredoxin found on one side of the clusters in ferredoxins (ct Fig. 9, See. a n t i p a i ~ ~ l13e sheets l

2.4.2) have been exchanged by helical structural elements in BdB. The three ironsulfur clusters are ranged in an almost line= chain with cent~r-to-cen~er distances of 13-14 This electron transfer chain is extended on one side by the active site FAD , which i s 13 A from the Fe2S2cluster and on the other side by the proximal at a distance of 11 from the Fe&$ cluster, ~ e n a ~ u i ~ i in o nthe e m e ~ ~ r a anchor ne

A.

A

y 2.14.I .

~~ ~ ~~ ~ ~e d ~~ x ti ~

~

~

Occurrence and Biological Role

Fyruvate~~er~edoxin oxidoreductase (PFOR; pyruvate synthae, EC 1.2.1.1) is a key enzyme in the energy metabolism of anaerobic microorganisms. It catalyzes the final step in carbohydrate f e ~ e n t a t i o nin which ~ ~ u v ai st oxidized e to a ~ e ~ y l " ~A ~ n 3-CO-COOH CoASH --3 CH,-CO-SCoA c GO2 2H 2e3, coupled to the reduction of femedoxin or Aavodoxin as p h ~ s i ~ l o ~electron cal acceptors. The enzyme is found in archaea, anaerobic bacteria, and a m i t . o c ~ o n ~ i aeukaryotes. te It contains Fe4S4clusters with electron transfer function and a thiamine ~ ~ o p h o ~ ~ h a (TPP) cofactor whose 6-2 aturn is supposed to be the reactive center for the oxidative decarboxylation of pyruvate. PFORs are complex enzymes with different oligomeric state and s u ~ u n icompositio~ t in difTerent o ~ ~ a n i1801. s ~ sThey occur as ~ o ~ o d ~ m c r a dimer of a heterodimer ((010)~)~ or a dimer of a heterotetramer ((olPy6>,). There has between the different been a great deal of interest in the evolutionary ~el~tionships ~ s ~to the conclus~onthat four ancestral genes were rearranged types of P ~ O leading and eventually fused to form the single large subunit typically present in enzymes from m~sophilicorganisms IS1,SZl.

+

+

+

lecular Structure of Desulfovibrio africanus P y r ~ ~ ~ a ~ e : ~ e r ~ e d o x i n ~x~~ore~~c~as&

Recently, the crystal structure of the ffee homo&meric PFOR from the sulfateu l~f o~ ~a~ ~~r i ~o was c d ae t e~~ m~~ ~ ate~ad resolu ti:n of 2.3 reducing b ~ ~ t e~ re i~ ~ ~ ~ B together ~ F ) with the structure o f its complex with pymvate (3.0 A resolu1. The D,a ~ r ~ c enzyme ~ n ~ shas a ~ o l e c u mass ~ r of nbout 266 kDa and is currently the only PFQR whose three-dimensional structure is known 183J. Due to high sequence homology it can be considered as representative of other homo~ sm .o n o ~ e rconsists of a single chain of 1231. amino acid dimeric ~ ~ O Each residues; this is presently one of the longest continuous polypeptide chains invesp h yare . threc Fe4S4 clusters tigated at high resolution by X-ray c ~ t ~ l ~ ~ aThere and one TPF cofactor per subunit of PFOR. Each subunit o f the hornodimer consists of seven structural domains of different size and function (Fig. 24). ~ o ~I ~ a r ~~s i d su eWZSS>, s 11 (residues 259-4151, and VI (residues ~ S 6 - 1 ~ ~ 0 > form the core of the enzyme; they are responsible for the majority of the tight mbunit ~ n t e r a ~ i o nand s For the binding of TPP which is deeply buried in the

A

--." FIG. 24. One subunit of thc h o ~ ~ o d i ~p ~e v~act e : f e ~ e d o x i on ~ j d o r ~ u c t a sfrom e ~ e s u ~ africanus f ~ ~ (PDB ~ ~ code ~ o1BOP) conskts of a single polypeptide chain of 1231 residues that i s folded into seven domains (indicated by roman numerals). Each subunit contains t h e e

Fe4S4 clusters and a t ~ i ~ i ~n e~ ~ o p cofactor. ~ o ~Two ~ ofh the a clusters ~ ~ are bound by a ferredoxin-like part of domain V, whereas the third one i s situated in domain VL, close to the thiamine ~ ~ o p h ~ s pcofaetor ~ a t c (depicted in a stick r e p ~ e s e n t a ~ iat o ~the ) interface o f domains I and VT. The disulfide bond between Cys-1195 and Cys-1212 In domain VII is aho shown because of its importance for the oxygen stability of the enzyme (see text,.

active site pocket at the inter€ace of domains I and VI. A large part o f the t h e e core domains exhibits a fold similar. to the ~ o ~ o d i ~ eenzyme ric tr~nsketol~~e ~ 1which ~ also contains ~ TPP. ~ The ) accessory ~ domains IIf (residues ~ 1 ~ - ~ 2 7 ) (residues 6 2 ~ 6 ~ $anti ) , Y (residues 669-785) do not contribute to the subunit interf~cebut lie at the exposed surface o f the protein. Part of domain V has a c ~ o ~ ~ ~e ~~~ . ~ e d io xa~%Id ~~ - l iand ~ e contains two Fe&, clusters bound by eight cysteines in a ~ e q u ~ ~motif c e t ~ ~ o~f 8Fe-8S c a ~~ e (see Sec. ~ 1.21. e

Domain VII of D,afrzcanus PFOR (residues 1171-1232) constitutes a C-terminal extension with respect to other homodimeric PFORs and is responsible for the unusually high oxygen stability of the D.africaaus enzyme. Except for a C-terminal cx helix it does not form any secondary s t ~ c t ~ r ea ll e ~ e n tbut ~ has an extended loop structure that partially embraces the other subunit. Domain VII of one monomer almost reaches the active site of tlie other and shields the Fe4S4 cluster in domain VI, which is very close to the pyrophosphate group of the TPP cofacclor and about 13 from the reactive C-2 atom of TPP. The protection effect of domain VII critically depends on the stabilization of its loop structure through a disulfide bridge between Cys-1195 and Cys-1212. Reduction of this diof the enzyme to sulfide bridge with sulfhydryl reagents leads to high ~ensit~vity oxygen and at the same time to a higher enzymatic activity [84]. The PFQR crystal structure suggests that both effects are caused by a ~onformation~l change of domain VII upon reduction of the disulfide bridge that would (1) leave the domain VI Fe& cluster (proximal to the active site) solvent exposed and therefore prone to oxidative degradation, and (2, open a new short channel for diffusion o f substrate and products between the active site and the protein surface 1851. The Fe4S4 cluster in domain VI is coordinated by four cysteines in a ~ y ~ ~ ~ ~ y ssequence ~ ~ ~ motif y s (Cys-812, x ~ ~ 815, y s 840, 1071) and has a distance of a ~ p r o ~ m a t e 16 l y from the closest Fe4S4 cluster in the ~ e r ~ e d o ~ n - ldomain ike V, which in turn is about 12 A away from the other (distal) Fe4S4 cluster of PFQR in the same domain. All distances between the redox cofactors of PFOR are compatible with efficient electron transfer from the pyruvate-TPP binding pocket to the distal FeBSd cluster and from there to the ~ h y s i o l o ~electron c~ acceptor ferredoxin. Recently, the complexes between PFOR and three ferredoxins from I), ~ ~ r were ~ characterized ~ a ~ by ~ measurem~nts s of electron transfer rates f861. The implications of the structure of I?. ufricaaus PFOK, for the molecular organization of PFOR enzymes with different oligomeric states have been discussed by Chabriere et al. 1851.

A

A

Ribonuc~eotidereductases (EC 1.17.4.-; R N b ) catalyze the reduction of ribonucleoin all organisms and thus play a key role in the regulatides to deox~ibonucleotid~s tion of DNA synthesis and replication. There are three differencl classes of ~ ~but ~ all of them use a protein free radical for activation of the ribonucleotide substrate (see ($71 or 1881€or recent reviews). Class I RNRs contain nonherne iron and use a t ~ o s y l / cysteinyl radical pair (see Chapter 111, class I1 adenosylcobalamin RNRs use a cysteinyl radical, and class I11 RNRs use ~-adenosyI~nethionine and an i r o n ~ s u l ~ ~ ~~~~

cluster to generate a protein glycyl radical. Only the latter class will be discussed in this p ~ a ~ a pwith h , emphasis on the role of the cluster. Glass Ilf RNRs or genes for these enzymes are found in strict and facultative anaerobic microorganisms (eubacteria and archaebacteria) and in the bacttjlrioe this class. It is phage T4. The anaerobic RNR from E. euli is the ~ r o t ~ ) t y pfor a ribonucleoside triphosphate reductase (EG 1.17.4.2) that is different from the ribonucleoside diphosphate reductase (EC 1.17.4.11 employed during aerobic ~ o w oft E. ~ eoEi, The anaerobic enzyme in its active form i s an gap2 heterotetramer protein complex in which the large a2 subunit (2 x 80 ma; NrdD) harbors at Gly-Cj81 the stable, ox~gen-sen~itive glycyl radical. The tightly as~oc~ated pa subunit (2 x 17.5 kDa; NrdG) contains an Fe4S4 cluster that is assumed to link together the two f3 polypeptides l89,901, similar to the cluster in nitrogenase Fe ~ R ~ are R strictly related protein (see Sec. 2.81. The c a t ~ ~pi rc~ p e ~ofj eanaerobic to the presence of the Fe& cluster auld it was demonstrated that reduced by the cluster during activation of the enzyme, which leads to f o ~ a t i o n of ~ethionine,5 ' " ~ ~ u x y a ~ e n ~ sand i n e ,the glycyl radical in a2. In the ~roposed reaction mechanism, SAM binds t o the reduced [Fe4S4]+cluster, accepts one electron from the iron-sulfur center, and is cleaved to methionine and a 5'-deoxyadenusine radical which then generates the glycyl radical in a2 1911. Thus, the small iron-sulfur pz subunit serves as anaerobic RNR-activating protein through a redox reaction at its iron-sulfttr cluster. The ciystal structure of the metal-free az subunit of the anaerobic RNR from bacteriophage T4 was recently solved to a resolution o f 2.75 (PDB entry IB8B). PwmvatrJformate-lyase activating enzyme (EC 1.97.1.4; PFL activase, formate acetyltransferase activating enzyme) is a functional analogue of the small Ba subunit (PFL; EC 2.3.1.54) is an important enzyme of anaerobic RNR. Pynrvate formate~~yase ~ ~ scatms of the anaerobic glucose f e ~ e n t a t i o nin E. coli and other m i c ~ ~ o ~ g that alyzes the CoA-dependent cleavage of pyruvate to acetyl-CoA and formate. The active This radical is enzyme contains a stable glycyl radical ~ G l yrequ~red - ~ ~ for ~ catalysis. ~ introduced posttranslationally through PFL activase, which abstracts one from My-734 of PFL in the following reaction:

A

+

f -NH- CH, - CO - l p ~~-adenos~~l-L-methioni~e ~ t dihydro~avodoxin--+ ( -NH - 6 H -CO -ppL 3- 5 '-deoxyadenosine ~ ~ e t h i o n+i ~ e av~~~oxin

+

As in the case of anaerobic RNR, a 5'-deoxyadenasyl radical, generated by reductive cleavage of SAM, has been proposed as the species that actually abstracts the hydro~ c e dacid sequences of PFL activase are gen atom in the PFL system. ~ ~ A - d e d ~amino known for a number of microorganisms (E. coli, C. pasteuriaizurn, ~ ~ @ ~ influenzae, ~ t ~ e ~ t o mutans) ~ ~ ~ cand u s revea1 an approximately 25~-residueprotein with a conserved ~ ~ ~ ~ motif ~ near y the ~ N~terminus. C y Thes same sequence motif exists in the p2 polypeptide (about 155 amino acids) of anaerobic RN activase was recently shown to be an iron-sulfur protein c o n ~ a i ~ imost i n ~ probably an Fe4S4cluster in its active form i92,93 I. Whereas Broderick et al. [92J found that the Fe4S,, cluster can be reduced to the I Fe4SJS state with dithionite only in the presence

o

of adenosylmethionine and suggested a s u b u n ~ t - b r ~ d ~Fe4S4 n g cluster that can undergo o~gen-media~ed conversion to an FezS2 cluster, Kiilzer et al. [931 reported that the iron-sulfbr cluster of reconstituted PFL activase is reducible by dithionite in the absence of a d e n o ~ ~ l m e t ~ ~and i o ~that ~ ~ nthe e ~econstitutedenzyme is ~ n ~ n o ~ e r i The latter group also showed that the iron-sulfur cluster is a prerequisite for effective to PFL activase aid p ~ o p ~ ~ one the d basis of sitebinding of a~enosylmethion~~e directed mutagenesis experiments that the thrce cysteine residues in the ~ y s ~ ~ sequence ~ y smotif ~ serve ~ ~as sligands to the i r o ~ ~ s u l f ucenter r of PFL activase.

Biotin i s an essential vitamin synthesized by microorganisms and plants and t o r can czrry activated GO2. The find step of biotin bioserves as a c ~ ~ f ~ cthat synthesis consists of the insertion of sulfur into detliiobiotin to form a tliiopbene ring via a radical mechanism. This reaction formation of two C-S bonds) req~~ires the iron-sulfbr protein biotin synthase, SAM as a source of the initial 5'-deoxyadenosyl radical, and a reducing system [94. Thus, biotin synthase (EC 2B.1.6; bioB gene product) belongs to the same family of Fe-S enzymes as anaerobic ri~nucleotidereductasc and pymvate forrnate-lyase activase (see Sec. 3.11, The r ibiotin c ~ synthase s ~ protein was isolated from E. coli and Bacillus ~ ~ ~ ~ ebut sequences from 15 different organisms (mchaea, bacteria, yeast, plants) are currently known hi the ~ ~ S datn Sbank. -They ~ o ~ r ~e s ~ oto~n~d ~ ~l ~ e poft ~ d about 40 kDa and all contain a putative cluster-ligating sequence motif, ~ y s ~ ~ G ~that s ~ is~ also ~ y found s , in a~aerobicENR and PFL activase (Sec. 3.1). R e c o m b i ~ ~ nE.t coli biotin synthase in the as-isolated form after aerobic p u r ~ ~ c a t is i o a~ bomodimer of subunits with 346 amino acid residues and contains one Fe2S2 cluster with incomplete cysteine ligation in each monomer. Anaerobic reduction with dithionite in the presence of 2 55% (vh1 glycerol or ethylene glycol, but without addition of iron or suEde, converts the form with two Fe2S2 clusters into a species that has one Fe4S4 cluster with complete +2e- 4 fFe4S4]2", This cluster c o ~ v e r s ~iso ~ cystejiie coordinat~on: 2[Fe2S2I2+ reversible, i.e., upon exposure to air the Fe4S4 cluster i s reconverted to the original Fe& clusters ~ 9 5 ~These 9 ~ ~results . were i ~ t e ~ p rin e tthe ~ sense that the Fe4S4 cluster is formed at the dimer interfbce via reductive dimerization of two Pe2S2clusters and that it is therefore a s u b u n i t ~ b ~ d ~ Fe4S4 n g cluster like the one in nitrogenase Fe protein (See. 2.8). It was also suggested that the presence v aed a t i o nof an of Fe2S2 clusters in purified biotin synthase i s due to ~ ~ ~ a tdie ~ ~ n t e r s u b u ~Fe4S4 t cluster during aerobic isolation [961. As far as the mechanism of biotin syynthase is concerned, there i s increasing center ~ r o ~ dthe e s sulfur evidence that it is not, a catalyst E971 but that its ir~n-sul€ur for biotin formation [98-100f; W and free cysteine were ruled out as sulfur donors in an in vitro assay [loll.

Very recently, lipoic acid synthase, which catalyzes the final step of lipoic acid s ~ t h e s i in s microorganisms and plants via the insei~ionof sulfur into the octanoic nt was shown to be an iron-sulfur protein acid backbone in a S ~ ~ d e p e n d ereaction, with properties similar to those of biotin synthase. The reconstituted recombinant e n ~ y c~o en t a i ~an Fe2,Szcluster per protein that can be reduced to an Fe4S4center by reduetion under anaerobie conditions f102l. This protein also contains the sequence motif CysX~CysX~Cys, which seems to be one of the d e t e ~ ~ of~ t s F e ~ ~ ~ / F ecluster 4 S 4 conversion. The same sequence motif is found in lysine 2,3-aminornutase (EC 5.4.3.2) from Clostriddiumsubterminale, another 8M-dependent iron-sulfur enzyme that catalyzes the isomerization of t-lysine and ~3~)-3,~-diaminohexanoate via a substrate radical r e a ~ ~ g e m e ~n et c h a n ~ s that r n is suggested to initiate with the abstraction of a hydrogen from C-3 of lysine by a 5'-deoxyadenosyl radical. It is postulated that the ~etweenthe one-electron reduced Fe4S4cluslatter is generated through interac~~on ter of the enzyme and SAM [ladl.

~e~edoxin:t~oredo reductase x~n ~F~~~is a 4Fe-4S protein functioning as disulfide reductase in the light regulation of carbon metabolism in oxygenic photos~thesis [104,105].It is found in the stroma of plant chloropl~stsand catalyzes the twoelectron reduction of the active site disulfide bridge in thioredoxin by sequential one-electron oxidation8 of a 2Fe-ZS ferredoxin, which had been reduced before through light-d~vennoncyclic electron flow from the chlorophyl1"containingthylakoid membrane. The reduced thioredoxin then activates or deactivates several carbon assimilation enzymes by reduction of regulatory disulfide bridges. FTR is a heterodimor that consists of a variable subunit (7-13 kDa) and a highly conserved (13 XnlSa). The lattcr contains seven conserved cysteine residues c a t ~ ~subunit ic whose functional role was deduced from biochemical modification e ~ e r i m e n t swith spinach FTR [1061: Cys-27 is a free cysteine without catalytic function, Gys-54 and $4 are disulfide-b~"id~ed in oxidized FTR and constitute the active site for thioree reaction, and Cys-52, 71, 73, and $2 doxin reduction in a t h i ~ l - d i s u ~ dinterchange coordinate an Fe4S4 cluster in a unique ~ y s X ~ ~ y s X C ~ s X sequence , ~ C y ~motif. This distributioii of the cysteines along the sequence clearly implies close pro^^^ of the active site cysteines (or the cystine disulfide formed by them) to the Fe& cluster because only one residue separates each of these cysteines from a cluster ligand. state by The [Fed&$+ cluster of FTR can be partially oxidized to the lE'e4?34]3rp o t a s s i u ~ferricyanide, ~ but it is not reducible to the CFe,&34f" state by dithionite or other strong reductants. Therefore, the clusker i s most likely not involved in electron transfer from 2Fe-2S ferredoxin to the active site disulfide of oxidized FTR. FTR reduces thioredoxin in a reaction in which cysteines 54 and 84 of the reduced enzyme are oxidized to a disulfide while the active-site disulfide of thioredoxin i s reduced to two cysteine thiols. Spectroscopic studies strongly suggest that

the Fe4S4 cluster of FTR serves to stabilize a one-electron reduced intermed~ate generated by initial reduction of the Cys-54iCys-84 disulfide after the first electron t ~considered as a cluster-b~se~ donation by a 2Fe-2S ferredoxh. This i n t e r m e ~ ais radical species in which the cluster is formally in the [Fe,S4]3t state and coordinated by five cysteines through a novel p3-S-S{Cys) finkagc. While one of' the cysteines tCys-84) in the one-electron reduced disulfide i s attached to the cluster in this manner, the other (Cys-54) is free for nucleophilic attack of the substrate disulfide in thioredoxin and forms a hetero~is~lfide. The second electron then cleaves the cluster-associated p3-S-S(Cys-84) bond so tpM) could have been available in the seawater during the first billion years of biological evolution. The sudden capability of the photosynthetic machinery to use H20 as a reductant 2-2.5 billion years ago led to the large-scale production of 0 2 and a fundamental change in the conditions for life on Earth. Oxygen-mediated oxidative damage disturbed many cellular processes, and the global precipitation of iron as ferric hydroxides, induced by the rising levels of oxygen in the atmosphere, made iron a rare element for living organisms. Thus, at present atmospheric redox

N O ~ ~ L ~

464

potentials, the iron availability in sea and wetlands is lower than M. Nearly all living o r g ~ i s mhave s iron as an essential element and the harvesting of iron from the environment constitutes a major challenge for most organisms Fl,2l. Aerobic microorganisms have evolved "iron mining" systems where they excrete siderophores into their surroundings, which are low-M, organic compounds serving as high-affinity ligands for ferric iron (Fig. 1).A large variety of siderophore complexes are synthesized by different bacteria to perform this function, many with iron dissociation constants in the pM range. Uptake of the iron siderophore complexes is mediated by specific receptors in an energy-dependent process. M e r active transport over the outer membrane the periplasm and the inner membrane, release of iron from the siderophores appear to be mediated by reduction and possible ligand degradation. Anaerobic microorganisms, on the other hand, can access iron directly as Fe", which is sufficiently soluble to be imported without chelators. In mammals iron is extracted from foodstuff in the gut. It can be stored in cells or transported to other cells by transferrins, which are proteins serving as intercellular transport vehicles for iron in the body. Transferrins are monomeric bilobal proteins with high affinity for ferric iron (Fig. 2 ) . The iron-loaded transferrin binds to the transferrin receptors exposed on cell surfaces. When sufficient amounts of t r a n s f e ~ i n " ~ r a n sreceptor f e ~ ~ ~complexes are assembled, the formation of intracellular vesicles is induced. These contain the loaded transferrin-transferrin receptor complexes in their interiors. These so-called endosomes have a lowered p leads to the release of iron. After iron is released apotransferrins are subsequently exported to the exterior of the cells and can be involved in further iron transport. In animals, plants, and some bacteria, iron is stored inside the coat of ferritins as cores of hydrous ferric oxides ~ ~ emixed with ~ phosphates ~ ~ [31. Ferritin ( ~is in most organisms formed by a 24-mer hollow protein shell, with the capacity to store

(a)

Iron loaded transferrin

FTG.1. Iron homeostasis and metabolism in (a) mammals and (b) bacteria.

~

XYG ENlNITROGEN PROTElNS

465

FIG. 1, continued

several thousand iron atoms by oxidizing ferrous iron by molecular oxygen. The mechanism for the ferritin storage process appears to involve two types of oxidation processes, one at a protein-coordinated, so-called ferroxidase site, and the other being an autooxidation of iron on the ferric oxide core. The mechanism for unloading iron from the ferritin shell as well as regulating the amount of free iron by redox control and ligation is largely unknown. The ferritin biomineralization process for storing iron probably also serves as an important means for detoxifying intracellular iron by making ferric adducts unavailable for unspecific binding and for keeping ferrous iron levels low in order, to limit uncontrolled dioxygen-derived radical chemistry. Another form of iron is heinosiderin, which is a term used for several different nonproteinaceous deposits of iron found in certain cell types and tissues. Biomineralized iron in the form of magnetite can be formed by, for example, bees, homing pigeons, and magnetotactic bacteria, where the magnetite nuclei play direct functional roles in the navigation of these organisms.

1.2. Iron and The selection of iron as the dominant redox metal in biological systems may, in addition to the large availability of iron during the early days of terrestrial life, be derived from its versatile redox and coordination properties. Variation of the Iigand field of iron can strongly alter its structural and ligand exchange properties and allow

466

NO~DLUND

FIG. 2. Iron transport in mammals is carried out by members of the transferrin family. Structure of the iron-binding lobe of two transferrins: (a) human transferrin in the iron-free form; (b) horse lactoferrin in the iron-containing form; (c) the coordination sphere of iron in lactoferrin. The presence of bicarbonate as an exogenous iron ligand renders the iron binding pH-sensitive, allowing the release of iron in the low pH of the endosomes [generated from PDB files: (a) lBTJ and (b), (c) lBlX].

fine-tuning of redox potentials as well as provide appropriate coordination environments for electron transfer or enzymatic reactions. According to the hard and soft acid-base principle, hard oxygen ligands are preferred coordinating groups for iron, but chclates of nitrogen ligands as well as the soft sulfur ligands are important in proteins. Typical coordination distances for imidazole nitrogen and carboxylate oxygenase with iron are about 2.2 and 2.1 but significant deviations of this are present due to local hydrogen bonding. Ligands for tetrahedral iron are typically cysteinyl thiols, having remarkable similarities of bond angles and distances among the iron-sulfur clusters found in proteins.

A,

IRON-OXYGEN/NITROGEN PROTE1NS

467

Nonheme ferrous and ferric complexes with proteins are nearly always found as high-spin iron, in contrast to heme iron which can form diamagnetic complexes due to strong ligand field interactions. High-spin iron is preferably coordinated as octahedral or pseudooctahedral complexes, but bulky side chains, such as thiol ligands, can allow the formation of tetrahedral complexes. Binuclear iron clusters can form that are most often antiferromagnetically coupled, whereas mixed valent diiron clusters display a nonzero net spin. The potential of the Fe(IIj/Fe(III) pair is highly sensitive to the ligand type, where, for example, CN-, aqua, and phenol ligands give potentials E' = 0.36V, 0.77V, and 1.12V, respectively. Thc polarity as well as the flexibility of polar groups in the second-sphere environment of the iron site in the protein matrix will effect the redox potentials of the site, as well as the pKa values of coordinating groups 141. The differences in spin state between O2 and most organic compounds render them inert to reactions with 02.Fe(I1j compounds, on the other hand, are notoriously reactive with 02,although some salts are reasonably stable against air (e.g., Mohr salt). The ability of the Fe(I1) ion t o break the kinetic barrier for reactions between 0 2 and organic compounds, by initial activation of the Oz molecule, renders it a major participant in living systems to harvest the oxidation potential of the dioxygen molecule for transformation o f organic substrates. Dynamic properties of the ligands of metal sites are important determinants for their reactivity. Conformation change, including ligand exchange, controls the coordination environment o f the metal site and allows the generation of binding sites for substrates and exogenous ligands. In general, the rearrangement of iron does not occur at the fast rates seen for some other metal ions. In biological systems, this property might be beneficial for the steric controlling of the catalytic events. In nearly all cases, Fe-S clusters in proteins coordinate iron in a tetragonal geometry and only minor reorganization takes place upon changes in the oxidation state [51. In contrast, the coordination of iron in the Fe-O/N proteins discussed below normally displays significant distortions from symmetrical coordination environments and often contains open coordination sites for binding of substrate and exogenous ligands. The coordination environment of most Fe-O/N proteins displays significant flexibility, and upon redox changes or substrate binding the coordination number and/or coordination geometry of the Fe-ON site changes. Carboglate ligands can easily vary their mode of coordination, and protonation of tyrosine ligands can modulate their bond lengths and even make them dissociate. Binding of solvent and substrates depends on the redox state and protonation state of the metal and the local environment in the active site. However, histidine ligands normally maintain their coordination geometry in Fe-O/N proteins, upon redox changes, and appear to serve as a more rigid framework for nonheme iron coordination, as discussed below.

N~~DLUND

468

2.

I

The classification of Fe-O/N proteins can be made in several different ways, emphasizing various aspects of their biology and Chemistry. For the discussion in this review, the Fe-Q/N proteins are classified into four main groups based primarily on the mechanistic role of the iron ion(s) (Table 1):(1)proteins with substrate-activating mononuclear ferric Fe-O/N sites; (2) proteins with 02-activating mononuclear Fe-O/N sites; (3) proteins with O2-activatinghindingdinuclear Fe-OIN sites; and (4) hydrolases with Fe-QIN sites. Within these four classes, the subclassification is made primarily on the structural homoloffyievolutionary origin within each individual subclass, a classification that in most cases closely coincides with mechanistic similarities. A majority of the Fe-O/N proteins have as their main function the harnessing of the oxidative power of 0 2 . The preferred mechanism for solving the kinetic problenis of activating O2 for organocheniistry in nature is by using Fe(I1) sites. The different families of 02-activating Fe-QIN enzymes, using mononuclear or biiiuclear Fe(I1)O/N sites (groups 2 and 3 above), provide a fascinating spectrum of detailed implementations of this concept. The catalytic machineries of the different enzymes generate activated iron species, with different catalytic potencies. The activated iron species can in different contexts catalyze a spectrum of different oxidative substrate transformations, such as desaturations, oxidative cycliaations, mono- and dioxygenations, hy~roperoxidation,epoxidation, and one- and two-electron transfer reactions. In some Fe-QIN proteins substrate binds in a highly specific manner, whereas in others a broad substrate specificity is provided. Some families provide access for highly charged substrates to their active site, whereas others nearly exclusively act on noncharged highly hydrophobic substrates. A thorough understanding of the evolutionary implementation of this important chemistry into different biological functions is now emerging from the available sequence, structural, spectroscopic, kinetic, and mechanistic data on the Fe-QIN protein families. This emerging picture not only demonstrates the power of divergent evolution in varying and refining hnctionalities on the same structural and catalytic theme but also gives examples of convergent evolution, where, for example, very similar iron coordination environments have evolved onto different protein frameworks for catalyzing related chemistry. The convergent scenario is particularly evident in the 2-His-l-Asp/Gluproteins (group 2 ) extensively discussed below, wherc the same iron coordination environment has been introduced into different three-dimensional structures, with interesting similarities and differences in their meclianism of action. '%'hisevolutionary view of the mechanistic aspects of these enzyme families also assists in determining their true molecular mechanisms by allowing the identification of common and distinct mechanistic features of the catalyzed chemistry through comparative studies. Of particular interest is understanding the mechanisms of Q2 activation, how thc different reduced O2 adducts are coordinated and controlled on the iron sites, and how the oxidative power of these adducts is directed into producing the right product, without damaging the protein environment. In the

469

I RON-OXYG EN/NITROG EN PROTEINS

TABLE 1 Iron-Oxygen/NitrogenProtein Families

Primary reaction Substrate-activating mononuclear FeCIII) enzymes: Lipoxygenases Intradiol dioxygenases

02-activatingmononuclear Fe(TI) enzymes (2-His-lAspiGlu proleins): Pterin-dependent liydroxylases 2-Oxoglutarate-dependent oxidases" Isopenicillin-N synthase" Type I extradiol dioxygenasesb 4-Hydro~plienylpy~vateb dioxygenase Type TI extradiol dioxygenases Rieske-type as-dioxygenases

Hydroperoxidation Oxidative catechol ring cleavage

None None

Representative

lLOX 3PCA

Aromatic hydroxylation Tetrahyrlrobiopterin lDMW IDRY, lRXG Alkane hydrorrylationi2e- oxidations 2-Oxoglutarate Double oxidative ring formation Oxidative catechol ring cleavage Coupled decarboxylation and aromatic hydroxylation Oxidative catechol ring cleavage Aromaticialiphatic cisdihydroxylation

02-activatiigibirLding diiron proteins (diiron carboxylate proteins): Hemery thrin Ribonucleotide reductases R2" Methane monooxygenasec

Reversible O2 binding le- tyrosine oxidation Nkane hydroxylation

Stearoyl-carrier A"-desaturase'

Alkane desaturation

Rubrerythrinl' Ferrit insibacteriofe~tins",'~ Iron-dependent hydroluses: Purple acid phosphatases Nitrile liydratase

0210,

a.6.c

Secondary electron donors

/FIJI2 scavenger (?I Ferroxidase Phosphoryl transfer Nitrile hydration

None None None

lBLZ lHf?a, lMPY

None NADPH-flavin reductase + Rieske center

IBFU lNDO

lCJX

Fc(I1) (?) NADP flavin reduclase + Fez& site NADP flavin 1AFR reductase --+ Fe& ferredoxin NADPN,Fe(I1) (?) 1RYT Fe(1I) lBFR IKBP, LUTE 2AHJ

The iron binding domains of these proteins are distantly related in structure (and evolution) to the other protein with the same footnote label. All other protein families have unique structural folds. "Not extensively covered in this chapter; see Chapter 12.

470

NORDLUND

case of hemerythrin, the 0 2 molecule i s bound reversibly and the O2 bond is not cleaved, a reaction that requires other types of kinetic control functions. The monoand binuclear 02-activating enzymes, however, have proven to be diffkult targets for obtaining conclusive mechanistic evidence; therefore? many uncertainties remain regarding the roles and structures of intermediates in the reactions catalyzed by these enzymes. The 02-activating iron(II1) proteins (group 1) constitute an interesting exception to the use of iron ions in biological systems. In lipoxygenases and intradiol dioxygenases, the Fe(1II) form is the active species. According to the generally accepted mechanism for these enzymes, the roles for the Fe(lI1) sites are in activating the substrates for O2 insertion by delocalizing electron density from the substrate. Although this concept for handling the O2 activation problem might provide a safer path of 0 2 activation, it appears to have found much less application in nature than the Fe(I1) strategy discussed above. One could speculate that the reason for the poor applicability of the Fe(l’i1) concept could be its limited potential for evolution due to the high dependence of the reaction on the redox properties of the substrate. One of the most common functions of metals in biological systems is in hydrolases, where they enhance the nucleophilicity of water molecules by coordinating them to the metal sites. The coordination and the charge of the metal shift the pKa of the water molecule to favor the more nucleophilic hydroxide form. Metal-coordinated nucleophiles are made in proteins by a number of different metals that are in most cases M2+ions such as Zn2’ and M 2 + . Interestingly, iron is rarely found in hydrolases, and one could speculate that the reason for this is the facile redox toggling of iron, which alters its charge and therefore limits its usefulness for catalyzing nonredox chemistry. Another potential problem with using iron in these systems i s also the high reactivity of Fe(1I) with 0 2 , which could lead to the generation of hazardous radicals. However, there are exceptions, and two hydrolase families using iron will be discussed below, In these cases homologous proteins exist in which the biological metal is Zn, Mn, or Co, and it appears that the selection of iron as the biologxal metal in these systems partly could be related to the control of activity by the direct or indirect use of the redox properties of iron.

2.1. Structural and Mechanistic Studies of l ~ o n - ~ x y ~ ~ ~ ~ i t r ~ roteins During the last decade, crystal structures of most of the known Fe-ON proteins have been elucidated, and they have revealed a detailed structural framework for discussing the mechanism and geometries of the reactions catalyzed. Most Fe-QIN proteins have been structurally characterized in multiple forms, and in many cases some of the most important states of the reaction cycle have been structurally characterized [4-61. In general, Fe-O/N proteins offer significantly less generous chromophoric properties for spectroscopic and mechanistic studies than do the Fe-S and heme proteins.

IRON-OXYGENINITROGENPROTEINS

471

However, a battery of spectroscopic methods has been applied to the Fe-O/N proteins, and together these methods have provided extensive information on the magnetic and electronic structure of the metals and their ligand environment. The most informative methods have been electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), resonance Raman (RR), Mossbauer, extended X-ray absorption fine structure (EXAFS), and magnetic circular dichroism (MGD) spectroscopies. Extensive kinetic data are also available on these proteins, and particularly useful information has been obtained from time-resolved studies of the reaction paths of dioxygen reactions using stopped-flowand freeze-quench methodologies. The spectroscopic and kinetic data on many of these proteins have recently been thoroughly reviewed (5,291. Modeling the Fe-O/N protein sites using low-Mr iron ligands has turned out to be a great challenge due to the asymmetrical and flexible properties of the metal sites in Fe-OIN proteins. Still, some very important results have been obtained in modeling the chemistry and spectroscopic features of Fe-0,” proteins. Particular useful information has been provided on the reactivities of different activated iron-oxygen cores, including peroxide and high-valent iron complexes [7,8]. Theoretical methods are also in the process of making an important impact on an understanding of the structurefunction relationship of Fe-0,” proteins. Most importantly, in the last couple of years density functional calculations have provided accurate energetic information for transition metal complexes [91, and ongoing extension of these methods to encompass the electrostatic properties of the protein should allow good evaluations of proposed reaction mechanisms for Fe-ON enzymes to be performed within the near future. Together, these methodological developments for studies of Fe-O/N proteins promise, in the coming years, to provide the required information for the formulation of true molecular level mechanisms for Fe-ON proteins that encompass structural, electronic, and dynamic features.

S IRON-OXYGEN/ 3. STRUCTURE AND M ~ ~ H A N I S MOF N I T R O G PROTEINS ~~ 3.1.

Lipoxygenases

Lipoxygenases (LOs) are mononuclear iron proteins found in plants and animals, as well as in some fungi, algae, and cyanobacteria. They catalyze the hydroperoxidation or polyunsaturated fatty acids by the addition of molecular oxygen to l,4-cis-cis-diene units of the substrates (Scheme 1) 110-131. LOs are normally labeled by their positional specificity in the peroxidation of arachidonic acid substrates (Scheme 2). In most instances the lipoxygenases are part of synthetic pathways producing secondary metabolites. In mammals the LOs are involved in the synthesis of prostaglandins, thromboxanes, leukotrienes, and lipoxins with important functions in inflammatory response TlO,l11. The effects of these mediators appear to be primarily achieved through 6-protein-coupled receptors or nuclear receptors. Leukotrienes, produced

472

NOR~LU~D

OOH Scheme 1

through the oxidation of arachidonic acids by mammalian &LO, are critical mediators of the inflammatory events of arthritis, allergy, and asthma [lLl. The 12-LOs appear to contribute to the chemotactic and mitogenic responses in smooth muscle cells in the circulatory system and have been suggested to play roles in i n f l ~ m a bowel ~ o ~ an can oxidize low-density lipoproteins disease and psoriasis 114,lti1. ~ a m ~ a l i 15-LOs and have been linked to the pathogenesis of atherosclerosis 1161. Due to the key positions of the mammalian lipoxygenases in the synthesis of inflammatory rnediators, they constitute important targets for drugs aimed at controlling a variety of inflammatory conditions.

12-LO

-vN

0 0

OOH

x

0 0 Scheme 2. The peroxidation o f arachidonic acids catalyzed by 5-L0, 12-L07 and 15-LO. Hydrogen atoms are abstracted from positions 7, 10, and 13, respectively.

~ / N I ~ R O ~PE~NO T ~ I ~ S

473

In plants, unsaturated fatty acids, such as linoleic and linolenic acid, are substrates for LOs and plant LOs have been implicated in pathways producing compounds effecting growth, development, and pathogen protection, such as jasmonic acid and other plant hormones [131. The best characterized plant LO is the soybean L1 isoenzyme (SLO-11, a 15-LO for which a wealth of biochemical and spectroscopic data has been recorded. The soybean 20-3 enzyme (SL -3) and several of the mammalian LQs have also been studied in some detail. All known LOs have a common evolutionary origin, as they display conserved sequence motifs including the residues involved in coordinating the mononuclear iron ion. The active form of LO has been shown to be the high-spin Fe(I1I) form [17]. However, the resting form of LO, as isolated, is in the Fe(1I) form but can be converted to the active Fe(IIIj form by exposure to the peroxy fatty acid product of catalysis by active LO [18]. 3.1.1. Structure of Lipoxygenases

The structures of several LQs have been determined revealing lipoxygenases to be ellipsoidal two-domain monomers (Fig. 3). The first three-dimensional structure of an LO was that of soybean LO determined at 2.6 fi by Amzel and co-workers in 1993 E191. The small N-terminal domain (-150 aa) is formed by an eight-stranded antiparallel p barrel with a jelly-roll topology, and the catalytic domain (-700 aa)contains a bundle of 23 a helices and two small antiparallel p sheets. The N-terminal domain displays structural similarities to the C-terminal domain of human pancreatic lipase, and a function for this domain in mediating membrane interactions in plants, in a Ca'"-dependent manner, has been proposed [20],

FIG. 3. Structure of rabbit 15-lipoxygenasewith the iron site buried in the subunit. An aryl inhibitor is bound in the proposed sub&rate binding sit,e (generated from PDB file 1LOX).

NORDLUND

474

The structure of the catalytic domain reveals the mononuclear iron site to be buried deep within the helical bundle. The iron is in the inactive Fe(I1) form and coordinated by three histidine residues-His504, His499, and His690-and the carboxylate of the C-terminal residue (Ile839) (Fig. 4). In addition, the side chain of Am694 is positioned as a potential iron ligand, but the distance is 3.3 A from the iron ion to the 0 6 ofAsn694, and therefore too long for a proper ligand interaction. No water molecule was found to coordinate the Fe(1I) ion in the original SLO-1 structure. However, in a more recent 1.4-A structure of SLO-1, solved in a different crystal form, a solvent molecule is found with a coordinating distance of 2.5 to the Fe(I1) ion, also forming a hydrogen bond to the dangling oxygen of the iron-coordinating C-terminal carboxylate [211. The Asn694 in this high-resolution structure is found 3.1 from the iron ion, which is still too far for proper iron coordination. Including Asn694, the coordination of the Fe(I1) ion in the SLO-1 can be seen as an octahedron.

A

A

32 -term HiS504

FIG. 4. Coordination sphere of (a) soybean-1 lipoxygenase and (b) rabbit 15-lipoqgenase in complex with an aryl inhibitor.

475

I RON-OXYGEN,” ITROGEN PROTElNS

The three histidine iron ligands and Asn694 all sit on two a-helices lining the active site. These helices display severe distortions in the region of the iron-binding residues, with irregularities in the main chain hydrogen bonding. The reason for these distortions is unclear: they might be due to the steric requirements for the observed coordination geometry, but they might also imply that some flexibility in the iron site is required during the reaction cycle. The region around the iron ion has been described as containing two internal cavities. Cavity 1 forms a channel leading from the surface of the protein to the active site and has been proposed to provide a route for molecular oxygen to reach the iron site 1191.The second cavity was proposed to constitute the binding site for substrates. The structure of a soybean LO-3 (SLO-3), which is a less specific LO than SLO-1, has been determined at 2.6 A resolution [221. SLO-3 has 72% sequence identity to SLO-1 and the structures of the two enzymes are very similar. In SLO-3, however, an additional cavity (cavity 3) is found reaching from the surface to the active site, which has been proposed to partly explain the differences in substrate specificity of the two enzymes 1227. The iron coordination sphere is similar in SLO-1 and SLO-3, and Asn713 in SLO-3, corresponding to Asn694 in SLO-1, is now 3 from the iron. A water molecule is found 4 .& from the iron ion in the SLO-3 structure, resulting in a four-coordinated iron in the Fe(I1) form. SLO-3 has also been structurally characterized with a bound 4-nitrocatechol inhibitor [23]. The catechol inhibitor does not coordinate directly to the Fe(I1) ion but the compound docks in the suggested substrate-binding cavity close to the iron ion. The structure of a mammalian 15-lipoxygenase (rabbit 15-LO) has been determined at 2.4 A resolution 1241. Mammalian LOs show around 25% sequence identity to the plant enzymes and are some 150 residues shorter in sequence. The structure of the catalytic domain of rabbit 15-LO is similar to that of the plant proteins but is more compact, containing 18 helices instead of the 23 found in SLO-1. The N-terminal domain of tho mammalian LO is also structurally related to that of the plant protein but has a more extensive structural relationship to /3 domains of other lipid-metabolizing enzymes such as mammalian lipases 1251. It has been suggested that a common fbnction of these domains is to localize the enz.ymes to their substrate-containing membranes or lipoproteins, and possibly also in the case of 5-LO t o the activating protein FLAP [24]. The structure of the mammalian 15-LO was determined in a complex with a bound competitive aryl inhibitor B41. The coordination environment of the Fe(I1) ion is similar to the one found in SLO-1 and SLO-3, including the 6-terminal carboxylate and three histidine ligands. In the position of the Asn694, however, is a fourth is5451, as was expected from sequence alignments. No coordinating water molecule was identified in the crystal structure of rabbit 15-LO. The earboxylate of the aryl inhibitor does not coordinate directly to the iron but instead makes close interaction with several of the iron ligands without forming proper H bonds. The inhibitor is conformationally constrained and is not expected to bind in the same mode as the arachidonic acid substrate. However, the space filled by the inhibitor probably demarcates the substrate-binding pocket and has allowed an interpretation

A

of the structural basis for the substrate specificity of the mammalian 5-,12- and 15-LO (Fig. 5). Based on the structural data and mutagenesis studies, binding is rationalized based on two factors: (1) an interaction with the carboxylate moiety of the substrate with a positively charged residue at the channel entrance (Arg403) and (2) the size of the pocket where 5-LO is predicted to have the largest pocket and 15-LO the smallest. 3.1.2. Reactioiz Mechanism of Lipoxygenases

Lipoxygenases catalyze the hydroperoxidation reaction with relatively high regio- and stereoselectivity. A distinguishing feature of the Fe(I1I) iron in LOs is the unusually high redox potential, which is around 0.6 V vs. NHE 1261. Based on XANES and MCD studies of the Fe(II1) form of soybean SLO-1, a six-coordinated Fe(II1) has been suggested for the active SLO-1 [27,281, which would leave no open coordination position for substrate or 02.One short coordination distance of 1.88 A has been

i

Felll

I

LYS

OH

/c1\

-3

i0 /

Arg

Felll

I

OH

0

\ 1

Arg

otein surface

Fell1

1

OH

y d r Q ~ h Qcavity ~ic

FIG. 5. Positional specificity of the lipoxygenases (LOs) could be determined by the size of the hydrophobic pocket in the different LOs [24]. From top to bottom: 5-L0, 12-L0, and 15-LO.

IRON-OXYG~N/NlTROG EN PROTElNS

477

obtained from EXAFS data, proposed to be due to a hydroxide ligand l281. Extrapolating from the Fe(1I) crystal structure and supported by spectroscopic data [29], it is possible that in the Fe(II1) form of SLO-1 Asn694 is also an iron ligand and that the charge of the iron site upon the redox change is compensated for by deprotonation of the water ligand to a hydroxide ion. The asparagine has been mutated in SLO-3 to alanine and threonine with loss of activity, while a mutation to histidine retains most of the wild-type activity, consistent with a requirement for an His ligand in this position. A remarkable deuterium isotope effect of labeled substrates of > 50 has been observed for the lipoxygenase reaction 130,311.This large isotope effect is explained by isotopic quantum mechanical tunnelling of a proton in an initial C-€I bond cleavage step. Anaerobic incubation of the Fe(II1) enzyme with substrate has been shown to result in the formation of a substrate radical [32,331, which can dissociate and dimerize. Isotope labeling methods have also been used to study the or diene substrate and 0 2 1341, revealing that O2 is bound after the step. Two solvent isotope sensitive steps have also been identified; one preceding the C-€3 bond cleavage and one following 0 2 binding. The most likely mechanism for the LO reaction is outlined in Scheme 3. This mechanism involves an initial hydrogen abstraction step from the substrate, yielding a substrate radical as the first reaction intermediate 1351. The hydrogen abstracting species is the six-coordinated Fe(1II) form with a hydroxide ion as the proton acceptor. The dangling oxygen of the coordinating C-terminal carboxylate may help in promot-

Scheme 3. Proposed reaction mechanism for lipoxygenases.

478

NORD~U~D

ing the proton transfei- of this step. The six-coordinated iron does not offer any free coordination position for direct O2 binding to the iron site. Instead, in the next step the activated radical substrate is attacked by molecular oxygen trans from the iron site [341. This leads to the formation of a peroxy radical intermediate, which can reoxidize the Fe(II) to a Fe(III) site. There is no obvious conserved Oa interaction pocket in the active site that could help in guiding 0 2 to the reactive position of the substrate. The relatively high stereo- and regiospecificity of the enzyme have instead been rationalized on the basis of the enzyme imposing a bent conformation on the substrate when bound in the active site pocket, a conformation that would only expose the right conformer for 0, insertion at the active site pocket. Plausible detailed proposals for the geometries for the LO reactions, including positions of possible proton transfer groups in the final steps of the reaction, have to await the structural characterization of the Fe(I1I) form of the enzyme with bound substrate. Incubation of protein with excess of peroxo product has been shown to lead to the formation of a strongly purple-colored complex 1361. In this complex the peroxo product appears to be coordinating directly to the iron ion with its peroxy moiety. Assuming the mechanism described above, this complex is not likely to be the peroxo complex of the reaction path, as the point of O2 addition into the substrate should be trans to the iron site according to the above mechanism. Alternative reaction mechanisms have also been discussed. Reaction mechanisms involving initial hydrogen or proton abstraction leading to the formation of organo-iron complexes have been suggested (371. This scenario is attractive as it would allow stricter control of the stereospecificity of the reaction. However, it appears that the formation of an organo-iron complex could be obstructed in the active site by the iron ligands. Also, the properties of the observed substrate radicals do not support an organo-iron intermediate. The possible involvement of a protein radical site in the reaction has also been discussed, but the lack of conserved potential radical sites in the region around the active sites argues against such a proposal.

The ring-cleaving dioxygenases arc found in microorganisms that use aromatic compounds as their main source of carbon and energy [38-40].In thesc organisms, enzymatic pathways are present to degrade aromatic compounds to succinate or other inetabolieally available carbon and energy sources. Much of the interest in the dioxygenases derives from their key role in the aerobic degradation of aromatic compounds and the possible application of these enzymes in bioremediation of environmental pollutants [39]. In these processes the diol dioxygenases attack dihydroxylated aromatic compounds in an 02-dependent reaction, which results in cleavage of the aromatic ring. The dihydroxylated substrates are often produced from a previous aromatic dioxygenation step catalyzed by Rieske dioxygenase (discussed in Sec. 3.6). Two families of ring-cleaving dioxygenases have been identified: the intradiol dioxygenases (IDOs) and extradiol dioxygenases (ED&) (discussed in Sec. 3.5). The

IRON-OXYGEN/NlTROGEN PROTEINS

479

two families are distinguished by their strategy for fission of the aromatic substrates: the intradiol dioxygenases cleave the substrate between two hydroxyl groups whereas the extradiol dioxygenases cleave the substrate adjacent to the diol (Scheme 4). Both families have mononuclear iron in their catalytic site and neither of the dioxygenase families utilizes an external source of reducing equivalents. However, there exists a distinct chemical difference between the two families in that the intradiol dioxygenases are active in their Fe(II1) form while the extradiol dioxygenases are active in their Fe(I1) form. The I D 0 reaction leads to ring fission between the two catechol hydroxyl groups producing a &,cis-muconic acid (Scheme 4). The best-studied intradiol dioxygenases are catechol 1,2-dioxygenase (CTD), protocatechuate 3,4-dioxygenase (PCD), and chlorocatechol dioxygenase (CCD). CTD and PCD show high substrate specificity for their respective physiological substrates, catechol and protocatechuic acid, whereas CCD has a less stringent substrate specificity, accepting a number of different methylated and halogenated substrates, with important applications in biorernediation [41I. The intradiol dioxygenases have been shown to be active in their Fe(II1) form [42,431. They exhibit purple color from a charge transfer to the Fe(II1) from coordinating tyrosine phenolates and substrate catecholates. The presence of the chromophore and an EPR-active high spin E’e(II1) in the intradiol dioxygenases has made them more amenable to spectroscopic characterization than the Fe(I1)-dependent dioxygenases. E W S studies of the resting enzyme suggested a five-coordinate center containing one hydroxide ligand. The ferric site in complex with substrate was suggested from EXAFS to be five-coordinate1441 and EPR provided support for a bidenLate substrate coordination [451, Steady-state kinetics of PCD have indicated that substrate binding precedes 0 2 binding [46].

lntradiol dioxygenase

I

OH

Scheme 4

\

Fe” Extradiol dioxygenase

OH

480 3.2.1. ~ t r u c ~of~ Intradiol re Dioxygenases

The first structure of an intradiol dioxygenase was the structure of PCD, determined at 2.1 1471. The functional common denominator for the intradiol dioxygenases, an ~ p - ~ e ( unit I ~ ~that ) is present in different oligomeric forms in the different enzymes, was shown to consist of CI and fl subunits with similar structures. The subunits are each formed by a sandwich of mixed flsheets (Fig. 6 ) and are related by a pseudotwofold axis, sugesting that they have evolved through a gene duplication event. The active site of PCD is found close t o the subunit interface and is formed by a solventcrevice. The Fe(I1I) ion in PC is coordinatBd by Tyz-447, Tyr4O&,His460, 62 (Fig. 7a). A solvent molecul hich is most likely a hydroxide, completes the t ~ i ~ bipyrimidal o n ~ coordination. The ligands of Fe(III), and other residues of the active site, are contributed from loops of the end regions of p strands of the f3 sandwich of the f3 subunit. A series of elegant structural studies of CD in complexes with inhibitors and substrate analogues has contributed the structural basis for a detailed geometrical description of the reaction mechanism of intradiol dioxygenases (Figs. 7b and 8) 148,491. In the anaerobic cornphx of PCI) with the substr 3,4-dihy~o~henylacetate), the catechol moiety is coordin ion with both diol oxygens, as predicted from spectrosc interacti~nsare made with the substrate by Gly14, the carboxylate moiety o f the substrate is hydrogen- bond^ to Tyr324 and active site water molecules. The substrate binding induces the release cif the TyrPQ7 ligand, and the side chain of this residue is rotated some LOO". The coordinated hydroxide ion is also released concomitant to substrate binding. The substrate has been shown to coordinat~in its dianionic form and the two released ligands could serve as proton acceptors during substrate binding [48,491, in support of a scenario where the overall charge of the site is conserved upon substrate binding. A more general charge eons , been suggested as n key servation behavior, also including reaction i n t e ~ ~ e d i a t ehas feature of the PCD active site.

FIG. 6. Structure of protocatechuate 3,4-dioxygenase with bwnd 3,4-dihydroxybenzoatt? (generated from PDB tile 3PCA).

I RON-OXYEEN/N1

~ PROTEI ~ IVS

Hism2 A4457

0

~

~

~

481

Oti,

FIG, 7. Coordination cnvironment of protocatechuate 3,4-dioxygenasein (a) the resting diferric form and (b) in complex with the substrate protocatechuate.

The five-coordinated substrate complex leaves an open coordination position trans to His460 which is lined by a small pocket providing a good site for dioxygen Lo approach the ~e(I~I)-substrate complex. The 02-binding pocket is formed by 61~14, Prol6, Tyrl6, Trp4Q0,and T'yr447, which are conserved in nearly all intradiol dioxygenases. PCD does not contain any obvious active site residues besides Tyr.447 that can serve as a cata1,ytic acid or base in the reaction. However, Arg457 is well positioned in the active site to participate in electxostatic catalysis when it is hydrogenbonded to the 0 3 o f the substrate. The 03-Fe bond is longer than the 04-Fe bond, which has been suggested to be due to a trans eff'ect of Tyr334.However, this could also be an effect o f 0 3 being an acceptor of' a hydrogen bond from Arg457. Structures of the substrate analogue I N 0 (2-hy~ro~sonicotinic acid M-oxide) and NNO (6-hydroxynicotinic acid N-oxide) have been determined as a n ~ o ~ ofe s potential monoanionic ketonization reaction intermediates 1481. In the complex with INO, the substrate analogue binds in a very similar mode as PGA, with the exception

482

NOR~LUND

FIG. 8. Coordination of inhibitor complexes to protocatechuate 3,4-dioxygenase as described in the text. (Reprinted with permission from [481.)

IRON-O~YGEN/~ITROGEN PROTEINS

483

that a solvent molecule remains coordinated to the Fe(II1) site. CN- was shown to be capable of coordinating in place of the solvent molecule (Fig. 8e). The charge of the dianionic substrate could in this case be substituted by the binding of the monoanionic NNQ and I N 0 together with CN-, or a solvent-derivedhydroxide ion. The structures of 4-hydroxy-3-halogenatedbenzoate inhibitors have been determined as models for an initial monodentate coordination mode of the substrate to the Fe(II1) ion [491. The 4-oxy group of these intermediates is coordinated to the iron without inducing the release of the Tyr447 or solvent ligand. The 3-halogen (iodine (Fig. 8b), chloride, or fluoride (Fig. 8c)) is hydrogen-bonded to Arg457 and 6 1 ~ 4 1which , suggests a role for these residues in a multistep substrate binding scheme. The crystal structure of catechol 1,2-dioxygenasehas recently been determined at 1.8 A, confirming the essential structural features for the intradiol dioxygenase reaction determined from the PCA system [501. 3.2.2. Reaction Mechanism of the Intradiol Diox.ygenases

The intradiol dioxygenases are active in the Fe(II1) form in contrast to most other Fe0," proteins, but similar to the LOs. The low dioxygen reactivity of the Fe(III) site has provided the impetus for the formulation of the currently accepted mechanism for the intradiol cleavage reaction (Scheme 5) 18,511. The inital role of the Fe(III) accord-

product H20

Scheme 5. Proposed reaction mechanism €or intradiol dioxygenases.

NO~DLU~D

484

ing to this mechanism is in activating the substrate for the dioxygen reaction. By inducing a build-up of negative charge andor radical character on C4 of the substrate, and a concomitant ketonization of the four-carbon-oxygen moiety, the substrate is made accessible for the O2reaction. The binding mode of the I N 0 analogue, which is very similar to the substrate, is expected to model the structure of such a ketonized inlermediate. The attack of 0 2 is suggested to be made on the substrate without the involvement of the iron ion when no iron-02 intermediate has been detected in the reaction cycle and the Tyr408 ligand is expected to stabilize the Fe(I1I) form of the site. However, in light ofthe available data, the direct involvement of the metal in an l?e(TI)-likeintermediate in the O2 binding step cannot be ruled out. The concomitant formation of a peroxo-substrate intermediate as a tridentate coordinated complex to ion was judged to be feasible from modeling studies. The heterolysis of the the ~e(II1) dioxygen bond most likely leads to the formation of a muconic anhydride adduct by a Criegee type of rearrangement. The alternative dioxetane has been excluded based on the introduction of labeled water into the reaction product [52,531.An epoxide is also a possible early intermediate of the dioxygen heterolysis preceding the anhydride. Based on the inhibitor structures discussed above, a detailed geometrical scenario has also been proposed for the initial binding of the substrate to the Fe(III) ion C491. In the first step, the hydroxide ligand is released concomitant with the deprotonation and coordination of one oxy group of the substrate. In a second substrate binding step, Tyr 447 is protonated and released while the second oxy group of the substrate is coordinated to the Fe(II1) ion. All intermediates in the proposed reaction scheme contain a strict Conservation of charge in the region of the iron.

ent Hydroxylases The amino acid hydroxylases (AAHs) constitute the most important members of a family of pterin-dependent mononuclear iron-containing hydroxylases [54-561. Three different M s have been identified in higher organisms: tyrosine hydroxylase, tryptophan hydroxylase, and phenylalanine hydroxylase. All three A A H s are active in their Fe(I1) forms and catalyze important transformations with major biomedical implications (Scheme 6). Tyrosine hydroxylase (TyrH) is predominantly found in the nervous system and catalyzes the formation of dihydroxyphenylalanine (DOPA) by hydroxylation of free L-tyrosine 1571. This is the first step in the synthesis of catecholai~ineneurotransmitters, and deficiency in TyrH has been linked to LDOPA-responsive dystonia and juvenile parkinsonism. Tryptophan hydroxylase (TrpH) is found in the central nervous system, where it is responsible for the production of 5-hydroxytryptophan via hydroxylation o f tryptophan [561. This constitutes the first dedicated and rate-limiting step in the synthesis of the neurokransrnitter serotonin, which also serves as a precursor for the synthesis of melatonin. Phenylalanine hydroxylase (PAH) catalyzes the oxidation of phenylalanine to tyrosine, which is the first step of the degradative pathway of the phenylalanine pools in the body, ultimately leading to the complete oxidation of L-phenylalanine to carbon

I RON-QXY6;EN/" ITRQGEN PROTElNS

485

Scheme 6

dioxide and water "$371. PAH is mainly expressed in liver and to some extent in kidney. Deficiency in functional PAH from inborn genetic variation can lead to the inability to degrade phenylalanine. This condition, called phenylketonuria, leads to the production of neurotoxic metabolites and mental retardation. Additional pterin-dependent hydroxylases using Fe(I1) ions have been identified in microorganisms; such as anthranilate hydroxylase [571 and mandelate hydroxylase [58],as well as a tyrosine hydroxylase in the bacterium ChrornohacEeriurnviolaceurn [591. A pterin-dependent human glyceryl-ether monooxygenase [56,60] has also been identified, but these enzymes all remain poorly characterized. The amino acid hydroxylases catalyze the hydroxylation of the aromatic moiety of amino acids at mononuclear iron-containing active sites. Although the active form of the enzymes is the Fe(I1) form, the U s are normally isolated in the Fe(II1) form. T~trahydrob~opteri~ (B&) (6(~)-~,-e~hro-5,6,7,$-tetrahydropterin) is a cosubstrate for the reaction with molecular oxygen, where 4a-hydroxypterin and hydroxylated amino acids are formed as the products (Scheme 6). The BH4 is subsequently regenerated by the reduction of 4a-hydroxypterin by the enzyme dihydropterin reductase. BH4 can also function as an activator for the resting form o f AAH by reducing the active site FelIII) to the active Fe(II) form 161,621. PAH is the best characterized due to its high stability, but extensive structural and biochemical informatioii is also available on 'l'yrH. The three enzymes have partly overlapping substrate specificities hydroxylating each of the three amino acids to some extent, with the exception of PAN that does not hydrolyze L-TJT.Studies using alternative substrates have been important for obtaining mechanistic information, but often uncoupling of the reaction is observed, where only the pterin cofactor is oxidized [561.

486

NORDLUND

The activity of all three M s have been suggested to be attributed to regulation, but the physiological relevance of some of these effects are uncertain. PAH shows a behavior characteristic of an allosteric protein by the cooperative binding o f the substrate L-Phe r571, inducing the high-affinity states of the enzyme. Phosphorylation of Ser16 of PAH has been shown t o sensitize the enzyme to allosteric regulation by 1,-Phe. is feedback-regulated by dopamines, which are downstream products of the dopagenic pathway. Regulation of the activity of TyrH by phosphorylation ol' Ser40 and possibly other N-terminal serines has also been demonstrated 1631. The major effect of the phosphorylation of Ser4O of TyrH has been attributed to the protection against inhibition by dopamines. A role of members of the 14-3-3 class of regulatory protein in enhancing the effects of the phosphorylations has been suggested 1641. 3.3.1. Structure of the Pterin-Dependent Hydroxylases

The three amino acid hydroxylases can oligomerize as 222-tetramers. The monomeric subunit consists of three domains: an N-terminal regulatory domain, a central catalytic domain, and a C-terminal dimerizationltetramerization domain. The three different M H s show strong sequence conservation for the last two domains, with greater than 45% identities, while the N-terminal region is less conserved. Important progress has been made in the structural characterization of the s C55l. Extensive structural data have recently become available on the PAH YyrR families while so far no structure of a TrpR has been solved. The first structure of an AAW: was of a truncated form of T p H containing the catalytic and the C-terminal tetramerization domains r651. The structure of a similar construct of PAH as well as of the catalytic domain of PAH in isolation was later solved, which revealed great structural similarities between PAW. and TyrH [66,671. Recently, the structure of a construct containing the regulatory and catalytic domains of PAH has been solved and, together with the previous structures, a model for the complete three domain subunits of AAHs in the tetrameric complex have been assembled 1681. The catalytic domain of PAH has an u / p fold making a basket-like arrangement of helices and loops (Fig. 9). The active site is formed by a shallow solvent-exposed cavity some 10 A deep in the basket containing the nonheme iron site. The protein provides three coordinating residues to the mononuclear iron, Wis285, His290 and 30 in PAH (Fig. 10). The iron is most likely in the inactive Fe(Il[I) form in all structures determined so far. In the structure of PAI-I, three water molecules coordinate the iron, while in the structure of TyrH oiily two water ligands are found. The active site predominantly contains hydrophobic residues, but a conserved gluta) is found approximately 4.5 A from the iron ion, making a mate (Glu286 in hydrogen-bond to ater molecule found trans to Glu330. Also the OH group of Tyl-325 is strategically positioned making a hydrogen bound to the water molecule trans to His285. The crystal structures of TyrH in complex with the BH4 analogue 7,8-&hydrObiopterin (BHZ) has been determined at 2.2 A resolution [69]. According to this study, Hz is positioned in close proximity to the iron but not directly coordinating. The

IRON-OXYGEN/NITROGEN PROTEINS

487

FIG. 9. Sti-uctui-eof the catalytic domain of phenylalaine hydroxylaae with bound cosuhstratc BH2 (generated from PDB file IDMW).

ring system of BH2 makes direct n: interactions with aromatic residues in the active site. A 'II NMR study of PAJ3 has later provided a model for the binding mode of BRZ that is different from the one suggested from the crystallographic study of TyrH in complex with BH2 [701 (Fig. 12). The cofactor i s rotated 180" and the 0 4 atom of BH2 could be coordinating directly to the iron site. In a veiy recent crystallographic study of €'AH,a similar binding mode of BHz i s seen as in the 'H NMR study [711 (Figs. 11 and 121, indicating that BWZ binds in a different mode in TyrH, or that the electron density of the crystallographic study did not allow a proper interpretation of the orientation of the cofactor. In the crystal structure of PAH in complex with BHZ the 4a carbon, the site of hydroxylation of the cofactor, i s found some 6.1 A from the Fe(II1) ion. The 04 is not found coordinating to the iron as suggested from the 'W-

FIG. 10. Coordination cnvironment of the iron site of phenylalanine hydroxylase.

488

NO~~LUND

FIG. 11. B& coordination in the active site of phenylalanine hydroxylase. (Reprinted with permission from 1711.)

W, study, but rather 3.8 A from the iron ion. The 0 4 is instead involved in a hydrogen bonding network with two of the three iron-coordinated water molecules and the conserved active site residue Glu286. In the structures of T y H in complex with BH2 a hydroxylation of the active site residue Phe300 in the meta position was revealed 155,691. The -OH group of Phe300 was found 6.6 A from the iron. However, this hydroxylation does not appear to be a functionally relevant modification but rather is an artifact of the crystal preparation. ~atecholsinhibit all three M s , and in the ease of TyrH dopamines are important physiological feedback regulators. Structures have been determined of PAH in complex with several catechol analogues [721. The direct binding of the inhibitors was previously shown from spectroscopic studies [731. In the crystal s t ~ c t u r e sthe inhibitors make a bidentate coordination to the Fe(II1)iron and the two iron-bound water molecules trans to the two His ligands are replaced by the coordinating inhibitor. The binding of the aromatic ring of the inhibitors overlaps partly with the binding of the BH4 analogue in 'I'yrH, which i s consistent with the catechols being competitive

l R O N - O X Y G E N / ~ l ~ R ~PROTEINS G~N

489

inhibitors with B . In contrast, the catechols are not competitive inhibitors with the amino acid substrate and the binding volume of the inhibitor should therefore not correspond t o the region of the active site where the substrate will be binding. So far no crystallographic data have been obtained for the amino acid substrate-bound forms of the AAH. However, from the 'H NMR study of PAEI a detailed proposal was presented for how the substrate could interact with the active site [701 (Fig. 12). The proposed L-Phe interactions involve a consewed argininc and serine (kg270 and Ser349 in PAH) interacting with the amino acid moiety of the substrate. The aromatic moiety o f the substrate is positioned in a pocket lined by a tryptophan and a phenylalanine (Trp326 and Phe331 in PAH). This model of the substrate in the active site leaves the C4 hydroxylation positions of L-Phe about 4 from the iron ion. The described crystallographic and 'H NMR studies were all made on the Fe(II1) foim of the proteins. CD/IMCD studies of PAII in solution have investigated the structure of the active Fe(I1) form and suggested that the substrate- and gterinfree forms of the F e W , as well as the Fc(I1T) form of PAH, are six-coordinated r741. The binding of the amino acid substrate to the Fe(I1) form has minor effects on the iron coordination, while the binding of the non-redox-activeBH4 analogue 5deaza-6-methyltetrahydropterin gives no significant effects on the Fe(I1) site [75]. However, the binding of both the BH4 analogue and the substrate induced a transformation from a six-coordinatedto a five-coordinated Fe(I1) site C751, with B possible open site for O2 coordination.

A

3.3.2. Reaction Mechanism of Pterin-Dependent Hyclmxylases As yet, no consensus view on the detailed mechanism of AAH has been obtained. Different orders for substrate binding have been reported from the different enz.ymes

FIG. 12. The 'H NMR model for the binding of BHa and phenylalanine to phenylalanine hydroxylase. (Reprinted with permission from [701.) See F'igure 11.12 in the color insert.

~ORDLU~D

490

under different conditions E56,571. This could be explained by the nonoverlapping binding sites of the BH4 and amino acid substrates, but it is clear that no product or intermediate is released before the binding of all substrates 156,571. It has been shown that in PAH, an 0 atom from molecular oxygen ends up in both the pterin and the amino acid products [76,7'7]. No direct observation of intermediates has been made in any of the AAH reactions. At certain conditions, uncoupled oxidation of XHe to hydroxy- and peroxypterin is catalyzed with no concomitant hydroxylation of amino acids, suggesting that the BH4 chemistry constitutes a first half-reaction. Possible reaction scenarios consistent with the structural data are shown in Scheme 7. Isotope studies suggest that the rate-limiting step of the reaction catalyzed by AAH[ is the reductive activation of molecular oxygen "781. Two distinct possibilities for the initial O2 activation have been considered: (1) the formation of an Fe(IIT)-superoxide complex by direct Ozreaction with the Fe(l1) ion, as is suggested for most other 0%-activatingFe(l1) proteins, or (2) the formation of a superoxo leading to the formation of 4a-peroxyhydropterin, analogous to the suggested mechanism of 0 2 activation in fiavin-dependent hydroxylases. The authors argue against the first scenario on the basis that the Fe(l1) iron has a low reactivity with

Glum

''L-

I

-'

02

H

0 arene oxide

Scheme 7. A possible reaction scenario for the amino acid hydroxylases.

I ~ O N - O X Y G E ~ / N I T R O GPROTEINS ~N

49 1

Q2 in the absence of BH4. However, in the light of the recent CDiMCU studies of Plaw

revealing conformational changes and the generation of an open coordination position upon L-Phc and pterin binding, an involvcment of the Fe(1I) ion in the initial 0 2 activation is an attractive possibility. The formation of a 4a-peroxytetrahydropterin species is a likely reaction path, but model studies have shown that such a species by itself is not sufficiently reactive for a direct hydroxylation of the amino acid. The geometry.of substrate and pterin binding suggested by '€3 NMR and crystallographic studies is consistent with both O2 activation scenarios above, and possibly both the iron and the pterin might play a role in the initial 0 2 activation. An ironcoordinated peroxopterin intermediate is an attractive option for the first product of the O2 activation reaction, but no direct observation of such an intermediate has yet been made. Significant uncertainties exist related to the structure of the hydroxylating species and the mechanisms of oxygen insertion used by the AAH 1561. However, a high degree of conservation of the active site residues in the three enzymes is also consistent with a common mechanism for these steps of the reaction. The hydroxylating species could involve a peroxide adduct, being inserted into the substrate concertedly with dioxygen cleavage 1701. Alternatively, an initial dioxygen heterolysis could lead to the formation of hydroxy-pterin and a distinct activated Fe=O core. In reactions with alternative substrates and reaction conditions, a broad range of reactions are catalyzed by thc AAH, similar to what is seen for cytochrome P450 enzymes. This suggests that, in addition to peroxo species, more reactive high-valent iron species exist, at least under the alternative reaction conditions. In model complexes, Fe(IV)=O intermediates have been shown to be capable of hydroxylating phenyl groups E791. However, no or small isotope effects are seen, excluding hydrogen abstraction as a major rate-determining step in the reaction. The migration of labels from the para to the meta position during the reaction, so-called NIH shifts, is observed in the PAH and TrpH reaction [80,81]. This is most consistent with mechanisms involving the formation of either a carbocation or an arene oxide intermediate, and support for both scenarios has been provided, although the carbocation reaction seems to be the more favored possibility. The presence of the conserved Glu286 close t o the meta position of the substrate in the proposed Phe binding site could play a role in stabilizing a substrate carbocation.

3.4. 2 - O x ~ g l ~ t a r a t e - ~ e ~ eOxidases n ~ e n t and Related A particularly versatile and ubiquitous family of oxidases is that of the 2-oxoglutarate (2-OG)-dependentnonheme iron oxidases E82,831. These enzymes are involved in the synthesis of a range of primary and secondary metabolites (Scheme 8). The substrates for these enzymes are normally relatively polar with some amino acid characteristics. They are found in plants and animals as well as in microorganisms. In plants they are involved in the synthesis of signaling molecules, whereas in microorganisms they constitute key components in several biosynthetic pathways for antibiotics. In

492

N~~DLUND

Scheme 8

animals the 2-QG-dependent hydroxylases prolyl and lysyl hydroxylase catalyze the post-translational modification of collagen of connective tissues, controlling the extension and stability of this biopolymer [79]. Aspartyl [3-hydroxylasescatalyze the posttranslational hydroxylation of the aspartyl residues of some receptors [711. Other 2OG-dependent hydroxyIases are involved in fatty acid metabolism and pyrimidine metabolism in higher organisms. The 2-06-dependent oxidases catalyze the two-electron oxidation of their primary substrate. In this reaction 0 2 is activated at the mononuclear ferrous site, which additionally leads to the oxidation of the cosubstrate 2-0G, transforming it into succinate and carbon dioxide (Scheme 9). Some members of the 2 - 0 6 structural family are not dependent on 2-OG as cosubstrate. A n important example is the acid oxidase (ACCO), which cataplant enzyme 1-~inocyclopropane-1-carbo~lic lyzes the formation of the plant hormone ethylene from aminocyclopropane-l-carboxylic acid in a reaction that is dependent on ascorbate and 0 2 1841. IsopenicillinN synthase (IPNS) is also structurally related to the 2-OG-dependent enzymes, although it does not use a cosubstrate but instead catalyzes a four-electron oxidation of its tripeptide substrate 1851. eacetoxycephalosporin C synthase (DAOCS) is one of the best characterized 2QG-dependent oxidases, which catalyzes the first committed step in the biosynthesis of cepliialosporins. In an Oz- and 2-0G-depcndent reaction the five-membered thiazolidine ring of penicillin N is expanded to yield deacetoxyceph~osporinC, containing a six-membered cephain ring IS6l. Another well-characterized member of this family is c l a v ~ i n a t esynthase (CAS), a major component of the biosynthetic pathway of clavulanic acid, an inhibitor of serine p-lactamases that is used clinically against bacteria resistant to p-lactam antibiotics 1871. CAS catalyzes three different steps in this biosynthetic pathway--a hydroxylation, a cyclization and a desaturation-illustrating the great functional versatility of the oxidation strategy employed by the 2 - 0 6 oxidases. An ordered sequential mechanism has been suggested for the 2-06-dependent oxidases, with an initial binding of 2-OG followed by the primary substrate and finally dioxygen. Product release is expected to be in the order carbon dioxide, succinate, and primary product 1881. Fe(1I) is the active form of the iron site, but iron binding


Prolyl ~ydroxylase

Clavamlnate Syntha~e

H

H

- R-NJx& 2-OG 02

0

COpH ~etoxycephalosporinC Synthase

,i,#J HY HS

R

0

02

%OOH

H 1

EOOH ~ s o p e n ~ ~ i ~ynthase llin~~

Scheme 9. Some of the reactions catalyzed by 2-oxoglutarate-dependentoxidases and related enzymes.

494

NORDLUND

appears to be relatively weak for these proteins, and excess iron and/or reductant in the reaction mixture normally complicates the assays. Spectroscopic data for CAS support the cosubstrate to bind in a bidentatc mode to the Fe(I1) [891. Isopenicillin-N synthase catalyzes the formation of the P-lactam framework of isopenicillin and cephalosporin antibiotics in some fkngal and bacterial species 1851. The substrate is the tripeptide 6-(L-ol-amino-S-adipoyl)-L-cysteinyl-D-valine (ACV) which is transformed in an (&-dependent reaction, where all four electrons needed for the oxygen reduction come from ACV and the oxygen atoms end up as water. IPNS was shown to bind the ACV by a direct substrate thiolate coordination to the iron ion 190,911. 3.4.1. Structures of 2-Oxoglutarate-Dependent. Oxidases

The first structure of a 2-QG-dependent oxidase was that of DAOCS 1921, which showed the jelly-roll motif with flanking helices characterislic of this structural family, previously seen in the structurc of IPNS [93J (see See. 3.4.2) (Fig. 13). The active site is found in R shallow pocket on the edge of the p sheet of the jellyroll. The mononuclear F e W site of DAQCS is coordinated by two histidines, His183 and His243, which are part of the central p sheet, and one aspartate, Asp185, at the end of one fi strand (Fig. 14). In the crystal a trimer is observed in which the Cterminal residues of one monomer project down into the active site of a neighboring monomer. However, in solution it appears that Lhe active form of DCPACS is a monomer [94 I with the C terminus probably located in the same position in the active site of the same monomer. The structure of DAQCS has been determined in three different forms: the ironFree apoprotein, the complex with Fe(II), and the complex with Fe(1I) and 2-QG C921. In the complex with iron, three water molecules coordinate the iron in addition to the three protein ligands, yielding a six-coordinatesite. In the structure of DAQCS with 2-

FIG. 13. Structure of isopenicillin-N synthase with bound AGV substrate (generated from PDB file 1BLZ).

495

i

2-oG

His243

FIG. 14. Coordination environment of deacetoxycephalosporin G synthasc.

OG bound, the cosubstrate is directly coordinated to the iron site in a bidentate mode, replacing two water molecules seen in the nonbonded form. The 5-carbowlate moietgr of the substrate makes hydrogen bonds to Arg258 and Ser260, which are part of an Arg-X-Ser motif conserved in most 2 - 0 6 oxidases as well as in IPNS (see next section). No structure is available for a complex with the cosubstrate 2 - 0 6 and the primary substrate penicillin N.In the apo form of the protein the C-terminal region covers the active site. The C-terminal lid is provided by the n e i g h b ~ ~ ~ molecule ng in the trimer. A conserved hinge is present between residues 298 and 302 (IGGNTI') of the arm, suppo~tingthe hypothesis that the flexibility of the C termini might be functionally important, presumably to protect the site at certain stages of the reaction cycle 1941. Clavaminate synthase does not show significant sequence homology to the other members of the 2-QG oxidase family. In a recent crystallographic study, GAS was indeed revealed to belong to the DAOCS structural family 1951, as the overall folds of the two proteins are very similar. The iron site in CAS is coordinated b-37 a 2-Hiscarboxylate motif, but instead of the aspartate found in the other family members, a glutamate is the iron ligand in CAS. It is suggested that the substitution t o Glu in C M might be due to the requirement for additional flexibility in performing the trifunctional role of this enzyme [95]. Two ligating water molecules are replaced by the bidentate coordination of the 2-0x0 acid group of 2-OG. The conserved motif €or binding the 5-carboxylate moiety of 2-OG is not present in the sequence of CAS. However, Arg293 is present at this position, making a similar interaction to 2OG. The structural role of the serine residue of this motif appears to have been substituted by Thr172, which is located in a different j3 strand. The conservation of the guanidino and hydroxo motifs of these very divergent proteins suggests a distinct binding mechanism for the 2-OG carboxylate. The crystallographic temperature factors are high for a lid covering the cofactor site, indicating that this lid might move for cofactor binding. The structure of CAS has been determined in two substrate complexes: (I)N-cx-r,-acetylar@nine(NAA), which is a stable analogue for the monocyclic

496

NO~DLU~D

p-lactarn substrate of the hydroxylation reaction; (2) proclavaminic acid, which is the substrate for the cyclization step (Fig. 15).The substrates enclosed the 2-OG molecule in the structures, consistent with initial binding of 2-OG in the reaction cycle. Arg297 coordinates the carboxylate moieties in both substrates, albeit in slightly different orientations. The two substrates have different nitrogen-containing terrnini-guanidin0 in the case of NAA and amino in the case of proclavamiiiic acid-and therefore the interactions of the substrates with the protein in this region are different. This

FIG. 15. Structure of clavaminate synthase in substrate complexes: (a) with N-a-L-acetylarginine; (b) with proclavarninic acid. (Reprinted with permission from (951,)

N NITROGEN PROTEINS

497

leads to a change in the positioning of the substrate with respect to a reactive 0 2 intermediate that can explain the difference in oxidation position and destination of the two reactions. CDIMCD studies of CAS support a change in coordination from six to five upon substrate binding, leaving an open coordination position for oxygen to bind [96,971. In the structure of the NAA complex, a water molecule i s coordinated to the Fe(1I) ion, leaving it six-coordinate, while in the complex with proclavaminic acid no water is bound, resulting in a five-coordinate iron. Somewhat surprisingly, the 2-OG coordination in DAOCS and CAS are made at difTerent positions, where the carboxylate of the cofactors i s found trans to the two different histidine ligands. This suggests that the geometry of O2 binding and activation is different in the two enzymes or that there will be rearrangements in the DOACS 2-OG binding mode when the primary substrate is binding into the active site. 3.4.2. Structures of' Isopenicillin-N Synth~ses

The structure of IPNS was the first structure determined of a member of the mononuclear iron family containing the jelly-roll motif, also present in the 2-OG-dependent oxidases 1931. The active site of IPNS is found in a very similar position as in the 2-OG oxidases, and the families are clearly evolutionarily related. In the initial structure of IPNS, where the active site iron is substituted by an Mn ion, His214, Asp216, and His270 are coordinating the Mn ion together with the side chain of Gln 330 (Fig. 16). The glutamine-330 is conserved in all known IPNS sequences, but mutagenesis studies showed it not to be essential for catalysis 1981. In addition to the four protein ligands, two water molecules coordinate the Mn ion. The structure of iron-containing IPNS was later determined in complex with the substrate ACV [991 (Fig. 17). In the structure of IPNS in complex with ACV, the 6terminal 6111330, previously seen to coordinate the Mn in the substrate-free form, i s replaced by the thiolate of ACV. The seven C-terminal residues of IPNS instead extend to the final helices to enclose the substrate in the active site [99]. The substrate thiol groxtp is coordinated directly to the iron and substitutes for one water molecule seen in the Mn structure. The substrate valine isopropyl group is in van der

FIG. 16. Coordination environment of the rnanganese-substituted, substrate-free isopcnicillin-N synthases.

498

NO~DLU~D

FIG. 17. Structure of isopencillin-N synthase with the substrate ACV.

Waals contact with the iron site and presumably blocks the binding of a second water molecule. ACV binds in an extended conformation in the active site. In the structure the Cp-hydrogen of valine is directed away from the iron. Arg279, previously directed away from the active site, is in the ACV-bound form directed into the active site and binding the ACV valine carboxylate group through a bridging water. This group is also coordinated by Serr281, Tyr189, and a water molecule. On the other end of the substrate the carboxylate of the aminoadipoyl group forms hydrogen bonds with the conserved Arg87. In the crystal structure of a ternary complex of ~e(11j:NO:ACV IPNS the NO molecule binds in the open coordination site trans to Asp216. Recently, the structure of IPNS has been determined with the reaction product isopenicillin N bound to the active site [loo]. The isopenicillin-N is found to bind in a very similar conformation as the substrate ACV. It makes most of the polar interactions with the enzyme made by ACV, and the thiazole sulfur atom is found 2.9 A from the iron. However, the cysteine carbonyl has shifted its position due to the steric constraints of the formation of the p-lactam ring. This conformational change appears to be facilitated by the lack of any protein hydrogen bonds to the cysteine carbonyl. The structure of the product formed by oxidation o f the substrate analogue ACmC (S-(L-~-aminoadiopoyl)-L-cysteinyl-L-S-methylcysteinej was also reported IlOOI. This structure showed the formation of a p-lactam-containingproduct binding in a similar

499

I RON-OXYGEN,” ITROGEN PROTElNS

geometry in the active site as the ACV and isopenicillin N. The methylcysteine position, corresponding to the valine position of ACV, is hydroxylated in the product of the ACmC reaction to form an iron coordinating sulfoxide. The formation of a monocyclic ring and a hydroxylated product supports a reaction mechanism for IPNS involving two half-reactions and possibly the existence of high-valent iron in the second-half hydroxylation step. 3.4.3. Reuction Mechanisms of 2-2-xoglutarate-L)ependentEn,zymes

Knetic studies on other 2-OG-dependentenzymes have suggested an ordered sequential binding of 2-oxoglutarate and the primary substrate, followed by dioxygen binding [BS]. The structure of 2 - 0 6 bound in the base of the active site of CAS is consistent with such a scenario. The subsequent binding of the substrate, with concomitant conformational change of the C terminus, may displace a water molecule and thereby assist in oxygen binding. No direct observation of intermediates of the reaction has been made for the 2-OG-dependent oxidases. However, based on substrate analogue studies and the observed uncoupling of the reactions after the 2 - 0 6 oxidation step, a scenario containing two subsequent two-electron oxidation steps can be outlined. In the initial step, 2-oxoglutarate is oxidized resulting in the oxidative decarboxylation (Scheme 10) of 2 - 0 6 and the formation of a ferryl intermediate. In the second half of the reaction, the primary substrate is oxidized by the ferryl site. Recent advances in the structural characterization of the 2-OG-dependent oxidases in different complexes allow a discussion of the detailed geometry of the reaction. An important feature for the control of the reactivity of these sites is the release of a water ligand upon substrate binding, although it is not obvious how this release is promoted by substrate binding. After the open coordination position trans to His279 (in CAS) has been generated, molecular oxygen can bind and form a ferric superoxo

R

substrate

substrate

product

2-0 c-product

substrate

‘\

I,”...J substrate

Scheme 10. Proposed reaction mechanism for 2-oxogluta~ate-dependentoxidases.

~ O ~ D L U N

500

intermediate. In the next step, the superoxide attacks the cosubstrate peroxo species, which then collapses to generate succinate, carbon dioxide, and a ferryl iron complex, Carbon dioxide has been shown to be the first released product and could possibly leave the site at this stage of the reaction [881. The proposed ferryl intermediate provides a general source for radical and oxygen insertion chemistry that is consistent with the rich chemistry catalyzed by the family of 2-OG-dependent enzymes. Highvalent iron species have been directly observed as intermediates in heme-dependent enzymes and binuclear iron proteins. Support for the existence of a ferryl intermediate of the 2-OG-dependent oxidases is provided by the observation of incorporation of dioxygen or water into the product in some enzyme reactions E101,1021. A discussion of the possible mechanism for the range of different substrate oxidation steps catalyzed by the 2-OG-dependent oxidases is too broad a subject for the present review. It appears, however, that the active site environment outside the metal coordination sphere plays a relatively small role in promoting the chemical steps but provides a suitable binding site for contact between the substrate and the ferry1 intermediate. 3.4.4. Reaction Mechanism of Isopenicillin-N Synthhase

Although IPNS is clearly evolutionarily related to 2-OG-dependent oxidases, it does not use 2-OG as a co-substrate but instead catalyzes the formation of penicillin N through the four-electron oxidation of the tripeptide ACV. IPNS is dependent on Fe(I1) and 0 2 for activity where 0 2 is fully converted to water in the reaction. No direct observations of reaction intermediates have been made for the IPNS reaction and the currently accepted mechanism, shown in Scheme 11, is mainly based

or Glnmo HOOE

HOOe

penicil1in.N

H+

,

EOOH

EOOH

Scheme 11. Proposed reaction mechanism for isopenicillin-N synthase.

I~ON-OXYGEN/NlTROGENPROTEINS

50 1

on substrate analogue studies and recent structural data [%I. The enzyme has a relatively relaxed substrate binding requirement for variations in the valine position. This has allowed a number of useful suhstituents to be used for the mechanistic studies. These studies have led to the proposal of a mechanism containing two halfreactions, where the initial oxidation chemistry is catalyzed on the thiol group leading to the formation of the fi-lactamring and, in a subsequent step, the thiazole ring [851. The substrate analogue studies have also provided support for substrate radicals, but no racemization is seen either in the products of the substrate analogues or of ACV, requiring that the geometry of the suggested substrate radicals be tightly controlled in the active site. Also, a high-valent iron species has been inferred from the substrate analogue studies to be involved in the second step of the reaction, i.e., formation of the thiazole ring. The structural data are consistent with the suggested mechanism and allow a detailed geometrical description of the reaction in the active site of IPNS. In the initial step of the reaction the substrate ACV binds to the active site, coordinating the ferrous iron with its thiolate group. The ACV-bound structure provides one open coordination position trans to the Asp216 ligand, as inferred from the crystal structures of ACV in complex with NO. 0 2 binding at this position leads to the formation of an Fe(II1) superoxo intermediate, which is juxtaposed to the pro-3-53 hydrogen of the ACV cysteinyl residue. In the subsequent step, the superoxo species can perform a formal hydrogen abstraction, resulting in the formation of a thioaldehyde, or related species, and an iron coordinated peroxide. In the next step of the cycle, a proton from the valine amide allows the heterolysis of the peroxide, concomitant with the closure of the p-lactam ring. The structural study of the ACmC analogue supports the view that p-lactam ring formation can occur with only minor realignments of the substrate in the active site ilOO]. As a product of the first ha-reaction a ferryl iron is formed, which in the subsequent step can abstract a hydrogen from the valine, concomitant with formation of the thiazole ring. However, the p hydrogen of the valine in the ACV-bound structure is positioned in the wrong direction and a side chain rotamer rotation of this group is required for the correct hydrogen abstraction geometry. This appears feasible considering the relaxed substrate specificity of the valine position. Although significant differences exist in the chemistry catalyzed by the IPNS and the 2-OG-dependent systems, some interesting similarities can be noted. Both systems bind peptide-like substrates or cosubstrates, which activate the iron site for the 0 2 reaction. Both reactions progress through a two-step mechanism in which the second step most likely involves ferryl-like intermediates with similar properties. However, the initial oxidation steps, including O2 activation, are very different. In the 2-OG-dependent systems the direct attack of O2 on the 2 - 0 6 succinate facilitates dioxygen heterolysis, while in the IPNS system the key to the O2 activation step should be the presence of the soft sulfur ligand. The direct availability of the amide proton at the site of dioxygen activation might also be important. The structural data provide support for that the O2 binding sites and the position of the 0x0 group of the ferryl intermediate is different in the two systems. In IPNS, these groups are most

NOR~LUN~

502

likely binding trans to coordinating carboxylate, whereas in the 2 - 0 6 system they are located trans to one of the histidine ligands of the 2-His-1-carboxylatemotif, suggesting that the trans effects from the protein ligands are not essential for 0 2 activation or stabilization in these proteins.

ioxygenases and Related Many organic compounds from natural or industrial sources are biodegraded through oxidative pathways, as has been discussed in the section on intradiol dioxygenases, In the degradation of aromatic compounds by soil bacteria, catecholic compounds can be formed as intermediates [40,103,104]. These are subsequently degraded by the ring cleavage dioxygenases. The extradiol dioxygenases are in contrast to the Fe(II1)-containing intradiol dioxygenases discussed above, active in their Fe(I1) form [lOS]. Extradiol dioxygenases degrading a vast range of different aromatic compounds have been isolated from a variety of soil bacteria. Most bacterial EDOs appears to have relatively relaxed substrate specificity and are more tolerant to substitutions in the aromatic groups than are the IDOs [103]. EDOs therefore serve as important targets for engineering improved enzymes for applications in bioremediation. The extradiol dioxygenases have been divided into three classes based on their primary sequence [106]. Structural studies have revealed that class I and class I1 enzymes actually belong to the same structural family, while class I11 enzymes constitute a distinct structural and evolutionary extradiol dioxygenase family. The division of the (class IDI) and type I1 (class 111) enzymes is therefore more appropriate and will be used in the present discussion [106]. The type 1 extradiol dioxygenases have been extensively studied and shown to contain an Fe(I1) ion in their active form. Catechol2,3-dioxygenase(also called meta(MPC)), protocatechuate 4,5-dioxygenase, Z,~-dihydroxybiphenyl HBD), and gentisate 1,2-dioxygenase,are Enetically and spectroscopically well-characterized members of the type I E D 0 family [29] (Scheme 12). Spectroscopic data provide support for the substrate being coordinated directly to the iron ion in its anionic form [107]. Some EDOs of type I also appear to use manganese instead of iron as the catalytic metal, but very little information on these enzymes is available.

Scheme 12. Reactions catalyzed by extradiol dioxygenases.

~ R O N - ~ X Y G E N / ~ I T R OPROT~INS ~EN

503

Much less information is available on the type I1 EDOs, but they also use a Fe(1I) ion in thc active site. Protocatechuate 4,Ei-dioxygenase is the best studied member of this family. Spectroscopic studies suggest great similarities of the metal site to the type I EDOs. As for type I, the substrates of type ZI dioxygenases coordinate directly to Fe(II), most likely in their monoanionic form 11081. Another dioxygenase that belongs to the type I ED0 family is homogentisate 1,2-&oxygenae, which is found both in bacteria and in higher organisms and is involved in the catabolism of tyrosine and phenylalanine. Deficiency in homogentisate 1,2-dioxygenaseactivity in humans leads to alkaptonuria, a rare condition in which the substrate homogentisic acid is deposited in connective tissues, thus causing a debilitating arthritis [1091. ~ " E y d r o x ~ h e n y l p ~ v dioxygenase ate (HPPD) is another Fe(l1)-dependent enzyme of tyrosine catabolism (1101. This bacterial enzyme is related to eukaryotic enzymes involved in tyrosine degradation in mammals and the biosynthesis of photosynthetic pigments in plants. In humans, deficiency in HPP activity can lead to tyrosinemia, a rare condition that causes severe mental retardation. HPPD does not catalyze a ring cleavage reaction (Scheme 15) but rather a coupled oxidative decarboxylation and hydroxylation reaction, i.e., a similar reaction to the 2-OGoxidases. HPPD was recently shown to be structurally related to the 0 s and will therefore be discussed in this section although it is not an extradiol dioxygenase [ill]. 3.5.1. Structure of Type I Extradiol Dioxygenascs

Several structures o f members of the type I extradiol dioxygenase superfamily havc been determined (Fig. IS). The first crystal structures of type I extradiol dioxygenases determined were those of two highly related Pseudomonas 2,~-dihydr~)xybip~enyl dioxygenases (DEB , also called BphC) [112,1131. These enzymes prefer polyaro-

FIG. 18. Structure of the extradiol dioxygenase metapyrocatechase with bound substrate (generated from PDB file 1MPY).

504

~OR~LUND

matics as substrates, as well as their halogenated analogues. The 2,3-diphenyl subacid. strate is transformed t o 2-hydroxy-6-0~0-6-phenylhexa-2-4-dienoic The structure of DWBD revealed a homooctamer of 422 symmetry. The 33k subunits are themselves formed by two distinct domains with similar structures, although the sequence identity is less than 20%. Each domain contains an 8- to 10stranded mixed sheet with two adjacent ct helices. The two domains are related by pseudo-twofold symmetry and the active site with the coordination sphere of the mononuclear iron site is localized in the 6-terminal domain. Some extradiol dioxygenases are significantly smaller than DHBD and in these enzymes only the C-terminal iron binding domain appears to be present 11061. The iron ion in DHRD is coordinated by thrce protein ligands: His146, His210, and 6111260(numbering from DHBD in 11133)(Fig. 19). All three of these residues are rigidly positioned on the sheet, in the region where the opposite side of the sheet, makes h e r interactions with the N-terminal domain, The Fe sites in the crystal structures are probably in their Fe(II1) form and therefore inactive. In addition to the three protein ligands, two solvent molecules coordinate the iron ion in the substratefree forms. In DHBD the Fe ion is bound deeply within the core of the 6-terminal domain. Two channels leading to the surface from the iron site are found, one approximately 10 A wide and the other about 6 wide, which constitute paths for substrate and 0 2 access to the active site. Structures of D ~ have ~ also D been solved in two different substrate-bound complexes, one with 2,3-dihydroxybiphenyl (2,3-DGBP) and one with S-methylcatechol (3-MCT) 11323. DHBD is relatively specific for 2,3-dihydroxybiphenyl and has a low activity toward catechol. The catechol moieties of the two substrates bind in a very similar manner in the two structures. The diol group coordinates directly to the Fe(1ID ion, probably in the diianic dioxy form, by replacing the two solvent molecules in the substrate-free structure. The catechol moieties of the two substrates are located in a mainly hydrophobic pocket, with highly conserved residues contributing to the ring interactions. The interaction pocket for the second ring of the biphenylic substrate 2,3-DGBP appears to be more relaxed. This interaction pocket also has low conservation among the EDOs and forms an important determinant for the differ-

FIG. 19. Coordination environment of 2,3-dihydroxybiphenyIdioxygenases.

505

I RON-OXYG E N / ~ l T OGEN PROTEINS

ences in specificity. Three active site residues, in addition to the iron ligands, are conserved in the EDOs: His195, His241, and Tyr250. His195 is localized on the more accessible side of the substrate and makes a hydrogen bond to one of the coordinating waters in the non-substrate-bound Form. His241 and Tyr250 are positioned on the opposite face of the iron. His241 makes a stacking interaction with the catechol substrate, whereas Tyr250 is positioned for making hydrogen bonds to His241 and possibly the dangling oxygen of Glu260, as well as to a catechol oxygen of the substrate. Only minor conformational changes occur in the active site upon substrate binding, of which the most significant is for His241 (about 1A) which stacks to the catechol ring. His194 shifts about 0.5 A toward the catechol upon substrate binding. The structures of the ferrous DHBD and ferrous DWBD in complex with subhave been determined but not yet fully described [291 (Fig. 20). The coordination in the diferrous protein is very similar to that seen in the ferric protein, containing two coordinating water molecules. The ferrous substrate complex reveals a six-coordinate iron where the substrate binds in a similar position to the ferric complex, but where a water is also bound with a long coordination distance (2.4 to the iron ion. This i s in contrast to the solution studies supporting a five-coor&nate form r1141. Recently, the structure of a catechol2,3-dioxygenase(metapyrocatechase, MPC) fiom Pseudomonas putida mt2 was solved at 2.8 A resolution [115]. This enzyme preferentially catalyzes the oxidation of monocyclic substrates and catalyzes the ring fission of 4’-substituted catechols such as 4-chlorocatechol or 4-methylcatechol more efficiently than catechol11161. The structure of MPC reveals a hoinotetramer formed by a 222 symmetry. The sequence homology to DHRD is around 20%, and the overall structure of the two-domain subunit is very similar to that of DHBD, as is the iron coordination environment. When the active Fe(I1) ion in MPC is oxidized to Fe(II1) it i s known to be released from the protein. Nevertheless, an iron ion is found in the active site in the crystal structure. Additional electron density is found at the iron site at a coordinating position, which was interpreted to be an acetone molecule from the

A)

FIG. 20. Coordination environment of ferrous of 2,3-dihydroxybipbenyldioxygenases in complex with the substrate 2,3-d~y~oxybipheny~.

NO~DLUND

506

crystallization liquid. The iron ion in the MPC structure is therefore presumably in the Fe(I1) form. The acetone binds in a coordination position in between where the two water molecules bind in DNBD, yielding a four-coordinate metal site with a close to tetrahedral geometry. 3.5.2. Structure of 4 - ~ y d r o x y ~ h e n y ~ pDioxygerzase-A ~ruu~~

Type 1

Exlradiol Dioxygenase Harnologue Recently, the structure of Pseudomoizas 4-hydroxyphenylpyruvate dioxygenase (HPPD) was solved [1111.HPPD is a bacterial enzyme related t o eukaryotic enzymes involved in the tyrosine degradation in maminals and the biosynthesis of photosynthetic pigments in plants [1171. HPPD catalyzes the formation of homogentisate from 4-hydroxyphenylpyruvate (4-IIPP) (Scheme 13). This reaction includes an oxidative decarboxylation step as well as a hydroxylation step in which molecular oxygen is incorporated at positions 6-1 and C-8 of the substrate. The bacterial enzyme is a homotetramer while the human enzyme is a homodimer and the sequences of the two enzymes display around 20% identity. The enzyme depends on Fe(I1) for its activity [ l l O ] . Surprisingly, the structure of' HPPD was found to be related to the structures of MPC and DHBD, although the sequence identity is less than 15%. The two-domain structure of the type 1 EDOs is present also in HPPD, with the 2-His-l-Glu iron being conserved in the 6-terminal domain (Fig. 21). However, the three active site residues Hisl95, His240, and Tyr250 in DHDB, which are present in all EDOs, are not conserved in HPPD and in the corresponding positions Phe332, Gln309, and Phe311 are found. No other obvious catalytically essential residues are found in the active site of RPPD. The iron ion in the HPPD structure is presumably in the ferric state and no coordinating water molecules are present. Instead an acetate molecule from the crystallization liquid is coordinated in a monodentate mode. While the coordination in is square pyramidal, the iron ion in HPPD, as in MPC, exhibits a distorted tetrahedral coordination due to the coordination of crystallization additives. The active site in NPPD exhibits a general hydrophobic character, as do the EDOs, but appears to be more deeply buried and has no channel leading to the outside. A model for the binding of the substrate 4-hydroxyphenylpyruvatein the active site of HPPD has been proposed where the carboxylate and carbonyl oxygens coordinate trans to the two histidine ligands.

02

OH

Scheme 13

OH


507

Acetate

FIG. 21. Coordination environment of 4-hydroxyphenylpyruvate dioxygenase.

3.5.3. Structure of Type II Extradiol Dioxygenase Recently, the structure of the type I1 ED0 LigAB from Sphingomonas paucimobilis SYK-6 was determined 11181. LigAB is a protocatechuate 4,5-dioxygenase and a key enzyme in the lignin degradation pathway, metabolizing several different aromatic ligniii derivatives. Previous spectroscopic studies of the iron site of LigAB have suggested similarities to the type I EDO, but the primary structures have not revealed any Similarities. The structure of the uzpz LigAB revealed a catalytic subunit with a novel fold formed by a 300-residue rxlp-type structure (Fig. 22). The active site iron is found on one side of a central p sheet where a cleft is formed. The a subunit is involved in covering the entrance of the cleft in the p subunit and the iron is therefore buried about 15 A from the surface in the R-0 heterodimer. The iron ion in the substrate-fi-ee form is coordinated by Hisl2, His61, Glu242, and one

FIG. 22. Structure of the type 11 extradiol dioxygellase LigAB (generated Don1 PUB file 1BFU).

NORDLUND

508

solvent molecule in a distorted trigonal pyramid geometry. The solvent molecule is found in the axial position and has a short coordination distance (1.8 indicating that it is a hydroxide ion. The structure of ferric LigAB in complex with PCA has been determined (Fig. 23) Ll18l. The two diol oxygens of the substrate are directly coordinated to the iron ion, whereas the solvent molecule present in the uncoordinated form of 1.igA.B is released upon PCA binding, as are several other water molecules of the active site. Iron moves towards the substrate in the substrate-bound form by 1.4 A, a movement that is accompanied by the movement of Hisl2. In this process, two conserved active site histidines, His195 and His127, form hydrogen bonds to one diol oxygen each. The residues interacting with the PCB are mainly hydrophobic residues of the p subunit contributed by loops and two helices. The interaction of the negatively charged PCB carboxylate group is made through main chain amino groups and alcohol side chains. In some type I1 EDOs, the 01 subunit covering the active site is missing, and instead a long insertion of 42-44 residues is present in the f3 subunit, which might be performing a similar lid function 11181. The iron coordination sphere of LigAF3 has been superimposed with the one of Bphc based on the substrate binding (Fig. 24) 11181. Although the positions of the 2His-Glu ligands are different for the two enzymes in this superimposition, the presence of two possible catalytic histidines in that active sites of both enzymes and the similar mode of substrate binding in the active sites support a common catalytic mechanism and reveal a beautiful case of convergent evolution of the two catalytic machineries.

A),

,GgSerOH 270NH

.. U

FIG. 23. Coordination environment of the type I1 extradiol clioxygenase Li@ with protocatechuate.

in complex

509

FIG. 24. The active site of the type I extradiol dioxygenase DHBD in complex with the substrate 2,3-DWBP (dark), superimposed on the type I1 extradiol dioxygenases LigAI3 in complex with PCA (light). (Reprinted with permission from [llal.)

3.5.4. Reaction Mech,anism of Extradiol Dioxygenases and Related Enzymes Steady-state kinetics indicates an ordered mechanism for the type I ED0 reaction where substrate binds before 02.No reaction intermediates of type I EDOs have yet been trapped for direct spectroscopic studies. The mechanism most consistent with the available data and based on the DMDB structures is shown in Scheme 14 14,8,104,1191. The initial substrate binding will replace one of the two water molecules of the ferrous form, leaving the other as a weakly bound water. EXAFS studies suggest that, the substrate binds in its monoanionic form 1107). This requires a proton to be released upon substrate binding, a process that might require catalysis and could involve one of the active site histidines, is194 or His241. The coordination of the protonated diol oxygen is expected to be weak and this coordination is likely to be stabilized by a second-sphere hydrogen bond to one of the active site histidines, most likely Nis194. EPR studies of NO binding to ED0 shows that the NO binding is promoted in a cooperative manner (100-fold) by the binding of substrate 11203. Similar results are obtained with azide, suggesting that this effect is not an electronic but rather a geometrical effect, and it is likely that O2 binding is effecting substrate binding similarly. The O2 insertion step could proceed through a nucleophilic attack of a ferric superoxide complex. Alternatively, the recombination of a substrate radical and a dioxygen adduct could give the same product. Using a cyclopropyl radical trap a reversible opening of the propyl ring during the reaction was observed that supports the radical path in the scheme 11191. The formation of an arylperoxy intermediate could be either through the hydroxo position of the substrate, as in the intradiol dioxygenases, or through the ortho position. Isotope studies have demonstrated that water can be inserted into the carboxylate moiety of the product, supporting a reaction path where the distal oxygen atom of dioxygen is inserted into the ortho

NORDLUND

510

B.

2

.

q

product

OH

Scheme 14. Proposed reaction mechanism for extradiol dioxygenases.

position and the proximal oxygen atom is inserted into the product carboxylate through a nucleophilic attack. The formation of a lactone intermediate is supported and the alternative path through a dioxyethane has been excluded based on isotopic exchange of water into the substrate (see discussion on the mechanism of the intradiol dioxygenases in Sec. 3.2.2) 152,531. The seven-membered a-keto ester lactone intermediate could possibly be preceded by an epoxide-type intermediate. The intradiol and extradiol dioxygenases catalyse the ring lysis at difrerent positions. Although several factors probably contribute to this difference, it may to a large extent be controlled by the position of the dioxygen insertion, to the hydroxyl position in intradiol dioxygenases, and to the ortho position in extradiol dioxygenases. Considering the stereogeometry of the ortho-alkylperoxo intermediate form in this reaction mechanism, it appears to be significantly less likely to form a tridentate coordination as expected for the hydroxoalkylperoxy intermediate of the intradiol dioxygenases. It is therefore not unlikely that the protonatedl diol oxygen is released from the iron ion in the reaction path to allow the required stereogeometry for an attack on the ortho group, and the histidines of the active site could also play important roles in the stabilization of these adducts through hydrogen bonding. The intriguing local structural similarities of the active site of type I and type 11 extradiol dioxygenases, engineered on two very different three-dimensional peptide folds, includes, in addition to the 2-His-Clu ligand motif, also two strategcally positioned histidine residues (Fig. 24). Most likely the reaction mechanism of the type I1 EDOs is very similar to the one described for type I E D 0 above, although some minor geometrical diKerences are present. One such difference is that the position for 0 2

IRON-OXYG~N/NITRO~EN PROTEINS

51 1

binding appears to be different in the two families; in the type I E D 0 the dioxygen is most likely bindifig trans to the glutamate, whereas in the type I1 E D 0 it appears to interact trans to the second histidine ligand. Very little mechanistic information is available on the HPPD family. However, the surprising evolutionary relationship between the active site of HPVD and the type I ED0 can serve as a basis for a mechanistic discussion. Despite the similarity in the first coordination sphere, HPPU lacks the two active site histidines present in both ED0 families. In fact, the HPPD active site does not appear to provide any suitable catalytic residues but rather forms a hydrophobic pocket for harboring the phenyl moiety of the substrate. The binding of 4-HPP in the active site of HPPD was modeled assuming a similar binding as the biphenol in DHBD and a direct coordination of the pyruvate moiety to the iron 11181. The substrate would occupy a hydrophobic pocket partly filled by water molecules in the unbound structure. Based on this structural model of the stereogeometry of the hydroxylation step, 0 2 was suggested to bind trans to the Glu ligand as is suggested €or the type I EDOs. The following reaction scenario could then proceed with reaction intermediates and geometries similar to that suggested for the 2-oxoglutarate-dependent2-His-GluIAsp proteins above.

ioxygenases Containing Rieske Centers Rieske centers and mononuclear Fe-QIN sites have been identified in dioxygenases from a large number of soil bacteria [40,1211. The cis-dihydroxylation activity of the Rieske dioxygenases (RDOs) provides substrates that are utilized by intradiol and extradiol dioxygenases in the degradation of aromatic compounds (Scheme 15). Most RDOB exhibit a broad substrate specificity and oxidize both aliphatic and aromatic C-H bonds. The range of different oxidation reactions catalysed by RDQs is similar to the cytochrome P450 family Ll221. However, the cis-dihydroxylation activity of RDOs is unique and can be catalyzed with high stereospecificity. The RDO reactions require dioxygen and NAD(P)H. Spectroscopic studies have revealed that the dioxygeii reaction takes place at a mononuclear iron(l1) site and that the Rieske l2Fe-ZSI center stores electrons required during the reaction. The biological redox partner for the Rieske center are NADPH- and flavin-containing reductases r1211. Phthalate dioxygenase (PDO) and naphthalene 1,2-dioxygenase(NDQ) are two wellcharacterized members of the RDO family [1041.

Scheme 15. cis-Dihydroxylationcatalyzed by naphthalene dioxygenase.

512

N~RDLUN~

3.6.1. Structure of Rieske-Type Dioxygenmes

The structure of the a& hexamer of a Pseudomonas NDO has been solved in two diRerent crystal forms 1123,1241.The structure of the p subunit of NDO is formed by an antiparallel p sheet lined by three a helices (Fig. 25). The a subunit consists of a catalytic and a Rieske-type domain. The catalytic domain i s formed by a ninestranded p sheet lined by a helices that extend through the whole a subunit. The mononuclear iron site and substrate-binding pocket of NDO are found in the bottom of a 30-A channel that leads to the protein surface. Two short distorted a helices line the active site and contribute the iron ligands. The specific residues that coordinate the iron include His208, His213 and Asp362, with Asp362 making a bidentate iron coordination (Fig. 26). His208 and His213 are stabilized by second-sphere hydrogen bonds with Asp205 and Asp361, respectively. The crystal structure might be the reduced form of the protein as X-rays have been shown capable of reducing the iron sites in the crystal [1251. The first NDO structure revealed a density in its active site that is consistent with an indole-peroxide adduct. The presence of this adduct is suggested to arise from an uncoupled oxidation reaction with indole that is present during the recombinant overexpression of the protein in E. coli [1231 (Fig. 27). A direct coordination between the iron ion and the peroxy-moiety of the adduct is observed. Subsequent structures o f NDO with and without substrate bound in its active site have also been solved a water molecule coordinates to the iron, resulting in a five11241. ~ i t h o usubstrate, t coordinate metal site, In the structure of NDO in complex with the substrate indole, the substrate binds in a very similar position as the indole moiety of the trapped peroxy complex. In addition, a water molecule is coordinated to the iron ion at the

n

FIG. 25. Structure of the naphthalene dioxygenase trimer showillg the Rieske and mononuclear iron site (generated from PDB file 1NDO).


513

.CYSSl

FIG. 26. Coordination environment of the mononuclear and Rieske iron sites in naphthalene dioxygenase. The two iron sites are found in neighboring subunits in the trimes.

FIG. 27. The active site of naphthalene dioxygenase with bound indole substrate. (Reprinted with permission from L1241.)

51 4

NORDLUND

position of the peroxide coordination. No large structural changes are observed upon substrate binding but Phe224 at the entrance of the pocket moves by 1A. Few polar groups are found in this pocket in addition to the iron site, including some main chain groups of residue 201-205 in the region faced by the indole nitrogen. All of the available crystal structures of NDO show a five-coordination of the mononuclear iron site but the oxidation states of iron in these structures are not clear. Rased on spectroscopic studies of phthalate dioxygenase, the Fe(I1) form is considered to be six-coordinated and upon binding of phthalate changes to five-coordinate [126-1281). Am201 is a potential ligand, but the distance (3.9 in the present structures is too long for a proper coordination. Asn2Ol could be a coordinating ligand in the Fe(l1) form, but a direct involvement of this residue in iron coordination is less likely because it is not conserved in all RDO sequences [1231. No other obvious additional catalytic residues are found in the active site that could be involved in providing protons for the reaction. However, Am201 appears to be in a position to hydrogenbond with dioxygen-iron species and could play a role in stabilizing reaction intermediates in some RDOs. The Rieske domain is made up of a p sandwich, with two fi hairpins forming fingers that coordinate the 12Fe-2S1 cluster. Two cysteines, Gys81 and CyslOl, coordinate iron 1 and two histidines, His83 and HislO4, coordinate iron 2. The catalytic domain of a neighboring CI subunit provides residues-Asp205 and 61~410,respectively-which hydrogen-bond to His83 and His104 (Fig, 26). This means that Asp205 hydrogen-bonds to the mononuclear iron site and a histidine ligand of the Rieske center, thereby linking the two iron sites. The distance between the two metal centers is about 12 A and is therefore the normal range seen for biological electron transfer [129]. A similar Rieske domain is observed in the cytochrome bcl complex [1301. However, the histidine ligands of the cytochrome bcl Rieske center do not hydrogen-bond with carboxylate residues, which might explain the difference in their redox potentials of about 400 mV. The interaction between the reductase and the Rieske domain in NDO is suggested to involve the surface lining the Rieske site, where the main chain atoms of CyslO1 are exposed. A possible role for the !3 subunit of RDO could be to participate with the reductase for this interaction surface. 3.6.2. Reaction Mechanism of Rieske-Type Dioxygenases

Limited mechanistic information is available for the RDOs. Based on steady-state kinetics and recent single-turnover experiments [131,2651, a feasible scheme for the overall reaction has been outlined. In this scheme, the generation of a fully twoelectron reduced form of the enzyme allows the substrate to bind. Substrate binding is suggested to enable the enzyme for the concomitant 0,reaction. Subsequent turnover of the enzyme results in cis-hydroxylation of the substrate and generation of the oxidized mononuclear and Rieske centers. To complete the cycle, the oxidized enzyme is reduced by two external electrons from the reductase system.

515

I RON-OXYGEN/NITROGEN PROTEINS

MCD data on phthalate dioxygenase suggest that the Fe(I1) site is six-coordinate, and becomes five-coordinate when substrate binds I126I. This is different from the crystallographic data of NDO where both the Fe(I1) and the Fe(I1) substrate forms are five-coordinate. These differences could be due to the fact that MGD studies of the mononuclear center are complicated by the presence of the Rieske site or differences between the two enzymes, or that real differences exist in the solution of the proteins due to the different solvent conditions used in the crystal and MCD experiments. Several possibilities exist for the reaction path and geometries of the O2 activation and substrate hydroxylation steps catalyzed by RDOs. However, the chemical requirements and the structural data place some restrictions on the possible mechanisms, outlined in Scheme 16. When the cis-hydroxylation requires a two-electron oxidant, one electron should be transferred to a dioxygen species at a relatively early stage of the reaction. This generates the key hydroxylating species, which could be either a formal Fe(II1)-peroxy or an Fe(I1)-superoxy species. A ferric-peroxy species have, for example, been shown to directly oxidize aliphatic 6-H bonds in model compounds 11321. The structure of a hydroxylating peroxy species could be either an end-on or a side-on dioxygen-iron complex. Stabilization of an end-on peroxy complex appear to require protonation or strong hydrogen bonding. Alternatively, the peroxide is generated concertedly with insertion into the aliphatic bond. A side-on peroxo, on

+

2e'from

reductase,

product

2Hc

or

+/

Scheme 16. Possible mechanism for the cis-dihydroxylatingactivity of the Rieske-type dioxygenases.

516

tlie other hand, could in principle be stabilized by the iron site per se, if a kghly accessible iron site is available. The active site of NDO might provide this accessi~ility in a four-coordinate form, if the water molecule is released. Although a cis-liydroxylation mec~anismcan be envisaged without the involvement of ferry1 intermediates, such intermediates are attractive for the monooxygenation, desaturation, and d e ~ e t h y ~ a t i oreactions n catalyzed by RDQs. No hydrogen isotope effects have been reported for the cis-hydroxylation catalyzed by RDOs, consisteiit with hydrogen abstraction not being a rate-limiting step. However, NI shifts have been observed, suggesting the possibility of epoxide or carbocationic intermediates in the reaction. The active site does not contribute obvious additional key residues for stabilization of reaction intermediates although Asn2Ol and the hydrogen bonding of the polar main chain groups to the indole nitrogen might play a role in the reaction.

e ~ e ~ h (Hr) ~ nare s nonheme Fe-O/N proteins that act as 0 2 carriers in some marine worms U331. They constitute one of three known structural solutions to l~ The other the problem of O2transport and storage seen in m u l t i c e l ~ u organisms. which mediates O2 binding by tlie reversible solutions are hemoglobi~niyo~lobin, formation of heme-superoxo complexes, and the hernocyanins, which carry dioxygen as a bridging peroxo-&copper core El341. H e ~ e ~ t is h most ~ n ofken found as an octamer, but other subunit compositions m e also known. The first well-characterized diiron Fe-O/i?J center in a protein was observed in r, and it became an important paradigm in the study of other diiron proteins. The reduced Fe(l[ll-FeUI) form of Hr, deoxy-Hr, is e form available for 0 2 binding. an ~ e a s u r e m e n t have ~ , shown scopic studies, in particular resonance binding forms a hydro~eroxo-Fe(III)"Fe(III}intermediate E1351. In most cases, the binding of O2 to hemerythrin does not appear to be a cooperative process, as is the case for O2binding to hemoglobin. Wemerythrin can also be isolated in a resting ~ e ( ~ ~ ~state, ) - met-Hr, ~ e t where ~ ~ ~the~ peroxy moiety has been lost or reduced. A cytoehrome b5 reductase has been shown to reduce metits active F e ~ I I ~ - F e ~form. l I ) The spectroscopic features of the iron c o ~ t ~ n i forms n g of the protein are characterized by an 0x0 bridge that results in an antif~~omagnetic coupling of the two iron ions. ntly, the c h e m o t ~ i spr ein DrcH has been shown to be s t ~ c t u r a l l y hernerythrins 1361. DRc contains a diiron site with similar spect p i " ~ p e ~ ito e sHr. The 0x0 form of DRcW is significantly less stable than deo suggests a possible functional role for this protein as an O2 sensor.

3.7.1. Structure of Hernsrythrin ost structural studies of Hr have been performed on the octameric Hr. The related m y o h e m ~ ~ h r ihas n also been structurally characterized L1331. The fold of the

I RON-QXYGEN/NITRQGEN PRQTEINS

517

hemei-ythrin subunit is a compact four-helix bundle (Fig. 28). In an otherwise hydrophobic core, the diiron center is buried within this bundle. The protein provides five histidines and two carboxylate residues that coordinate the diiron core (Fig. 29). In the structure of the reduced Hr (deoxy-I-Ir)one of the irons-Fel-has two terminal histidine ligands and the other-Fe2-has three terminal histidine ligands. The two iron ions are bridged by one glutamate, one aspartate, and one hydroxo group. This results in Fel being five-coordinated and Fe2 being sixcoordinate and leaves one open coordination position on Fel for binding dioxygen. The structure of oxo-Hr shows an identical coordination of the protein ligands as seen , with only minor adjustments of the coordination distances [1371. The dioxygen molecule is bound end-on to Fel with its free oxygen atom hydrogen bonded to the bridging 0x0 group of the Fe(III)-Fe(III) center. The dioxygen molecule faces a hydrophobic pocket and makes direct van der Waals contacts with residue Leu98. ~ u t a t ~ oto n sthe corresponding Leu98 residue in a myohemerythrin have been shown to severely affect the stability of dioxygen binding and easily lead to protein autoxidation [1331. The structure of a Hr-azide complex has also been determ to the diiron core in a similar mode as the dioxygen molecule of When compared with the dioxygen-activating diiron proteins discussed below, the order of the helices of the four-helix bundle is different. It i s clear that these families do not share the same evolutionary origin and that the h e ~ e ~ t h r i conns stitute a unique structural class 11391.

FIG. 28. Structure of oxo-hemerythrin (generated from PDB file 1HMO).

518

NORDLUND

nrs 181

FIG. 29. Structure of the diiron site of hemerythrin in the (a) diferrous deoxy form and (b) the diferric(-peroxy)oxy form [generated from PDF file: (a) lHMD and (b) 1HM01.

I R ~ N - ~ X Y G E ~ / ~ I T RPROTEINS ~GEN

57 9

3.7.2. Reaction Mechanism of Herrterythrin The currently accepted mechanism for reversible O2binding to hemerythrin is shown in Scheme 17 11331. The binding of O2 to Hr induces no significant conformational changes in the metal cofactor, as is also the case for the other two O2 transport proteins that have heme and copper centers.

Scheme 17. Reaction scheme for thc reversible O2 binding to hemerythrin.

A recent theoretical study using DFT methods of the Hr 02-binding mechanism may have shed some light on its electronic and protonic details 1140,1411. In the twostep reaction mechanism proposed, initially one electron is transferred from 0 2 onto the iron 2 ion. This brings about an elongation of the Q-O bond and a transfer of charge onto the iron core. It also changes the proton affinities of the peroxo and hydroxo groups and sets the system up for the subsequent step. In the second step, a proton tunnels from the peroxo group to the 0x0 bridge in a reaction that is coupled to an electron movement from the dioxygen molecule to Fez. The electron transfer step involves the whole Fe-O-Fe unit as a superexchange pathway. It is argued that a bent conformation of the 0x0 bridge is responsible for the observed strong antiferromagnetic coupling and is essential for promoting this electron transfer. As the electron and proton travel along different paths, these movements should be seen not as a hydrogen transfer but rather as a proton-coupled electron transfer I141I.

3.8. Large Diiron Carboxylate Proteins 3.8.1. Ribonucleotide Reductase R 2 Protein

Ribonucleotide reductase (RNR) catalyzes the formation of the deoxyribonucleotides needed for DNA synthesis through the reductive cleavage of the Z’-carbon oxygen bond of ribonucleotides [142,1431 (Scheme 18).The same enzyme catalyzes the reduction of all four deoxyribonucleotides in a highly regulated manner. Due to their key position in DNA synthesis, ribonucleotide reductases are potential targets for the development of antiproliferative therapies such as antiviral or antitumor drugs [144-1461. In all known RNRs, the substrate is activated for reduction by a protein-radical intermediate, generated from a radical cofactor. Three classes of RNRs have been identified based on their respective radical cofactors [1471: the class I RN

NO~DLUND

520 2Ht,2e’

B HO

H

-t H20

OH OH

Radical cofactor

OH H

Scheme 18. The R2 reaction cycle catalysed by ribonucleotide reductase.

radical system, the class I1 RNR cobalamin radical system, and the class I11 RNR glycyl radical system. The class I system is found mainly in higher organism but also in bacteria and DNA viruses, while the class 11 and I11 systems me only found in bacteria and viruses. All three classes of RNR have related catalytic subunits, but the class I enzymes contain an additional homodimeric subunit, the R2 subunit [2541, which is involved in generating and harboring the tyrosyt radical site. The stable tyrosyl radical of RNR R2 is capable of activating the substrate that is bound in the active site of the catalytic R1 subunit. a t e dsite and the neighboring The R2 subunit contains an ~ ~ ~ - c o o r ~ ndiiron tyrosyl radical site on Tyr122 [142,148,149].The diiron site is directly involved in the is days at 4°C) in an oxygengeneration of the highly stable free tyrosyl radical dependent reaction. The best characterized R2 proteins are those from E. eoli and mouse. The active protein is the diferric and radical-containing form (activeR21, which can be reduccd to give the radical-free diferrous form [150].The radical can also be scavenged without the reduction of the diiron site; this yields the resting diferric radical-free form (metR2). Charge transfer through a bridging 0x0 group produces characteristic absorption bands of the diferric site, related to those seen in hemerythrin. EPR and magnetic susceptibility stu s reveal an antiferromagnetically coupled diferric site. The different forms of the diiron core have been further characterized by a variety of techniques that include resonance Etaman, ~ N D ~ MCD, and Mossbauer f29,135,151].The tyrosyl radical has been shown to be magnetically coupled to the iron site but this coupling displays s ~ i ~ differences c ~ t between different R2 species. The diiron site catalyzes the (&-dependent generation of the tyrosyl radical on Tyr122. In il. pioneering single-turnover study on E. coli RNR R2, reaction intermediates in the radical generation reaction were revealed using time-resolved techniques 11521. Fudher studies on the E. c d i protein, and later on the mouse RNR R2, now provide a plausible reaction sequence for the radical generation reaction of RNR R2 (Scheme 19). The 02-reactive form is the Fe(I1)-Fe(II)center. No direct evidence for a peroxide intermediate has been obtained in the radical generation reaction of wildR2, but in the D84E mutation of E. coli RNR R2 a peroxide species is observed 12551. Optical, Mossbauer and resonance Raman parameters suggest that it is a cis-p-1,2 peroxo species that accumulates in this mutant. The reduction of the O2 bond leads to the formation of an activated formal Fe(III)-Fe(IW core, labeled intermediate X, and is concomitant with the transfer of an electron from an external

I ~ O N - O X Y ~ ~ N / N I T R OPROTEINS ~EN

521

Reduced R2

Peroxo ln~~ediate

l n t ~ ~ e d i aXt e

Active RZ

Scheme 19. Proposed intermediates in the 112 reaction cycle catalysed by the diiron site of ribonucleotide reductase.

electron donor [256]. EXAFS data on intermediate X suggest an unusually short iron12571. Intermediate X can drive the transfer of an iron distance of around 2.5 electron from Tyr122 to generate the stable tyrosyl radical. Fe(11) is the source of the external electron in the in uitro generation system. A possible intermediate species in the electron transfer has been suggested to be a tryptophan cation radical, most likely on Trp48 1153-1551. 3.8.2. Structure of the Ribonucleotide Reductase R2 Protein

The first crystal structure of an R2 protein to be determined was that of the E. coli RNR R2 homodimer 1154,1561. The fold of each subunit is dominated by a bundle of eight long M. helices (Fig. 30). The diiron site is buried in the core of the bundle, about 10 from the nearest surface. The R2 dimer interface is formed by extensive contacts

A

FIG. 30. Structure of the E. coli ribonucleotide reductase R2 protein (generated from PDB file 1RIB).

522

A

from four a helices in each subunit and the two diiron sites are positioned about 25 from each other. The protein ligands for the diiron site are His118 and the four carboxylate residues are Asp84, 61~115,6 1 ~ 2 0 4and Glu238. All of these ligands reside on the four helices that surround the diiron site (Fig. 31). These four helices display an internal pseudo~twofoldsymmetry that is also observed in the s of the iron ligands. Asp84, Glu115 and is118 are pseudo-twofold Glu238, and His241, giving the sign re amino acid sequence structures of several different forms of 141. coli R2 have been ). The original structure was of the resting radical-free diferric , determined at 2.2 A resolution [156]. In this structure, the irons are 115 and one 0x0 group, and the Fe-Fe distance is 3-3 6 1 ~ 2 3 8coordinates in an asymmetrical ~ o n o d e n t a t emode to Fe2. Each iron is also terminally coordinated by one histidine and one carboxylate. The carboxylate ofAsp84 is in this structure coordinating in a semi-bidentate mode. In a recent 1.4-A cry0 structure the a t e Each iron also coordination of Asp84 to F e l can be described as ~ o n ~ d e ~ t[ZSS]. coordinates a single water molecule, both of which face a hydrophobic patch. This 12 and Ile234, is formed by the conserved residues Phe208, h y d r o ~ ~ o bpatch ic and at the edge of this patch the OH group of the radical Tyr122 is found about 5.2 from Pel. The coordination of the diiron core of metR2 shows significant asymmetry; in the room t e ~ p e r a t u r estructure both irons are six-coordinate, while in the highreso~utioncry0 structure Fel is five-coordinate. The structure of the diferraus reduced ( r e d ~ 2E. ) coli protein has also been determined and reveals a considerably more symmetrical iron site than in the differric form [157]. In redR2, the two irons are four-coordinate, with an Fe-Fe distance of 3.9 A, and no solvent molecules are

A.

HNTrpll I I II

FIG. 31. The coordination environment of the diferrous E. coli ribonucleotide reductase R2 protein.


523

j

FIG. 32. The structure of the diiron site of B. coli ribonucleotide reductase R2: (a)apoR2, (b) diferrous redR2, and (c) Merric metR2 (2.2 A structure). [(a) from [362], (b) and (c) generated from PDB files IPFR and lRIB, respectively)].

524

bound to the site. A comparison of the diferric and diferrous sites shows that large conformational shifts are made by 6 1 ~ 2 0 4and Glu238. Both residues change their side chain rotamer conformations, and their carboxylate-iron interactions involve different binding modes in the two forms. This unusual conformational change is probably made energetically feasible by stabilizing second-sphere hydrogen bonding patterns for dangling oxygen groups as well as the presence of relatively low protein density around these residues, allowing the conformational change to take place with.. out steric hindrance. Glu204 is surrounded by two water molecules and 61~238is found in the hydrophobic pocket lining the iron site. Based on sequence and structural considerations, Trp48 was identified as a potential participant in electron transfer events related to the dioxygen activation and the radical transfer to the R1 subunit [154,1561. The conserved Trp48 i s linked to the iron ligand His118 through conserved hydrogen bond with Asp237. A set of hydrogen-bonded conservcd residues on the R1 subunit have also been identified, which are potential candidates for an extended transfer path of radical properties to the substrate bound in the active site on the 1 subunit about 30 A away [1581. Mutations of these residues on the R2 subunit have been shown to severely affect the dioxygen reaction and to virtually abolish radical transfer to the R1 subunit C155,l59-16Ilt The structure of the E.coZi R2 protein has also been determined in the iron-free form tl621 (Fig. 32a). When compared to the diferrous form this structure revealed only very small positional changes of the iron ligands, resulting in a clustering of four carboxylate residues in the apoR2 structure. Rased on the observed hydrogen bonding pattern in this structure, it is implicd that four protons have been introduced into the ligands sphere, two on the histidine ligands and two on the carboxylate ligands; that is, the charge lost from two ferrous iron ions is fully compensated by four protons in the apoprotein. Several structures of metal-substituted and mutant fornis of the E. coli H2 protein have been determined. The coordination spheres o f Mn(lI)- and Co(II)-substituted R2 have been shown to be very similar to the coordination of the diferrous site [ 157,163j, with the exception that the dimanganese site has a water molecule that coordinates to Fe2. Mutations to residues in the hydrophobic pocket that lines the iron site have been shown to have severe effects on the dioxygen chemistry. One example is the substitution of Phe208 in the hydrophobic pocket to tyrosine, which leads to its meta-~iy~roxylat~on and the formation or the d ~ h y d r o ~ h e n y l a l ~ (DOPA) 208 residue [164]. A second example is the mutalion of the flexible 6 1 ~ 2 3 8 , exposed into the hydrophobic pocket, to an alanine, which leads to the meta-hydroxylation of Phe208 11651. The structures of these mutant proteins have been determined and in both cases the hydroxylated residues coordinate the iron site. The exact mechanisms of these reactions remain to be elucidated, but these mutations clearly illustrate the sensitivity of the dioxygen reaction to structural perturbations in the region of the iron site. Another example of resteering of the dioxygen reaction is the Y122F mutation where new protein radicals on tyrosine and tryptophan residues are seen L259l.

The structure of a mutant E. coli RNR R2 (F208A) has been determined in a complex between azide and the diferrous form of the protein (2601. h i d e binds to Fe2 in this structure, and Glu238 changes the coordination mode from a symmetrical carboxylate bridge in the noncoordinated structure, to an asymmetrical monodentate bridging mode (see discussions below). This is concomitant with the shortening of the Fe-Fe distance to 3.4 A. The structure of RN R2 from two additional organisms has been determined, i.e,, mouse RNR R2 and SalrnonelEa typhimuriurn R2 f166,1671. The respect sequences of these proteins are around 45% and 25% homologous to the E. cedi protein and their overall structures are v e n similar. The mouse protein in its nonradical form easily loses one of its irons, and the structure determined contains only mononuclear iron sites. The lability of the iron ion in mouse RNR R2, as well as the higher sensitivity ofthe tyrosine radical to radical scavengers, can be explained by a channel that leads from the iron binding site to the surface of the protein 11661. The structure of the Salmonella R2 protein has been determined in both the diferric and diferrous forms. Both of these structures we similar to the corresponding structures of the E. coli protein. However, a significant difference of the S. typhirnuriurn protein is that the OH of the tyrosine radical site is found 7.5 A from Fel, instead of 5.2 as in the E. coli protein, and that an additional water molecule is found hydrogenbonding to the tyrosine OH group in the S. typhirnuriurn protein [167].

A

3.8.3. ethane ~ ~ n o o ~ y g ~ n ~ e s

Methane monooxygenase (MMO) promotes the first and rate-limiting step of carbon fixation in obligate methanotropic bacteria. The enzyme catalyzes the hydroxylation of methane to methanol in an N (P)H-dependent reaction F168,169I

CH4-I-NAD(P)W+H++O2 ---f CH30H+NAD(P)f -I-H 2 0 A consequence of methane oxidation by methanotrophs is the reduction of this greenhouse gas which is released into the atmosphere [l?Ol. MMOs have also attracted attention by their unique ability to oxidize a broad range of hydrocarbons in addition to methane [171-1731. Saturated, unsaturated, cyclic, and halogenated hydrocarbons are oxidized Lo yield oxygen insertion products such as alcohols, epoxides, and phenols, as well as two-electron oxidation products. Potential industrial applications for MMO include the bioremediation of land contaminated by organic pollutants and the oxidative removal of trichloroethylene from drinking water. Two forms of MMO exist: a soluble form and an unrelated integral membrane form. 'i'he soluble MMO is a diiron protein made up of three protein components: the hydroxylase (MMOH), which harbors the iron-containing active site; the reductase (MMOR),which is the site of NAD(P)H oxidation; and protein €3, which is an essential activator of MMO. Several other hydroxylases that are evolutionarily related to ~~O exist, including toluene 2-monooxygenase, toluene .?l-monooxygenase,phcnol hydroxylase, xylene monooxygenase, and alkane hydroxylase 151. The mechanistic information on these systems is still limited, although it appears likely that their catalytic

NORDLUND

526

strategies are similar to those of MMO. They are less potent hydroxylases, and none of them have been reported to hydrovlate methane. The MMO hydroxylase is an a2(3,y2 protein, with an M,. of about 245 ma. Each u subunit houses a catalytic diiron center that is the site of methane oxidation. The reductase and protein B bind noncompetitively to MMOH. The reductase MMOR contains an Fez& center and an FAD cofactor that can store two electrons, taken from NAD(P)H, and transfer them to the diiron site during the reaction cycle 1171,1741. Protein B does not contain any cofxtors, and its effects on the reaction are complex 1175-1771. These effects encompass effects on the chemistry at the active site as well as the comunication between the reductase and MMOH. The structural basis for these effects is uncertain, but it has been shown by MCDiCD studies that the binding of protein B to MMOH induces conformational changes, most notably changes to the coordination of one of the active site irons [175,178,1791. The resting diferric form does not show any chromophoric feature which excludes the existence of a bridging 0x0 group at the diiron site. Magnetic studies of the site reveal the diferric site to contain two antiferromagnetically coupled high-spin iron ions 11801, while the diferrrous O2 reactive form has been shown to contain a ferromagnetically coupled site [181]. Pioneering single-turnover kinetic studies has provided a detailed understanding of the reaction cycle of MMO (Scheme 20) C182,1831 and has identified several istence of an initial iron-bound 0 2 adduct, unique reaction intermediates. T has been indirectly inferred from the kine and an early peroxy intermediat

Reduced MMO

Fe"-Fe" 02

*

Compound Q

Compound T

Fe'"-Fe'"

Fe"!Fe"f

Scheme 20. The methane monooxygenase reaction cycle.

I RON-OXYGEN/NITROGEN PROTEINS

527

data [184]. The first directly observed intermediate is a peroxy intermediate, has been characterized using optical and Mossbauer spectroscopy. It has been proposed to contain two F’e(II1) ions and a peroxide, most likely coordinated in a p1,2 geometry with similar features to the peroxo intermediate found in RNR R2 D84E e next step of the reaction, the peroxo intermediate is converted to the which is a formal Fe(lV)-Fe(TV) cluster, and best described as two antiferromagnetically coupled high-spin Fe(IV) ions [1881. This unique species is directly responsible for the substrate oxidation and reacts the substrate in a bimolecular fashion. Based on EXAFS data, the structure of s been predicted to contain a short F’e-Fe distance of around 2.5 A and Fe-0 dist of 1.7 A and 2.2 A [189]. This has been the basis for suggesting that intermedi as a diamond core structure, but other core structures are also consistent with the data (see See. 3.8.7) [1891. Substrate hydroxylation leads to the formation of a product complex, and the release of the product is the rate-limiting step in the reaction. The presence of substrate in the single-turnover experiment does not affect the lifetime of the observable reaction intermediates in the MMO reaction cycle, exce for the hydroqlating species [261J. On the other hand, the presence of protein enhances the reactivity of the differous site to O2 by a factor of 1000, and in the absence o f protein B none of the reaction intermediates described above are observed in the single-turnover experiments 12621. 3.8.4. Structure of Methane Monooxygenases

The structure of the azP2y2MMO hydroxylase from M. capsulatus 11901 was originally determined, and later the same form of M. trzchosporium Mil40 [1911. The ironbinding @ subunit of MMOH is formed by two domains: the N-terminal domain, which is composed of an eight a-helical bundle and shares significant structure homology to the RNR R2; and the C-terminal domain, which displays a unique a/P fold (Fig. 33).

FIG. 33. Structure of the M . capsulatus methane monooxygenase hydroxylase (generated from PDB file 1MTY).

The B subunit of MMOH shares structural similarities with the N-terminal a-helical bundle domain of the 01 subunit, although no sequence homology exists between them. The y domain is formed by eight short c1 helices that interface with both the a and the p subunits. The structure of the regulatory protein B has been recently solved and reveals a one domain c1-8 protein. No structure has been determined for ~0~ complexed with protein B or the reductase 1192,1931. The diiron site of MNIOH is buried in the C-terminal domain of the a subunit and coordinates with two histidine residues, His147 and His246, and four glutamate residues, G1u114,G1u144, and Glu209, and Glu243 (Fig. 34). The positions of the iron sites within the a-helical bundle of MMOH and RNR R2 are very similar. The pseudotwofold symmetry of the iron ligands previously observed in the structure of RNR R2, is also found in MMOH. In the original structures of the M. capsulatus diferric site, in addition to the protein ligands, three solvent molecules were bound to the diiron site. They were assigned as one terminal water molecule, and bridging hydroxy group and a larger bridging group, most likely an acetatc ion that had been recruited from the crystallization solution. This acetate ion extends into a hydrophobic pocket, which most likely constitutes the substrate binding site. The active site pocket of MMO consists, in addition to the iron-coordinating residues, of mainly hydrophobic residues. Exceptions are Thr213 and Cys151.61~243is also found in this pocket where it makes a hydrogen bond to a coordinating solvent molecule on Fel with its dangling oxygen. The structures of the diferric M. capsulatus and M. trichsporiurn proteins, without the diiron coordinating acetate have also been determined. The distances between the two six-coordinate iron ions in the acetate-free structures are 3.0 and 3.1 respectively. In these structures, a hydroxide or a water molecule replaces the acetate in either a bridging, or a semibridging, coordination mode [191,1941. The structure of the diferrous M. capsulatus NTiVO hydroxylase protein has also been determined and reveals a major conformational change of the side chain of

A,

Thr7.13

,

?H% ,

HNTrpll

-SH CYs1sr

FIG. 34. Coordination environment of the diferrous site of the M . capsulatus methane monooxygenase hydroxyjase.

IRON-OXYGEN/N ITROGEN PROTElNS

529

6 1 ~ 2 4 3as compared to the diferric structure (Fig. 35). This side chain makes bidentate interactions with Fez, and one o f its carboxylate oxygens bridges the two irons, which results in an Fe-Fe distance of 3.3 A. Two water molecules occupy coordination sites on Fel, but only one of these waters is within a proper coordination distance, and both iron ions can be considered to be five-coordinate. The iron coordination sphere of MMOH displays significant stabilization by second-sphere hydrogen bonding. Both iron-coordinating histidine residues are making hydrogen bonds to aspartates (Asp242, Asp143). Glu144 and 6 1 ~ 2 0 9are hydrogen-bonded by Gln140 (Fig. 34). The dangling oxygen of 6 1 ~ 1 1 4also makes hydrogen bonds to coordinating water molecules in all structures, while the dangling oxygen o f

I

FIG. 35. Structure Qfthe diiron site of Ibl. capsulatus methane monooxygenase hydroxylase in the (a) diferrous form and (b) diferric form [(a)from [1941 and (b) generated from PDB file 1MTY).

611.1243 makes a hydrogen bond to coordinating solvent molecules in the diferric structures. A hydrogen bonding network of water molecules extends into the substrate binding pocket to Thr213. The stnicture of the x2&y2MMOH is unusually flat and it is possible that the reductase and B component could bind on the extended surface close -to the interface of the 01 and the p subunits. The binding of the B component has been shown to induce large conformational changes on the coordination of at least one of the irons [175,178,1791. These effects are most likely mediated through interactions with rx helices exposed on these surfaces and could either directly influence the conformation of the iron-liganding residues or do so indirectly by altering the local hydrogen bonding pattern.

3.8.5. A' Desuturuses The fatty acid desaturases insert a cis double bond into fatty acids in an NAh)PH and 02-dependent reaction (Scheme 21) 11951. This is an essential step in fatty acid biosynthesis. and the desaturation of lipids strongly affects their physical properties. The desaturases exist as both membrane bound and soluble enzymes, and different enzymes show large variations in stereo and regio selectivity. Both m e m b r ~ ~ - b o u n d and soluble desaturases use a diiron cluster in their active site but they are not structurally related. The best characterized soluble desaturase is the diiron protein stearoyl-acyl carrier protein (ACP) A' desaturase (n9D)from castor seeds [196,1971, which is found in the plastids, Free fatty acids are not substrates for the enz.yme; instead, the substrate for the desaturation reaction i s a complex of the fatty acyl chain covalently attached to the acyl carrier protein, The A% catalyzes the 02-dependent insertion of a cis double bond between the 9th and 10th position of the fatty acid to produce oleoyl-AC. The electrons needed for this reaction are contributed by NADPN through ferredoxin and ferredoxin oxidoreductase. A9D i s isolated as a diferric protein with absorption features similar to Hr and RNR R2 due to an 0x0-bridged diiron site. The two ferric ions are strongly antiferromagnetically coupled, and EXAFS data have suggested two different forms, with iron distances of 3.1 and 3.4 A [198]. The diferric protein can be reduced either chernically or by the biological ferredoxin system. owever, the two procedures produce

A

NADPH. Ferredoxin Oxidoredudase

[2Fe-2S] Ferredoxin

Stearoyl-ACP a9-ciesaturase

Scheme 21. The stearoyl-acyl carrier protein A9 desaturase reaction cycle.

IRON-OXYGEN/NITROGEN PROTElNS

531

proteins with very different properties. The biologically reduced protein has not yet allowed extensive mechanistic studies and most physical data obtained so far relate to the chemically reduced protein. This form shows a lower reactivity with dioxygen than the biologically reduced form and does not catalyze the desaturation reaction but rather works as an oxidase 11991. Addition of the substrate ACP increases the reactivity with O2 by lOOO-fold, and MCD studies suggest that the coordination of the diferrous site changes from two five-coordinate ions, without substrate, to one fourand one five-coordinate iron with substrate present [2001. When the chemically reduced protein is reacted with dioxygen a long-lived peroxide intermediate is observed that has spectroscopic features similar to those of the MMO intermediate and the peroxo intermediate observed in D84E RNR R2. Azide also hinds the chemically reduced protein where binding at pH > 7.8 is suggested to be in an ql-terminal mode while at pH lower than 7 the azide is suggested to bind in a bridging p1,3 mode l20ll. A large KII/KD isotope effect of around 7 has been observed for substrates labeled at the %position, while no significant isotope effect is seen for substrates labeled at the 10-position 12021.

3.8.6. Structure. of dgDesaturases The crystal structure of the stearoyl-ACP A9D from castor seeds have been solved at 2.4 A resolution L2031. The structure reveals a subunit fold containing eight long a helices that is very similar to the one seen in RNR R2 and MMO, although no significant amino acids sequence homology exists among the different proteins (Fig. 36). The structure was solved in the reduced diferrous state, which had serendipitously been generated by X-ray irradiation of the protein crystal. The diiron corc is bridged by Glu229 and Glu143 and the terminal iron ligands are provided by Hisl46, His232, and the two bidentate carboxylates, 6 1 ~ 1 9 6and Glu108 (Fig. 37). This yields a very symmetrical structure with a distance between the two iron ions of 4.2 A (Fig. 38). No bridging or terminal solvent molecules were observed to coordinate the hgD diiron site, which results in two 5-coordinate ferrous ions. A predominantly hydrophobic pocket is found at the diiron site and in this pocket a solvent molecule is hound that is close to being coordinated to the diiron site in a bridging position. The protein ligands of the A9D iron site are in the same positions in the four-helix bundle surrounding the iron site as are the ligands in MMO and RNR R2. The second-sphere coordination of the two histidines also hears similarities to MMO and RNR R2, and in A9D both histidines are hydrogen-bonded to aspartate side chains. A deep, long hydrophobic channel, containing several solvent molecules, extends from the surface of the subunit, passing the iron site and presumably constitutes the pocket for substrate binding. Electron density found in this pocket was suggested to be due to the hydrophobic acyl tail of a bound octylglucoside. Modeling of the fatty acid substrate in this pocket places the site of desaturation close to the diiron site [203].

532

FIG. 36. Structure of the stearoyl-acylcarrier protein A'-desaturase (generated from PDB file 1AFR).

FIG. 37. Coordination environment of the diferrous site of A'-dewaturase.

FIG. 38. Structure of the diferrous site of Ag-desaturase (generated from PDB file I.AFR).

XYG E N / N ~ T R O EN ~ PRQTEINS

533

3.8.7. Geometly of 0, Activation in the Large Diiron Carboxylate Proteins

The formulation of plausible mechanisms and geometries for the 0 2 activation reaction catalyzed by MMO, RNR R2, and A9D poses a delicate problem, and will therefore be discussed in some detail. In contrast to the mononuclear iron and heme proteins, the large conformational space available to the diiron enzymes, through carboxylatc shifts of the iron ligands, allows these proteins to potentially access a range of coordination modes and therefore open coordination sites on the irons which can harbor 0 2 adducts. A key question in understanding these dioxygen reactions are: to what extent are the O2 mechanisms are similar among the different diiron proteins? The chemistry catalyzed by the diiron enzymes, after lysis of the dioxygen bond, shows significant differences, and includes hydroxylations by MMO and related proteins, desaturations by A’D, and two consecutive one-electron oxidations in RNR R2. Still, the structural similarities of the ligand sphere, the observed peroxide adducts and high-valent iron oxidation products Q and X in MMO and RNR R2, imply a significant degree of similarity in their O2 activation mechanisms. The existence of mechanistic similarities among these proteins is also consistent with their common evolutionary origin. An important issue related to the mechanism of 0 2 cleavage is the role of protons in the reaction. In the P450 system, the dioxygen molecule is suggested to coordinate in an end-on mode to the heme iron before it is heterolytically cleaved to generate a ferry1 intermediate 12041 and water. Protonation of the “leaving” oxy group, to yield water, is probably a kinetic requirement for the P450 reaction. In the diiron proteins, on the other hand, the negatively charged product from either homolytic or heterolytic cleavage of dioxygen could in principle be stabilized by both iron ions, i.e., protonation may be less of a requirement in these systcms. No solvent isotope effects are seen in the 0 2 cleavage step of the E. coli RNR R2 reaction (W. Blodig, unpublished). Protons have, however, been implied in the h4MO reaction on the observation of K ~ / K effects H of 1.3 and 1.4 for the formation of the intermediates, respectively [184]. Although these observations may eorrespo to essential proton transfer steps, the values are small and may instead result from unspecific solvent effects. The RNR R2 system appears to lack an obvious proton source at the djferrous site. No strongly bound water molecules and no additional proton donors other than Y E 2 are present. However, it is unlikely that the OH of Y122 is essential €or the Q2 cleavage step, since RNR R2 Y122F catalyzes the reaction relatively efficiently [152]. In MMO, the conserved T h r l l 3 is a possible source of a proton. However, mutagenesis studies of the corresponding residue in the homologous toluene 4-monooxygenase have no major effects on the activity of the enzyme 12051. Protons may also be provided by non-metal-bound waters, as is the latest proposal for the source of protons in the cytochrome P450 reaction [ZOfiI. However, an end-on dioxygen cleavage geometry in RNR R2 that involves only a single iron ion does appear unlikely after a consideration of the ENDOR data on intermediate X, which suggest that both oxygen atoms end up on the diiron site-one as a bridging 0x0 and one as a terminal bydroxy

N~RDLUND

534

or water molecule (2071. The following discussion will therefore focus on possible dioxygen activation geometries involving both iron ions. Another key to understanding the dioxygen chemistry of the diiron proteins is a proper understanding of the structural features of the &-reactive diferrous forms of the diiron proteins. At present, the structures of diferrous forms of RNR R2, MMO, and A'% have been determined. However, the diferrous forms that have been structurally characterized of MMO, without protein B, and A%, without substrate, have low 0 2 reactivity, and it has been shown by MGD studies that the corresponding structures in solution exhibit significant coordination difference, in comparison with the 02-reactive forms [291. In contrast, the diferrous RNR R2 forms that have been structurally characterized should correspond closely to most 02-reactive forms of these proteins. It has also been confirmed that the crystals of E. coli R2 are highly efficient in catalyzing the tyrosyl radical generation reaction (20Sl. An additional key to the dioxygen activation mechanism is the structures of the in R2 and intermediate in MMO. These high-valent diiron forms, intermediat t clues to the geomet of the dioxygen intermediates may also contain imp activation reaction as they most likely correspond to the direct product structure of the Q2 cleavage reaction. The paradigm for the high-valent iron structures has been s a symmetry in the the symmetrical diamond core, which by its geome actual O2 cleavage reaction [189]. The EXAFS data are, however, consistent with other coor nation geometries for these intermediates E2091 (Fig. 39) and the ENDOR data of rather imply an asymmetric mode of O2 cleavage I2071. Although the coordinative flexibility of the diiron site is large, the available structural data on different forms of R2, MMQ, and A$ desaturase provide support for some basic restrictions to be placed on this flexibility; therefore, some predictions can be made for possible accessible coordination geometries along the reaction path.

I

FIG. 39. Structure of diiron cores consistent with E W S data on X and

I RON-OXY~EN/NlTROGENPROTEINS

535

Based on such an analysis, a maximum of three different open positions on the diiron center appears to be available for the coordination of solvent and oxygen adducts. These positions have been labeled B1, B2 and T and are shown in Fig. 40. Glu238 (and the corresponding residues in the other diiron enzyme) has been seen to coordinate in three different geometries. Tnterestingly, these three geometries appear to directly control the availability of the open coordination sites. The terminal ligands, Asp84 and Glu204 in RNR R2, also control the availability of the open coordination position, and when in their bidentate coordination mode, they inhibit adduct binding to the B1 and T positions. When thesc terminal ligands make monodentate coordinations, their dangling carboxylate oxygen can also influence solvent binding to B2 and T, by contributing stabilizing hydrogen bonds to the solvent molecules. Clearly, these control features have significant asymmetry. Which are the available geometries for the dioxygen cleavage step? Excluding an end-on cleavage scenario, as discussed above, it is reasonable to assume that both oxygen atoms of the dioxygen cleavage product coordinate to the diiron center. This effectively excludes a symmetrical bridging coordination of the two irons by 6 1 ~ 2 3 8 (Fig. ~ O C ) ,since only the €31position is available for coordinating the lysis product. However, an uncleaved peroxy group might reside in only the Bf. position. The bridging bidentate chelate coordination of 6 1 ~ 2 3 8has two open coordination sites (Fig. 4Ob), the B1 and T positions, which makes it a possible candidate for participating in the dioxygen cleavage step. In the monodentate coordination of 61~238,all three coordination positions are in principle available (Fig. 40a). However in the nonbridging geometry of Glu238, it is very likely that a water or hydroxide is required to stabilize a hydrogen bond with the dangling free oxygen of 61~238,as no other suitable group is available for hydrogen bonding. This assumption leaves only two free coordination positions on the iron site. Depending on the position of the solvent molecule coordinated, the free coordination positions may be any combination of B1, l32, and T. Based on these geometrical assumptions, only four possible reaction scenarios are available, outlined in Scheme 22. All four schemes predict a high coordination number of' the high-valent iron product and therefore imply a low probability for reorganization of the coordination after dioxygen

FIG. 40. A proposed generalized scheme for the control of the available open coordination sites on the diiron carboxylate proteins RNR R2, EMO, and A9D. The maximal availability of the sites is fully controlled by the three observed positions of Glu238. T and R1 are accessible in the hydrophobic pockets, whereas B2 is not.

536

~~RDLUND

Scheme 22. Four potential dioxygen activation geometries for the large diiron carboxylate proteins.

cleavage, i.e. the observed structures of should he very similar to that of the direct 0 2 cleavage products. Interestingly, the four outlined pathways predict only two possibilities for the principal coordination environments of the high-valent iron cores. Both of these structures are consistent with the experimental data. The distinguishing geometrical features between these schemes is whether 6 1 ~ 2 3 8will go through a major conformational change during the reaction or not, and whether the O2 cleavage geometry will be symmetrical or not. The three first schemes predict a large conformational change o f 6 1 ~ 2 3 8from a bridging to a terminal coordination. This conformational change includes breakage of' the interaction between one carboxylate oxygen and the iron site, as well as the change of two rotamer orientations of the side chain of Glu238, which appear to require significant energetic costs. However, the fourth reaction pathway contains no carboxylate shift hut predicts an asymmetrical dioxygen cleavage mode. This mode does not allow a mctal/iron-bound solvent molecule to provide a proton to the reaction and is


537

inconsistent with a diamond core structure. It does, however, provide a reaction path without obvious energetic hurdles in the form of conformational changes. In addition, it provides a strict control of the dioxygen chemistry and could explain why only oxygen atoms from O2 are inserted into the products of the MMO reaction. In pathways 1 and 2, water molecules coordinate to the two positions, B1 and T, that are exposed into the hydrophobic pocket, and could potentially end up in the hydroxylated product. Pathway 4 also has the attractive feature of a well-accessible terminal 0x0 group in position T that could serve as the hydroxylating species, in a manner similar to that of the Fe(IV)=O species predicted to exist in cytochrome P450 [2041. MMO was one of the first systems where density functional theory ( culations were shown to have the potential to contribute significantly to the understanding of complex metal cluster chemistry [ZlOI. To date, several DFT calculations have been made on the MMO system, which provide some insights into some of the possible Oz cleavage mechanisms for diiron centers, as well as the mechanism of hydroxylation [210-2121. These studies have focused mainly on a symmetrical dioxygen cleavage scenario, based on the diamond core paradigm. The stability of some potential oxygen intermediates has been analyzed, although leading to con~icting conclusions. In two studies, fully Symmetrical four-bridged sites were preferred, while in another study such a fully symmetrical four-bridged site was rejected as a possibility. As yet no calculations have been made for any of the intermediates proposed in reaction pathway 4. In conclusion, the structural data of the three different diiron proteins provide support that only a limited set of geometries are available for the O2 cleavage step catalyzed by these enzymes. The lated data are consistent with related dioxygen cleavage geometries in at lea and RNR R2, and where pathway 4 appears to provide the best control of, and the smallest energetic hurdles for, this reaction. 3.8.8. Reaction Mechanism of Radical Generation in RNR R.2

RNR R2 catalyzes what appears to be the simplest reaction of the three diiron enzymes discussed. The R2 radical generation reaction can be described as two consecutive one-electron transfer reactions, with the first electron provided by an external source and the second from "yr122. Free ferrous iron can serve as the external electron donor in vitro. The generation of a radical on Trp48 has been suggested to be an initial intermediate in this reaction [1541. Trp48 resides close to the protein surface and a radical located on this residue should be reducible by exogenous reductants. The electron from Trp48 could either be injected directly into the peroxide intermediate to assist in O2 cleavage or may be transferred to a hypothetical transient Fe(IV)Fe(IV) intermediate, to generate intermediate X [153]. A proton transfer from the surface of the protein, in connection with this electron transfer event, would conserve the net charge of the iron site. However, no obvious path for such a proton transfer has been identified in the R2 structures.

538

NORDLU N D

In the second half of the reaction, an electron is transferred from Tyr122 to . If not kinetically coupled, this electron transfer should be at least thermodynamically coupled to a proton transfer from the OH of Tyr122 to the diiron core. In the E.coli system, a kinetically coupled electroniproton transfer could be in effect as Asp84 can make a direct hydrogen bond between the coordination environment of the iron core and Tyr122. However, in the structure of Salmonella lyphirnurium R2 i1661, no direct link is present. Instead a water molecule exists between the iron coordination sphere and the tyrosine radical site, which implies a significantly more complex transition state for a kinetically coupled electrodproton transfer. Alternatively, intermediate might be a sufficiently strong oxidant to drive the electron transfer directly from a protonated tyrosine side chain, which is subsequently deprotonated followed by a reorganization of the local hydrogen bonding. This last scenario is consistent with the observed lack of isotope effects on the radical generation step. 3.8.9. Reaction Mechanisms of the Methane Monooxygenase Hydroxylation

The mechanism for the hydroxylation reaction catalyzed by MMO has attracted significant interest due to its potency and scope for commercial applications. Much of the mechanistic discussion on the hydroxylation step of MMO has been made by analogy with the cyctochrome P450 monooxygenase system. In this system, a radical rebound mechanism has been proposed, involving the generation of a substrate radical in an initial hydrogen abstraction step 12131. More concerted scenarios can be outlined for the P450 system, as well as reaction schemes based on carbocationic intermediates. Insights into the mechanism of the hydroxylation step of MMO have mostly been obtained from studies using isotope-labeled and radical clock substrates, single-turnover studies of the reaction between Q and the substrate, and theoretical investigations. A very large deuterium KIE effect of KEliKIj = 50-100 was discovered to affect the oxidation rate of methane by Q, which is suggestive of a proton tunneling event as the rate-limiting step in the reaction with this substrate 11761. For other substrates that have weaker hydrogen-carbon bonds, this isotope effect is reduced or absent, which has been explained by a rate-limiting substrate binding step for other substrates, as opposed to a rate-limiting chemical step for methane [1931. Several studies have addressed the question of the extent to which the &EM0 hydroxylation reaction is a concerted one, or whether distinct substrate radicals are generated in the reaction. Radical clock studies of chiral ethane reveal a racemization of its configuration which favors a radical mechanism [2141. The oxidation of a chiral methylcubane by M. trichosporium MMQ also reveals rearrangements in its products. In contrast, similar experiments that used a chiral methylcubane in the M. capsulatus MMO system did not reveal such rearrangements and are suggestive of a more concerted reaction mechanism in this case [215]. DFT studies of the reaction mechanism offer results that favor the existence of distinct methane radical species [210,2161. The methane radical has been suggested to first coordinate to the iron core through a weak iron-carbon bond [210]. Alternatively, the substrate radical is proposed to

I RON-OXYGENlNITROGEN PROTEINS

539

directly recombine with a terminal oxyl radical on the iron core [2113. Other theoretical studies have provided support for a concerted reaction mechanism with a strong coupling of the hydrogen abstraction and oxygen insertion steps (see discussion and references in f291). Most of the recent mechanistic discussions of the MMO reaction, including the theoretical studies, have been based on the diamond core paradigm for the hydroxylating unit of intermediate . However, as discussed above, the bridging position B2 of a hypothetical diamond core structure is not likely to be an active participant in the hydroxylation chemistry since it is not accessible from the hydrophobic pocket of the active site. Instead, groups in position B1 and T are possible participants (Schemes 22 and 23). As the coordination spheres are most likely to be saturated (six-coordinate) ,the formation of an iron-carbon bond as a reaction intermediate is unlikely, since it would require the dissociation of a ligand or 0x0 group from these cores during a critical chemical step. Several possible hydroxylation geometries, based on the asymmetrical core as a hydroxylating unit with an 0x0 group in position T and R1, as a hydroxylating unit, have been outlined in the scheme. The direct involvement of both 0x0 groups in the chemistry is an attractive possibility as it allows the formulation of a triangular transition state.

CHR

Scheme 23. Four potential reaction geometries for the hydroxylation reaction catalyzed by an asymmetrical intermediate Q in methane monooxygenase.

The binding of protein B to MMO has complex effects on substrate activation rates, product distributions, redox potentials, and electron transfer rates. studies have provided evidence for a dramatic change in the coordination environment of one of the ferrous ions upon protein B binding [175,178,1791. Probably, most of the effects on the chemical and physical properties of ~ M Owhich , are induced by protein B binding, could be rationalized by the effects on the iron coordination environment. Binding of protein I3 could change the equilibrium between two or more accessible coordination environments to favor a catalytically more optimal conformation of one or more of the iron ligands. A possible structural mechanism for this effect is a change in the local hydrogen bonding pattern in the active site, which appears to be directly involved in stabilizing the coordination modes of carboxylate ligands and solvent molecules. Gys413 has been implied in the hydroxylation reaction of MMO, as it i s found in a similar position to the radical tyrosyl site in the owever, since it i s at a distance about 7 from the substrate, it is probably too far away for a direct involvement in the catalytic step. ~~~

A

3.8.20. Reaction ~ e c h a n i ofs ~d9 Desaturase The observed large isotope effect at the 9-position of the fatty acid substrate A9D suggests that a hydrogen abstraction at this position is a key step in the desaturation reaction L2021. The key question is whether peroxo or superoxo adducts are sufficiently good hydrogen acceptors to drive the initial step of the reaction. The alternative scenario is the lysis of the oxygen-oxygen bond before the hydrogen abstraction step, leading to a formal diferryl adduct as the initial hydrogen abstraction species. The strong similarities of the first and second coordination sphere of the diiron core of A9D, and those of RNR R2 and MMO, argue for a similar faith of the dioxygen reaction and that high-valent iron inlermediates are also involved in the A' tion. However, the presence o f a stable peroxo species in the chemically protein can be seen to indicate that such a species is important for substrate transformation. The fact that the chemically reduced protein does not catalyze product~ve desaturation argues against the peroxo being responsible for the initial hydro~en abstraction step. No isotope effect is seen on the transfer of the second hydrogen, but a good proton acceptor should also be required for this step, ~ron-coord~nated hydroxy/oxo groups could also serve as hydrogen acceptors in this step of the reaction, but another possibility is that is203, which is well positioned in the active site pocket, helps in proton handling in this step of the reaction. In a recent study the lack of incorporation of a 018-labeleddioxygen into the oxobridge of the diiron site of A'D was used to argue for a significantly different reaction scenario in this enzyme as compared with RNR R2, where the 0x0 bridge 1s labeled by 0'' [2171. Although this is a possible interpretation of these results, other inte~~retations are also possible, such as a faster exchange rate of inter media^ 0x0 ' the A'D site when it is significantly more exposed to solvent molecules than thc R2 site.

The observed conformational changes of the chemically reduced form of AgD upon substrate binding provide evidence for the ability of the substrate Lo induce significant local structural changes at the diiron site. Such a mechanism of substrate-induced activation is attractive for the control of O2 binding7and would minimize unproductive and potentially damaging autooxidation. However, the detailed implementation of such a conformational change in the biologically reduced protein could be significantly different from that observed in the chemically reduced protein.

re ryth rin Rubrerythrin (Rr) i s a nonheme iron protein found in the cytoplasm of anaerobic sulfate-reducin~bacteria, The rubrerythrin subunit is formed by about 190 resi and contains two types of iron sites: one rubredoxin-like Fe-S4 site and one Fe-Q/N type oxo-bridged diiron site containing the signature sequence including the tandem motif 1218,2191 seen in the large diiron carboxylate proteins. The redox potential of both the rubredoxin and the diiron sites are unusually high (>200 mV) r2201. The physiological role of rubrerythrin is uncertain, but it has been shown to have a ferroxidase activity with similar rates of iron oxidation as those seen in femitins [22l]. Molecular oxygen, as well as peroxide, can oxidise the iron sites of rubrerythrin, and it has been suggested that the biological role of rubrerythrin is to work as a scavenger of different dioxygen species. Recently, NADPH has been shown to work as a direct reductant of the oxidized rubrerythrin, suggesting a catalytic cycle of oxygen scavenging followed by NADPH reduction of the oxidized enzyme [222]. This would be an expensive solution to the problem of scavenging dioxygen adducts. Nevertheless, this solution may be attractive in an anaerobic context, as it would bypass the productio~of toxic 0 2 , which is normally generated from the two dioxygen scavengers of' aerobes: catalases and superoxide dismutases. 3.9.1. Structure and Function of Rubrerythrin

A

The structure of ~esulfov~brio vulgaris rubrerythrin has been determined at 2.1 resolution [2231. The rubrerythrin subunit f o m s a two-domain structure that in the crystal structure interacts to make a potential tetrameric complex with a 222 symmetry. The first 146 residues of the subunit form a compact four-helix bundle that coordinates the diiron site, while the last 45 residues form the rubredoxin domain (Fig. 41). The diferric site in the structure is coordinated by 611.153 and 61~128,which are bridging in a bidentate mode ( igs. 42 and 43). The two iron ions are also bridged by an 0x0 group. Termin bidentate carbosylates from Glu20 and 61~194,and one terminal histidine ligand, is131, also coordinate the diiron site, while the other iron al monodentate carboxylate from Glu97, yielding two six-coordinateiron iferric rubrerythrin structure shows significant differences to the diferric R2, ~ ~ and Q A9D, although , all of the principal ligmds present in the larger diiron proteins are seen in Fir. Surprisingly, His56, which belongs to the

542

NORDL~ND

FlG. 41. Structure of rubrerythrin (generated from PDB file 1RYT).

first EXXH motif, does not coordinate t o an iron. Another major difference is the presence of the 611197 ligand in Rr, which has no corresponding coordinating residue in RNR R2 or MWIO and which is probably the main reason for the distorted coordination environment in Rr. An additional effect of this distorted geometry is that the bridging 0x0 group is in the trans orientation as related to the His ligands, while in the 0x0 group is cis t o the histidine residues. Although the diiron site in the structure is coordinatively saturated, the ligand sphere of the diiron site of rubrerythrin is significantly more exposed than the sites of ribonucleotide reductase and MMO. It contains on one face a solvent-exposed crevice

IRON-OXYGEN/NITROGENPROTEINS

543

R

FIG. 43. Structure of the diferric site of rubrerythrin (generated from PDE a e 1RYT).

which consists of mainly hydrophobic residues and may serve as an entrance for O2 or ferrous substrates to the iron. The diiron-coordinating four-helix bundle of Rr bears great similarity to the four-helix bundle of ferritins and bacterioferritins. In addition, the ferroxidase sites of these proteins are also exposed to a similar extent as is the diiron site of Rr [223]. The structure of the FeS binding domain of rubrerythrin is very similar Lo that of rubredoxin. The residues Cysl.58, Cysl61, Cys174 and Cys177 coordinate the iron ion, and all four cysteine sulfurs make N-H, - .S main chain hydrogen bonds in a way similar to that of the rubredoxins. The iron site of the rubredoxin domain is connected across the closest dimer interaction of the crystal structure with hydrogen bonding from His56 and the carbonyl of Cys161. The distance between the enough for an outer-shell diiron site and the rubredoxin site is about 12 &--close electron transfer between the two sites. A structure of a non-recombinant rubrerythrin has been reported that has significantly different coordination of F e l as compared to the earlier reported Rr structure, which was of a recombinant protein [2241. In the nonrecombinant protein structure, His56 is now coordinating the Fel position whereas 6 1 ~ 9 7is not, and the metal site has moved about 2.5 A in the direction of His56. This indicates that large conformational changes can be allowed in this Rr dimetal site, It was proposed that the reason for this conformation change is the presence of a Zn ion instead of a Fe ion in the Fel site. A similar change in the metal binding position of the Fel metal binding site was seen in the structure of the E97A mutation of the recombinant protein E2251. The structure of the diferrous site of Rr is not known, and t o what extent the coordination is changed in this form of the protein is uncertain, although changes of the coordination of Fe2 appear to be possible also in the case of the diferrous protein 12231,

Even though the exact biological role of Rr is unknown, it appears likely that the diiron site plays a role in coordinating dioxygen adducts for further reduction. The dioxygen reduction could be directly coupled to electron transfer to the rubrcrythrin rubredoxin site, or to an externally bound reductant such as ferrous iron or NADPH. An importafit issue to be revealed by further studies is to what extent the dioxygen chemical reaction catalyzed by rubreryth and the related ferritinslbacteriofe proceeds with a similar geometry as in MRfIO, RNR R2, and A’D, and whether ferry1 iron is involved.

3.10. ~i~rile Hydratases Nitrile hydratases (NHases) catalyze the hxdration of aliphatic and aromatic nitriles to their corresponding arnides through the reaction r2261: R-CN + HZ0 ---+RCONH These enzymes are found in bacteria, plants, and fungi, where they are involved in the degradation of toxic compounds. The NHases are of considerable biotechnological interest with regard to the industrial production of acrylamide [2271. Also, bacteria with NNases could have a use in the bioremediation of nitrile-containing wastes. Since several herbicides contain toxic nitriles, efforts are made to develop transgenic crops that express NHases. e bacterial nitrile hydratases that contain iron cofactors are the best, studied of this family. These enzymes contain a and subunits, with a mononuclmw iron site in the 01 subunit. A unique feature of this NHase family is a nonheme low-spin ferric iron [2281. E M S data suggest a mixed N-S-0 coordination of protein ligands to the iron [229,230], with the enzymes exhibit in an absorbance at about 710 nm, which is ~ ~ o b due a bto~ a~cysteine-to-iron charge-transfer. A hydroxide ion has been suggested to coordinate the iron 12311. The presence of the enzyme in either an excess of substrate or inhibitors has been shown to induce a shift in its EPR parameters 12281. However, EXAFS studies did not provide support for a direct coordination of inhibitors to the iron ion [230]. The activity of iron-dependent NHases has been shown to be photoinducible, yme being inactive in the dark 1232-2341. The inactive state is associated with NO binding to the enzyme, which upon photoactivation results in the release of NO. A noncorrinoid cobalt-dependent nitrile hydratase also exists wit.h homologies greater than 50% to the iron-containing NHases [2351. X-ray absorption gest that the iron and cobalt cofactors have very similar ligand environments 12361 and that the enzymes probably catalyze the nitrile hydratase reaction by a very similar mechanism. The cobalt enzyme is, however, not subject to photoregulatory control.

I RON-OXYG~ ~ / N l T R O G EPROTEI N N

545

3.10.1. Structure of Nitrile The structure of NHase from Rhodococcus sp. R312 was first determined at 2.65 A resolution [237].The structure of the a subunit is a compact a/P domain formed by four layers, two central P sheet layers lined by layers of a helices (Fig. 44). The f! subunit is formed by two subdomains: the N-terminal domain, consisting of five a helices, and the C-terminal domain, consisting of a p roll and a short a helix. The iron site is found in a central cavity formed at the interface between the a and p subunits. All of the iron ligands and the residues with which they make second-spherehydrogen bonds are provided by the a domain, except for Arg56 which is provided by the 13 subunit, The ferric iron is bound to a conserved stretch o f residues composed of the side chains of Cys109, CysllZ, Cysll4 and the amide nitrogens of Serll3 and Cysll4 (Fig. 45). The axial nature of the ligand field and the strong-field amide nitrogen coordination has been suggested to be the reason for the presence of the low-spin iron. A significant sequence variation is seen in the active site residues of different NHases that is consistent with their difyerent substrate specificities, and in addition to the ligand sphere, only T ~ r 7 2is conserved. In a more recent 1 . 7 4 structure of NHase in complex with NO, the detailed coordination of the iron site was revealed [2381. This structure shows a post-translational modification of Cysll2 and Cysll4, by oxidation to form Cys-sulfenic and Cys~e~ sulfinic acids, respectively, that is supported by mass spectrometry ~ e a s u r ets. The mod~cationsare stabilized by hydrogen bonds to the conserved Arg56 and Argl41 residues. The modiiied cysteine residues and the side chain of Ser 113 were described to form a claw setting around the NO [238]. The mechanism for the covalent modification is still unknown, but an autocatalytic process involving the metal site appears to be feasible. 3.10.2. Reaction Mechanism of Nitrile Hydratase

The two most likely reaction mechanisms are outlined in Scheme 24. These mechanisms imply a key role for a hydroxide ion coordinating the iron. A possible role for the claw setting around the sixth coordination position is to stabilize the hydroxide. Upon

FIG. 44. Structure of nitrile hydratase (generated from PDB file ZAHJ).

546

PIG. 45. The environment of the iron site of nitrile hydratase. (Reprinted with permission from 12381.)

light + H20

HB

NO

substrate

Scheme 24. Two proposed mechanisms Tor the nitrile hydratasc reaction.


547

substrate binding, nucleophilic attack by the hydroxide at the nitrile moiety is expected to be concomitant with protonation of the substrate nitrogen. In one of the two proposed mechanisms, a metal-bound hydroxide directly attacks the substrate, while in the other an intervening water molecule is activated for attack by the hydroxide ion. In the subsequent step, substrate binding leads to the nucleophilic attack by the hydroxide ion on the substrate nitrile moiety concomitant with protonation of the substrate nitrogen. In a final reaction step, a proton is provided by a water molecule. The claw setting could also mediate this proton transfer by acting as an acid-base catalyst. It is likely that the same oxidation of the claw-setting cysteines exists in the cobalt enzyme and that they share a common enzymatic mechanism, since the residues Arg56 and Argl41 involved in stabilizing this structure are conserved.

urple Acid Phasphatases Acid phosphatases catalyzes the hydrolysis of monophosphate esters and phosphoryl transfer between phosphoesters and alcohols. Purple acid phosphatases (PAPS) are members of a structural family of acid phosphatases that contain NiO-coordinated dimetal centers in their active site. The PAPs have either Fe(II1)-Fe(1Ijor Fe(II1)Zn(I1) sites and are characterized by their intense purple color caused by tyrosineFe(II1) charge transfer. Members of the PAP fmily are found in animals and plants but are also present in bacteria, The exact in uiuo role of these enzymes largely remains largely unknown although their function as protein phosphatases, iron transport molecules, or antimicrobial radical generators has been proposed [239,2401. Kidney bean PAP has been extensively studied and is shown to contain a Fe(Il1)Zn(I1) site. This enzyme forms a dimer in which the subunits are larger and contain an additional M-terminal domain as compared with the mammalian enzymes. Mammalian PAPs have been identified in spleen, lung, placenta, and at high levels in osteoclasts of growing bone 12411. These enzymes are monomeric glycoproteins and although enz.ymes purified from different tissues show somewhat different properties, they are probably all are expressed from the same gene. The tartrateresistant phosphatase (TRAP) is the human PAP expressed in osteoclasts, and several lines of evidence support TRAP having a function in bone resorption, bone mineralization, and bone composition [2401. The mammalian PAPs contain a diiron site which can be isolated in either the inactive Fe(~I~)-Fe(IIIj form or the active Fe(I1Ij-Fe(1I) form. The 35-kDa protein is proteolytically cleaved to yield one 20-kDa and one 16-kDa unit linked by disulfide bridges. This cleavage has been shown to activate the enzyme for the in uitro phosphoryl transfer reaction, but it is uncertain if this cleavage event plays m y regulatory role in uiuo 12421. Spectroscopic evidence supports the presence of a p-hydroxo bridge in the active form of the diiron site as well as a terminal hydro-oxyl coordinated to the 1243,2441. Tetrahedral 0x0 anions such as molybdate and tungstates are good inhibi-

548

s. The plant enzyme can be converted t o an Fe-F spectroscopic features very similar to those of the mammalian 3.1 1.1, structure of Purple Acid Phosphatases

The Fed kidney bean purple acid p h o s p h a t ~ ewas the first structure of a PAP to be determined. In addition to the catalytic domain, this enzyme contains an N-terminal ~ i ~[2 ain of unknown function that is not present in the n ~ a m m PAPS of the catalytic domain of red kidney bean PAP contains two central lined by 01 helices on both sides (Fig. 46). The active site crevice is found on one edge of the sheets, and most of the active site residues are contributed by connec~ngloops ated by residues of the C between (3 strands and 01 helices. The Fe-Zn center is co termini of four of the central p strands (Fig. 47). The Fe atom is liganded by Tyr167, is325 and by Asp135, and a solvent molecule, proposed to be adjacent Zn is terminally coordinated by two histidines, €€is323 and agine ligand, Asn201, and a water molecule. The two metal ions monodentate Asp164 and a solvent molecule which is suggested from the spectroscopic data to be a hydroxide bridge. in complex with phosphate and tungstate has The structure of the plant P been d e t e r ~ n e dand reveals the ons to be coordinated in a very similar way to ent molecules, and hy onded to the side , as well as the main cently, structures of iron-c~nta~ning m a ~ m a l i mined in several forms 1247-2491. The overall structure of the protein and the p r i ~ coordinatio~ ~ ~ ~ ~ isl very similar e catalytic domain of the plant enzymes. ~na z.z-W structure of mammalian T a sulfate and Zn ion pair, recruited from the ~ ~ ~ t a l l i z a tbuffer, i o n is found in the active site L247J. The s t ~ c t u r is e ~robabl~ that of the diferric form of the enzyme, and the sulfate coord~atesdirectly to the n ~ while the Zn ion is coordinated by the sulfate, a iron site in a t e ~ i mode

FIG. 46. Structure of kidney bean purple acid phosphatase with bound phosphate (generated

49

Tyr167

FIG. 47 " Coordination environment of kidney bean purple acid phosphatase.

histidine (Wis216j, and two solvent molecules [2471. The active site of mammalian appears to be somewhat less accessible €or solvent and substrates than that of the plant enzymes. The basic fold of the PAPs and related metal binding sites are also seen in the large family of serine/threonine protein phosphatases (PP) as well as in 5'-nuclootidases I250l. These enzymes use ~ ( I I ) - ~ ( sites, I I ) but the coordination of these sites are very similar to the ~ e ( I ~ ~ )site - ~o(f PAPS, I ~ ) except that the tyrosine ligand of the PAPs is replaced by histidine. ch,anism of Purple Acid Phosphatases The Fe(I1I)-Fe(J1)and Fe(II1)-Zn(l[Ijforms have been shown to be the active f o m s of the mammalian P s and the plant enzyme, respectively. Inversion of c o n f o r ~ a ~ i o n of the phosphate has been observed l251l which is consistent with a direct attack from a metal-bound solvent molecule rather than the involvement of covaIent phosphoenzyme ~ t e ~ e d i a t eThe s . currently accepted mechanism is outlined in Scheme 25 [252], and follows the conceptual steps outlined far the phosphoryl transfer reactions catalyzed by other &metal sites 12531. The phosphate in the catalytically active form is coor~natingin a terminal manner to the MUI) ion. The nucleophilic solvent molecule is activated at the metal site, which promotes deprotonation, mainly by charge effects. The subsequent nucleophilic attack on the substrate p~osphatemost likely intermediate. i.e. associative inechanleads to the f o ~ a t i o of n a penta~coor~inated ism, with concomitant buildup of negative charge. The positive charge o f the metal site together with hydrogen bonding from protein residues can compensate for this charge buildup an possibly also play a role in polarizing the P-8 bonds for facilitating Eormation of a p~~ta-coordinated adduct. In the final step o f the reaction, the leaving g alkoxide group has to be pro~onated,where the His296, and the c o r r ~ s p o n ~ nconserved histidines in the other phosphatase are the most likely proton donors, although other groups have been suggested [2471. B ~ d g i n gc o o ~ d i ~ a ~oi foanphosphate ion was seen in the structure of the plant enzyme where the n o ~ ~ ~ i solvent d ~ n g ligands axe replaced by the phos~hate.

550

NORDLUND

His2BBH

P04H+ROH HZO

His%

R

R

Scheme 25. Proposed reaction mechanism of purple acid phosphatases.

Alternative reaction paths involving the bridging hydroxo group as the nucleophilic agent are an alternative possibility for dimetal-mediated hydrolysis, but the bridging hydroxo group is most likely not sufficiently reactive for serving as a nucleophile in the reaction.

Due to the limited space, only a fraction of the vast amount of information obtained during the last decades on structural, spectroscopic, and kinetic properties of the FeOiN proteins has been discussed in the above sections. It is hoped that the selected information has illustrated the level of detail in our presenL understanding of the structure and mechanism of these enzymes, as well as some of the problems that remain. In nearly all of the systems, a good overall understanding of the reaction scenario has been obtained, i.e., as to which cofactors and components are involved, and what appears to be the basic sequence of events in the active site. In most systems, reaction intermediates have been observed, either indirectly by using reporter substrate analogues, or directly, by observation of kinetically competent reaction intermediates in the form of activated oxygen or substrate species. Spectroscopic features of the iron sites have been analyzed in detail, contributing extensive insights into electronic and magnetic properties of resting and functional forms of these proteins. The structLtra1 sludies have provided a picture of the catalytic machinery, thereby limiting the number of possible reaction geometries in the active site. As an important driving force toward the goal of formulating true reaction mechanisms,

IRON-OXYGENllVITROGEN PROTElNS

551

information from the model systems on general features of 0 2 and substrate activation chemistry catalyzed by iron sites has been instrumental. In fact, at this time, plausible and relatively detailed reaction mechanisms have been put forward for all of the enzymes discussed in Sec. 3. However, many ambiguities remain, many of which are related to the detailed electronic properties of the 0 2 activation process, the geometry of activated adducts, the exact timing of electron transfer events in the reaction, and the role of protons. Similar ambiguities also exist concerning the detailed mechanisms for the substrate transformation processes. A large part of the mechanistic studies are based on mutant proteins and substrate analogue studies, which introduces a possible complication that, at least for some enzymes, different mechanisms could be in effect in reactions with different substrates and protein mutants. For example, different substrates may encounter differently activated dioxygen species, depending on the binding mode and ease of activation of the substrate. The initial substrate activation events could also lead to the generation of different substrate intermediates, e.g., the formation of distinct substrate radicals in one case and the generation of a carbocation substrate intermediate in another. This is, for example, a possible explanation for some of the mechanistic ambiguities in the MMO system, as well as in the RDOs and M I . The emerging view is now that during the evolution of these enzymes, significant efforts have been made toward controlling the reactions and therefore tailoring the sites to generate one predominant oxidizing species for each oxidation stage of the reaction. The presence of structurally well-defined oxidative iron species should of course be highly beneficial for the efficient substrate transformation, as well as for the control of these reactive intermediates, blocking them from participating in alternative and damaging reaction paths. Mutations of residues in the region of the iron sites in RNR R2 illustrate the sensitivity of these systems for disruptions, where a range of different reaction paths are followed in these mutant proteins. Similarly, studies with a nonphysiolagical substrate can disturb the timing of the encounters of the activated iran care with the substrate leading to alternative, potentially damaging reaction paths, as seen, for example in the systems. Despite their obvious importance for the selected reaction scenarios, these control aspects are not often addressed experimentally. Although highly sible reaction scenarios have been formulated for lnost of the Fe-O/N enzymes, further insight into which structural (and dynamic) components of the metal sites are the major determinants for the mechanistic s remains to be elucidated. Such insights concern, for example, the following questions: To what extent do different Sigand types play different roles in promoting the chemistry? How do the coordination geometry and coordination number of the iron site affect the reactivity and the fate of reduced O2 intermediates? To what extent are other, nonmetal ligand residues in the active site involved in catalysis? Comparative studies between related enzymes are ver;y usehl in this process. Particularly rewarding in this respect are the 2-His-l-Glu/Asp iron proteins, which are found in four different nonevolutionary-related stmctural superfamilies discussed above, and where the functional and structural differences and

between these systems can pinpoint major s t ~ c t u r a l ~ n c t i o nof these enzymes. I following section, aspects of the chanism re~ationshipof the F proteins will be summarized with is on such co~parisons.

lu, and Tyr have different sizes, charges, and ing ~ o t e n t tio~surroun residues and solvent. Therefore, they can pote roles in the formation of the iron site and promotion of chemis are only found in iol dio~genasesand purple acid phosphatases. The main reason for this low

side reactions such as t ~ o s i o ~ ~ed a t i o nand rmation. ~ysteineresi~uessuffer from simil steine residues are seen in nitrile hydratase w

ion framewor~sfor most of the

species. versatile iron li

de several possible c 3. They also provide x i ~ l eiron ligands, due to fle~ibilityis particularly

ntate t o mo~odent at

lates has yet been observed. n of mono~entatelycoordinated c~boxylatescan for sta~ilizinghydro~enbonds to iron-coordin~te

N/NITROG~NPROTEINS

553

substrate binding. In PAPS, on the other hand, the asparaghe ligand is probably coordinated throughout the reaction cycle and might be preferred at this site when it binds anionic substrates. The aspasagine side chain will not repel the negative charge of the substrate, as would a carboxylate side chain, and the asparagine can also serve as a potential hydrogen bond donor t o the phosphate group of the substrate. An aspect of metal sites that is often ignored in discussions of their structure and function is the second-spherecoordination of the metal site. Hydrogen bonding to protein and solvent ligands appears to be evolutionarily conserved within each enzyme family and the effect of these hydrogen bonding patterns on the structure and dynamic behavior is probably significant. The extent of hydrogen bonding could also potentially affect the resulting charge state of the site, including the protonation state. Based on second-sphere hydrogen bonding, most histidine side chains can be assumed to coordinate in their neutral, monoprotonated form. In some enzymes, i.e. M ,RDO, and R R2, second-sphere hydrogen bonding of histidine ligands with carboxylate residues is present. Such hydrogen bonding could allow these histidine ligands to attain anionic properties, i.e., partial deprotonation, and potentially build up more negative charge on these ligands. Interestingly, these three enzyme families are ~ s t ~ n ~ i s from h e dthe other Fe(I1) proteins by the fact that they do not coordinate anionic substrates or cosubstrates, and the role of the “anionic histidines” could therefore provide additional negative charge needed during the 0 2 activation process and/or to stabilize high-valent iron intermediates. The coordination distanceshtrength of the coordinating carboxylate oxygens can also be controlled by second-sphere hydrogen bonding. In such cases, where the coordinating oxygens are serving as hydrogen bond acceptors, they often coordinate with a longer Fe-O distance. It is of considerable interest is to understand to what extent the protein provides a restricted metal coordination geometry. Together with the particular metd-ligand affinities, this may determine the physiological specificity for iron binding. Most proteins discussed in this chapter are most likely not dependent on iron for their proper folding, since they can be directly isolated as relatively stable apoproteins. Instead, the empty metal binding site is “preformed” and the ligands are in close spatial proximity before iron binds. In two cases, DAOCS and RNR R2, both the structures of metal-containing protcins and apoproteins have been determined and the met& ligands are in both cases found in virtually identical positions in the apoproteins as in the metal-containing proteins. Also, no water molecules are bound instead of the metals in the apo site, which further minimizes the required reorganization upon metal binding. Rather protons, through protonation of histidine and carbowlate side chains, bind to the RNR R2 site, and possibly also to the DAOCS site, compensating for the charge difTerence between the metal-containing proleins and the apoproteins [92,1621. The small conformational changes of the metal ligands during metal binding in these proteins could be of importance in determining the speci€icityfor iyon binding. Most Fe-OIN sites are easily substituted with other metals, such as Mn, Co, and Ni, and in some cases Cu, but few quantitative data are available on the: relative

NO~DLUN~

554

binding constants of these metals to Fe-ODT proteins. However, the in viuo metal binding specificity will certainly be highly dependent on the effective metal concentrations in the cell. Also, yet-to-be-discovered iron chaperone systems might play a role in determining the specificity of iron loading into certain proteins. The observed reorganizations of the iron coordination environment in the Fe-O/N proteins studied to date may explain why these proteins are not selected as pure electron transfer components, as the reorganization requirement may severely alter the electron transfer rates and the reversibility of the reaction.

harge and Charge Distributions The minimization of net charge buildup and the prevention of abnormal charge distributions in the active site of Fe-QIN enzymes are certainly important components of the catalytic strategy of these enzymes, as for most other enzymes. The energetic costs for net charge buildup depends on the capability of the surrounding medium to respond by polarization effects, i.e., its effective dielectric constant. In the proteins where the metal sites are shielded from the solvent, effective polarization is hard to obtain, particularly when hydrophobic residues are surrounding the site. Even when mainly polar residues are surrounding the metal site, which is the case for most metalloproteins, it does not necessarily mean that these polar residues can change their orientation to execute significant polarization effects. At solvent-exposed sites, on the other hand, the water dipoles can potentially shield variations in ch‘arge distribution by reorienting their dipoles. The control of charge as a major determinant for directing the catalytic fate of reactions has now been postulated for a number of metalloproteins. However, in many cases ambiguities exist when the overall charge states are unknown due to undetermined protonation states of some groups. In some cases, direct experimental data exists, such as for assigning solvent ligands to be hydroxide groups. In other cases, assignment of the protonation state o€ solvent molecules and coordinated substrates is less certain, although local hydrogen bonding patterns are helpful for such analysis. 1t would, however, be expected that particular structural features are required t o significantly shift the pK, values of coordinating groups. Such features could include the charge effects of metals or other local charged groups, the local hydrogen bonding to the group, but also the potential buildup of local net charge in either the protonated or unprotonated form of the group. In the case of intradiol dioxygenases, strict charge conservation For all intermediate stages of the reaction has been postulated 1491. In the diiron proteins, observation of a strong preference for a “charge-neutral” diiron core has been derived in support for at least a “thermodynamic preference” for charge conservation [157,162,1901. For the 2-His-GluiAsp proteins, in most cases, the substrate-free ferrous forms are only partly charge-compensated by one anionic carboqlate ligand (assuming the solvent ligands to these sites to be water molecules). This could partly explain the weak binding of iron to several of these proteins but might also be an

IR0~-OXYGEN/NlTROGENPROTEINS

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important feature to allow the recruitment of anionic substrates and cosubstrates for the initiation of the reaction cycle. Among the 2-His-GluiAsp proteins, the exceptions to this scenario are the amino acid hydroxylases and Rieske-type dioxygenases where no anionic substrate binds the site. It might be that hydroxide ions coordinate these sites, but the presence of the “anionic histidine residues” in these proteins might also serve to allocate additional charge to the site, as discussed above. Although nearly all reaction steps for the Fe-O/N enzymes could probably be envisaged to work under a strict conservation of local charge, proteins employing electron transfer reactions pose a particular problem in this respect. In the diiron proteins and Rieske-type dioxygenases, electron transfer to other redox sites is probably not directly coupled to proton transfer when no obvious proton transfer paths are present in the structures. However, thermodynamic coupling might be obtained through alternative proton transfer paths involving water molecules, as suggested for the P450 system f2061, or other reorganization reactions. In the case where activated iron cores are the electron acceptors, the charge separation might be driven by the strong oxidation potential of these cores. In the case of RNR R2, tryptophan and tyrosine radicals can be generated by intermediates containing high-valent iron. In the Rieske-type dioxygenases, the electron transfer from. the Rieske cluster to the mononuclear iron site is probably driven by a superoxy-Fe(II1) intermediate. 4.3.

Conformational Flexibility and the Control of

O2Reactivity

Conformational changes are observed in several Fe-O/N proteins that can be categorized as large-scale movements of secondary structure elements upon substrate binding, as seen in IPNS and DAOCS, or as local conformational changes induced into the ligand environment of active sites during the reaction cycle. The large conformational changes seen in IPNS and DAOCS between substrate-bound and non-substrate bound-forms could be an important mechanism to protect the metal site from chelation as well as from autooxidation leading to damaging oxygen and radical adducts. Functionally important local conformation changes of the metal binding environment have been observed from crystallographic and spectroscopic studies in several of the Fe-ON enzymes. Of particular interest are observations of structural changes induced upon substrate binding, as, for example, in the AAH, Ag-desaturase, and 2OG-dependent hydroxylases. Substrate binding in most of the enzymes precedes O2 binding, and the binding of substrate hence directly enables the iron site for the O2 reaction. The direct binding of an anionic substrate to the metal will clearly have important effects on the site, but even more subtle effects are induced by noncoordinating substrate. The emerging scenario is that substrate binding, in most of the enzymes, affects the reactivity of 0 2 with ferrous sites, by opening free coordination posilions on the iron ion where &oxygen can bind or react, i.e., a kinetic mechanism 1291. Alternative thermodynamic explanations implying redox potential changes of the iron ion to explain the higher 0 2 reactivity appear less important. In the case

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of MMO, the effects of binding of protein B bears significant similarity to the effects of substrates in the other system, where one major effect of protein B is to enhance the reactivity of the diiron site to 02. The exact structural mechanisms with which the release of the metal ligands is mediated t o obtain an additional open coordination site remains to be elucidated for the different enzymes. However, these mechanisms do appear to be complex, and effects on the second-sphere hydrogen bonding of the released ligands, through the binding of substrate, could be a major common component of this control. 4.4.

Geometries of the Activated O2Species

The existing structural and mechanistic information put significant constraintis on the possible geometries for the dioxygen reactions. The proper 0 2 binding most likely depends in all Fe(I1) enzymes on an initial electron transfer event from the ferrous ion to dioxygen to form a Fe(II1)-superoxospecies. For all 2-His-GldAsp proteins, except RDO and AAH,a terminal binding geometry appears to be the only possible geometry for intermediate dioxygen adducts. In all of these cases, except IPNS, the O2 cleavage appears to be preceded by a nucleophilic attack on the substrate by an intermediate dioxygen adduct. This constitutes a well-controlled mechanism to provide additional electrons to reduce the dioxygen molecule and, in the subsequent bond lysis reactions, to stabilize the terminal “leaving oxygen atom” of dioxygen onto a hydrocarbon skeleton. However, the proposed covalent dioxygen adducts in the Fe-O/N enzymes have either of the formal oxidation states: carbon-peroxo-Fe(II),i.e. two electrons are provided onto the dioxygen adduct, or carbon-superoxo-Fe(III),i.e., no electrons are provided from the substrate. Based on presently accepted mechanisms, the latter adduct is only formed in the oxoglutarate-dependent hydroxylase, which is the mononuclear iron protein that most likely uses high-valent iron in the reaction cycle. These two different reaction scenarios also pose questions as to whether there are eledrostatic requirements at the metal site for steering the 0 2 lysis into either of the two different heterolysis scenarios, or if this is mainly determined by the oxidation states of the covalent intermediates. The most intriguing dioxygen lysis reaction of the Fe-O/N proteins is the one catalyzed by IPNS where a terminally coordinated 0 2 is suggested to heterolyze, concomitant with oxidative p-lactam ring formation and the generation of an Fe(IV)=O core. This reaction is suggested to proceed without the formation of a covalent dioxygen-substrate intermediate. Instead, protons from the thiol p carbon and an amide group provide the required “leaving group” stabilization of the distal oxygen atom, in the form of a water molecule. The RDO reaction appears in several respects to be a potential exception in its mode of dioxygen activation. The formal dioxygen intermediate before lysis is a Fe(I1)superoxo species and the geometry of the mononuclear site is consistent with both end-on and side-on reaction scenarios. It is possible that a more concerted reaction mechanism is in action in the RDOs than in the other iron enzymes and that the


557

dioxygen lysis is directly coupled to the hydroxylation step. The geometry of the AAH metal site does also appear to allow either an end-on or side-on geometry. Also, the diiron proteins provide sufficient open coordination positions for a side-on coordination, which appears to be the most likely lysis geometry in these enzymes, although an end-on geometry cannot be excluded. Several of the Fe-0,” enzyme reactions have been suggested to involve Fe(IV)=O adducts as key intermediates, and in the large carboxylate diiron proteins direct observation of formal high-valent iron intermediates have been made. The detailed structures of these intermediate iron cores are of significant interest to reveal the possible reaction geometries for a range of oxidation reactions catalyzed by these sites. Although an Fe(lV)=O unit is a likely candidate for the hydroxylating species, additional structural features surrounding this unit might be required, for example, to handle proton transfer in the reaction. In the diiron sites, an additional 0x0 group derived from dioxygen might be serving as a proton shuttle (base) in the reaction, while in some of the mononuclear iron proteins possible proton shuttles are basic groups neighboring the suggested Fe(N)=O unit, in particularly carboxylates. Finally, an interesting question is to what extent the distinct organization of oxygen and nitrogen ligands around the iron site directly determines the reactivity of the iron ion with O2 and the downstream fate of the reaction. Although the specific effects of the iron ligands on the orbitals of the iron ion might be significant, no clear preferences of’ ligands in certain positions, either neighboring the reactive oxygen species, or positioned trans to the reactive O2 adducts, appears to be in place.

ACKNOWLEDGMENTS Many thanks to Deborah Berthold, Derek Logan and Matthew Bennett for linguistic corrections and discussions and to Pa Stenmark and Martin Andersson for help with pictures. Also thanks to other people in my group for help and advice, as well as for patience during the preparation of this manuscript, and to the Swedish Research Council for Natural L%iences.(NFR), the Swedish Research Council for Engineering Sciences (TFR) and EU contract No FMRX-CT98-0207 for financial support.

ABBREVIATIONS AND DEFINITIONS AAH ACCO ACmC ACP ACV BH2

amino acid hydroxylase l-amino-l-cyclopropanecarboqdic acid oxidase 6-(L-a-aminoadipoyl)-L-cysteinyl-L-S-methylcysteine acyl carrier protein 6-(L-a-amino-6-adipoyl)-L-cysteinyl-D-valine 7,8-dihydrobiopterin

558

G(R)-~-erythr0-5,6,7,8-tetrahydropterin clavaminate synthase DAQCS deacetoxycephalosporine C synthase chlorocatechol dioxygenase CCD circular dichroism CD catechol 1,2-dioxygenase CTD density firndional calculation DFT 2,3-dihydroxybiphenyl dioxygenase DRBD dihydroxyphenylalanine DOPA extradiol dioxygenase ED0 ENDQR elec%rondouble resonance electron paramagnetic resonance EPR EXAF'S extended x-ray absorption fine structure flavin adenine dinucleotide FAD Fe-O/N iron-oxygednitrogen proteins 3-fluoro-4-hydroxybenzoate FHB 5-lipoxygenase-activatingprotein FLAP 4-hydroxyphenylpyruvic acid dioxygenase HPPD hemerythrin Hr intradiol dioxygenase IDQ 3-iodo-4-hydroxybenzoate IHB 2-hydroxyisonicotinic acid N-oxide IN0 isopenicillin-N synthase IPNS lipoxygenase LO magnetic circular dichroism MCD 3-MCT 3-methylcatechol MMO methane monooxygenase MMOB methane monooxygenase protein B MMOH methane rnonooxygenase hydroxylase MMOR methane monooxygenase reductase metapyrocatechase MPC N-a-L-acetylarginine NAA NAD(P)H reduced nicotinamide adenine dinucleotide (phosphate) NDO naphthalene 1,2-&oxygenase NHase nitrile hydratase NHE normal hydrogen electrode NNO 6-hydroxynicotinic acid N-oxide 2-OG 2-oxoglutarate PAH phenylahnine hydroxylase PAP purple acid phosphatase PCA protocatechuate PCD protocatechuate 3,4-dioxygenase PDO phthalate dioxygenase PP protein phosphatase

BH4

CAS

NORDLUND

I RON-QXYGEN,” ITROGEN PROTElNS

RDO RNR Rr RR SLO

TRAP TrpH

TYrH XANES

559

Rieske-type dioxygenase ribonucleotide reductase mbrerythrin resonance Raman soybean lipoxygenase tartrate-resistant acid phosphatase tryptophan hydroxylase tyrosine hydroxylase X-ray absorption near edge structure

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Iron Storage and

Magnetic Resonance Center, University of Florence, Via L. Sacconi 6,I-50019Sesto Fiorentino, Italy

1. INTRO~UCTION

572

2. PROTEINS WITH KNOWN STRUCTURE 2.1. Ferritins: Occurrence and Biological Role 2.2. Ferritins: Structural Classification

573 573 573 575 575 576 577 577 578 578 579 579 579 581

2.3. Structures of Ferritins 2.3.1. Description of Eukaryotic Ferritin Structures 2.3.2. Description of Bacterial Ferritin Structures 2.3.3. Assembly of Subunits 2.3.4. The Fcrroxidase Site 2.4. Transferrins: Occurrence and Biological Role 2.5. Transferrins: Structural Classification 2.6. Structures of Transferrins 2.6.1. Description of Eukaryotic Transferrin Structures 2.6.2. Description of Bacterial Structures 2.6.3. Iron Binding Sites 3. ~ R O T WITH ~ I ~~ ~ 3.1. The Iron Pathway

J STRUCTURE ~

4. S T R U C T U R E - F ~ ~ T I ORELATIONSHIPS N 4.1. Expression Systems 4.1.1. Ferritins 4.1.2. Transferrins 57 1

~

~

O 584 584 585 585 585 585

ARNESANOANDPROVENZANI

572

4.2. Ferritins: Structure-Function Relationships 4.2.1. Biological Function 4.2.2. Assembly Pathways 4.2.3. Ferroxidase Activity 4.2.3. Iron Release 4.3. Transferrins: Structure-Function Relationships 4.3.1. Biological Function 4.3.2. Iron Uptake and Release 4.3.3. Iron Binding Sites

5. PERSPECTIVES

586 586 587 587 589 590 590 590

591 593

ACKNOWLEDGMENTS

593

ABBREVLATIONS

594

REFERENCES

594

1. INTRODUCTION Iron is required in almost all organisms for growth and crucial metabolic pathways. The redox properties of iron favor its use in a large number of protein complexes. Sonie require iron in the form of heme (Chapter 9) or iron-sulhr clusters (Chapter lo), but other forms are also known (Chapter 11).The major functions of iron are in electron transfer reactions, oxygen transport, and a variety of catalytic processes. Although iron is an essential element, excess iron is hazardous because it produces free oxygen radicals that damage DNA, proteins, and lipids. In humans, accumulation of' cellular iron can result in cirrhosis, arthritis, hcart failure, diabetes mellitus, and increased risk of cancer. Several diseases, such as anemia and hemochromatosis, are correlated with iron overload and deficiency disorders ([l] and references therein). Thereforc, the maintenance of iron homcostasis in the body ac:well as in the cells must be balanced to supply enough iron for the metabolism and to avoid excessive, toxic levels. This occurs primarily by regulation of iron absorption; excretion has a more passive role. Intestinal absorption is the primary mechanism regulating iron concentration in the body. The exact mechanisms by which the body informs intestinal cells of the organism's requirement of iron are not fully delineated. However, intestinal absorptive cells contain iron in proportion t o the state of iron repletion and requirement for iron. In the presence of oxygen, ferric iron (Fe"+) is the favored species, but in the organism ferrous iron (Fe") is needed. The uptake and transport of iron under physiological conditions require special mechanisms because the solubility of hydrated Fe3+ is very low at neutral pH [a]. In daily diet two distinct forms of iron are present: nonheme iron (prevalently Fe3+)and heme iron. Different pathways have been identified for absorption in intestinal and nonintestinal cells for nonheme Fe2 ', Fe", and heme iron. In

IRON STORAGE AND TRANSPORT PROTEINS

573

nonintestinal cells most iron uptake occurs via the pathway utilizing transferrin receptors or the poorly defmed transferrin receptor-independent pathway (TRIP) [1,3-51. The vast majority of iron in plasma is bound to transferrin. Transferrin maintains iron soluble at the near-neutral pH of plasma and also binds the iron, thus avoiding the formation of free oxygen radicals. Transferrin accomplishes the transport of iron between the sites of absorption, storage, and its use by all organs of the body. Approximately 80% of iron bound to transferrin is used for the production of hemoglobin. The remainder is delivered to nonerythroid cells, which utilize iron for production of iron-containing proteins and place excess iron in ferritin and hemosiderin, a watcr-insoluble degradation product of ferritin. Ferritin is a protein that serves predominantly as a storage protein that incorporates iron to protect cells from oxidative damage of ionic iron and to supply it on demand [el.

2.

2.1.

PROTEINS WITH KNOWN STRUCTURE

Ferritins: Occurrence and Biological Role

Ferritins are vesicle-like assemblies of 24 polypeptide (four-helix bundle) subunits that concentrate iron in cells by directing thc formation of a ferric mineral in the hollow protein interior 17,81. Effective cellular iron concentrations 10’l times the solubility of the ferric ion are achieved by ferritins, which are found in microorganisms, plants, and animals. The complexity and the sophistication of the genetic regulation of the ferritins, involving both DNA and mRNA L9-121, emphasize the central role of iron and ferritin in life. Rates of Fez+ oxidation, translocation of Fez+and Fe3+ (10-20 A), and mineralization arc all controlled by ferritin [71. Fe2+ release from ferritin following reduction of the mineral is slow and poorly understood 113,141, but it is important for the biosynthesis of iron proteins, such as those required in respiration, photosynthesis, nitrogen fixation, and cell division, [7] and as dietary iron [15]. How and where the iron exits from ferritin in vivo is not known. Two types of subunits, H or M (fast) and L (slow), are identified in vertebrate ferritins. They differ in rates of iron uptake and mineralization and assemble in vivo t o form heteropolymeric protein shells. H/L subunit ratios reflect cell specificity of Hand L-subunit gene expression. Ferritin molecules in cells containing high levels of iron tend to be rich in L chains and may have a long-term storage function [16,17], whereas H-rich ferritins are more active in iron metabolism [18,19].In frog, the ratio between H and M subunits varies among tissues; for example, M is more abundant in liver while H is more abundant in red blood cells [18].

2.2.

Ferritins: Structural Classification

Ferritin is a collectivc name for a family of iron-binding proteins. On the basis of the SCOP classification, the fcrritin family belongj to the class of all-r proteins.

ARNESANO AND PROV~NZANI

574

Vertebrate amino acid sequences of either R or L chains show about 9045% and 8085%amino acid identity, respectively, but the identity between the two groups is only about 50% C2Ol. For this reason, H and L chains are thought to have diverged from a common ancestor some 200-300 million years ago [211. Structures are available for the following species: human (Homo sapiens), horse U3yuus caballus), bullfrog (Rana catesbeiana), Escherichia coli, Listeria innocua. PDB references of these structures are reported in Table 1.

TABLE 1 PDB References of the Available Ferritin and Bacterioferritin Structures Solved by X-ray Diffraction Protein

Structures available

(Apo)ferritin Human (Homo sapiens) orse

2fha:1-3chain complexed with Ca; mutant [ZZI lfha: H chain complexed with Ca, Fe; mutant [I061 laew: L chain complexed with Cd [22] (Equus caballus) ldat: L chain complexed with Cd 11591 lier: L chain complcxed with Cd 11601 lies: L chains a-f complexed with Cd 11601 lhrs: L chain complexod with Cd, heme 11611 Bullfrog lbg7 Complexed with Ca; mutant [31J (Rana catesbeiana) lrc : L chain complexed with trimethyl glycine f1621 L chain complexed with trimethyl glycine; mutant [1621

lrcg: L chain complexed with trimethyl glycine 11623 lrce: L chain complexed with trimethyl glycine; mutant [I621 lree: L chain complexed with trimethyl glycine; mutant

Listeria innocua Ferritin homologue Escherichia coli Bacterioferritin Escherichia coli

[1621 lmfr: M chains a-x complexed with Mg 1191 Iq : Chains a-1 1271

laps: Chains a-1 complexed with Na; mutant la91

lbfr: Chains a-x complexed with heme, Mn [251 lbef: Chains a-b complexed with heme, Mn [261


575

2.3. Structures of Ferritins 2.3.1. Description of Eukaryotic Ferritin Structures

All types of ferritin subunits show a considerable degree of structural homology in respect t o both subunit folds and inter-subunit interactions. Each subunit contains about 175 residues, and most of them belong to helical regions. The a helices are named A, B, C, and E. Helices A-D form a four-helix bundle. Helices B and C are connected by a long loop L 1221 (Fig. 1A). Twenty-four subunits (24-mer) constitute the protein shell of relative molecular ,> ranging from 450,000 to 500,000 with internal and external diameters of and 120 A, respectively. The geometry of the shell is approximately that of a rhombic dodecahedron (432 point-group symmetry), the faces of which consist of two subunits related by a twofold symmetry axis at its center. Channels that traverse the protein shell are present at the fourfold axis, where the E helices interact, and at the threefold axis, near the N termini of the subunits (Fig. 1B). Iron i s stored in the protein shell of ferritin as a hydrous ferric oxide nanoparticle with a structure similar to that of the mineral ferrihydrite. Eight hydrophilic channels at the threefold axes are thought to be the primary avenues by which iron gains entry to the interior of eukaryotic fenitins ([231 and references therein). The H chain contains a dinuclear

FIG. 1 . (A) A ribbon model of one subunit of bullfrog apoferritin showing the packing of the four main CI helices (A, B, C, D),the connecting T i loop and the E helix. Residues in the M-chin ferroxidase site are indicated. (B) The protein coat of bullfrog apoferritin as deduced from X-ray diffraction of crystals of the protein (PDR code: lmfr). The outer surface o f the protein coat shows the arrangement of the 24 ellipsoidalpolypeptide subunits. Channels at the fourfold axis, near the C termini of the subunits, and at the threefold axis, near the N termini, are indicated. See Figure 12.1 in the color insert.

576

ARNESANO AND PROVENZANI

ferroxidase site that is located in the four-helixbundle of the subunit; it catalyzes the The L subunit lacks this site but oxidation of f e m u s iron by 0 2 , producing H202. contains additional glutamate residues on the interior surface of the protein shell that produce a microenvironment that facilitates mineralization and the turnover of iron(I1n at the H subunit ferroxidase site 1231. 2.3.2. Description of Bacterial Ferritin Structures

Bacteria possess two types of iron storage proteins: the heme-containing bacterioferritins and the hemefree fenitins. These proteins are widespread in eubacteria and archaebacteria [241. Bactcrioferritins and vertebrate ferritins are distantly related but retain similar structural and functional propcrties. The crystal structure of the bacterioferritin from Escherichia coli has been recently determined (PDB code: lBFR, 24 subunits complexed with heme) [251. Bacterioferritins are composed of 24 identical or similar subunits (approximately 19 kDa) and contain up to 12 protoporphyrin M heme groups located at the twofold interfaces between pairs of twofold related subunits. The tetragonal crystal form of the protein has also been determined (PDB code: lBCF, two subunits complexed with heme) C261.The role of the heme is unknown, although it may be involved in mediating iron core reduction and iron release. Some bacterioferritins are composed of two subunit types, one conferring heme binding ability (a) and the other (PI bestowing ferroxidase activity. Bacterioferritin genes are often adjacent to genes encoding a small [2Fc-2S]-ferredoxin (bacterioferritin-associatedferredoxin, Bfd). Bfd may interact directly with bacterioferritin and could be involved in releasing iron from (or delivering iron to) bacterioferritin or other iron complexes. Some bacteria contain two bacterioferritin subunits, or two ferritin subunits, that in most cases coassemble. Others possess both a bacterioferritin and a ferritin, while some appear to lack any type of iron storage protein [241. An exception to the general assembly of ferritins is the dodecameric ferritin from Listeiia innocua [27]. The structure of L. innocua ferritin has been determined by molecular replacement, using as a search model the structure of Dps from Escherichiu coli [28]. Dps is a DNA-binding protein that protects DNA from oxidative damage. It has essentially the same fold as ferritin but forms a dodecamer (12-mer) with 23 (tetrahedral) point-group symmetry which also has a hollow core and pores at the threefold axis [29]. The L. innocua 12-mer too is endowed with 23 symmetry and displays the functionally relevant structural features of the ferritin 24-mer, namely, the negatively charged channels along the threefold symmetry axes that serve for iron entry into the cavity and a negatively charged internal cavity for iron deposition. Analysis of the nature and stereochemistry of the iron-binding &an& reveals strong similarities with known fcrroxidase sites. The L. innocun fcrroxidase site, however, is the first described that has ligands belonging to two dif'erent subunits and is not situated in a four-helix bundle E271.

577

IRON STORAGE AND T R A N S ~ O ~PROT T

2.3.3. Assembly of Subunits

Ferritins are tightly packed molecules in which each subunit interacts with six neighbors. The folded subunits have the ability to form inter- as well as intraspecies hybrids [301 consistent with a relatively high degree of conservation of residues participating in inter-subunit contacts (75% identity across the vertebrate sequences). Studies on M- and L-chain interactions from bullfrog ferritin have allowed separation of subunitspecific differences from species-specific differences. These interactions include several hydrogen bonds, salt bridges, and hydrophobic contacts, and one-third of residues of the polypeptide chain are involved in such contacts The packing of ferritin subunits generates two types of channels at the fourfold and threefold axes. It has been suggested that iron ions enter and exit through the threefold channels, which are similar among human and frog ferritins [31-341. In addition, electrostatic forces may drive cations, like M$* in bullfrog, through the fourfold channel [19].

[la.

2.3.4. The Femxidase Site

The first ferritin structure described was from horse spleen and contained 85% of L and 15% of H-type chains 135-381. In accordance with the usual pattern of apolar residues in helical bundles, most buried residues in human H chain and horse L chain are apolar, although not highly conserved, because their interactions with apolar residues from a neighboring helix are not strongly directed. Unusually, in both H and L ferritin, the four-helix bundles do not have a uniformly hydrophobic interior; instead they have a central hydrophilic region sandwiched between two hydrophobic cores. The hydrophilic region at the center of the human H chain, aa well as that at the center of the M chain (in bullfrog), is able to bind metal ions 1321. Furthermore, based on mutagenesis, it has been shown that pairs of Fe(11) atoms bound at this site are rapidly oxidized [39,401. For this rewon, this hydrophilic region has been addressed as the femxidase center and comprises seven amino acids in human H chain (61~27,Tyr34, Glu61, Glu62, His65,Glu107, Gln1411, all essential for maximal activity. These catalytic residues are also present in the ferritins of plants and bacteria, and five of them are found in heme-containing bacterioferritins [7]. In the ferroxidase site of the M-chain ferritin crystals from bullfrog, two metal ions (Mg)from the crystallization buffer were found that interact with Glu23, Glu58, €€is61,Glu103, Gln137, and, unique to the M subunit, Asp140 [191. A completely different pattern of residues is found in the central region of Lchain ferritins. The L chain lacks the ferroxidase center activity. In fact, the central region comprises seven hydrophilic residues as in the W chains, but the only invariant residues are Tyr34 and Glu61. In horse L chain the residue Glu62, which is present in the H chain, is replaced by a lysine whose positive charge forms a salt bridge with the negative charge of Glu107, adding stability to the L chain [22].

ARN~SANOAND P ~ O V ~ N Z A N I

578

2.4.

Transferrins: Occurrence and Biological Role

Transferrins are a group of bilobal glycoproteins that contain two homologous metal binding sites with high affinities for ferric iron [41]. Although originally thought to be restricted to vertebrates, transferrin-like proteins have now been identified in several invertebrate species where they occur in the hemolymph [421. In vertebrates, the transferrins are found in a variety of body fluids, including the serum transferrins found in blood; the ovotransferrins found in avian egg white; the lactoferrins found in milk, tears, saliva, and other secretions, and the melanotransferrins found anchored to the membrane surfaces of melanocytes and other cells via a glycosyl-phosphatidylinositol linkage. Each of these vertebrate transferrins consists of a single polypeptide chain with an M , of about 80,000 containing two structurally similar binding sites for ferric iron and other metals and a binding site for a synergistic anion 1411. The proteins of the transferrin fknily play a crucial biological role by sequestering and solubilizing iron, thereby controlling the levels of free iron in body fluids f431. Serum transferrin, in particular, has the role of binding ferric ions in the bloodstream and transporting this bound iron to cells where it is released by a process of receptormediated endocytosis [44]. Iron release occurs at the reduced intracellular pH (-5.51, apparently with the active participation of the receptor 1451, and the iron-free apotransferrin is returned to circulation without degradation. A function analogous to that of serum transferrin has been proposed for ovotransferrin, although an in vivo iron transport function for ovotransferrin has not been proven. In support of this hypothesis, specific transferrin receptor interactions have been demonstrated for the protein 146491. Lactoferrin is an iron-binding glycoprotein of the transferrin family, first isolated from milk. Lactoferrin exerts its effects on glandular epithelia, secretions, mucosal surfaces as well as in the interstitiurn and vascular compartments where it has been postulated to participate in iron metabolism, disease defense, and modulation of inflammatory and immune responses [50-521. The many reports on its antimicrobid and antiinflammatory activity in vitro identify lactoferrin as important in host defense against infection and excessive inflammation. Human and bovine lactoferrin contain peptides of 47 and 25 amino acids, respectively, named lactoferricin H 1521 and B [51], that are thought to be responsible for their antimicrobial activity.

2.5.

Transferrins: Structural Classification

On the basis of the SCOP classification the transferrin family belongs to the class of rx and proteins. Determination of the amino acid sequence of human transferrin revealed extensive sequence identity between the amino and carboxyl terminal halves of the polypeptide chain [531. This sequence similarity has been found in other transferrins, such as rabbit transferrin, human lactoferrin, and hen ovotransferrin [54,551. These data suggest that the modern-day transferrins have evolved by means of a gene


579

duplication event from an ancestral gene coding for a protein with an M , of about 40,000containing a single metal-binding site. Subsequent duplications of this larger gene have given rise to the transferrins, lactofemins, and melanotransferrins found today. The organization of the human transferrin gene is consistent with this evolutionary history /561. Structures are available for the following species: human (Homo sapiens), rabbit (Oryetolagus euniculus), cow (230s taurus), domestic: water buffalo (Bubalus arnee bubalis), horse (Equus caballus), duck (Anus platyrhynchos), chicken (Gallusgallus). The PDB references of these structures are reported in Table 2.

2.6. Structures of 2.6.1. Description of Euhuryotic Transferrin Structures

Transferrins me soluble glycoproteins consisting of a single chain of about 700 amino acids. Crystallographic studies of human lactoferrin gave the first detailed view of a three-dimensional transferrin structure [57,581. This structure, subsequently refined to 2.2 A resolution [591, showed that the polypeptide is folded into two homologous lobes (referred to as the N lobe and the C lobe) representing the N- and C-terminal halves of the molecule. The two lobes are joined by a connecting peptide, which in lactoferrin forms a three-turn helix. Each lobe is further subdivided into two similar domains (1and 2) separated by a deep cleft that contains the binding site for ferric iron; in each lobe, the ligands to iron are the same: two tyrosine residues, a histidine residue, and an aspartie acid residue together with two oxygen atoms of a bidentate carbonate ion that is bound synergistically with iron. Subsequent crystallographic analyses have led to the determination of the three-dimensional structures of other transferrin molecules. These are listed in Table 2 and include the diferric forms of rabbit transferrin 1601, chicken ovotransferrin [61], duck ovotransferrin [621, and an 18-kDa iron-binding fragment of duck ovotransferrin 1633, and bovine lactoferrin (641, as well as various fragments ke., separate N and C lobes), mutants, and metal- and anion-substituted proteins. Although there are some subtle diff’erences, the folding and iron binding sites of all these species are essentially similar to those originally described for lactoferrin [411. A schematic structure of the protein, deduced from crystal structure analysis, is shown in Fig. 2. 2.6.2. Description of Bacterial Structures

The first crystal structure of the iron transporter ferric ion-binding protein from Hwmophilus influenme (hFBP) was recently determined (PDB code: lMRP, complexed with Fe and phosphate; 1 D9V, complexed with phosphate), revealing the structural basis for iron uptake and transport required by several important bacterial pathogens 1651. A homologous protein From Neisseria gonorrhoeae has also been determined (PDB code: 1D9Y,complexed with Fe and phosphate). Paradoxically,

580

ARNESANO AND P R O V ~ N ~ A N I

TABLE 2 References of the Available Transferrin Structures Solved by X-ray Diffraction Protein Lactoferrin Human (€€ornosapiens)

Bovine (Bos taurus) DomeRtic water buffalo (&balun a n m bubalis) Horse (Equus caballus)

Ovotransferrin Duck (Anas platyrh.ynchos) Chicken (Gallus gallus)

Transferrin Human (Homo sapiens)

Rabbit (Oiyctolagus crcniculus) Ferric-binding protein Haemophzlus influermw

NeisserLa gonorrhoear

Structures available"

llct: complexed with COB,Fe [741 ldsn: complexed with C03, Fe; mutant 1831 llcf: region 1-334, 1-334 complexed with COB,Cu, nag, oxl 11631 lcb6: region:1001-1334, 1335-1691 complexed with Cl[84] leh3 chain a N-terminal lobe complexed with COB,Fe; mutant 1801 llfg: region 1-334,335-691 complexed with ' 2 0 3 , Fe, fuc, nag C1641 lb01: region 1-334, 335-691; mutant 11121 lhse: complexed with COB,Fe; mulant ll651 1 E region 1-334, 335-691 complexed with COB,Cu, fuc, nag [1661 lbka: region 4-334, 335-691 complcxed with Fe, oxl 11671 lvfe: complexed with COa, Fe; mutant [168] lvfd complexed with COB,Fe; mutant 11681 llfh: region 1-334,335-691 complexed with 61 11641 llgb: Chain c N2-fragment (one domain fragment) complexed with Ca, fuc, gal, man, Mn, nag 11691 1bW. region 5-333, 334-689 complexed with COB,Fe, man, nag [641 lee2: region 1-333, 334-689 complexed with COS, Fe 1661 Ibiy: region 1-333, 334-689 complexed with COB,Fe 1671

lblx: rcgion 1-333, 336689 complexed with C 0 3 , Fe 11701 lb7z: region 1-333, 334-689 complexed with Fe, 0x1 [149] lqjm: region 1-333, 334-689 complexed with CO3, Sm 11501 lb7u: region 1-333, 334-689 [171] lovb: domain 2 in the N-terminal lobe complexed with COs, Fe [a31 ldot: region 1-334,335-686 complexed with COs, Fe, fue, nag E621 laov: region 1,734,335-686 [1721 ltfa: Chain a N-terminal lobe only cornplexed with SO4 [79] lnft: Chain a N-terminal lobe only complexed with Fe, nta, SO4 1791 lovt: region 5-334, 335-686 complexed with ' 2 0 3 , Fe [el] lnnt: N-terminal lobe only complexed with COs, Fe 1731 laiv: region 1-334, 335-686 complexed with nag 11351 lase:N-terminal lobe complexed with COB,Fe I75f N-terminal lobe complexed wilh COS, Fe [751 : Chain a N-terminal lobe complexed with COB,Fe; mutant 1811 ld4n:Chain a N-terminal lobe complexed with COB,Fe; mutant 1811 lbp6: Chain a 4 N-terminal lobe 11441 lbtj: Chain a-b N-terminal lobe L1441 ldtg: Chain a N-terminal lobe El461 lb3e: Chain a N-terminal lobe complexed with 603, Fe Ill01 ltfd: N-terminal lobe cornplexed with GO:+, Fe [721 lmrp: complexed with Fe, PO, I651 ld9v: complexed with PO4 ld9y: cumplexed with Fe, PO,

aman, a-u-mannose;nag, N-acetyl-o-glucosamine;CQ3, carbonate; SO*, sulfate; POe, phosphate;

k c , fucose; gal, D-gdaCtOse; 0x1, oxidate; nta, nitrilotriacetic acid.

581

IRON STORAGE AND TRANS^^

R

FIG. 2. Three-dimensional structure of ladoferrin (PDB code: llfg): (A) A ribbon model representation of the N and C lobes. (B) The folding pattern of the N lobe. The protein iron ligands and the synergistic carbonate are also shown. See Figure 12.2 in the color insert.

although hFBP belongs to a protein superfamily that includes human transferrin, iron binding in hFBP and transferrin appears to have developed independently by convergent evolution [651. A structural comparison of hFBP with other prokaryotic periplasmic transport proteins and the eukaryotic transferrins suggests that these proteins are related by divergent evolution from an anion-binding common ancestor, not from an iron-binding ancestor. The iron binding site of hFBP incorporates a water and an exogenous phosphate ion as iron ligands and exhibits nearly ideal octahedral metal coordination. FBP is highly conserved, is required for virulence, and is a nodal point for €ree iron uptake in several gramnegative pathogenic bacteria, thus providing a potential target for broad-spectrum antibacterial drug design against human pathogens such as H. influenzae, N. gonorrhoeae, and I?. meningitidis I651. 2.6.3. Iron Binding Sites

Differences between various eukaryotic transferrin structures are found in the dimensions of the binding cleft and the interlobe orientation. The interlobe interactions are predominantly hydrophobic in nature and could act as a cushion for a change in orientation under the influence of external conditions, thus facilitating

the sliding of two lobes. The interdomain interactions are comparable in the N and C lobes [64,66,673. The two separate glycosylated N- and C-terminal lobes of buffalo lactoferrin have been produced by limited proteolysis using proteinase K. Circular dichroism studies indicated a high a-helical content in the native lactoferrin while comparatively lower helical structures were present in the N and C lobes. In addition, the iron saturations of the N and C lobes appeared to be lower than that of the native protein

[W. Proteins of the transferrin family control iron levels in higher animals through their very tight but reversible binding of iron. These bilobate molecules have two binding sites, one per lobe, each housing one Fe3+ and the synergistic carbonate ion. Upon uptake and release of Fe3+, transferrins undergo a large-scale conformaLional changc depending on a common structural mechanism: domains l and 2 rotate as rigid bodies around a rotation axis that passes through the two antiparallel fistrands linking the domains. However, the extent of the rotation is variable for different transferrin species and lobes [691. The transfedn N and C lobes are stabilized by many intrachain disdfides [691. The interdomain disulfide bond present in the C lobe of all the transferrins was postulated to restrict the domain movement (openinglclosure) resulting in the slow rate of iron uptake and release. A recent study analyzes the conformational stability and iron binding properties of a derivative of the isolated C lobe of ovotransferrin in which the interdomain disulfide bond was selectively reduced [701. Iron release kinetics showed no difference between the disulfide-intact and disulfide-reducedalkylated forms. The conformational stability, evaluated by unfolding and refolding experiments using guanidine hydrochloride, was greater for the disulfide-intact form than for the disulfide-reduceflalkylatecl form. This study demonstrates that the interdomain disulfide bond has no effect on the iron uptake and release function but significantly increases the conformational stability in the C lobe [701. X-ray crystallographic and solution X-ray scattering studies have shown that apotransferrins (serum transferrin, lactoferrin, and ovotransferrin) have a structure with open interdomain clefts, which close when iron is tightly bound by the coordination of the four protein ligands (AspGO, Tyr92, Tyrl91, and His250 in the ovotransferrin N lobe) and of the physiologically bidentate synergistic carbonate 158, 60-62,64,71-75 I, The closed conformation (holotransferrins) has been suggested as an important step in the receptor recognition 146-48,76,771. In the crystal structure of human apolactoferrin the N lobe binding cleft is wide open, following a domain rotation of 53”,mediated by the pivoting of two helices and flexing of two interdomain polypeptide strands. Remarkably, the C-lobe cleft is closed but unliganded C781. Domain-opened structures with the coordinated Fe3+ are demonstrated in fragment and intact forms. To solve the Fe”-loaded, dornain-opened structure, an ovotransfenin N-lobe crystal that had been grown as the apo form was soaked with F$+nitr~otriacetate.The Fe”-soaked form showed almost exactly the same overall open structure as the iron-free apo form. The electron density map unequivocally proved

583


the presence of an iron atom with the coordination by the two protein ligands of Tyr92-OH and Tyrl91-OH. This structure has been proposed as a possible intermediate for iron uptake and release E791. The bovine lactoferrin structure is notable for several well-defined oligosaccharide units that demonstrate the structural fwtors that stabilize carbohydrate structure. One glycan chain, attached to an asparagine residue, appears to contribute to interdomain interactions and may modulate iron release from the C lobe [64]. The structure of the monoferric N-terminal half-molecule of hen ovotransferrin revealed for the first time an unusual interdomain interaction formed between the side chain N< atoms of Lys209 and Lys301, which were 2.3 apart. This strong interaction was an example of a low-barrier hydrogen bond between the two lysine N1; atoms, both of which were also involved in a hydrogen bonding interaction with the aromatic ring of a tyrosine residue. The close proximity of the two resulting positive charges, and their location on opposite domains of the N lobe, might be the driving force that opens the two domains of the protein, exposing the Fe3" ion and facilitating its release 1731. Lactoferrin and serum transferrin combine high-affinity iron binding with an ability to release this iron at reduced pH. Lactoferrin, however, retains iron to significantly lower pH than transferrin, giving the two proteins distinct functional roles. The behavior of lactoferrin is due equally to interlobe interactions and to the absence of a dilysine trigger. In human transferrin, Lys206 and Lys296, which correspond to Lys209 and Lys301 in hen ovotransferrin, form a dilysine interaction that is missing in lactoferrin, where the first lysine of the pair is replaced by an Arg residue. The structure of the Arg2lOLys mutant of lactoferrin has been solved but, even in this case, the lysine trigger is absent 1801. The X-ray crystallographic structures of two mutants (Lys206Gln and His207Glu) of the N lobe of human transferrin have been determined. Both mutant proteins bind iron with greater affinity than the native protein. The structures of the Lys206Gln and His207Glu mutants show interactions (both H bonding and electrostatic) that stabilize the interaction of Lys 296 in the closed conformation, thereby stabilizing the iron-bound forms [811. X-ray solution scattering experiments of the mutated N-terminal fragment of human swum transferrin with Asp63Ser(Cys)provided direct experimental evidence for the existence of a trigger mechanism for the closure of the interdomain cleft well as evidence that this trigger mechanism is disrupted by mutation of Asp63, the only ligand o f iron from domain 1 [82]. The crystal structure of a site-specificmutant of the N-terminal half-molecule of human lactoferrin, in which the iron ligand Asp60 has been mutated to Ser, was determined in order to evaluate the erfects of the mutation on iron binding and domain closure. The domain closure is changed, with the Asp6OSer mutant having a more closed conformation. Consideration OP crystal packing suggests that the altered domain closure is a genuine molecular property and both the iron coordination and interdomain contacts are consistent with weakened iron binding in the mutant [831. Some structural data show anion binding sites in the interdomain cleft that can

a

play crucial roles in the domain opening and synergistic carbonate anion release in the iron release mechanism [84,851. A mechanism for the Fe3' release in the presence of a nonsynergistic anion has been proposed on the basis of the sulfate-bound apo crystal structure of the ovotransferrin N lobe [%I.

3. PROT~INSWITH UNKNOWN STRUCTURE 3.1. The Iron Pathway During the last decade, various iron transporters have been identified for lower or~anisms,including the Fe2' transporters, feoB from E. coli 1861, FET4 from yeast t871, the plant IRTl [SSl and the Fe3+ transporter FTRl from yeast [SSl. However, the transporters that mediate direct uptake of iron and metd ions into mammalian cells remain elusive. Recent molecular genetics studies on hemochromatosis have brought new information in the physiology of iron homeostasis, a key problem in biology and medicine. The protein encoded by the HFE gene appears to serve as a regplator of iron absorpE is mutated in the iron overload disease hereditmy hemochrornatosis 1901. ds at the cell surface to the transferrin receptor (TfR) 1917 and reduces its affinity for iron-loaded transferrin [92]. The crystal structure of HFE reveals the locations of hemochromatosis mutations that prevent cell surface expression of FE, suggesting a mechanism by which mutated HFE might enhance the system uptake of iron into the tissues [931. After the identification of HFE, the physicochemicalproperties of iron hampered the discovery and isolation of iron transporters. Recently, an expression cloning system using Xenopus oocytes has been used to identify a protein capable of transporting divalent transition metal ions, like Fe2+, Zn2+, Mn2+, Co2+, Cd2+, Cu2+, and Ni2' 1941. This protein, Nramp2, renamed divalent metal transporter-1 (DMT-1) is localized to the brush border of human duodenum where it is predicted to serve as the principal transporter of ferrous iron from the intestinal lumen. Mutations in t h murine Nramp2 gene were implicated as the cause of the rnicrocytic anemia C951. Since DMT-1 expression is up-regulated in hereditary hernochromatoeis and the HFE protein can be crystallized as a ternary complex with the transferrin receptor and iron-saturated transferrin, these proteins are likely to interact in a common pathway for human iron homeostasis 1961. Ferric iron is absorbed via a p-integrin and mobilfenin (IMP) pathway that is not shared with other nutritional metals. Other proteins were recently described which are believed to play a role in iron absorption. SPT (stimulator of iron transport) is postulated to facilitate both ferric and ferrous iron uptake E971. Other steps participate in the pathway of iron delivery across the mucosal membrane, and the recently identified ferroportinlflREG1 protein that is involved in the egress of iron across the basolateral membrane of the enterocyte must also play a role in the net incorporation of iron from the intestinal lumen into the body [98,991.

IRON STQRAGE A N

TRANSPORT PROTEINS

585

IREGl mediates iron efflux in the presence of the ferroxidase hephaestin, a transmembrane protein that shares homology with the plasma multicopper ferroxidase ceruloplasrnin 14,1001 and is important in the transfer of iron from enterocytes into the plasma. The mouse membrane-bound transferrin-like protein (MTD cDNA was cloned. MTf was originally found in human melanomas and was called melanotransferrin 1101-1031. Phylogenetic analysis indicated that the MTf gene diverged from the common ancestor gene earlier than the genes of the other transfedns, such as serum transferrin, lactoferrin, and ovotransferrin, and that the divergence occurred after the divergence of vertebrates and invertebrates C1041. MTf, as well as the other transferrins, consists of two repeated domains. The similarity between the N-terminal and the 6-terminal domains of MTf is much higher than that of the other transfeains, although the five amino acid residues required for iron binding were not conserved in the C-terminal domain of MTf, in contrast to the conservation of these residues in both domains of the other transferrins. The sequence characteristics and the expression pattern suggested that during development and in adult tissues, MTf has some functions that are M e r e n t from those of other transferrins [1041.It may act as a eapture-and-hold mechanism to prevent toxic frtu? iron deposition on membranes. Alternatively, MTf may limit the presence of excessive amounts of free iron released from the damaged tissues TlO51.

.

ST

URE-FUNCTl0N RELATI

4.1, Expression Systems 4.1.I. Ferritins

The three-dimensional structure of a human ferritin H-chain homopolymer was obtained by genetically engineering a change in the sequence of the intermolecular contact region to obtain crystals isomorphous with the homologous rat L ferritin and of high enough quality for X-ray diffraction analysis [106].Horse ferritin L chain was isolated from the organism (spleen) or expressed in E. coli using several plasmids 11221. The bullfrog native femitin was overexpressed in E. coli from complementary DNA sequences of bullfrog red cell femitin 11071. Various mutants of this protein were also expressed in E. coli using several plasmids. The ferritin from L. innoeua was isolated and purified from packed bacterial cells I28l. Dps protein was obtained from starved E. coli cells 1291. 4.1.2.

Transferrins

Most of the transferrin structures described in the previous sections have been obtained by isolation of the native proteins from serum, milk, and egg white. Amino terminal and carboxy terminal half-molecules were prepared by enzymatic

586

digestion and purified by gel filtration and column chromatography [l uman recom~inantproteins were generally expressed in baby hamster kidney ) cells plasmid as expression vector [1091 ew expression systems have been developed using Pichia pastoris, Zans, and AspergiZZus awarnori. The ferric form of the N lobe of human serum transferrin has been expressed at high levels in P. pastoris. The cornparison of the structures of recornbinant protein expressed in P. pastoris and mammalian cells with serum transferrin shows that the polypeptide folding pattern is dentical [1101. Overexpression of human lactoferrin in the fungi A. nidu. awamori has resulted in the availability of very large quantities of this ~. of the latter recombinant structure onto the native protein [ 1 1 1 , 1 ~ 2~uperposition and milk human lactoferrin structure shows a very high level of corres~ondence~ there are no significant differences in side chain conformations or in the iron binding sites. This shows that the structure of the protein is not affected by the mode of ression, the use of strain improvement procedures, or the changes in glycosylation due to the fungal system [112].

idogical Function cal function of iron storage omplished in ferritin essentially by the uptake and oxidation of and the production of a crystalline to enter into ferritins through eight mineral similar to ferrihyd~ite.Fez+ is sup negatively charged channels and it occupi rroxidase centers in the middle of the four-helix bundles of H (or M) chain where it is oxidized. ecent spectroscopic studies have shown that a peroxodiferric intermediate, in ribonucleQtidereductase 1 and methane monooxygenase hydroxylase (MM ), is produced at the ferroxidase site followed by formation of a p-oxobridged dimer, which then fragments and migrates to the nucleation sites to form incipient mineral core species [23]. chains lacks the ferro~idaseactivity and seem to have evel loped an increased cy in ~ine~alization. The key to L-chain function~lityis thought to reside in their four glutamate residues [113-115], that point into the iron storage cavity, forming a diamond-shaped cluster, and bind metal ions. The L chain in homopolymers has a significant role in iron storage in the absence of chains, whereas in heteropolymers it has a cooperative role promoting iron core nucleation, thus avoiding nonspecific hydrolysis outside the cells 1221. Once sufficient core has developed, iron f the growing crystallitee, oxidation and mineralization occur primarily on the sur thus ~ i n i ~ i z i the n g production of potentially harmful


587

4.2.2. Assembly Pathways

Concerning assembly pathways in both horno- and heteropolymers, it has been predicted that the parallel dimer, with its mainly hydrophobic interface, is the first and most stable assembly intermediate. Experimental evidences for dimer intermediates have been found from a variety of methods, including site-directed mutagenesis r1161. The results support the hypothesis that the symmetrical subunit dinners are the building blocks of the ferritin assembly and are consistent with a reassembly pathway involving the coalescence of dimers, probably around the fourfold axis, followed by stepwise addition of dimers until the 24-mer cage is completed [1161. A feature of ferritin molecules is their high stability. A heat step is normally used in purification of both native heteropolyrners and recombinant hornopolymers, and the assembled shells require harsh conditions for denaturation. The high stability of the ferritin shells may be related to their low surface area to volume ratio, resulting from the tight subunit packing into an almost spherical molecule. H and L homopolyrners were studied under different denaturing conditions by varying pH or denaturant concentration E1141. The study on H-chain variants showed that ferritin stability was not affected by alterations of regions exposed t o the inner or outer surface of the shell and not involved in intra- or interchain interactions. Stability was reduced by alterations of sequences involved in inter-subunit interactions such as the deletion of the N-terminal extension or substitutions along the hydrophobic and hydrophilic channels. In addition, stability was increased by the substitution of two amino acids inside the four-helixbundle with those of the homologous L chain. One of the residues is involved in a salt bridge in the L chain, and the stability difference between H and L ferritins is to a large extent due to the stabilizing effect of this salt bridge on the 1,-subunit fold I114I. The L. innocua 12-mer ferritin shows an increased stability at low pN in comparison to the classic ferritin 2Pmer and preserves its quaternary assembly at pH 2.0, despite an altered tertiary structure. Several hydrophilic and hydrophobic interactions play a role in determining the resistance to dissociation of L. innocua femitin at low PH r m i . 4.2.3. Ferroxidase Activity

Recently, the mechanism for iron mineralization in €3 and M chain type has been almost completely clarified. The detection of a peroxodiferric intermediate in the ferritin ferroxidetse reaction [118,119] firmly establishes the ferritin ferroxidase site as very similar to sites in the 02-activating diiron enzymes such as NLMOH or RNR [l20]. It is likely that the distinction between diiron cofactor sites (02-activating enzymes) and diiron substrate sites (ferritins) depends on the structures of the peroxodiferric intermediates, and thus on the identity and the geometric arrangement of the amino acids hound t o the iron. In MMOH, RNR, and other 02-activating enzymes, each iron ion is ligated to Glu and His in the conserved, duplicated motif G l m i s . In frog M chain, one iron is ligated to a GluXXHis motif and the other i s ligated to

588

A R N ~ S A N OAND PROV

GI

p 119,221. The data suggest that Gln and Asp are a vestige of the second G1ssite, resulting from single-nucleotide mutations of Glu and His codons. In all plant and animal H-type ferritins, Gln (in the GlnXXAsp motif) is conserved but Asp can be substituted with Ma or Ser by additional single nucleotide mutations C18,1211. This variability in Fe ligation may be important in determining peroxodiferric kinetics, biological specificity, and chain stability. In fact, frog M ( G l ~ p ) and frog H (GlnXXSer)chains differ in rates of peroxodiferric formation, decay, and in cell specificity, which is consistent with a biological role for these ligands in controlling ferritin activity 11221. Through X-ray absorption spectroscopy ( U S > ,the structure of recombinant frog M chain reacting with O2 was examined. It has been possible to observe an unusually short distance between the two iron ions in the peroxodiferric intermediate of 2.53 A. This distance suggests a unique triple-bridge strudure, never observed in iron chemistry, with two single-atom bridges between the two iron ions in addition to the peroxo bridge [122]. The two single-atom bridges could derive from ligands in the femxidase center (Fig. 3). Different geometries of the peroxo intermediates yield a high-valence iron oxidant in the Q2-activating enzymes, like MMQH and RNR, but give diferric oxohydroxo biomineral precursors in ferritin. In this latter case, the triple-bridge structure of the peroxo intermediate leads to a stronger 0-0bond that could favor the observed release of HzOz 1123,1241and the formation of a p-0x0 or p-hydroxo diferric biomineral precursor over 0-0bond cleavage and the formation of high-valence intermediates, as seen for Q2-activatingenzymes.

FIG. 3. The peroxodiferric complex in the catalytic ferroxihe site of ferritin. The peroxodiferric complex decays to diferric 0x0 or hydroxo precursors that are translocated between the catalytic and biornineralization sites, and *H202is released. The iron-iron distance in the peroxodiferric complex was found to be 2.53A. (Adapted from 11221.)

IRON STO

ANSPO~TPROTEINS

589

In contrast with other femitins, the ferroxidation reaction in L. innocua ferritin proceeds more slowly than the following step of oxidation/mineral~zation. H20 is the final product of dioxygen reduction in the L. innocua 12-mer femitin 11251 and in the E. coli 24-mer bacterioferritin 11261, whereas € 3 2 0 2 is produced in 24-mer mammalian ferritins. Different kinetic values among ferritins undoubtedly reflect differences in the detailed structures of their ferroxidase centers; most notably, the centers of the E. COZZ and L. innocua ferritins contain two histidine residues, whereas mammalian fenitin centers have only one [23,24,27,127,128].The failure to observe H202production in L. innoeucr ferritin is perhaps due to H20z being a better oxidant for bound Fez'- than is 02,as also found for E. coli bacterioferritin 11261 but not for mammalian ferritin 11241. H2Q2produced at one ferroxidase center could be rapidly consumed at another. Another possibility is that iron oxidation occurs via intermediate dinuclear F e W ) species in both L. innocua ferritin and bacterioferritin 11261. The latter possibility is consistent with the failure to observe iron turnover at the dinuclear center, as suggested by the lack of regeneration of ferroxidase activity in both L. innocua ferritin and bacterioferritin 11291. In contrast, iron turnover occurs at the ferroxidase centers of animal ferritins, whore 2 0 2 is produced [122,124]. Inertness of bacterial ferritins appears to be characteristic of high-valent dinuclear iron centers in enzymes such as ~~0~ that completely reduce Qz during their catalytic cycles. The stability of the diferric reaction centers in MMOH and RNR contrasts with the instability of diferric centers in ferritin, which are precursors o f the femic mineral L191. The observation of'the GlnXXAsp site in the frog M ferritin accounts for both the instability of the diferric oxy complexes in ferritin compared with MMOH and RNR and the observed kinetic variability of' the peroxodiferric species in different ferritin sequences. The reason for this difference from a biological point of view lies in the different function between ferritins and 02-activating enzymes. In the first case, iron ions bind to the protein transiently as substrates and must vacate the site upon its oxidation. In 02-activating enzymes, iron ions are essential for cycles of oxygen activation and substrate turnover, which requires the iron ions to be tightly bound (191.A structural reason for this difference lies in the missing His in the second iron binding site 11301. 4.2.4. Iron Release

Localized unfolding in assembled ferritin, at sites of cooperative subunit interactions, can increase the rate of release of iron from ferritin. When conserved Leu134 is replaced by Pro, the protein assembles, oxidizes Fe2+, and mineralizes Fe3.', but the time for complete dissolution of the mineral in vitro is greatly decreased E311. X-ray diffiaction studies of crystals of H-chain Leul34Pro ferritb showed a flexible region localized near the termini of two subunit helices ( C , D), which form the interfaces of subunit trimers and a channel. The results indicate that iron can exit from ferritin at the trimer subunit junction. A possible mechanism for regulated iron release in viva could be a localized disorder in the assembled protein, enhanced by

590

ARNESANOAN~PROV~N~ANI

cytoplasmic changes with effects analogous to the effect of the H-chain Leul34Pro mutant r3I.1. 4.3. Transferrins: Structure-Function Relationships 4.3.1. Biological Function

Serum transferrins and lactoferrins deal with different aspects of the metabolism of iron. Serum transferrins transport iron from the bloodstream to the cytosol via receptor-mediated endocytosis and lose their iron in mildly acidic media [131,132]. In contrast, lactoferrins sequester iron from biological fluids and do not lose it in mildly acidic media 11331. This is assumed to be the result of the higher affinity of lactoferrin for iron compared with that of serum transferrin. The high affinity of lactoferrin for iron is also assumed to be responsible for its diff'erent biological function [1331. 4.3.2. Iron Uptake and Release

In vitro, iron uptake by serum transferrin and lactoferrin occurs by similar mechanisms, which involve exchange of Fe3+between a chelate and the C lobe of the proteins in interaction with the synergistic anion. This first step is followed by a series of proton dissociations, probably triggering changes in the conformation of the protein which affect the N site and allowing the capture of a second iron, if available. The last step is a very slow conformational change of the Globe monoferric, if iron is not available, or of the Globe and N-lobe diferric proteins to achieve their final state of equilibrium E1341. The occurrence of such monoferric forms along with the closed (diferric) and open (ape) forms may be related to the observed modes of transferrin receptor interactions. Several lines of evidence have demonstrated that the diferric form is essential for the full binding of transferrin to the receptor [46-48,76,77], indicating that the closed conformation is that recognized by the receptor 11351. Cooperativity for iron binding to the N and C lobes, whether positive or negative, has been detected by iron stability analyses for the two sites of human serum transferrin 11361 and those of ovotransferrin 1137,1381.The iron binding stability of lactoferrin is due primarily to the C lobe, which functions cooperatively to stabilize iron binding in the N lobe, as found in lactoferrin mutants that selectively lack the iron binding function in either lobe [139]. The cooperative interactions upon iron release at acidic pH are modulated by binding to the receptor 11401. Therefore, the cooperativity between the two iron sites has an important functional role in the uptake and release of iron. The sequence of steps leading to iron release is not well understood, although kinetic studies point t o several key protonation events 11321, and biophysical studies 1141,1421show that iron release is associated with a major conformational change that appears to be broadly similar in all transferrins. The crystal structure of the iron-


591

bound N lobe of human transferrin has identified protonation and dissociation of the synergistically bound anion as a likely first step in iron release 1751, consistent with the kinetic studies "21. A trigger mechanism, involving protonation of a pair of lysine residues from opposing domains of the N lobe, has also been suggested from the crystal structure of the N-lobe half-molecule of ovotransferrin 1731. Crystals of the protein were grown at pH 5.9, which is well below the usual pK, of approximately 10 for a lysine side chain. It has been suggested that the pKa value of one or both of these residues lies below the pH of the structure determination and that therefore the lysine side chain is not positively charged f731. Uptake of the Fe3+-transferrin complex into an acidic endosome (where the pH is approximately 5.0) via receptormediated endocytosis results in the protonation of both lysine residues, determining the disruption of the hydrogen bond and, consequently, the opening of the N lobe "731. The detailed nature of the conformational change in transferrins has been defined in the crystal structure of apolactoferrin [781, where a domain rotation of 54" gives rise to an open N-lobe binding cleft; this depends on a hinge movement in two antiparallel polypeptide strands that pass behind the iron site, which allows one domain to move as a rigid body relative to the other [1431. There are, however, distinct structural and functional differences between lactoferrin and transferrin, in particular the fact that transferrin releases iron from its N lobe at a distinctly higher pH than lactoferrin (5.5, compared with 3.0). Amino acid substitutions may also influence the conformational change 11441. 4.3.3. Iron Binding Sites

The two iron binding sites are located within the interdomain cleft of each lobe. Crystal structures of the diferric forms [58,60-62,64,711 and the monoferric M lobes [72-75] of several transferrins reveal that the two domains are closed over an Fe3' ion. Four of the six FeS+coordination sites are occupied by the protein ligands of two tyrosine residues, one aspartic acid residue, and one histidine residue (Asp60, Tyr92, Tyrl91, and His250 in ovotransferrin N lobe), and the other two by a synergistic anion, namely, the physiologically available bidentate carbonate [58,60,61,64,72751, However, for the iron-free apo form, all of the transferrin lobes, except for the lactoferrin C lobe in the crystal, assume a conformation with an opening of the interdamain cleft. This implies that transferrin initially binds Fe3+ in the open form before being transformed into the closed holo form. Extensive studies in vitro indicate that iron release from transferrin is very complex and involves many factors, including intralobe interactions, receptor binding, pH, and an anion effect. Differential domain and hinge locations of the four protein ligands (Asp in the domain I, one Tyr in the domain 2, and a second Tyr and His in different binges) [58,60,61,64,72-751 inevitably require an alternative Fe3+ coordination geometry for the Fe"-loaded, domain-opened intermediate. In the Fes+-loaded, domain-opened structure of the ovotransferrin N lobe, the bound iron atom is coordinated by the two Tyr ligands. Other Fe3+ coordination sites are occupied by nitrilotriacetate,

592

SANO AND P R O V ~ N Z A N ~

which is stabilized through hydrogen bonds with protein groups [791. This observation suggests that the two tyrosine residues are the initial Fe3+-bindingligands in the open transferrin. The two tyrosine ligands of the human serum transferrin N lobe have been demonstrated to be inequivalent by means of site-directed mutagenesis, metal release kinetics, and absorption and electron paramagnetic resonance (EPR) spectroscopies. The mutant Tyr95Phe showed a weak binding affinity for iron, whereas mutant Tyr188Phe completely lost the ability to bind iron. Other studies have demonstrated that mutations of the other two Iigands, histidine and aspartate, did not completely abolish iron binding [1451. The structure of the AspGOSer mutant deviates in two important aspects from the parent lactoferrin (N lobe) molecule. At the mutation site the Ser side chain neither binds to the iron atom nor makes any interdomain contact as the substituted Asp does; instead a water molecule fills the iron coordination site and participatcs in interdomain hydrogen bonding [SSl. The release of iron at low pW involves the protonation of the histidine ligand. The ~ i s 2 4 ~ mutation ~lu in the N lobe of human transferrin determines the metal ligand substitution from a neutral histidine residue to a negative glutamate residue and the disruption of the dilysine trigger. The loss of the dilysine interaction does make the protein more acid-stable, but this is counterbalanced by the replacement of a neutral ligand (His) by a negatively charged one (Glu), thus disrupting the electroneutrality of the binding site [146,1471. As a result, the mutant releases iron slightly faster than the native protein. Two alternative conformations of the synergistically bound carbonate anion have been found in the crystal of the N lobe of human transferrin. Arg124 moves to accommodate the different carbonate positions and several water molecules appear to stabilize the carbonate anion. These structures are consistent with the protonation of the carbonate and the resulting partial removal of the anion from the metal; these events would occur prior to cleft opening and iron release [751. Some studies highlighted the importance of nonsynergistic anion sites in carbonate and iron release. From the electron density map of the N lobe of apoovotransferrin, the existence of four bound sulfate anions was detected 185I. Three of them are located in the opened interdomain cleft. They directly interact with an Fe3+-coordinating ligand, i.e., forming hydrogen bonds with Nz2 of His250, or indirectly through HaO molecules with EunctionaUy important protein groups, such as the other Fe3+coordinating ligands, Tyr92-OH and ‘X’yr191-OH, and a dilysine trigger group, Lys209 NC. Moreover, one sulfate anion binds Arg121 NE and Nq2, the consensus anchor groups for the synergistic carbonate [851. Analogous results were found in human transferrin bound to chloride anions [148]. These data are consistent with the view that the anions have a positive effect in opening the holo and stabilizing the apo conformation. Lactoferrin binds two FeSt and two carbonate ions with high affinity. It can also bind other metal ions and anions. In order to determine the perturbations in the environments of the binding sites in the N and C lobes and elsewhere in the protein, the crystal structure of oxalate-substituted diferric mare lactoferrin has been deter-

IRON STORAGE AND TRA~SPORTPROTEINS

593

mined E1491. The substitution of an oxalate anion does not pertwb the overall structure of the protein but produces several significant changes at the metal binding and anion binding sites 11491. The structural consequences of binding a metal other than Fe3+ have been examined by crystallographic analysis of mare samarium-lactoferrin [150],The samarium geometry (distorted octahedral coordination) is similar in both lobes. However, the anion interactions are quite different in the two lobes and a smaller number of hydrogen bonds is found in comparison with iron-lactoferrin. The binding of Sm3+ by lactoferrin shows that the protein is capable of sequestering ions of different sizes and charges, though with reduced affinity [150],Many other studies (nuclear magnetic resonance, circular dichroism, ultraviolet) focused on metal-substituted transferrins with Go(III), Co(II), Cu(II), Al(III), Tl(III), V(III), VOz, lanthanides, and other metal ions, indicating that metal-induced conformational heterogeneity can affect the transferrin-receptor recognition process, resulting in a different metabolic fate of these metals in the organisms [151-1581.

5. PE

CTlV

During the past few years, the identification of novel genes and the isolation of novel proteins in iron regulation pathways has dramatically increased our understanding in mammalian iron homeostasis. Many questions can be posed regarding how these novel iron transporters are regulated and how iron absorption is regulated by the HFE-transferrin pathway. Identification of the molecular interactions of HFE with DMT-1 and other key components of the iron transport pathway has implications for a mechanistic understanding of the pathophysiology of human iron storage diseases, like hereditary hemochromatosis, as well as the regulation of normal iron balance. Treatment of disturbances of metal ion homeostasis often involves supplementation of the missing metal ion, or metal ion chelation. Now an alternative strategy of medical treatment could be thought of by directly acting on these proteins. Nevertheless, many important issues need to be clarified both in the general overview of iron homeostasis and in the intimate molecular mechanism of the agents involved. The similarity in iron uptake by serum transferrin and lactoferrin is very striking and cannot alone explain the differences in the metabolic behavior of the two transferrins. These differences may be the result of a series of dynamic events induced by the slight differences in the intimate structure of the proteins.

The authors thank -of.

Ivano Bertini for helpful discussions and suggestions.

594

ABBREVIATIONS AND DEFINITIONS Bfd BHK DMT-1 DPS hFBP HFE IMP IREGl MMOW Mr MTf Nrarnp2 RNR

sco

Tfr TRIP XAS

bacterioferritin-associated ferredoxin baby hamster kidney cells divalent metal transporter-1 DNA-protecting protein Haemophilus influenzae ferric ion-binding protein hemochromatosis protein p-integrin and mobilferrin pathway iron regulator transporter methane monooxygenase hydroxylase relative molecular mass rnembrane-bound transferrin-like protein (melanotransferrin) natural resistance-associated macrophage protein 2 ribonucleotide reductase structure classification of proteins (http://scop.life.nthu.edu.tw) transferrin receptor transferrln receptor-independent pathway X-ray absorption spectroscopy

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onnanni, and J. Hirose, Biometals, 10, 303313 (1997). 159. €3. Gallois, €3. 2. d'Estaintot, M. A. Michaux, A. Dautant, T. Granier, 6. Precigoux, J. A. Soruco, F. Roland, 0. ChavasAlba, A. Herbas, and R. R. Crichton, J. Biol. Inorg. Chem., 2, 360-367 (1997). 160. T. Granier, B. Gallois, A. Dautant, B. L. d'Estaintot, and G. Precigoux, Acta Crystallogr. D Bid. Crystallogr., 53, 580-587 (1997). 158.

602

ARNESANO AN

161. G. Precigoux, J. Yariv, B. Gallois, A. Dautant, C. Courseille, and B. L. d'Estaintot, Acta Crystallogr. D €301. Gystallogr., 50, 739-743 (1994). 162. J. Trickha, E. C. Theil, and N. M. Allewell, J. Mol. Riol., 248, 949-967 (1995). 163. C. A. Smith, B. F. Anderson, €3. M. Baker, and E. N. Baker, Acta Crystallogr. D Bid. Crystallogr., 50, 302-316 (1994). 164. 6. E. Norris, B. F. Anderson, and E. N. Baker, Acta CrystaElogr. B Struct. Science, 47, 998-1004 (1991). 165. H. Nicholson, B. F. Anderson, T. Bland, J. W. Tweedie, S. C. Shewry, and E. N. Baker, Biochemistry, 36, 341-346 (1997). 166. 6. A. Smith, B. F. Anderson, H. M. Baker, and E. N. Baker, Biochemistry, 31) 4527-4533 (1992). 167. H. M. Baker, B. F. Anderson, A. M.Brodie, M. S. Shogwe, C . A. Smith, and E. N. Baker, Biochemistry, 35, 9007-9013 (1996). 168. W. R. Faber, C. J. Baker, C. L. Day, J. W. Tweedie, and E. N. Baker, Biochemistry, 35, 14473-14479 (1996). 169. Y. Bourne, J. Mazurier, D. Legrand, P. Rouge, J. Montreuil, G. Spik, and C. Cambillau, Structure, 2, 209-219 (1994). 170. A. K. Sharma, M. Paramisavan, A. Srinivasan, and T. P. Singh, J. Mol. Biol., 289, 303-317 (1999). 171. A. K. Sharma, K. R. Rajashankar, M. P. Yadav, and T. P. Singh, Acta Crystallogr. D Biol. Crystallogr., 55, 1152-1157 (1999). 172. A. Rawas, K. Moreton, H. Muirhead, and J. Williams, J. Mol. Bid., 208, 213-214 (1989).

1t i

ts hn

S*

tt

Department of Chemistry Imperial College of Science, Technology and Medicine South Kensington, London SW7 2AY, UK

1. INTRODUCTION 1.1. Outline of Dates, Structures and Reactions 1.2. Aims, Scope, and Organization of the Review 1.3. Nomenclature of Corrinoids 1.4. Basic Coordination Chemistry: Oxidation States, cis

and trans Effects 1.5. Why Go? 1.6. Why Corrin? 1.6.1. COO)and Co-H Complexes 1.6.2. Photophysical and Related Properties of the Conjugated Chain 1.6.3. Steric Effects of the Exocyclic Ring 1.6.4. h i d e and Nucleotide Side Chains

605 605 610 610 611 613 615 616 616 617 618

2. ENZYNIES WITH KNOWN STRUCTURE: Biz-DEPENDENT

MUTASES 2.1. Reactions Catalysed by Mutases (Isomerases) 2.1.1. C-Skeleton Mutases 2.1.2. Eliminases: Diol Dchydratase and Ethanolamine Ammonia Lyase 2.1.3. Aminornutases 2.1.4. Ribon~cleo~ide (Ribonueleoside Triphosphate) Reductases 'Possible non-corrinoid Co-containing cnzymes arc discussed in Chapter 23, Section 3.6.

603

618 619 619 62 1 621 621

PRAIT

604

Available Structures of Mutases Major Structural Features: Domains, Modular Construction, and Evolution 2.4. Structural Features Relevant to the Mutase Reaction Mechanism. 2.4.1. Comparison of Protein-Free and Protein-Bound AdoCbl 2.4.2. Nature of the Substrate-Induced Conformation Change and Its Role in Co-C Bond Fission 2.4.3. Evidence Relating t o Formation and Rearrangement of the Substrate Radical 2.5. Summary of Main Points 2.2. 2.3,

~Z-BIND~NG E N ~ E S / P R O T E I N SVVITH U N ~ O ~ STRUCTURES 3.1. Distribution and Diosynthesis 3.2. Absorption, Transport, and Transformation of Co Corrinoids 3.3. BIZ"Dependent Methyltransferases 3.4. Other Known or Possible B12-DependentEnzymatic Reactions 3.4.1. Other Methyl Transfers 3.4.2. Reductive Dehalogenation 3.4.3. Reduction of Epoxyqueuosine to Queuosine 3.4.4. Anaerobic Methane Oxidation

4. 4.1.

4.2. 4.3.

4.4.

S, M E C ~ N I S M SAND S T R ~ C T U R E - F U ~ C T I O ~ ONSHIPS OF TJ3E MUTASES Fission of the Co-C Bond: Mechanism for Applying Steric Distortion 4.1.1. Proposed Mechanisms 4.1.2. Studies on Protein-Free Corrinoids 4.1.3. Co-C Bond Labilization on Binding the Substrate (Stage 11) 4.1.4. Co-C Bond Labilization by the Protein Alone (Stage I) The Co-Radical Charge Transfer Complex Rearrangement of the Substrate-Derived Radical 4.3.1. Glutamate Mutase: Migration of the sp3 C Atom 4.3.2. Other Mutases: Migration of the sp2 C Atom Summary of Main Points

5. P ~ R S P E C T ~ ~ S 5,1. Blz-dependent Mutases: Precis of Present Results 5.1.1. Wow Does the Protein Promote Womolytic Fission of the Co-C Bond? 5.1.2. How Do the Substrate-Derived Radicals Undergo Rearrangement? 5.1.3. Why Go? M y Does Co(I1I) Form Such Stable Co-C and CO-HBonds?

622 623 627 627 628 630 630 631 63 1 632 634 636 636 636 637 637 637 638 638 640 641 645 646 647 648 651 6F2 653 654 654 655

655

COBALT IN VITAMIN 812 AND ITS ENZYMES

5.1.4. Why Corrin? 5.1.5. Why Ado as the Ligand? 5.1.6. Lessons Learned 5.2. B12 in the mote Past: Insights into Evolution Future: Problems and Prospects

605

656 656 656 65 7 659 660 660

REFERENCES

662

The Co eorrinoids* (derivatives of vitamin Biz) probably provide the best opportunity among metalloenzymes to seek answers to the following sequence of questions: (1)Why that particular metal? (2) Why those particular ligands and what is the nature of the metal-ligand interactions? (3) How does the protein manipulate those interactions l o modify and control the enzymatic reactions? The underlying theme of this chapter is to provide a progress report to answer such questions with reference to the best studied (mutase) family of Blz-dependent enzymes. Structural data on B12dependent enzymes have only become available since 1994 but have already (as from 1999) provided a uniquely detailed insight into the protein machinery which uses a conformation change to link substrate binding with changes to the active site. Structural and mechanistic studies on the B12-dependentmd other ancient enzymes offer a tantalizing glimpse into the evolution and mechanism of enzymes before the appearance of oxygen and even below the base of the evolutionary tree as we now know it. The year 1999 also saw publication of the multiauthor Chemistry and Biochemistry of B12 [ll as successor to the earlier (1982) B12 [ZI in providing an up-to-date reference to much of the field,

utline of Dates, Structures, and Vitamin 1312 or cyanocobalamin (CN-Cbl) is the only known metal-containing vitamin and the only vitamin (for humans) that is synthesized solely by microorganisms. It was isolated in the form of dark red crystals by research groups at both Merck

* Abbreviations used for the corrinoids include Cbi Icobinamide), Cbl (cobdarnin), and DMB (5,6-dime~ylbenziminazole),which are more fully described in See. 1.3 on page 610, while those used for the corrinoid-dependent mutases include DiolD (diol dehydratase), EAZ, (ethanolamine ammonia lyase), GluM (glutamate mutase), IvIMCM (methylmalonyl-CoAmutase), and RNR (ribonucleotide reductase), which are more l l l y described in Sec. 2.1 on page 619; note also the methyltransferase MetS (methionine synthase).

606

PRATT

(Folkers) and Glaxo (Smith and Parker) in 1948 and by three other groups shortly thereafter 131. X-ray studies by Dorothy Hodgkin’s group in Oxford revealed a novel structure for the inner corrtn ring, far more complex than the porphyrin ring, in 1964 and the structure of the whole B12 molecule in 1957 [4].The molecular structure is shown in 1”ig.l;for the atomic positions, see Sec. 1.6 and Fig. 3 below. It is a diamagnetic, six-coordinate complex of the d6 Co(II1) ion with the formidable empirical forN , 4cyanide P G 0 ligand . is an artifact resulting from the use of mula C ~ ~ ~ ~ ~ ~ ~ 4 The cyanide-containing solvents in isolation and purification. In 1958, Barker’s group at Berkeley isolated a very light-sensitive derivative of BI2 as the true coenzyme for an unusual mutase or isomerase that catalyzes the reversible C-skeleton rearrangement of glutamate 1 to 3-Me-aspartate anaerobic CEostridium;the reaction involves migration of the sp“-hybridized C atom [51. In 1961, Lenhert and Hodgkin showed that it possessed the extraordinary 5’deoxyadenosyl (Ado) ligand shown in Fig. 2 with a unique G o 4 bond 161.

FIG. 1. Molecular structure o f the six-coordinate Co(ll1) vitamin BIZor cyanocobalamin (CNChl) with rings A-D, side chains a-g and ring carbon atoms 1-19 numbered. The positive charges on the Co(1II) ion are balanced by negative chargcs on the cyanide, the corrin ring, and the phosphate ester in side chain f.

COBALT IN VITAMIN

BIZ

AND ITS ENZYMES

607

Evidence for the involvement of a B12 derivative with a Co-Me bond in Me transfer from N-Me-tetrahydrofolic acid 3 to homocysteine t o form methionine 4 was reported in 1962 [7]. This is one of thc two enzymatic reactions for which BIZ is required by man and other mammals as a vitamin from external sources; the other is the C-skeleton rearrangement of the Me-malonyl 6 to the succinyl 6 derivative of CoA (denoted by RSH). Me and Ado are the ligands normally observed in vivo; their role is explained below. For several reasons (see Sec. 1.4) they can best be described as carbanions coordinated to the Co(II1) ion, although they will also be written here as Me-Co, Co-Me, etc., without indicating the oxidation state of the metal or ligand. The foundations of both the chemistry and biochemistry of B12 were really laid in the period 1957-1962 and the demonstration that the naturally-occurring coenzyme (AdoCbl) possessed a thermally stable (but very light-sensitive) Co-6 bond, challenging the prevailing dogma that such metal-alkyl bonds were inherently unstable (e.g., toward water), triggered a dramatic development of the coordination and organometallic chemistry of the Co corrinoids (derivatives of BIZ),as outlined in the author’s book [Sl. The year 1961 can be taken as the starting point for the bioinorganic chemistry of Blz. This was given a clearer sense of direction in the 1970s as evidence mounted that the Co-C bond in AdoCbl underwent reversible homolytic fission to generate a free radical during enzymatic activity; see Babior’s 1975 review 191. The first structures of B12-containingproteins to be determined were those of the inactive MeCbl-containing fragment of rnethionine synthase (MetS), reported by Martha Ludwig’s group at Ann Arbor in 1994 [lo], and of a mutase, reported by Evans’s group at Cambridge in 1996 [lll. This was followed by the structures of several other forms of the same mutase and of two other mutases, which have revealed the protein machinery operating through a significant conformation change (see Secs. 2.3-2.4). Their often very low cellular concentrations (which may be below 10 molecules per cell, see Sec. 3.2) has meant that X-ray structure determinations had to wait over 30 years for the introduction of molecular biology to provide adequate amounts of material for crystal growing and also for improved spectroscopic data and

FIG. 2. Structure of the 5’-deoxyadenosyl (Ado) ligand coordinated to the Co(1II) ion in the coenzyme forms such as AdoCbl.

PRATT

608

kinetics. However, the increasing interest in the very primitive anaerobic bacteria, which form the B12 “heartland” and may contain high concentrations of BI2 (up to 0.7 mM; see Secs. 3.1 and 3.31, is rapidly adding to our knowledge of B12-dependent enzymes. The combination of molecular biology and interest in anaerobic bacteria could well promote a new boom in B12 studies.

i““

HOOC.CH2.CH2.CHNH2.COOH

HO0C.CH CHNH2.COOH 2

1

Me.S.CH2.CH,.CH”,.COOH 4

HOOC.CH.CO.SR (COA) 5

HOOC.CH2.CHpCO.SR(COA) 6

There are two main families of BIz-dependent enzymes, both of which depend on the making and breaking of a Go-C bond:

(A) The mutases or isomerases (see Table 1 in Sec. 2.1), in which the coenzyme possesses an Ado ligand, catalyze a rearrangement of the substrate, e.g., the reversible interconversion of the pairs 1-2 and 5-6. The initial step involves reversible homolytic fission of the Co-C bond to form Go(I1) and the (protein-bound) Ado free radical according to the simplified equation (l), followed by abstraction of an H atom Co-Ado + Go(II) + Ado’

(1)

from the substrate to form the substrate radical Co-C fission only occurs, however, on binding of a substrate or substrate analogue (denoted here by HZ), which triggers a significant eonformational change and applies steric strain to break the Co-C bond (see Secs. 2.4 and 4.1). This produces the substrate-derived radical, with no evidence (e.g.,from ESR) for the Ado radical, and leaves the radical C atom close enough to the Co(I1) ion to form a nonbonded “charge-transfer complex”, characterized by a distinctive ESR “signature” (see See. 4.2). The simplified equilibrium (1)should therefore be expanded to (21, where P* and P denote the two protein conformations and square brackets enclose the protein and any protein-bound species.

ITS ENZYMES

609

[P*.Co-Ado] HZ + [P.CO(II).Z.A~OH]

(2)

12 AND

+

The substrate radical (Z) then rearranges by a 1,2 shift of a C atom (tetrahedral in 12 and trigonal in S 4 > ,an N or 0 atom under the influence ofthe Co(I1) ion (see Sec. 4.3). The rearranged product is released aRer uptake of the H atom and reformation of the Co-C bond. The B12-dependentribonucleotide reductases, which combine rearrangement with reduction, are an atypical subgroup of the mutases (see Sec. 2.1). (B) The Me transferuses (see Sec. 3.3) catalyze the transfer of an Me+ cation from one nucleophile to another (eg., from 3 to the precursor of 1 via the cyclic formation of Co(1) and Co-Me intermediates according to equation (31, where B denotes a neutral (e.g., pterin) or anionic (e.g., thiolate) nucleophile. Although the Me transfer reactions appear simple on paper, the enzymes are complex and difficult to study. They usually consist of readily dissociated subunits or modules for the separate binding of donor, acceptor, and corrinoid and often an “activating” module (see Sec. 3.3). Co-Me + B ;;;t Co(1) + BMe+

(3)

The mutases have hitherto attracted the most widespread interest. They involve not only a Co-C bond, which was unexpectedly stable in an aqueous environment, but also (protein-bound) free radicals, which challenged the then-prevailing dogma that enz-ymesavoided free-radical intermediates, and C-skeleton rearrangements, which at that time had no precedent in organic chemistry. The problem for the enzyme is to produce the very reactive Ado radical (the true catalyst) from the stable AdoCbl by causing a massive change in the equilibrium constant for Co-C bond fission from log R = -18 in equation (1) to logK = 0 i 1 in (2). The Me transferases are also of considerable general interest because the Co(1) corrinoids are both some of the most active nucleophiles known in chemistry (so-called supernucleophiles) and some of the strongest reductants known in biochemistry; the midpoint potentials may be more than 100 mV more negative than the usual physiological limit of about -500 mV 1121. As far as is known, the C-skeleton rearrangements remain the unique preserve of BIZ-dependent enzymes. However B12-independent enzymes are known that possess 8-adenosylmethionine (AdoMet) as cofactor; they generate the Ado radical by equation (4)and can also act both as a ribonucleotide reductase and an aminomutase (see Sec. 5.2).Many Me transferases are known with no obvious cofactor apart from a Zn ion, including a BIZ-independent MetS 1131. AdoMet+ + e- +=+ Ado ’

+ Met

(4)

Other Blrdependent enzymes, including the recently discovered family of reductive dehalogenases, which catalyze, say, the reduction of tetrachloroethene to cis-1,2-dichloroethene and even ethene and are attracting interest in the field of bioremediation, as well as possible BIZ-dependent enzymes, which may be involved in the anaerobic “oxidation” of methane, are mentioned in See. 3.4. The ultimate source of B1, required as a vitamin is the limited range of micro-

610

PRATT

organisms that have retained the genes for biosynthesis (see Sec. 3.1). Other BIZbinding proteins are involved in the absorption (e.g., from the gut), transport, and conversion of Co corrinoids into the coenzyme forms by reduction and alkylation (see Sec. 3.2).

1.2. Aims, Scope, and Organization of the Review The occurrence of a limited range of Co corrinoids (with a rare metal and complex equatorial ligand) and of enzymatic reactions (with a Go-C bond as common denominator) has provided a useful focus for the B12 field and poses a small number of simple questions of broad relevance. Co is a rare metal in the environment, so why the special need for Co? The biosynthesis of corrin shares initial steps with that of porphyrin (see See. 3.1); Co porphyrins have been found in prokaryotes and one enzymatic reaction (i.e. decarbonylation of aldehydes to alkanes) tentatively identified in an alga f141 and a mechanism proposed [151. So why the need for the much more complex corrin ligand? Since the Co-C bond is generally very stable in neutral solution in the dark, then how does the protein labilize these Co-C bonds according to equations (1)and (2), respectively? Each family poses additional questions, such as, in the case of the mutases, what is the mechanism of rearrangement and why the choice of Ado as ligand? Our knowledge and understanding of the mutases have advanced much more rapidly than that of the more complex Me transferases, rounded off by the recent structural determinations of the mutases, which have served both to clarify the role of the protein and to curtail speculation. No structure has yet been reported for any complete Me transferase. This chapter will therefore focus on the rnutases and address the above five questions involving why (Co, corrin and Ado) and how (Co-C bond fission, rearrangement of the substrate radical). The mutases and their reactions are discussed in Secs. 2 and 4,basic coordination chemistry in See. 1.4, the properties of Co in Sec. 1.5, and those of the corrin ring in Sec. 1.6. The Me transferases are treated more briefly in See. 3.3, other B12-dependentenzymes in Sec. 3.4, and the BIZbinding proteins involved in absorption, transport, and transformation in Sec. 3.2. Details of individual enzymes, biosynthesis, physical properties, and certain medical aspects can be found in the (1999) ‘Chemistry and Biochemistry ofB12’ 111, to which frequent reference will be made for hrther information. Trying to follow the Biz literature is made difficult, even for insiders, by the problems of nomenclature (see Sec. 1.3) as well as by the confusion and shadow boxing common to any area where rival theories compete.

1.3. Nomenclature of Corrinoids The Rules for Nomenclature, published by the IUPAC-IUB Commission on Biochemical Nomenclature in 1976 [lSl, are designed to accommodate the many

COBALT IN VITA MI^

B12

61 1

AND ITS ENZYMES

variations to the corrin structure and side chains produced by synthesis and degradation rather than variations in oxidation state and axial ligands of greater interest to bioinorganic chemists. Not surprisingly, the resulting names for corrinoids may be inconveniently long or even conflict with the rules for naming coordination compounds. Many trivial names and abbreviations remain in common use. All of the naturally occurring corrinoids (derivatives of BIZ) possess the same corrin ring and substituents except that they may differ in the presence or absence of the nucleotide on side chain f and, if present, in the nature of the base. Those that possess the same side chains as in 12 itself (officially vitamin i n ~ o Z e DMB), and differ only in the nature the base 5 , 6 - d i m e ~ ~ ~ ~ ~ e n z z m(unofficial of the axial ligands are termed cobalumins (Cbl’s), whereas the cobinamides (Cbi’s) lack the whole nucleotide. A variety of other nucleotide bases or “aglycons” are found among bacteria; they may be (1)derivatives of benziminazole, (2) purines (adenine, guanine, hypoxanthine, S-methyl- and 3-methylmercaptoadenine) or, strangely enough, (3)phenol or p-cresol 1171. The DMB base may be displaced from coordination by, say, protonation, substitution by stronger ligands such as cyanide or the presence of a ligand with a very strong trans effect in the transpoe protein (see Sec. 2.3). Cbl’s are (unofficially) termed “base-on” is coordinated and “base-off ’ when displaced. Corrinoids where the amide side chains have been converted to esters are (unoficially) termed cobesterti. The “upper” and “lower” or (officially) @ and (x coordination sites, which are occupied by cyanide and DMB, respectively in BIZ, are nonequivalent because the corrin ring is nonplanar. The Me and (official) Ado ligands occur o in the upper position in naturally occurring corrinoids, but axial isomers of corfinoids can also be prepared. The most common forms of the two coenzymes are MeCbl and AdoCbl. Aqua- and hydroxocobalamin can officially also be known as vitamins BrLaand Bl2bZb,respectively, although they are often both treated together as BIza (with pK 8). Reduction of BlZaproduces first cob(II)alamin, vitamin B12,. (both official), Bla, or Co(II)-Cbl(both unofficial) and then cob(Ijalamin, vitamin B12s (official), BIzs or Co(1)-Cbl (unofficial); so also, by analogy, cob(1I)inamide or Co(I1)-Cbi,etc.

-

tates, cis and trans Effects The oxidation states and coordination numbers of the Co corrinoids relevant to the enzymatic reactions are: iarnagnetic d6 Co(III), which may be either (a) reddish and six-coordinate, as in Q12 itself and the so-calledbase-on forms of MeCbl and AdoCbl[18], or (b) yellow and five-coordinate, as in the “base-ofl” forms of Me-Co and AdoCo corrinoids (e.g., where the nucleotide base is either missing or displaced from coordination by protonation) [191.

61 2

PRATT

2. Low-spin paramagnetic d7 Co(11) with one unpaired electron (hence ESRactive), which is normally (a) yellow and five-coordinate 1181 but may become (b) six-coordinate with weak coordination of, for example, pyridine [201. The transient formation of (c) an unstable four-coordinate Co(I1) has been detected on photolysing of the five-coordinate MeGbi 121I. 3. The very air-sensitive, diamagnetic d8 Co(1) is normally (a) gray-green, four-coordinate and base-off in the case of the Cbl[221, but (b) the unstable five-coordinate base-on form of BLZs,which has never been observed at room temperature, appears to be produced by the one-electron reduction of AdoCbl in frozen MeOH glass using y-irradiation 1231;the UV-Vis spectrum of the product is similar to that of the chemically produced BIZ8 but shows the expected bathochromic shift of the y-band (from 387 nm to > 400 nm). The CotI) becomes protonated with pK 5 1to give a Co(1II) hydride [24,251.

More highly oxidized and reduced species, such as Me-Co(n/-) and probably e-Co(II) corrinoids, may be obtained using electrochemical methods, among others. There is no evidence that they are involved in enzymatic reactions, but their likely occurrence may complicate the interpretation of, for example, reactions observed in the presence of metallic zinc. Co corrinoids can also form face-to-face dimers, which can open up additional mechanistic pathways (see Secs. 1.6.3 and 3.4.4).AdoCbl and other alkyl-Co corrinoids are very light-sensitive. This property delayed their earlier isolation but has also proved usefbl in providing a specific test for &-dependent Me transferases in vivo (inhibition by PrI, reversed by light) and a method for producing free radicals in the presence of Co(1I) (see Secs. 4.2 and 4.3). Co(I1I)complexes in general exhibit a pronounced trans efiect [261, i.e., changing one axial ligand affects the equilibrium and rate constants for ligand substitution in the opposite (trans) axial site. Replacing an axially coordinated H 2 0 in a Co(II1) corrinoid by Me, for example, reduces the binding constant for cyanide in the transposition by more than lo1* (a thermodynamic trans effect); furthermore, it reduces the binding constants for all Iigands in the transposition such that formation of the five-coordinate form becomes feasible. Conversely, the rates of Me transfer are affected by the nature (and presence or absence) of the ligand in the transposition (a kinetic trans effect). Reactions of the axial ligands may also be affected by a cis efiect, it?.,by changes to the neighboring (cis) equatorial ligand; rates of comparable ligand substitution reactions in the axial positions may be more than 10" times faster with corrin than with the saturated cyclic tetramine cyclam as the equatorial ligand (a kinetic cis effect). Co(II1) corrinoids are also very sensitive to the steric effects of increasing compression and distortion around the coordinated ligand atom (whether in amines, carbanions, or phosphite esters) which, by analogy, might be termed a thermodynamic f i n t eflect. The mutases and Me transferases share coenzymes that can be identical except for the choice of Ado or Me as the axial ligand and the use of homolytic or heterolytic

COBALT IN V I T A ~ I N812 AND ITS ENZYMES

613

Co-C cleavage as the reaction path. The Ado ligand is large enough for equilibrium (2)

to be readily controlled by the protein through manipulation of the steric front effect, while the Me ligand is too small to be subject to steric effects and equilibrium (3) appears t o be controlled through manipulation of the electronic trans effect and probably also the cis effect. Selection of the ligand almost automatically entails selection of the reaction path. Additional background information on the coordination chemistry of the Go corrinoids relevant to their enzymatic reactions is available in reviews on their redox potentials C27,281 organometallic chemistry 1291, and cis and trans effects [26].

1.5. Why Co? The reasons for nature’s choice of Co are now understood in its essentials, which are summarized here; for additional references and further discussion, see [261. For several reasons, complexes such as MeCbl, AdoCbl, and the simpler [Co(NH&CH$+ or [CO(CN),X]~, where X = H or Me, are best treated as complexes of the Co(II1) ion with a semi-covalent bond to a coordinated hydride or carbanion ligand; for example, the d-d spectra of the pentaammine and pentacyanides [30-33 I are similar to those of typical Co(II1) complexes, while the graded series of ligands used to study the trans effect and the spectra ofCo(II1) corrinoids (i-e., cyanide, ethinyl or acetylide, vinyl and methyl/ethyl) links methyl to cyanide with no obvious discontinuity 115,261. Corrinoids that probably possess a Co-H bond can be observed in, for example, glacial acetic acid and are formed by protonation of the Co(1) corrinoid with pK 5 1 [24,25] according to equation (5); this can bc written most simply as (5a) but also as (5b) to emphasize the analogy between protonation and methylation of Co(I) according to (3), where B can be HzO or as (6) bclow.

Co(1) + H+ + Co-H co-

2 0

* Co(1) f H 3 0 +

(54 (5b)

Comparing these Co(II1) corrinoids first with other Co(II1) complexes and then with complexes of other transition metal ions shows that the ability to form such Co-C and Co-H bonds is common to the Co(1II) ion and almost independent of the other ligands (see examples above), and that the Cr(II1) and Co(II1) ions together share a virtually unique ability among ions of the first transition series t o form M-H and M-C (alkyl) bonds that are reasonably stable in combination with ligands and redox potentials typical of‘a biological environment. Since the B12-dependentenzymes rely on the reversible formation of Go-C bonds, the question “why GO?” can be rephrased as “how and why can the Co(II1) and Cr(1II) ions form such stable M-C (also M-H) bonds?” This can only be answered by discussing the role of the outer 4s orbital, Recent work on the simple binary MH molecules and MH’ ions in the gas phase [34,351 reveals an excellent linear correlation for the first-row metals between increasing M-H“ bond dissociation energies (BDEs) to give Mf and the H atom

PRATT

614

and decreasing d-s promotion energies (i-e.,from the 3d to the 4s atomic orbital). This confirms that the formation of a strong semi-covalent metal-ligand bond requires the use of the outer s and possibly even p orbitals (4s and 4p in Co). The linear correlation extrapolates to a M-H BDE of about 57 kcaVmol for zero promotion energy, which is regarded as the “intrinsic” M-€3bond energy both in the gas phase and in fully ligated complexes. The finding of a similar Co-H BDE of about 58 kcal/mol in the pentacyanide 1361 provides direct experimental evidence for extensive use of the 4s orbital in such Co-H bonds and, by extension, Co-C bonds. It can also be shown C261, that for six-coordinate low-spin complexes of the first transition series the d-s promotion energy is expected to be unusually low, and the M-H and M-C BDEs therefore unusually high, for reaction between a neutral H atom or methyl radical and both the d4 Cr(1I) and d7 Co(II) ions; this is equivalent to the formation of complexes of an anionic hydride or carbanion with the d3 Cr(1II) and d6 Co(II1)ions-where unusual stability is found experimentally. Since enzymatic activity requires access to lower oxidation states as in equations (1) and (Z),the difficulty of producing the highly reducing Cr(II), let alone Cr(I), in a biological environment leaves Co as nature’s choice for the widest range of‘organometallic chemistry in a metalloenzyme. In these Co-C(Me/ Ado) and Co-H bonds one can, to a first approximation, consider the 4s orbitals “overlap-active”,while the 3d are “overlap-inactive” and the 4p “overextended” [3Eil.

L.Co1*’RR-+ H30+ + L.Co%H2 L.C~IIIR-;=t- L.COII

L.Col”R-

-t-

+ RH

R-

+ 130- e L.Cd f ROH

There is now sufficient information available to test the stability of the Co-C bond in the six-coordinateMeCbl toward decomposition according to each of the three paths (6 - €9,where L = Cbl and R = Me. BDEs of 36 rt 3 1371,36 f 4 [381 and 37 k 3 L391 kcal/mol have been determined for reaction (7). Sauer and Thauer [40] used a B12-dependent Me transferase to study the reversible methylation o f Co(1)-Cbl by to determine the free energy of hydrolysis at pH 7, from which one can 61 a value o f logK = + 5.8 for reaction (8). This value can then be combined with the redox potentials of the Co(lII)/Co(I) and MeOWmethane couples to calculate approximate values of logK = -27 45 and about 4-24 for reactions (6a) and (6b), respectively r261. MeCb1 is, therefore, thermodynamically very stable toward hornolytic fission (71, stable toward hydrolysis by hydroxide (8) only below and unstable toward protonation to give methane (6) at any pH--yet the mains kinetically stable (in the dark) even in concentrated sulfuric acid! represents the first case where we can explain nature’s choice of metal and trace this back to the electronic structure of &helow-spin Co(1II) ion. The most i ~ p o r t apiece ~ t of evidence has heen the semiquantitative measure ol4s involvement obtained from gas phase studies on simple MH+ ions in a field far rem The data relating to reactions (6)-(8), together with additional Co-C

ALT IN VITAMIN €332 AND ITS ENZYMES

615

Ado and other alkyl ligands (see lag]) and certain other equilibrium constants 1261, provide a collection of equilibrium data involving hydride, methyl, and other carbanions, which is unique among transition metals.

I .6. Why Corrin? The molecular structure of the parent BIZ was shown in Fig.1 and the atomic positions of a corrinoid that lacks the nucleotide side chain are shown in Fig.3. What can the corrin ligand offer, relevant to enzymatic reactions, that a porphyrin ligand cannot? Although the overall corrin structure is roughly spherical, it is best considered as four concentric rings radiating out from (a) the central Co ion with its axial ligands through (b) the conjugated chain, which interacts strongly with the Go ion, to (c) the saturated and hydrophobic C-C and C-H bonds of the exocyclic ring and (d) the terminal hydrophilic amide groups, together with the nucleotide attached to side chain f. Comments can be made under each heading. Structures have been determined for more than 50 protein-free corrinoids, natural and synthetic, mostly with Co, some with other metals, one metal-free. Reviews with comparisons of the observed variations in, for example, the buckling of the corrin ring and the orientations of the side chains in all of these compounds are available [18,42,431. The main features of their UV-Vis spectra have recently been reviewed and some of the factors that determine their distinctive properties and variability (cf. Fig. 4 below) discussed [15L Like the metalloporphyrins, the Go corrinoids are highly colored and exhibit intense UV-Vis spectra due to TC-TC transitions within the conjugated ring. This has made W-Vis spectrophotometry the work horse for studying the corrinoids. The spectra also open a window into the electronic machine room of the corrin (and porphyrin) ligand and, together with the subsequcnt emission spectra and photochemical reactions, may shed some light on

FIG. 3. Projection of the atomic positions in the so-called cobyric acid, where side chain f terminates in a carboxylate and the upper tfi) and lower (a)ligands are water and cyanide, respectively, viewed roughly along the C-ii/C-16 axis. The downward-projecting Me group on C1 is circled for clarity. (Reprinted with permission from D. C. Hodgkin, “Tho structure of the corrin nucleus from X-ray analysis”, Proc. Roy. Soc., A288, pp. 294-305 (1965), figure 3@), published by the Royal Society.)

616

PRATT

the workings of this machinery and possible differences between the corrin and porphyrin ligands. 1.6.1. Co(2) and Co-H Complexes

Me transfer via equation (3) requires that the highly reduced Co(1) state can be reached with biologically available reductants and is not then protonated in the physiological range to form a Co-H bond, since this would be equivalent to “poisoning” the catalyst. Conjugated macrocycles, such as corrin, porphyrin, and cobdoxime, appear to have a comparable ability to stabilize the Co(1) state, but there is indirect evidence that COO)porphyrins may be protonated in neutral solution 1151, whereas Co(1) corrinoids are only protonated below pH 2 124,251. 1A.2. Photophysical and Related Properties of the Conjugated Chain

The conjugated chain possesses 14 electrons spread over 13 atoms, i.e., it is a cyanine and not a polyene. The single bond between the tetrahedral C: atoms in rings A and D and the substituents on the exocyclic ring restrict the extent of conjugation and cause severe and variable buckling of the chain. The electronic transitions with their vibronic structure are seen clearly in the spectrum of the dicyanide at low temperature (Fig. 4).The spectra are surprisingly insensitive to steric changes in the conjugated ring, but sensitive to changes in the ring substituents and very sensitive to changes in the axial ligmds (Fig. 4). The first three electronic transitions in order of increasing energy (decreasing wavelength) are designated the ap bands (500-600 nrn), the DE bands (low bands about 400 nm), and the y band (usually an intense band in the region of 350-380 nm); the y band of the dicyanide has a molar absorbance of 3 x lo4. The absorption and fluorescence spectra of both synthetic and naturally occurring

FIG. 4. Comparison of the W-Vis spectra of the six-coordinate Co(II1) dicyanocobinamide (-) and rnethylcobalarnin (---) in ethanol at -180°C to illustrate their vibronic structure and sensitivity to changes in the axial ligands. (Reprinted with permission from R. A, Firth, H. A. 0. Hill, J. M. Pratt, R. J. P. Williams, and W. R. Jackson, “The circular dichroiein and absorption spectra of some vitamin B12 derivatives”, Biochemistry, 6, pp. 2178-2189. Copyright (1967) American Chemical Society.)

ALT IN ~ I ~ A Bj2 ~ AND I N ITS ENZYMES

617

corrinoids, with and without a metal, and the photochemistry of MeCbl all reveal several unusual features [lSl: (1) Dual fluorescence (Le., emission not involving a triplet state) has been observed with a synthetic corrin (where the only ring substituent is CN on C-151, both with and without a metal, and from the naturally occurring metal-free corrins (the naturally occurring Co corrinoids do not fluoresce). This can be explained in terms of an adiabatic photoreaction, which involves a radiationless transition or equilibration between the initial photoexcited singlet state and a second excited singlet state, both of which fluoresce and show the same excitation spectrum. (2) The photolysis of MeCbl shows an anomalous wavelength dependence [451, which has been further studied using femtosecond time-resolved transient absorption spectroscopy L46 I. Excitation at 400 nm (but not at 520-530 nm) results in the prompt formation with rate constants @eater than 0.05 ps-l of two species with very different absorption spectra. One shows a spectrum typical of Co(1I) and must therefore be accompanied by a Me radical; the other exhibits an intense y band at 340 nm and may represent a Co(I11)complex with a weakly held methyl anion (perhaps as an ion pair). Since their rates of formation are much faster than those normally observed for any singlet-triplet intersystem crossing, they are probably both singlet species. (3) The absorption spectra reveal several features “intrinsic” to the corrin ligand, with or without a metal. These include well-defined vibronic progressions in at least the ap and DE bands (see Fig. 4) and a temperature-dependent ratio of intensities. (4) Comparable vibrational splittings are observed for both the a@ bands and the higher energy fluorescence and a different set of comparable splittings for the DE bands and the lower energy fluorescence.

None of these unusual features have been observed in metal porphyrjns. Both (I)and (2) provide fairly good evidence for the existence of a second excited singlet statc and, by extension, for a second (metastable) ground state. Items (3) and (4)provide further compatible evidence about the potential energy curve of the excited state and, in addition, suggest that the previously unexplained DE bands may be connected with some “mixing in” of the metastable state. Since the ground state structure and pattern of bond alternation in the polyenes is determined by the balance between the one-electron resonance interaction and coulomb repulsion terms, which favor equivalent and alternating bond lengths, respectively 1471, it seems likely that these factors are even more delicately balanccd in a strained and buckled cyanine such as corrin. This could make the corrin ring more polarizable and more open to manipulation by the protein (cis effect). 1.6.3. Steric Effects of the Exocyclic Ring

As Figs. 1and 3 show, the conjugated ring is asymmetrical and very buckled (contrast

the planar porphyrin ring) due to the C-C single bond linking rings A and D, substituents on the exocyclic ring, various repulsions involving both ring and exocyclic

618

PRATT

substituents and, in the case of the Cbl’s, between the coordinated DMB and the ring between 65 and C6. The type and degree of buckling can vary widely and folding along the CoiCiO axis is common [i8,42,431. The Go ion is surrounded in the upper (PI coordination site by four equally spaced hydrophobic “hills” or “sentinels” (i.e., the Me groups on C12 and C17 and the methylene groups of the acetamides on 6 2 and 67) with “valleys” bckween them and in the lower ( a ) coordination site by another four sentinels (i.e., the Me on 61 and the methylenes of the side chains on C3, C8 and C13), which are, however, not equally spaced. These groups provide an area of low dielectric constant around the active site and can also offer steric hindrance to incoming reagents (see Sec. 4.2) and to the lateral shear motion involved in the mutase reaction (Sec. 2.4.2) and can promote interaction or “docking” with other reagents. The symmetrical arrangement of the four hydrophobic groups on the upper (p) face allows the formation of a p-face-to-p-face dimer. Structures determined for two such dimers, i.e., an iodide-bridged dimer of a five-coordinate Co(1I) cobster f8,421 and the dimer of a six-coordinate ~ y l - C o ( ~ ~ ~ )with - C b al Co-(GHz)4-Co link [48l, show an interloc~ngP-to-b arrangement without voids, with the planes parallel and Co . . . Co distances of 5.65 A in the first structure and 6.95 A in the second. NMR shows that such dimem can be formed in aqueous solution even without a bridging ligand 1491. Such h e r s must be involved in the reversible Co-to-Co Me transfer, which has been well studied with protein-free corrinoids [SO-521 and may open up new mechanistic pathways [29] (see also See. 3.4.4). 1.6.4. Amide and Nuckotide

Side Chains

The side chains naturally play a major role in binding to the protein. The long nucleotide side chain allows the corrin to be firmly held from below, while the upper face remains free to interact with different catalytic and substrate binding domains in contrast to the Fe porphyrins (Sec. 2.3). In addikion, the nucleotide side chain of the Gbl’s appears to be used as a “molecular key” to unlock a conformation change for binding to the Cbl-specific intrinsic €actor (see Sec. 3.2). Other points for possible ma~pulationby the protein are discussed in 1151. There are clearly several significant differences between eorrin am d porphyrin. The most fundamental difference is the unusual electronic structure of the corrin ring (see Sec. 1.6.2). It would be strange if this difference was not exploited in, say, Me transfer reactions, but this remains to be established (See. 3.3).

2.

OWN ~ T R U C ~ U

Structures have now been determined for three mutases in about 10 different forms (see Sec. 2.2). Identifying the electron density corresponding to the Ado group both before and after Co-C bond fission, which is needed to “visudize” the mechanism of

COBALT IN VITAMIN B12 AND ITS ENZYMES

619

hond fission, was finally achieved in 1999 [531. The methyltransferases usually comprise separate subunits or modules for binding each of the two substrates and MeCbl, as well as for reactivating the enzyme after inadvertent oxidation to Co(I1) (see See. 3.3). Structures have been reported for the B12-bindingfragment and the AdoMetbinding “activation” module [10,541, but not of either of the substrate-binding modules. Except for a brief comparison with the structure of the B12-bindingfragment from MetS, we focus here entirely on the mutases.

utases (Isornerases) The 10 currently known B12-dependent mutase reactions, together with the BIZdependent ribonucleotide reductase, are listed in Table 1. Much of the available information on the individual enzymes is summarized in the relevant chapters of [l] indicated for each group below. The reactions all involve the 1,2 interchange of an H atom in one direction and a C, N, or 0 atom in the other and the following successive steps (a)-(e): (a) Homolytic fission of the Co-C(Ado)bond to give Co(1I) and the Ado radical, which is associated with a substrate-induced conformation change and probably proceeds via the intermediate formation of a strained, five-coordinate form of AdoCbl (see Sec. 4.1.31, (b) Abstraction of H by the Ado radical from the substrate to form the substrate radical (but see Sec. 2.1.4 below), and (c) Rearrangement of the substrate to the product radical, which may involve a transient and partial redox reaction with the Co(I1) ion to generate some carboniwn ion character (see Sec. 4.3).

This is followed by (d) as the reverse of (b) to form the product and then by (e) as the reverse of (a) to regenerate AdoCbl. There are, therefore, two unusual types of reaction to consider: (a) Co-C bond fission and (c) rearrangement of the substrate radical. These are considered in more detail in Secs. 4.1 and 4.3. H atom abstraction in (b) and (d) can be taken as facile and well understood. The mutases can be divided into the following four groups in order of increasing number of additional steps involved: 2.1.1. C-Skeleton Mutasss

These reactions require no additional cofactors and a minimum of’ additional steps. They involve the migration of a trigonal C atom or, in the case of glutamate mutase (GluM), of the tetrahedral C atom of a glycyl group and are all reversible; the equilibrium constant favors the less branched partner in MMCM (-- 20), methyleneglutarate mutase (- 17) and GluM (111, but is probably nearly balanced in the case of isobutyryl-CoArnutase [55-571. GluM was the first mutase enzyme to be discovered. MlMCM is the only mutase present in humans and other mammals. The role of the enzyme in mammalian liver is to convert methylmalonyl-CoA, produced in the degra-


621

dation of branched chain amino acids, odd-chain fatty acids, and cholesterol, into succinyl-CoA for inclusion in the citric acid cycle. PVlMCM and GluM are both well studied and X-ray structures of both are available (see Sec. 2.2). The rearrangement step (c) of GluM probably involves no discrete intermediate, while those of the others almost certainly proceed via a cyclopropyl intermediate (see Sec. 4.3). 2.1.2. Eliminases: Diol Dehydratase and Ethanolamine Ammonia Lyase These reactions tend to be irreversible, usually require a monocation such as K+, and involve the two distinct steps of migration of a tetrahedral 0 or N atom, followed by the elimination of water or ammonia (58,591. In the case of propane 1,2-diol, it has been shown that one OH is transferred from C2 to C1 and then, in a subsequent step, the protein catalyses the stereospecific loss of one OH to form propionaldehyde. Less direct evidence suggests an analogous two-step migration and elimination in the case of ethanolarnine. The overall activity of diol dehydratase > Et < i-Pr, showing how the effect of increasing steric hindrance toward the external reagent (Me >> Et) may eventually be outweighed by steric weakening of the Co-C bond (Et < i-Pr) [651. For elucidating the mechanisms of the two key steps of Co-C bond fission and radical rearrangement, GluM and MMCM are potentially the most instructive and GluM, which involves migration of a tetrahedral C atom, the most challenging. The RNRs are of particular interest in providing another perspective on the initial Co-C bond fission, but the subsequent steps appear to share little in common with those of the other mutases. As far as we know at present, the reactions of the C-skeleton mutases are uniquely dependent on AdoCbl as the cofactor, while Blz-independent enzymes are known for both aminomutases and RNRs (see Sec. 5.2). 2.2.

Available Structures of Mutases

Structures have been reported for the following three mutases (a)-(c)in a total of 10 different forms. The accession codes for coordinates deposited with the Brookhaven Protein Data Bank, the Cbl and ligands used, the resolution and references are listed below for each enzyme. All but the two mutase structures indicated as “open” have the so-called “closed” conformation (see Sec. 2.4). Considerable disorder is usually observed for the upper ligand, which may reflect a large B factor or, more likely, the presence of the five-cooordinate Co(I1) complex formed, for example, by X-ray radiation damage; see e.g. the calculations in [66]. Details have also been included for the inactive B12-bindingfragment of (d) MetS.

(a) Methylrnalonyl-CoA mutase @fMCM), as the ap heterodimer from Propionibacterium shermanii, which binds one Cbl (and one substrate or analogue) in the a subunit; the mammalian form is an a2 homodimer that binds one Cbl on each subunit. MMCM has been crystallized in the presence of AdoCbl, both with and without substrate; because of the difficulty of locating the AdoH formed after fission of the Co-C bond, crystals containing a range of other substrate analogues were also studied. Electron density corresponding to the Ado group has been identified only in 3REQ (“open”) and 4REQ (“closed”), i.e., in one form each with an intact and a broken CoC bond. *NB. The Y89F mutant was used for 5REQ, the wild type for all others. 1 REQ 2 REQ 3 REQ 4 REQ 5 REQ 6 REQ 7 REQ

AdoCbl + desulfo-CoA (2.0 6) “open” (2.5 A) AdoCbl + CoA “open” (2.7 ..k AdoCbl alone (substrate-free) AdoCbI + succinyl-CoA (2.2 A) AdoCbl + succinylcarbadethia-CoA(Y89F)* (2.2 A) (2.2 A) AdoCbl + 3-carboxypropyl-CoA (2.2 A) AdoCbl +2-carboxypropyl-CoA

[lll [671 [671

[531 [681 [531 [531

COBALT IN VITAMIN BIZ AND ITS ENZYMES

623

(6) Glutarriate mutase (GluM), as the E ~ C tetramer T ~ from Clostridium cochlearium, which binds one Cbl (and one substrate or inhibitor) in each CT subunit. Crystals of GluM were grown in the presence of the inhibitor tartrate (exchange of the m i n e and methyl groups of methylasparate by hydroxyls yields tartrate) and, in order to reduce the problems caused by fission of the Co-C bond in AdoCbl, in the presence of the inactive CN-Cbl and MeCbl; considerable disorder was still observed (see Sec, 2.4). 1 CCWNC-Cbl f tartrate 1 CB7 MeCbl itartrate

(L6h (2.0

A)

[661 1661

(c) Diol dehydratase (DiolD), as the ( C I ~ Ydimer )~ of heterotrirners from Klebsiella pneunzoniae, which binds one Cbl per tximer. This way also crystallized in the presence of CN-Cbl as well as the substrate propane 1,2-diol and potassium phosphate buffer to give the composition (cxf3y.CN-Cbl.diol.K+),.

1 DIO NC-Cbl + propanediol + K’

(2.2 A)

[SS]

ldl Methzonzne synthase (MetS), only the inactive B12-binding fragment (27 kDa of the total 137 kDa bolo-enzyme) from E.coli. 1 BWI’MeCbl MeCbl

__

(3.0 A) (2.4 A)

1101 [ti41

The recent review by Ludwig and Evans [54] summarizes the work of Phil Evans’s group on the different forms of MMCM and that of Martha Ludwig’s group on the B12-bindingfragment of MetS, including unpublished data at 2.4 resolution; However, it does not include the more recent results on GluM and DiolD. The “closed” conformation of i.WMCM can be compared, firstly, with the closed conformations of GluM’andDiolD as well as with the Cbl-binding fragment of Met5 to provide a good illustration of the use of modular construction with well-defined domains (see Sec. 2.3) and, secondly, with the “open” conformation of MMCM t o demonstrate the nature and role of the substrate-induced conformation change in triggering fission o f the Co-C bond (see Sec. 2.4). 2.3.

Major Structural Features: Domains, Modular Construction, and Evolution

The mutases are virtually all multimeric; cf. MMCM mammalian a2 and bacterial ap, GluM E ~ c ~DiolD ~ , (CIP~)~,but the RNRs can be both monomeric (e.g., from Lactobacillus leichrnannii) or x2. No evidence €or cooperative interaction in the binding of substrates has been reported, but the isolated subunits often show only a limited ability to bind AdoCbl (see also Sec. 4.1.4). Figure 5 shows a schematic view of the whole MMCM heterodimer in the closed conformation with substrate bound. The active CI chain is colored; for the color coding of the different sections see the figure caption. The inactive p chain, which has a fold similar to that of the CI chain but binds neither Cbl nor substrate, is left uncolored.

624

PRATT

FIG. 5. Schematic view of the methylmalonyl-CoA mutase (MMCM) us heterodimer in the “closed” conformation with bound substrate and ruptured Co-C bond. The active CI chain i s colored as follows from the N-terminal end. Red: the N-terminal arm, which wraps around the p sub-unit. Yellow: the so-called TIM-barrel substrate binding domain. Green:a long linker chain, which includes a four-helix bundle and two other ix helices. Blue: the C-terminal Cbl binding domain. In addition, pink denotes the cobalamin, dark blue the deoxyadenosine (AdolI) derived from the ligand, and dark green the substrate succinyl-Cok The inactive p chain, which has a fold similar to that of the c( chain but binds neither Cbl nor substrate, i6 uncolored. (Reprinted with permission from F.Mancia, G. A. Smith, and P. R. Evans, “Crystal structure of substrate complexes of methylmalonyl-CoA mutase”, Biochemistry, 38, pp. 7999-8005.Copyright (1999) American Chemical Society.) See Figure 13.5 in the color insert.

The active CI chain includes two main domains, ie., the yellow N-terminal substrate binding domain (see below) and the blue 6-terminal Cbl binding domain (see below), together with the pink Cbl, dark blue deoxyadenosine (AdoH) derived from the ligand, and dark green succinyl-CoAsubstrate. Figure 6 shows the isolated yellow (substrate binding) domain and the blue (Cbl binding) domain in more detail. Figure 7 gives a schematic view of the MeCbl-bindingfragment of MetS, which clearly divides into two main domains, is., the N-terminal four-helix domain, which serves as a “cap” to the active site while in the resting state, with a short linker region which connects to the Cbl binding domain, which shows a striking analogy to that found in MMCM. The corrin ring is positioned between well-defined “upper” and “lower” domains (facing the and c1 sides, respectively) in all four proteins. In the three mutases the upper domain is the substrate binding domain and exhibits the same structure, but in the MetS fragment it is the “cap”. The lower domain is the Cblbinding domain in all four proteins, exhibiting the same structure in all proteins except DiolD. The Four-helix bundle, which is seen above the Me ligand in the Cbl-

FIG. 6. Schematic views of each of the two main domains within the MMCM ct chain. Left: The N-terminal TIM barrel domain (yellow), with the substrate analogue succinylcarbadethiaCoA (dark green) bound along the barrel axis. Right: The C-terminal domain (blue), which binds the cobalamin (red) in a Aavodoxin-like fold. (Figure kindly provided by P. R. Evans.) See Figure 13.6 in the color insert.

FIG. 7. Schematic view of the Cbl-binding fragment or module from MetS (methionine synthase), showing the upper N-terminal “cap”, the MeCbl, and the lower C-terminal Cbl binding domain, which reveals a striking analogy to that found in MMCM (see right-hand side of Fig. 6). Both Cbl binding domains consist of a flavodoxin-like fold of five parallel p strands and six a helices, although one CI helix in each structure (at bottom left in Fig. 6 (right) and behind the p sheets in this figure) has no counterpart in the other. The labeling of the sheets and helices in MetS has been retained but is irrelevant to the present comparison. (Reprinted with permission from C. L. Drennan, S. Huang, J. T. Drummond, R. G . Matthews and M. L. Ludwig, “HOWa protein binds BIZ:a 3.0 A X-ray structure of B,,-binding domains of methionine synthase”, Science, 266, pp. 1669-1674. Copyright (1994)American Association for the Advancement of Science.) See Figure 13.7 in the color insert.

626

PRATT

binding fragment of MetS (see Fig. 71, must be displaced when the active site is presented to any of the other three modules and probably serves as a cap only in the resting state of the enzyme. Positioning the coenzyme with a Cbl binding domain from below in order to present the upper face to a variety of domains, providing specific catalytic groups and substrate binding sites, may be a general principle of BIZ-dependent catalysts [54,711 and even of BIZ-transportingproteins (see Sec. 3.2). The lower Cbl binding domain exhibits the same flavodoxin-like fold, composed of five parallel fl strands and six a helices, in two of the three mutases (MMCM and GluM) and in the MetS fragment; the DMB, which is coordinated to the Co in proteinfree Cbl’s, has been displaced by an endogenous His and buried in a deep pocket between the p strands and cc helices. In other words, the cofactor has undergone a significant conformation change to the so-called base-off form in all three proteins. The most detailed description of this Cbl binding domain can be found in the reported structure of the MetS fragment [lo]. The underside of the corrin structure is in contact with three loops which each link a p strand to an cn helix; one of these loops provides the His ligand (see Fig. 7) and another contains the conserved Leu, whose carbonyl 0 atom interacts with the downward-projecting C20 Me group (see Fig.3), which appears to carry a slight positive charge (see [El). Ludwig has stressed the importance of this base-off structure for binding the Cbl; in the MetS fragment protein-Cbl contacts bury about 715 of the 990-x2 accessible surface area of MeCbl, about half of which represents contacts with the extended nucleotide tail [lo]. The Cbl binding domain of DiolD has a different structure, in which DMB remains coordinated and the cofactor therefore remains base-on; few structural details have been described except that a Rossmann-like fold is observed in the central part of the B subunit 1691. The main feature of the upper substrate binding domain in all three mutases is a so-called TIM barrel (named from triosephosphate isomerase), in which a core of eight twisted parallel strands is surrounded by eight ‘x helices. Although the TIM barrel is one of the commonest folds observed in proteins, the “mutase” version is so far unique both in possessing a hydrophilic inner lining and in being constructed in two halves that can close together onto the substrate and open up to release the product (see Sec. 2.4). The upper and lower domains may form part of the same or different subunits; inclusion in a single subunit occurs in MMCM and the MetS fragment, in two different subunits in GluM (I and a ) and DiolD (a and p). Furthermore, the Cbl-binding fragment from MetS forms part of a long (137-kDa, 1227-residue)single chain, which can be split by controlled proteolysis into four fragments or modules. Starting from the N-terminal end, these four modules separately serve to bind homocysteine, Metetrabydrofolate (i.e., the two substrates), MeCbl, and AdoMet (for reactivating the enzyme when oxidized to the inactive Co(1I) state). Ludwig and Evans [541 pointed out that sequence alignments show analogies both between the homocysteine-binding fragment of MetS and betaine-homocysteine methyltransferase and between the folate-binding fragments and dihydropteroate reductase; also that the structure of the latter enzyme has been shown to be a (flcl), TIM barrel. This suggests that the two

COBALT IN VITAMIN Biz AND ITS ENZYMES

627

substrate binding domains of MetS may have originally been adopted ready-made from other enzymes and that the folate binding domain of MetS may therefore also be a TIM barrel. The further parallels between MetS and MMCM are intriguing; cf. the N- to Cterminal sequences of [substrate-binding TIM barrel]-l linker with 4helix bundle][Cbl-binding domain] in IViMCM and of [folate-binding TIM barrel]-[cap of 4-helix bundle]-[Cbl-binding domain] in MetS. This is suggestive evidence that at least some of the mutmes may have evolved from Me trunsferuses. The origin of the Ado ligand may even be connected with the presence of the “activation” module, which is common to many Me transferases and exists to regenerate the CoU) and/or Me-Co forms after accidental oxidation to the catalytically inert Co(I1) state. Activation requires reducing equivalents and either ATP (to drive the reduction of Co(I1)) or AdoMet (to remove the Co(1) by methylation). AdoCbl is actually formed in vivo by the enzymatic reaction of ATP with Co(1) to form AdoCbl and triphosphate and, although no analogous reaction of’AdoMet has been reported, one could envision steric misalignment causing the transfer of Ado+ instead of Me’. Such a close working association between Co(1) corrinoids and both ATP and AdoMet in the “activation” modules could have provided an opportunity in the course of evolution to form the Co-Ado bond and exploit its mechanistic possibilities.

elevant to the Mutase Reaction Mechanism Comparisons can be made between the structures of‘ (1) protein-free and proteinbound AdoCbl, to test for any detectable “preconditioning” of the coenzyme, (2) protein-bound AdoCbl with and without substrate and substrate analogues, to characterize the nature of the substrate-induced conformation change, and (3) proteinbound AdoCbl with inhibitors selected (see below) to test for any conformation change associated with the rearrangement step. During enzymatic activity the closed conformation occurs only together with (protein-bound) free radicals, but over the much longer timescale of X-ray diffraction, the radical must clearly have taken up the extra H atom. 2.4.1. Comparison of Protein-Free and Protein-Bound AdoCbl

The most striking gross change observed when AdoCbl is bound by the protein in MMCM, GluM, and the MetS fragment is displacement of DMB as ligand by an endogenous His (see Sec. 2.3). Since DMB remains coordinated in DiolD and probably also in EAL, the prescnce of His is clearly not essential to the basic mutase mechanism. Burying the nucleotide tail in the flavodoxin-likefold (and displacing DMB as the ligand) may have functions in addition to that of firmly binding the Cbl (see See. 3.2). Binding AdoCbl to the protein in ?MMCM naturally induces minor changes in side chain conformation and also moves the adenine ring of the Ado ligand from a position over ring C to one over ring B r543.

628

PRATT

All the mutases show an apparent lengthening of the axial Co-N bond length to His and DMB, as pointed out by Evans [11,671; most of the relevant bond lengths are summarized in 1541. The following Co-N bond lengths (in A) are observed in proteinfree Co corrinoids: cyano-Cbl 2.01, MeCbl 2.19, AdoCbl 2.21, Co(I1)-Cbl 2.16, and imidazolyl-B12, in which the nucleotide DMB has been replaced by imidazole, 1.97; cf. also MeCbl in the MetS fragment 2.2(2).By contrast, all four closed conformations of M ~ with C CollI)-Cbl ~ reveal Co-N bond lengths of 2.4 or 2.5(1) and the two open conformations with AdoCbl 2.6(3) and 2.7(2); cf. also GluM with CN-Cbl 2.27 and 2.30 and with MeCbl 2.35 [661 and DiolD with CN-Cbl 2.50 [691. The lengthening would suggest a positioning of the base by the protein to facilitate a reduction in coordination number either from six- to five-coordinate AdoCbl or from five- to fourcoordinate Co(I1). The first would promote formation of a strained five-coordinate form of AdoCbl, which probably occurs as an intermediate (See. 4.1.3);lengthening of the Co-N bond per se is unlikeIy to promote bond fission since all of the evidence indicates that removal of the base from alkyl-Cbl’s increases their kinetic and thermodynamic stability toward homolytic fission [?2,?3].The second would serve to stabilize the normally four-coordinate Co(1) and promote the partial electron transfer from the substrate radical, which appears to be involved in the rearrangement step (Sec. 4.3). 2.4.2. Nature of the Subslrate-Induced Conformation Change and Its Role in Co-C Bond Fission

The N-terminal substrate binding domain of MMCM was shown in Fig, 6 in the closed Conformation. The eight-stranded TIM barrel is divided into two four-stranded halves, which close around the substrate. Strands 2-5 form the outer barrel, strands 6, 7, 8 and 1 the inner barrel. The two halves can open and close about a hinge between strands 5 and 6, and €3 bonds between strands 1 and 2 bind the two halves together in the closed form. In the substrate-free open form the Ado ligand protrudes into the bottom of the barrel with the adenine pushed up against Tyr A89, which i s attached to the end of strand PI of the inner barrel, while the other end of the barrel is open to solvent. When the substrate enters, it binds along the central axis of the barrel with the acyl end towards the Co and the two halves of the barrel close up, forming numerous I31 bonds with the substrate. The “open” to “closed” conformation change invohes the loss of many water molecules, which can be estimated as more than 30 [15] and is probably accompanied by a significant increase in entropy (See. 4.1.3).The C-terminal Cbl binding domain acts as a third rigid body and also moves toward the inner barrel when the substrate binds. The relative movements of both the outer barrel and the Cbl towards the inner barrel (including the attached Tyr A89) are shown in Fig. 8. The most clear-cut effect of reducing the “vertical” distance between the base of the inner barrel and the plane of the corrin ring is to force Tyr A89 (fixed to the base of strand PL)into the space previously occupied by the Ado ligand. As outlined in i53aI and further clarified by [53b], this closing up forces the adenine ring to rotate into a


629

FIG. 8. Key features of the substrate-induced “open” to “closed” conformation change in MMCM which, as represented here, involves a “horizontal” movement to the right of the outer half of the TIM barrel (strands 2-5) and a “vertical” movement upward of the Cbl binding domain (for simplicity, only the Cbl itself is shown), both toward the inner haK of the TIM barrel (strands 1and 6-81 with Tyr A89 attached to the foot of strand 1.The changes in position from the open to the closed conformation are denoted by changes in color: outer half of the barrel (green to gray); inner half (red lin‘es to gray) with Tyr A89 (black to red); Cbl (blue with Ado rnagenta to black with Ado not shown). The substrate bound within the closed barrel and protruding out of the top is black. The red compound at the top of the open barrel represents a molecule of coenzyme A that is bound in crystals of the open conformation without triggering the conformation change. (Reprinted with permission from F. Mancia and P. R. Evans, “Conformational changes on substrate binding to methylmalonyl-CoA mutase and new insights into free radical mechanism”, Structure, 6, pp. 711-720. Copyright (1998) Elsevier Science.) See Figure 13.8 in the color insert.

plane roughly normal to the corrin ring and the whole Ado ligand to move laterally and roughly parallel to the corrin plane, i.e., the vertical movement of Y’yr A89 toward the adenine ring is converted into a lateral movement of the adenine ring, which exerts sufficient leverage around the coordinated C atom, to rupture the Co-C bond. Rupture of the Co-6 bond appears to be driven entirely by steric compression between the Ado ligand and groups on the TIM barrel, in particular between the adenine and Tyr A89, which develops when the barrel closes around the substrate; “suggestions [see Sec. 4.11 that upward folding of the corrin ring might play a part in destabilising the Ado bond are not borne out by the structure” [741. Using Gerstein, Lesk, and Chothia’s classification of mechanisms for domain movement 1751, the cause of Go-G fission is a shear motion between the corrin structure and the base of the TIM barrel, linked to a hinge motion of the TIM barrel itself, Sufficient leverage can only be applied to the Co-C bond by preventing “slippage” either through lateral displacement of the Co and its ligand vis-a-vis Tyr A89 or

630

PRATT

through rotation of the Ado ligand around the Go-C axis. The corrin structure clearly provides sufficient contacts or anchors with the protein and the Ado group to prevent such slippage, but it seems unlikely that the large, flat, and “slippery” surface of a porphpin could prevent it so effectively7if at all. This suggests a further advantage of corrin over porphyrin which depends on differences in steric and H: bonding rather than electronic properties (cf. Sec. 1.6.3). The change between the two protein conformations, which include the open and closed forms of the TIM barrel, respectively, and are denoted by P” and P in equilibrium (2), seme to link together (1) the binding of substrate and loss of water molecules at one site and ( 2 ) fission of the Co-C bond at a diFferent site. The egress of a large number of water molecules provides a mechanism for building up the required energy (for Co-C fission) in small increments and allows the two halves of the TIM barrel to move together like hinged levers in order to gather and transduce the energy available €rom the large area ofthe cavity into energy focused at one point to break a single Co-C bond-a reciprocating “molecular nutcracker” I151 or, for those familiar with mining, a “molecular jaw-crusher”. 2.4.3. Evidence Relating to Formation and Rearrangement of the Substrate Radical

The closed form of lUMCM with the true enzymatic substrates shows that fission of the Co-C bond leaves “the C5’ carbon close to the substrate in a suitable position for €3 abstraction by an adenosyl radical” 1531. The X-ray results suggest distances from the radical centers of the substrate to the Co ion of 6.2-6.5 in MMCM [741,6.6b in G1uM [661, and 8 . 4 9 . 0 A in DiolD 1691, which agree with the ESR data but arc incompatible with suggestions (see See. 4.3) that rearrangement involves the intermediate formation of a Co-C bond between the substrate radical and the Co(I1) ion. Structures were also determined with 2-carboxy- and 3-carboxypropyl-CoA,that mimic the substrates Me-malonyl- and succinyl-CoA but lack the thioester carbonyl function essential for rearrangement. The close similarity of the two structures around the active site showed that “there is no significant conformational change between substrate and product complexes, in contrast to the enormous change on substrate binding” tLi31.

2.5. Summary of Main Points (1) The &,-binding proteins exhibit a clear modular construction, based on well-defined domains, with the corrin ring in both MetS and the mutases held between a lower (a)Cbl binding domain and an upper (0) substrate binding or capping domain (Sec. 2.3), in contrast to the better known hemoproteins, such as the Mb/Hb, peroxidase and P450 families, where the porphyrin ring is held in a hydrophobic groove between helices inside a single multihelix domain. This enables the Co corrinoids to exploit a strategy, not readily available to the Fe porphyrins, of attaching


631

ready-made domains with different potential functions in close proximity to the Co in order to evolve new forms of enzymatic activity. (2) Comparisons of the mutases with the Me transferases and non-B,,-dependent enzymes suggest that there has been an extensive interchange of domains and cofactors between different enzyme families, and that the typical mutases could have evolved from the Me transferases and the Co-Ado bond from their known reactions with ATP or AdoMet (Sec. 2.3). (3) The substrate binding domain in bolh the C-skeleton mutases and the eliminases is an unusual (p/cr), TIM barrel possessing a hydrophilic lining and constructed in two halves, which can open and close about a hinge between the two halves when binding the substrate (Secs. 2.3 and 2.4.2). (4) Opening and closing the TIM barrel represents a significant conformation change, which serves to link the binding of substrate (and loss of more than 30 water molecules) at one site with fission of the Co-C bond at another site. Co-C bond fission is caused by an essentially horizontal shear motion between the base of the TIM barrel and the irregular corrin structure (with no detectable upward pressure from the corrin ring), which would probably be difficult to generate with the large flat porphyrin ring (Sec. 2.4.2).The energetics and mechanism of this step are discussed further in Sec. 4.1. ( 5 ) By contrast, there is no detectable difference in conformation between forms containing inhibitors that model the substrate and product (Sec. 2.4.31, i.e., rearrangement is not associated with any detectable conformation change. (6) The structural data suggest distances of 6-9 A between the Co and radical C atoms in the three mutases, precluding Go-C bond formation to the substrate or product radicals (See. 2.4.3). (7) An apparent lengthening of the axial Co-N bond length, compared to that observed in the protein-free forms, is seen in all mutases in both open and closed conformations (Sec. 2.4.1).

3. Bf2-BINC)INGENZYMES/PROTEINS WITH UNKNOWN STRUCTURES

3.1. Distribution an B12 is unique among the vitamins in being synthesized only by certain microorganisms. These include representatives of the bacteria, both aerobic and anaerobic, and of blue-green, brown, and red algae [76]. Chlamydomonas appears to be the only reported example among the green algae [771. The very primitive and strictly anaerobic acetogenic, methanogenic, and sulfate-reducing bacteria form B12’sreal “heartland”; concentrations up to 0.7 mM have been recorded for wet cells of Clostridium th,errnoaceticum [781. B12 is required as a vitamin From external sources by many other microorganisms and probably by the whole animal kingdom, from mammals through tapeworms down to the photosynthesizing protozoan EugZena gracilis [76].

PRATT

632

Humans and other mammals require B12 for both MctS (in the cytosol) and MMCM (in mitochondria) 1791, while Euglena requires B12 for MMCM and MetS and also for the B12-dependentRNR CSOI. Claims for a €312 requirement by plants (see, e.g. [Sl]) remain controversial. Exclusion of fortuitous uptake or contamination is made difficult by the ubiquitous occurrence of B12 in water and soil and the very low concentrations that may be required (see Sec. 3.2). Unraveling the biosynthesis of Co corrinoids has been a major success story and is fully reviewed elsewhere 182,831. B12 shares a common biosynthetic pathway with the other tetrapyrroles from the five-carbon precursor S-aminolednic acid (&A) to uroporphyrinogen 111. The BIZpath then diverges and from precorrin-2 it divides into aerobic and anaerobic paths, which differ in the mechanisms used to form the Cl-Cl9 link between rings A and D.The anaerobic path (elucidated mainly with SaZmon,eZZa typhimuriurn) requires Co for the Cl-C19 ring contraction and produces only Cocontaining corrinoids. The aerobic path uses oxygen and produces metd-free corrinoids before Co insertion. The aerobic cobaltochelatase resembles Mg chelatase in being a complex ATP-dependent enzyme E841 and differs from the monomeric ATPindependent ferrochelatase. Scott et al. have commented that one cannot exclude “the remote possibility that another route to BIZ awaits discovery” C831. Significant contributions have been made by groups in France (Thibaut), the UK (Battersby), and the USA (Scott). Special mention should be made of the successfd “one-pot” conversion of L A to the complete (but Co-free) corrinoid, reported by Scott’s group in 1994 [85]; incubating a mixture of ALA with 12 enzymes and other essential cofactors H, NADPH) for 15 h at 30” gave the product of the 17-step sequence in 20%overall yield, which exceeds the eBciency even of the natural cellular synthesis! A range of &binding proteins is then involved in adsorption (e.g., from the human gut),transport, and possibly storage before conversion into coenzymes, such as MeCbl and AdoCbl (see Sec. 3.2), and incorporation into enzymes, such as the mutases (Sees. 2 and 4), Me transferases (Sec. 3.3), and other known or possible BI2-dependent enzymes (Sec. 3.4).

3.2.

Absorption, Transport, and Transformation of Co Corrinoids

Absorption and transport has been studied in mammals, E. coZi and EugZena. A useful comparison of all three is available 1801, together with separate reviews of the mammalian [86,871 and bacterial 1881 systems. In humans the uptake and transport of B12 involves three Blz-bindingproteins: haptocomin (HC), intrinsic factor (IF), and transcobdamin (TC). Intrinsic factor is present in gastric juice, whereas the External Factor (now called Blz) is obtained from food. IF binds to Cbl’s released from food by digestion, and the IF-Cbl complex is then bound by receptors on the surface of the small intestine and internalized. After conversion to the coenzyme forms, AdoCbl circulates mainly with TC before being bound by receptors on the cell surfaces and internalized. HC binds mainly MeCbl and con-

COBALT IN VITAMIN

832

AND ITS ENZYMES

633

stitutes the main pool of bound Cbl in the serum; however this is not readily available because most cells lack an HC receptor. At present, “haptocorrins have no known essential function” [89]. HC, IF, and TC show similarities in their exon/intron structure, which suggest a common origin [79]. Their binding constants (K)can be very high; a recent redetermination of K for B12 with HC gave a value of logK = 16.7 h 0.6 at 25“ f901, Varying the upper ligand (cyanide, methyl, Ado) has little effect on the value o f K for any of these proteins [91], but varying the nucleotide sidechain has a large effect on their binding by IF, a weak effect with TC, and hardly any effect with HC 1921. IF is fairly specific for the Cbl’s, although it will bind CN-Cbi in the presence of‘ components of the nucleotide sidechain (e.g., free ribazole), which appear to promote a conformation change required to expose the corrinoid binding domain 1931. These proteins provide further examples of the corrinoid binding domain interacting with the lower (CO part of the corrin, as already shown for MetS and the mutases (see Sec. 2.3). Mechanisms for B12 storage undoubtedly exist to counteract fluctuations in the external supply, but little is known about them. Man’s reserves of B12 in the liver are sufficient to maintain plasma levels of BIZ unchanged for several years after external supplies become deficient. The reserves of Lactobacillus Zeishmannii, whose BIZdependent growth is used to assay for BIZ, can be rapidly depleted by innoculation into a BI2-freemedium and this can be used to estimate the minimal requirement; the BIZ concentration was reduced to about one molecule per cell before growth became filamentous and unbalanced I941. Other workers reported approximately eight molecules of Bl2 per cell for normal growth [951. Several enzymes, which appear to be analogous in microorganisms and rnammals, are involved in preparing the corrinoids for their role as coenzymes [Sol. They include:

1. Aquacobalamin reductase (EC 1.6.99.81,which reduces BIza (but not Co(II) 1312rand, at least in the rat liver, i s the same as N ~ P ~ - c ~ o c h r o m c reductase [961. 2. Cob(I1)alamin reductase (EC 1.6.99.91, which reduces Co(l1) to Co(1) Bias; see also r971. 3. Cob(1)alamin adenosyltransferase (EC 2.5.1.171, which catalyzes the reaction of Co(1) with ATP to form AdoCbl and triphosphate; see also B41. 4. Other enzymes are required t o handle the more refractory CN-Cbl (B12 itselQ such as cyanocobalamin reductase (EC 1.6.99.12). 5. Circumstantial evidence (e.g., the presence of MeCbl as the main HC-bound species) suggests that enzymes exist t o form the Me-Co bond that are distinct from (but perhaps similar to) the activating subunit of the methyltransferases, but no such enzymes have yet been identified.

634

PRATT

12-Dependmt Methyltransferases The Me transferases catalyze the transfer of a Me cation from a donor to an acceptor by an apparently simple mechanism involving the cyclic formation of Co(1) and Me-Co corrinoids according to equation (3). Both species are diamagnetic but readily detected by their UV-Vis spectra. Both steps involve, where studied, a simple SNz substitution with inversion at the C atom, leading to overall retention of configuration between donor and acceptor. Our knowledge and understanding of the Me transferases has lagged considerably behind that of the mutases because of their greater complexity and the difficulty of culturing the extremely oxygen-sensitive, primitive anaerobes, which form the real Bla heartland. These are the acetogenic, methanogenic, and sulfate-reducing bacteria; for relevant background information, see [981001. Me’

+ CO + RS

;rt

Me-CO-SR

(10)

These bacteria alone possess a unique pathway for the formation and dissociation of acetyl-CoA according to equation (lo), which probably represents a metabolic pathway that was important early in evolution. Acetyl-CoA is formed by combining a methyl cation (delivered by corrinoids) and the thiolate anion of CoA with CO according to equation (10). This unusual reaction is catalyzed by CODWACS (GO dehydrogena;se/acetyl-CoA synthase), which contains an NiFeS cluster and can also catalyze the reversible interconversion of CO and C 0 2 (together with electrons). Acetogenic bacteria can convert organic compounds such as sugars and aromatic methyl ethers (ArOMe),hydrogen, carbon monoxide, and carbon dioxide into acetic acid. Most or all methanogens can grow on Ha and C 0 2 , many on acetate, some also on other onecarbon sources, such as MeOH, methylthioethers (RSMe), methylamines, and even the NMei ion, all of which require corrinoid-dependent Me transferases. They often live in tight symbiosis with the acetogens and use much of the H2, C 0 2 and acetic acid produced by the latter to form methane. ~TL(CHOH) -“I TzCH~COOH --+nCH4 + nCOz

(11)

Major contributions to the metabolism of these anaerobes come from Ihe exergonic disproportionation of carbohydrates first to acetic acid (by the acetogens) and then to methane and carbon dioxide (by the methanogens), as summarized in equation (1I), where (CHOH) denotes carbohydrate. In the acetogens acetyl-CoA plays the central role in both energy production (hydrolysis of acetyl-CoA to acetic acid yields ATP) and cell carbon synthesis. The methanogens have retained the central position of acetyl-CoA for cell carbon synthesis (but not for energy production) and the use of CODH/ACS (although run in reverse) but have developed a new path for energy production; this centers on the formation of Me-CoM (where coenzyme M is WS-CH2-CH2-S0;) by a corrinoid-dependent enzyme and its reduction to methane by an enzyme that contains coenzyme F430 (a Ni tetrapyrrole). The anaerobic “oxidation” of methane would represent a further stage beyond equation (11); it is, in fact, well established but not widely known [101,102]. The mechanism is not

ALT IN VITAMIN

12

AND ITS ENZYMES

635

simply the reversal of methane liberation, and the terminal oxidant may be sulfate in the presence of, for example, iron salts to precipitate insoluble sulfides and drive the overall reaction. The coenzyme involved in “activating” methane is not yet known but could be a corrinoid (see See. 3.4.4).The central role of ace+$-CoA and CODH/ACS in the metabolism of these primitive anaerobes, together with their heavy dependence on one-carbon sources, provides a large market for Bizdependent Me transferases. The Me transferases generally consist of readily dissociated subunits (or modules) for the separate binding of donor, acceptor, and corrinoid. They often possess a further “activating” module for regenerating the Co(1) or Me-Co corrinoid after adventitious oxidation to the inactive Co(l1) state, using ATP or AdoMet, respectively, to drive the reaction. Their structural organization further exemplifies the strategy (see See. 2.3) o f using well-defined and interchangeable domains, with the corrinoid presumably held on the lower (a) side by the corrinoid binding domain, leaving the upper ((3) side free to interact in turn with domains involved with the substrates and activators. The range of nucleophilic centers that the corrinoid-dependent enzymes can handle as either donors or acceptors include N in reduced pterins (as both donors and acceptors), mono-, di- and trimethylamine and NMe:; 0 in Me0 (phenylmethyl ethers); S in SMe (Me-thioethers) as donors, RSR (thiols) such as homocysteine and CoM as ceptors and, for reactivation, the sulfonium ion AdoMct; the Ni complex (exact atom not known) in CODH/ACS (see above) as both donor and acceptor. Methylation of the C5 atom of cytosine in vertebrate DNA has recently been reported 11031. There is no reason to suppose the list is closed. Our rapidly increasing knowledge of the many Me transferases present in the primitive anaerobes, together with thc recent availability of experimental data needed to quantify the rate enhancement by the protein (the rate of Me transfer from to homocysteine is enhanced by approximately 10” at pH around 7 ) and of a theoretical framework for understanding some of the factors that may influence Co activity, provides the basis for a rapid increase in our understanding of these enzymes; these have recently been reviewed elsewhere [as]. A theoretical framework i s provided by the observed linear correlation between the logarithm of the rate constants for rnethylation of a range of Co(1) complexes and their Co(II)/Co(lE)redox potential, as first emphasized by Costa et al. I3041 (see also L105-10611, their linking to trans effects in Co(1II) corrinoids [261, and the extension of the Marcus-Hush theory, as applied to methyl transfer, to the CoU) “suupernueleophiles” by Lewis et al. [107,1081. A true understanding of the Me transferases may depend on a better ~ n d e r s t a n d i nof~the unusual electronic structure of the corrin ring (see Sec. 1.6.2) and its possible manipulation (as a cis effect) by the protein.

636

PRATT

3.4. Other Known or Possible B12-DependentEnzymatic Reactions Several other groups of known or likely BI2-dependentreactions are mentioned here briefly, including other Me transfers that are of interest because of their environmental implications but may be only “incidental” reactions.

3.4.1. Other Methyl Transfhrs

There has been considerable interest in the biomethylation of metals and metalloids because of their environmental implications; some, but not all, may involve corrinoids. Best known is the methylation of Hg(I1) ions to form the toxic MeHg’ ion and the volatile HgMez by the simple transfer of a methyl anion from an Me-Co corrinoid that was discovered as an in vitro reaction in 1970 [65]. The organism responsible for these reactions in anoxic aquatic sediments has been identified as a strain of the sulfate-reducing Desulfouibrio desulfuricans, but it is not known whether this is a spontaneous or enzymatically catalyzed reaction; somewhat surprisingly, the Me transferases of many methanogens and acetogens are unable to methylate Hg(I1) [l091. For a discussion of the mechanisms of methylation of metals by MeCo corrinoids studied in vitro, see 1291. Reduction and methylation of arsenite to dimethylarsine, as well as that of selenate and tellurate to volatile and smelly compounds (probably dimethylselenideand dimethyltclluride), by Me cation transfer from MeCbl occurs in Methanobacterium [ l l O l .

3.4.2. Reductive Dehalogenatwn

Several enzymes have been identified from anaerobic bacteria that can catalyze the reduction of an olefinic or aromatic C-Cl bond to a C-H bond with formation of a chloride ion. Two reviews are available [111,1121. The enzymes all possess a corrinoid and an Fe-S cluster. Substrates include tetra- and trichloroethene, 3-6l-benzoate, and a range of chlorophenols. The first two produce cis-dichloroethene except in one organism, which can also reduce vinyl chloride to ethene. The usual ultimate redue tant is HZ and at least three organisms can use this “dehalorespiration” with H2 to produce energy. The active form of the corrinoid is assumed to be COG),but this has yet to be identified by its UV-Vis spectrum, and it has not been established whether the first step involves an outer sphere electron transfer from Co(1) to the substrate or formation of a Co-C bond. There is increasing interest in these enzymes because of their potential for the bioremediation of polluted soils and groundwater, Since chlorinated ethenes probably occur naturally, these enzymes may not have evolved simply in response to man’s use of chlorinated organic solvents!

COBALT IN V I T A ~ I NBj2 AND ITS ENZYMES

637

3.4.3. Reduction of Elpoxyqueuosine to Queuosine Queuosine (Q) is an unusual base that may replace guanosine at certain positions in tRNAs and is synthesized de novo in eubacteria. It was found that Q is produced by reduction of the epoxy group of epoxyqueuosine to the olefmie group of Q in an enzymic reaction that requires B12 11131. A mechanism has been suggested, based on the reactions of epoxycyclopentanewith Co(1) Cbl and cobaloxime to give an alkylCo(II1) intermediate that can undergo acid-catalyzed decomposition to form cyclopentene, Co(III), and hydroxide [114]. The protein presumably acts to overcome thc acid dependence of the protein-free reaction. 3.4.4. Anaerobic Methane Oxidation,

Anaerobic methane oxidation is well established and probably coupled to the reduction of sulfate (as the terminal oxidant) to sulfide, but the cofactor remains unknown (see Sec. 3.3). The mildest conditions for the "activation" of methane by any transition metal complex were reported by Sherry and Wayland in 1990 [115al. Benzene solutions of Rh(II) porphyins react reversibly with methane at 2 50" (also with hydrogen) according to equation (121, where M is Rh and L is the tetramesityl- or tetraxylyl-porphinato ligand selected to prevent formation of a direct Rh-Rh bond; the evidence points to a linear (Rh CH, . H - . Rh) transition state. The second-row transition metal Rh is the heavier congener of Co. We therefore suggested a mechanism for methane activation [116] involving a Co(I1) corrinoid dimer (see Sec. 1.6.3) according to equation (12), where M is Co and L is corrin, analogous to that shown by the Rh(I1) porphyrin dimer, except that the Co(II1)-hydridemay further lose a proton to form CdI).

2L.M" + MeH 4 L.MIIIHH+ L.M'*'Me-

(1%

An increasing number of cases of the anaerobic oxidation of higher alkanes are being reported; see, for example, [117,1181. The first step in their activation may also involve the formation of Co-alkyl and Co-H or CoU) corrinoids by an analogous mechanism. However, other mechanisms are possible, such as the simultaneous removal of two H atoms from vicinal C atoms to produce an olefin directly.

ELS, M ~ C H A ~ I S MAND S ~TRUCTUR~-FUNCTIO~ RELATIONSHIPS OF THE MUTASES The mutase enzymes, their abbreviations and substrates, together with the steps involved in their reactions, were summarized in Sec. 2.1. Further mechanistic evidence is given here on the two main steps of interest: 1. The reversible homolytic fission of the Co-C bond (See. 4.1) according t o equilibrium (21,which produces the substrate-derived radical in nonbonded

PRATT

638

association with the Co(I1) ion as a charge-transfer (CT) complex (See. 4.2). The substrate-triggered conformation change has been described in Sec. 2.4. 2. Rearrangement of the substrate-derived radical to the product-derived radical within the CT complex (Sec. 4.3). The Rl2 literature has resounded with the clash of competing theories about both steps as evidence has gradually built up in support of some novel and unexpected mechanism (e.g., Co-C homolytic fission to produce a free radical or the 1,2 migration of an alkyl group in GluM) and against more orthodox competitors. This accumulation of evidence has occured more slowly for the rearrangements, which have often been considered the central mystery of BI2. Suggestions for the form in which the substrate might undergo rearrangement include radical, protonated or deprotonated radical, carbanion, fully formed carbocation, partial charge transfer from radical to Go, o-bonded alkyl-Co(Il1)complex, carbene complex, n-bonded complex of Co(lI1) or Co(II), as well as mechanisms involving fragmentation and recombination. Fortunately, the application of molecular biology has now provided better supplies of the enzymes, the first structural data, improved spectra (UV-Vis and ESR), and more detailed kinetics. This will serve to curtail much of the speculation about both steps. Because of limitations of space, this chapter i s concerned mainly with those ideas that have survived through to Y2K and with those findings which have proved most informative, Readers may find it difficult to follow the literature-discult to see the forest for all the trees in Co-C bond fission, difficult to see the forest for the scarcity of trees in the rearrangement step.

4.1.

Fission of ‘the Distortion

ond: Mechanism for Ap

4.1.1, Proposed Mechanisms

Until the end of the 1960s it was assumed that the Co-C bond in AdoCbl underwent heterolytic fission t o give either Co(1) or Co(II1). The possibility that steric distortion, caused by “misf%ting between the enzyme binding and the Co binding of the adenosine in the presence of substrate”, might be used to promote Co-C bond fission was first proposed by Hill, Pratt, and Williams in 1969 11191 as a means to form Co(1) and a carbonium ion; this was based on the known greater lability of bonds to see-alkyl ligands such as isopropyl. During the early 1970s, it became clear that the Co-C bond underwent homolytic fission, since ESR could detect both Co(I1) and an organic radical; see Babior’s 1975 review 191. The concept of steric labilization was then extended to homolytic fission of the Co-C bond according to equilibrium (1)by considering possible mechanisms for overcoming the obviously very adverse e ~ u i l i b ~ u constant, and a mechanism for Co-C bond homolysis was proposed by the author in 1975 [120], based on three main points:


639

1. The main effect of the protein t substrate is to cause a significant increase in the apparent equilibrium constant K(= k f / k , ) for dissociation according to the simple equation (1)(i.e., to reduce the steep rise from reactant to product on the free-energy reaction profile), and not merely to increase both the forward (kf)and reverse (k,) rate constants without significantly increasing K. 2. The protein achieves this increase in K mainly by distorting the Co coordination sphere (probablythe Co-6-C bond angle) and destabilizing the initial AdoCbl. 3. A minor distortion (stage I) may be caused when the coenzyme is bound by the protein (to account for any slow inactivation of some mutases in air even in the absence of substrate) and the major distortion (stage 11) by a substrate-induced change in protein conformation (ensuring that the reactive radical is produced only when required).

Stage I1 involves coupling between three component equilibria: (a) binding of substrate by protein and (b) a change in protein conformation as well as (c) Co-6 bond fission. Large changes in the apparent equilibrium (c) for Co-C bond fission could therefore occur, even where the overall change in free energy for substrate binding (a) is small, if accompanied by large Compensating changes due to the change in conformation (b). Other proposals for promoting homolytic fission, put forward in 1974 [121], listed “(i) steric influences resulting from conformational changes in the corrin ring, (ii) Go-C bond weakening resulting rrom trans-axial ligand substitution. . .or (iii) an oxidative or reductive cleavage process.. .” but excluded the mechanism proposed above and that finally established by structural studies (Sec. 2.4). Readers attempting to follow the literature should be aware, firstly, that proposals related to ti) have been overzealously marketed by several groups, often under the label ‘“mechanochemical”or “butterfly” mechanism, right until publication of the structural results; this has caused considerable confusion. Secondly, the role of the protein and its conformation change have frequently been ignored despite early evidence for a conformation change associated with Co-6 bond fission presented by Babior for EAL in 1970 11221 and by Tamao and Blakley for RNR in 1973 [1231. As mentioned in See. 1.1,the Ado radical has never been detected, and the observed equilibrium must therefore include the subsequent H abstraction from the substrate as in the more complex equilibrium (2) as given on page 609, where HZ denotes substrate (or substrate analogue), P* and P denote the two protein conformations, and the square brackets enclose the protein and any protein-bound species. The atypical RNRs require the slightly different equation (7) (see Sec. 2.1). [P*.Co-Ado]+ HZ + [P.Co(II),Z.AdoH]

(2)

Studies on protein-free corrinoids had clarified both the problem and possible mechanistic solutions by 1985, but the necessary structural and solution studies on the enzymes had to wait until 1999.

640

PRATT

41.2. Studies on Protein-Free Corrinoids

The first protein-free model for sterically induced homolytic fission of the Co-C bond at ambient temperature was provided by np-Cbl (np = neopentyl, Co-C reported in 1980 by Chemaly and Pratt [1241 and subsequently by Schrauzer and Grate [1251. It was later shown that p elimination (e.g., from isopropyl-Cbl to give propylene, Co(1) and a proton) probably also proceeded via an initial homolytic fission W61. The effects of increasing steric distortion within the alkyl ligand on rate constants for Co-C fission, on equilibrium constants for coordination in the transposition, and on their UV-Vis spectra were studied systematically by Grate and Schrauzer and by Chemdy and Pratt; see summaries in [72,731. Starting from MeCbl, increasing distortion will cause a decrease in the stability of the Co-C bond and in the stability of the red six-coordinate alkyl-Cbl relative to the yellow five-coordinate base-off form, such that np-Cbl is mainly b a s e d and five-coordinate. Solution studies on the corrinoids were complemented by structure determinations of alkylcobaloximes (from Me to i-Pr and np) by Marzilli and co-workers [127], which revealed severe distortions of the bond lengths and bond angles around the coordinated C atom; this demonstrated that such distortions were due to “internal” strain between bonded atoms within the alkyl ligand and not to steric distortion from the equatorial ligand. The finding that Co-C bonds to np [1241, i-Pr, and cyclohexyl Cl28l couId be labilized by the transcoordination of imidazole as well as the bulky DMB, which causes buckling of the corrin ring (see See. 1.6.31, showed that the labilizing effect of DMB in the Cbl’s over the Cbi’s was due t o the increase in coordination number and not to any change in conformation of the conin ring. By 1982, protein-free alkyl-corrinoids had modeled labilization of the Co-C bond from that seen in, say, Et-Cbl by well over 10” through increasing strain within the alkyl ligand, while conversion of‘ the five-coordinate Cbi’s or base-off’Cbl’s into the more strained six-coordinate base-on Cbl’s could increase the rate by a smaller factor of up to lo3. It was concluded that (1)“the ribose and adenine parts of the ligand (could) provide a ready-made handle for pulling or exerting leverage around the coordinated C atom”, while (2) conformational changes in the corrin ring were unlikely to provide a mechanism for steric labilisation by the protein. It was further predicted (3) that the reaction would proceed through a spectroscopically distinct, strained form of AdoCbl (see See. 4.1.3) 1721. From studies on diol dehydrase, Toraya et d.also concluded that ”the tight interaction of the adenine moiety with the enzyrrie produces a kind of tensile force or angular strain between the adenine and the Go ion, which is at least one element of the force which weakens the Co-C bond” [1291.

Values for the forward (kf?and reverse (kJ rate constants, required to calculate the equilibrium constant for Co-C bond fission from the relationship K = k f / k , became available as follows. In 1979, Endicott and Netzel used picosecond flash photolysis of AdoCbl to obtain kr2 values of approximately 2 x 109M11s-l for the second-order rate of recombination of the freely diffusing species, close to the d i e sion controlled limit of about 10” NI-’ s-’ and of krl about 1.3 x lo9s-l for the first-

COBALT IN VITAMIN

Bj2

AND ITS ENZYMES

641

order rate of recombination of the geminate radical pair 11301. S e ~ q u a n t i ~ t i v e values of k f were estimated by extrapolation of kinetic results at around 9O"C, which were reported by the groups of Finke [1311, Hdpern [1321, and Pratt C731 in 1984-1985. In 1985, the author's group estimated a half-time of about 10 years for homolysis of AdoCbl at ambient temperature, which corresponds to a kf of about s-' [731; Hay and Finke later used their data to estimate the same value of k f at about lo-'*' 8-l [133]. This good agreement between the two groups gives cons-'. Combination with the published value of fidence in using a kf value of about Kr2 then gave logK of around -18 (in units of M)* for Co-C bond homolysis according to the simple equilibrium (1)involving free products L731. Since reaction (2) produces a bound (Co-radical) pair, krl should in principle be used in preference to k,; in practice this results in the same value of logK -18 (as a dimensionless ratio) or AG +24.5 kcal/mol. Further comparison with the reported rates of inactivation of some mutases in air in the absence of substrates (taken as a qualitative measure of labilization by the protein done) and with reported turnover numbers of up to 370 s-' with substrate indicated an overall labilization of the Co-C bond in AdoCbl by a factor of 2 lo1', built up in two stages: a smaller step (stage I) of 5 10' when AdoCbl is bound by the protein and a larger step (stage 11) of 2 lo6 when substrate is added "731. Hay and Finke first suggested an overall labilization by 2 lo1' [1311, later amended to >_ 1011 11331. By 1985, both the source of the problem and its magnitude had become clear; the enzymatic equilibria and rates can now be updated as follows. The enzymes increase the apparent equilibrium constant logK for Co-C bond fission by a massive factor of about +18 from -18 to 0 f 1 (see Sec. 4.1.3). This reflects the combination of an increase in the forward rate constant log k f by ? 11from about -9 to about -1-2 and a decrease in the reverse logk, by -7 from 9 to 2 (all s-l>. Steady-state turnover numbers span the range from 2 to 300 s--' (1341 but a pre-steady-state rate of > 60 0 s-' was reported for M ~ C M L135al. In spite of a similar increase in the overall logK and log kf in all mutases, the proportion of the increase in log f i f achieved on binding to the protein alone (stage I) varies from < 50% to about 75% and, conversely, the further increase on binding the substrate (stage 11) from > 50% down to about 25% (see See. 4.1.4).The key questions are therefore the mechanism of labilization at each stage, and the why and how of partitioning between the two stages. Evidence for the mechanism of stage I1 is now available and is considered first.

4.1.3. Co-C Bond Labilization on Binding the Substrate (Stage II) The recent structure determinations o f ~~C~ by Evans and co-workers, culminating in 1999 1531, have revealed the overall mechanism of Co-C bond fission associated with substrate binding (i.e., stage 11) in considerable detail (see See. 2.4). The "Note added in proof. Kinetic studies of Co-Cbond fission have now been extended down to 30T, using AdoCbl with a tritiated ligand IK. L. Brown and X. Zou, J. Inorg. Biochem., 77,1a5 (199911; extrapolation over 5" provides a similar value of logK -18.2 at 25°C.

PRATT

642

binding of substrate is associated with a major conformation change as the TIM barrel closes around the substrate, expelling more than 30 molecules of water, and knocks away the adenine of the coordinated Ado ligand. This, in turn, exerts sufficient leverage around the coordinated C atom to break the Co-C bond, with no detectable contribution from upward folding of the corrin ring, in agreement with the above results. Solution studies on GluM by Marsh and co-workers 1136,1371 and on RNR by Stubbe and co-workers C64,1341 in 1998-1999have established the third point (cf. Sec. 4.1.1), i.e., that Co-C bond fission according to equation (2) or (9) represents a genuine equilibrium involving substrate or allosteric effector, respectively. In addition, the initial species of each equilibrium was identified as the Michaelis (or analogous) complex; the values reported for the dissociation constant K , (in mM) are given here as the reciprocal formation constant (in M-’). The UV-Vis spectra observed in such enzymatic studies have generally been interpreted in terms of only AdoCbl and Co(II)-Cbland the possibility of a third species (see below) ignored; this may introduce an error into the values quoted. Stubbe’s group obtained a value ofK = 2.0 f0.3 at 37°C for equation (9) in RNR D341 with a binding constant of logK = 4.4 for the allosteric effector dGTP [64]. Marsh’s group found that 2-ketoglutarate is capable of promoting Co-6 bond cleavage in GluM (using a fusion protein of the two subunits) and of undergoing HID exchange with AdoCbl without subsequent rearrangement, allowing them t o study Co-C bond fission under true equilibrium conditions with a single pseudosubstrate. The W-Vis spectrum of the equilibrium mixture indicated the presence of about 35% Co(I1) and about 65%undissociated AdoCbl, corresponding to a value of K 0.5 for equilibrium (2) at 10°C with the Michaelis complex (no K , reported) as the initial species tl361. Marsh’s group also studied the enzymatic reation, where equilibrium may include the initial and final species derived from each of the two interconvertible substrates. They found that both substrates eventually produced the same “equilibrium” spectrum, corresponding to a value of (the composite) K 0.25 at 10°C for equilibrium (2), and determined values of logK = 3.2 and 3.9 for the Michaelis complexes with L-glutarespectively [137]. Methylmalonyl-CoA binds to matt! and E-threo-3-methylaspartate, M1MCM with a similar value of logK = 3.9 at 20°C [135bl. Equation (2) should therefore be expanded as in (2a + 2b) below to indicate (2a) the initial formation of the Michaelis complex, followed by (2b) the coupled conformation change and Co-C bond fission; equation (9) should be expanded in an analogous way.

-

-

[I?* .Co-Adol+ HZ * [P*.Co-Ado.HZ]* [P.CO~II),Z.A~O~]

(2a-t-2b)

In the reactions catalyzed by GluM, slightly higher concentrations of Co(II), and correspondingly lower concentrations of AdoCbl, were observed during the steady state than at equilibrium when using methylaspartate as substrate; with glutamate as substrate their concentrations remained almost unchanged r1371. This suggests that the steady-state concentrations observed during enzymatic reactions may be used to calculate approximate values of K for equilibrium (2). The apparent steady-

643


-

state concentrations of Co(Ii1) reported for other mutases with their physiological substrates (together with the value of K calculated here) include MMCM at 25"C, 22% (K 0.25) 1135al; DiolD at 3O"C, 45% ( K 0.8) [1381; ethanolamine ammonia lyase at 25"C, 58% (K 1.4) but rising to 3 95% (K > 20) with the unphysiological substrate r,-Baminopropanol 11391. A value of logK = 0 f 1can therefore be taken as representing the values for all five mutases with their physiological substrates. Comparison with the value of log K = -18 for free AdoCbl (see above) shows that the enzymes increase the apparent logK by a massive factor of about 18. This also demonstrates that the primary effect of the protein is, as expected, to increase the equilibrium constant for the dissociation of AdoCbl to form Co(I1) and a radical, which is associated with a smaller increase in the forward rate and a decrease in the reverse rate. Stubbe's group provided evidence that the increase in K is due mainly to a favorable entropy change that overcomes the adverse enthalpy change found in free AdoCbl, and they suggested that this might be associated with closure of the binding site and the release of bound water 11341; there is an obvious parallel with the conformation changes found for MMGM. It therefore seems reasonable to assume that a large increase in entropy, associated with the closure of a binding site and the release of water molecules, provides the common mechanism for achieving the large increase in logK observed for both equilibria (2) and (9).A n estimate of the entropy change associated with the known conformation change in MMCM would be useful. There is considerable evidence for an additional intermediate in the enzymatic reactions between AdoCbl and Co(IGCb1. Studies on free alkyl-Cbl's suggested 172,731 that a sudden distortion of the alkyl ligand would rupture the more labile Go-N bond, converting the red base-on Cbl ( m a . at 520 nm) to a yellow five-coordinate base-off form, before the more covalent Co-C bond and that increasing distortion would move the absorption band of the yellow form increasingly from 460 to about 440 nm or even beyond, before formation of the yellow five-coordinate Co(l1)-Cbl;the spectra of the five-coordinate forms with Ado, Et, and cyclohexyl as ligands of increasing strain are compared in Fig. 9. Figure 10 shows the direct conversion of proteinfree AdoCbl into Co(II)-Cbl, which should be sufficient to interpret the steady-state spectra of the enzymes if no other corrinoids are involved as intermediates; there is no significant change in the Co(I1) spectrum either with solvent [1411 or on replacing is as ligand Ll42J.The steady-state spectrum of EAL (Fig. ll), on the other a band 440 nm [59,1391, which clearly cannot be ascribed to either 475 nm) or the six-coordinateAdoCbl. Similar anomal nds at 440) nm can also be seen in the steady-state spectra of 11381, CM t135a1, and probably GluM 11361, while anomalous biphasic etics (derived from W-Vis spectra) have been reported for ~~C~ E1353.1, uM [1371, and RNR [123,134,1431. The observalion of similar anomalies in the three groups of mutases (the ~ n ~ m u t 3 . s have e s not yet been studied) suggests some common mechanistic feature, but the form of Co(11) detected by ESR (see Sec. 4.2) is unlikely to affect the kinetics or to exhibit a charge-transfer band at such low energy. Although other explanations are possible, the simplest assu must be that they reflect the ex ected occurrence of (possibly several) strained five-

-

-

-

-

-

-

-

644

FRAT?

COO

350

150

600 nm

550

500 Wavelength

FIG. 9. Comparison of the UV-Vis spectra OP aqueous solutions of the yellow five-coordinate (protonated “base-off’) forms of cobdamins with the ligands Ado (---I, Et (-1, and cyclohexyl (. . .) to show the effect of increasing compression around the coordinated C atom on the spectra (acting as a “molecular strain-gauge”). (Reprinted with permission from J. M. Pratt, “Coordination chemistry of the BI2-dependent isomerase”, in D. Dolphin (ed.), B,2, Val. 1, pp. 325-392. Copyright (1982) John Wiley and Sons, Inc.)

0-1

I

I

400

I

I

500 Wavelength (nm)

\

E ‘0

FIG. 10. UV-Vis spectra (recorded at 26°C) showing direct conversion of AdoCbl to Co(I1)-Cbl with good isosbestic points (by thennolysis in ethylene glycol at 100°C). (Reprinted with permission from B. P. Hay and R. G. Finke, “Thermolysis of the Co-C bond in adenosylcobalamin (coenzyme pJ1p)-IV77, Polyhedron,, 7, pp. 1469-1481. Copyright (1988) Elsevier Science.)

645

COBALT IN V I T A ~ 812 I ~ AND ITS ENZYMES 0.2 0.18

Cob(X1)alamin 0.16

0.14 0.12

0.1 0.08

0.06 0.04

375

425

475

525

575

626

avelength (nm)

FIG. 11. UV-Vis spectrum of the ethanolamine ammonia lyase steady state with propanolm i n e as substrate to show the presence of absorption at about 440 nrn (cf. Fig. 9) which does not belong to either AdoCbl or Co(lI)-Cbl (cf. Fig.10). (Reprinted with permission from V. Bandarian and G. H. Reed, “Ethanolamine ammonia lyase”, in R. Banerjee (ed.), Chemistry and Biochemistry of B I Z ,pp. 811-833. Copyright (1999) John Wiley and Sons, Inc.)

coordinate “base-off ’ forms of AdoCbl as intermediates between the red six-coordinate AdoCbl and the yellow five-coordinate Co(I1)-Cbl. 4.1.4.

Co-C Bond Labilization by the Protein Alone (Stage I)

The following rates of Go-C bond fission have been reported for enzyme-bound AdoCbl in the absence of substrate but in the presence of air or other reactant: 1. Ethanolamine ammonia lyase (EAL), 0.2 s.-’ at 23” [1441;cf. turnover number of 120s-1 in the presence of substrate [1451, ie., the protein alone causes an increase in logkf for Co-C bond fission by +8 and the binding

of substrate a further but smaller increase of +3. 2. DiolD, tllz 20 min, i.e. , 6 x lo-* s-l at 30” [1461. 3. RNR, 3 x s-’, probably at 37” ([1341, footnote 4).

-

No analogous rates have been reported for MMCM or GluM; they appear to be more s-’ (t1,Z > 2 h) can probably be assumed. The proportion stable, and a rate of < of the overall increase of about 11 in logkf which is achieved by the protein alone (stage I) therefore falls in the order: EAL (- 75%) > DiolD and RNR (- 50%) > MMCM and GluM (.= 50%). Since an increasing contribution from stage I will increase the risk of loss of enzyme from unwanted side-reactions in the absence of substratc, the observation of a large stage I contribution (e.g., in EAL) suggests this might be an unfortunate but inescapable part of the mechanism. In the case of EAL there is direct evidence that the initial binding of AdoCbl by the apoenzyme is fol-

PRATT

646

lowed by a relatively slow conformation change to produce the catalytically active and air-sensitive form D391. There is some evidence in both EAL [59,1471 and DiolD [148,1491 that the mechanisms of Co-C bond labilization in stages I and I1 share common features. It is unlikely that the binding of small substrates, such as ethanolamine or ethylene glycol, would be able to release as many water molecules as is seen when ~ M binds C its ~much larger substrate or t o generate the increase in entropy needed to increase logK to the required value. Therefore, one role for the other polypeptides in the multimeric mutases (Sec. 2.3) might be to control the partitioning of the required labilization between stage I (on binding to the protein) and stage 11 (on binding the substrate), perhaps acting as a device to use some of the AdoCbl-protein interaction energy from stage I to build up strain in the resulting “tense” (P*)conformation. This could then be released by conversion to the “relaxed” (P) form and used to assist Co-C bond cleavage on binding the substrate in stage 11.Brown and coworkers have reported the very high value of logK = 16.7 for the binding of CN-Cbl by haptocorrin at 25”, higher even than that of the biotin-avidin pair. They pointed out that, since binding constants with the mutases are much smaller, “a substantial amount of binding energy may be utilized to physically distort. the coenzyme” 1901, perhaps also indirectly via distortion of the protein. Labilization by the protein alone (stage I) is obviously important, especially for RNR and the eliminases, but has received little attention in comparison with labilization by the substrate (stage TI). Of the three main components of the original (1975)proposals, the existence and nature of a significant substrate-induced conformation change was established in 1998 [67], Co-C bond fission induced by distortion of the Co coordination sphere (more speciiically “leverage around the coordinated C atom”) in 1999 I531, and the primary role of the protein in changing the equilibrium constants of the coupled equilibria (2) and (9) in 1998-1999 1134,136,1371.

o-Radical Charge-Transfe r Homolytic fission of the Co-C bond in the enzyme generates a substrate-derived radical and thc? low-spin d7 Co(1I) ion, which is the only ESR-active form of the Cbl. When the solutions are rapidly frozen, the combination of these two species produces unusual ESR signals (see reviews in 1150,1511).These may range from (1) an organic radical centered at g = 2.0, which appears as a doublet due to weak coupling with the unpaired electron on Go(II), to (2) signals characterized by a g-value of 2.1-2.2, intermediate between those exhibited by a simple organic radical and by the Co(I1) alone, and exhibiting an hif structure with splittings that indicate strong coupling between the two species. Such unusual ESR signals were observed on the addition of substrate or substrate analogue to DiolD and EAT, (both weak coupling) or, in NR, an allosteric effector (strong coupling with a tKy1 radical) in the , and with the C-skeleton mutases MMCM, methylene glutarate mutase and (all strong coupling) in the 1990s. These unusual ESR spectra were successfully


647

modeled and interpreted independently by Coffman and Pillbrow and their co-workers in thc late 1970s by invoking both dipolar exchange and isotropic exchange, i.e., direct overlap between the atomic orbitals of both partners over a distance of 5-10 A. This is compatible with the more recent X-ray data, which suggest distances from the Co to the radical centres on the substrate and product of 6.2-6.5 A in MMCM, 6.6 A in GluM, and 8.44.0 Ain DiolD (see Sec. 2.4.3).The weaker coupling observed for DiolD may reflect the longer Co-to-C distance. The formation of a Co(II)-radicalpair with a similar ESR signature by the photolysis of protein-free AdoCbl at 77 K demonstrates that protein i s not required for their formation 11521. ESR was also used outside the BIZfield by Lerch, Mims, and Peisach in 1981El531 to demonstrate that a protein-bound free radical and a nonbonded metal ion in, for example, Compound I of the hemoprotein cytochrome c peroxidase may be coupled together in what they called a “CT complex” through sharing of the wave function. By measuring the electric field effect they showed that the wave function with its associated eledron density may be displaced (i.e., charge may be transferred) under the influence of an applied electric field. A similar transient redox interaction between the Co and radical C atoms would clearly be possible. Electron transfer between nonbonded sites in a protein is well understood; rates of approximately lo9 s--’ are observed over distances of 10 A, i.e., greater than that in DiolD, and electron transfer can be detected at distances up to 22 A 11541. Electron transfer from Co(1) corrinoids to organic alkylating reagents is also well established. Using an electrochemical technique in the presence of acrylonitrile to trap any free radicals formed, Walder and co-workers showed that Co(1)reacts with the less bulky reagents via a one-step S,Z substitution but with the bulkier neopentyl-,isopropyl-and sec-butyl iodide via an initial electron transfer from Co(I) to produce iodide and the radical, which then couples with Co(1I) in a second step to form the Co-alkyl bond 11551.The key question is whether rearrangement of the radical (see Sec. 4.3) requires assistance from the associated Co(I1)in the CT complex. We have adopted Peisach’s chemically more meaningful “CT complex” in preference to the commonly used “exchange-coupled” radical pair, which focuses on the physical properties but diverts attention from its chemical potential. Protein-free Co(IIbradica1 pairs can be generated as transients near ambient temperature by the photolysis or thermolysis of a preformed alkyl-Co corrinoid or cobaloxime. Following the report that certain free radicals undergo rearrangement only in the presence of Co(II) (see Sec. 4.3,many ingenious methods were devised to increase the residence time of the radical in proximity to the Co before difksing away and even to develop semicatalytic systems.

4.3.

Rearrangement of the Substrate-Derived Radical

Attention is here focused on mechanisms that can most easily accommodate experimental observations from the widest possible range of mutases and from analogous protein-free corrinoids. On this basis the many protein-free models and theories for the rearrangement of diols to aldehydes, which are proton- or hydroxide-dependent,

PRAlT

648

are excluded because the overall activity of DiolD is pH-independent over the pH range 6-10 [601, as is that of EAL over the range pH 6.6-8.2 1611. Conversely, the 1,2 migration of an sp2-hybridizedC or N, capable of forming a three-membered cyclic intermediate, is well known (cf. the likely scheme for the methylmalonyl-CoA radical in Fig. 121, while the requirements for the 1,2 migration of the sp3 C in GluM, the sp3 N in ethanolamine, and the sp3 0 in ethylene glycol are much more restrictive. 1,2Alkyl migrations form the core of this section. 4.3.1. Glutamate Mutase: Migration of the sp3 C Atom

The general observation of easy 1,2-alkyl migrations in carbocations but not in radicals or carbanions was explained by, among others, Zimmerman and Zweig in 1961 D561 and Zimmerman in 1972 11571.A strongly antibonding MO in the three-membered transition state carries 0, 1, and 2 electrons in the carbocation, radical, and carbanion respectively; where this MO is even singly occupied, rearrangements are energetically difficult. In principle, therefore, rearrangements might be promoted by reducing the electron density in this MQ through interaction with either proton donors (excluded in at least some mutases) or electron acceptors. The earliest examples of such 1,2-alkylshifts were provided by oxygen-containing diradicals in the 196Os, but in 1972 Walling and Cioffari [1581 reported the first clear-cut case of an intramolecular 1,a-alkyl shift in a monoradical without hetero atoms. They provided evidence for the reversible interconversion of the 2-phenyl cyclopentylmethyl and 2-phenyl cyclohexyl radicals and suggested “a transition state with considerable carbonium ion character” due to partial charge transfer to the phenyl group, as shown in 7. Following initial reports of the C-skeleton isomerization of alkanes over Pt and Pd films in the 1960s, Rooney’s group established one case in 1977 [159] and a second in 1982 11601 wherein an adsorbed alkane required the abstraction of only one W atom to undergo isomerization. They emphasised that a transition metal has orbitals of the correct symmetry to overlap with antibonding orbitals of the three-membered cyclic transition state which, if unfilled, can remove charge from the radical and allow it to react more like a carbonium ion [1611. In 1977, Salem et al. proposed an analogous mechanism for the Bt2-dependentisomerization of’ diols to aldehydes, which involves the pHindependent migration of an sp3-hybridized Q atom [162]. Their theoretical calculations indicated that “complexation by the Co atom (could provoke) electron transfer and consequent rearrangement in a pseudoCHZ HC-C-SR I 8

I

COOH

Q

e

43

HZC,

O ,

.-

SK H \‘!

I

COOH

HlC- -C-SR CHr 1

I

COOH

FIG. 12. The generally accepted mechanism for interconversion of the radicals derived from methylmalonyl-CoA (left) and succinyl-CoA (right), the substrate pair for MMCM, via a cyclopropyloxy intermediate, The radical intermediate probably acquires some carbonium ion character through transient and partial electron transfer to the Co(I1) ion (see text).

COBALT IN V I T A ~ I N 12 AND IT§ ~ N ~ Y ~ E S

649

cationic substrate” and that complexation could reduce the activation energy for rearrangement by 40 kcaVmol compared to that in the isolated radical and only 4.6 kcallmol above that in the carbonium ion. 1,2-Alkyl migration can therefore be promoted by removing electron density from the three-membered transition state by partial charge transfer either to a nonbonded organic group or to a bonded transition metal ion. It appears that 12 exemplifies the intermediate case of transfer to a nonbonded transition metal ion.

7 Protein-free models for 12-dependent C-skeleton rearrangements, involving migration of an sp”-hybridiz were reported from 1975; see, for example, L1631661. They usually involved the thermoIysis or photolysis of an initially formed Co-6 bond, which generates the radical in the presence of Co(I1). In 1975, RBtey’s group reported the rearrangement of a methylmalonyl radical possessing two ester groups (i.e., no thiol ester) prepared by photolysis of the cobaloxime [164]. In 1979, they showed that no rearrangement occurred in the absence of Co but that increased residence time in proximity to the Co increased the yield of rearranged product; they suggested a “catalytic role for the Go atom in the rearrangement” with ‘‘trimsient redox processes between Go(I1) and the substrate radical” [1671. In 1976, CPMbCbl in aqueous solution is Chemaly and Pratt showed that ~yclopropyl~ethyl( stable at room temperature (< 10% decomposition over one week) and will rearrange to allylcarbinyl(AC)-Cbl only on photolysis or heating (half-time of about 105 min at 60°C) [l65], even though the free CPM radical will spontaneously rearrange to the AC radical in solution at -140, i.e., formation of a Co-6 bond inhibits rearrangement. Combining these results, but not yet aware of the 1981 results of either Peisach (See. 4.2) or Rooney (see below), we suggested in 1982 [721 that the overlap of atomic orbitals revealed by the ESR spectra (See. 4.2)could allow the Co(I1) ion to remove electron density from (i-e.,place a partial positive charge on) the radical center during the act of rearrangement and in phase with the movement of the nuclei (equivalent to changing the applied electric field) to form a “Go-promoted carbonium ion”. The first example of a 1,2 migration of an sp3 C, catalyzed by protein-free corrinoids, was reported by Rooney and co-workers in 1981 studying rearrangements of the tetramethylcyclohexyl (TMC l,2-alkyl shift may involve either an Me group or a C atom of the cyc cyclopentyl derivatives) and found that the reaction of TMCH tosylate Cbl in EtOH (in the presence of excess borohydride) gave re ing from both possible migrations [1681, as did hydrolysis o oacetic acid which reacts via the carbonium ion [1691. They later showed that reaction

PRATT

650 of the xanthate 8 with tributylstan

ne to produce the radical in the absence of Co gave only the unrearranged product r1701. The authors assumed that rearrangement could occur only while coordinated to the Go, but this seems unlikely since steric hindrance prevents even the less bulky neopentyl (np) iodide from reacting directly with the Co(I) (Sec. 4.2). In fact, calculations using molecular mechanics [17U indicate that nonhonded steric repulsions rise to more than 50 kc4mol as the Co-toC(TMCH1 distance falls to 2.27 A, which is still considerably longer than that expected for np-Cbl (2.06 A) by analogy with the cobaloxime [l271; even allowing the full Co-C BDE of 24 kcal/mol determined for np-Cbl [1721 would leave a CoC ~ T ~ bond C ~unstable ) by about 25 kcalimol! It seems unlikely that the Co-radical pair could form a Co-C bond in any meaningful sense and it should therefore be considered as a CT complex (Sec. 4.2).

n & I X

a.x=O

d. X= xantliate

b. X= tosylate

e. X= H

c. X= OH

8

By the early 1980s the use of protein-free corrinoids and cobaloximes had (1) provided a model for the elusive 1,2-alkyl shift, (2) shown that substrate-related radicals where the migrating group was an ester required the presence of Co(II1, and ( 3 ) shown that formation of a Co-C bond inhibited rearrangement. In addition, Peisach and co-workers had shown (Sec. 4.2)that transient redox reactions of the type proposed by Rhtey and by Pratt could indeed occur in a CT complex between nonbonded partners, such as the Co(1I)-radicalpair. Results on the GluM enzyme and its Glu substrate were only added during the 1990s. The rearrangement of @-Me-aspartate2 to Glu 1 (as diethyl esters) in the presence of Co(I1) was successhlly achieved by Murakami’s group in 1987 [173] through photolysis of the relevant alkyl-Co corrinoid (with esterified side chains) embedded in hydrophobic vesicles in aqueous solution at 2 0 . In 1992 El741 they established that rearrangement was occurring through migration of both the glycyl group (7043%) and the ester group (17-30%); the enzyme is specific for glycyl migration. No glycine or acrylate, as required by an alternative fragmentation-recombination mechanism (see below), was detected when analyzing the products [175]. The ESR “signature” of the CT complex in GluM, which had not previously been observed, was reported in 1994 [1761; this establishes the existence of the required Co(Wradica1 CT complex. The structure of the enzyme was reported in 1999 r661; the data suggest a distance of about 6.6 between the Co and the radical C atom, which effectively excludes the other alternative mechanism involving Co-C bond formation to the substrate radical. The 1,2 shift of the sp‘-hybridized C observed in the enzymatic rearrangement of Glu can therefore be modeled and explained on the basis of a Co-dependent “noncla~sical’~ mechanism with adequate precedent, in which partial donation of electron

A

COBALT IN VITAMIN 8 1 2 AND ITS ENZYMES

651

density to the associated Co(I1) in the CT complex serves to stabilize the nonclassical three-membered transition state. No other proposed mechanism is compatible with the available data. One would expect an analogous Co-assisted nonclassical mechanism for the pE-independent migration of OH in DiolD and of NH, in EAL. Unfortunately, the various protein-free models are dominated by proton- and hydroxide-dependent paths and no pH-independent path has yet been convincingly demonstrated against this background. 4.3.2. Other Mutases: Migration of the sp2 C Atom

The three C-skeleton mutases other than GluM involve the 1,2 migration of an sp2 C atom. The aminomutases probably involve migration of the sp2 N atom in the Schiff base ketimine formed by condensation between the amino group and the pyridoxal phosphate cofactor 1621. There appears to be no 1,2-migration of any atom in RNR (Sec. 2.1). It is generally accepted that the classical path for the 1,2 migration of an sp2 C atom involves a cyclopropyl intormediate, such as the cyclopropyloxy radical shown for MMCM in Fig.12. R6tey’s group has prepared substrate analogues for MMCM by keeping the CoA “tail” constant and varying the succinyl “head” and have shown that the presence of the carbonyl group, but not the S atom, is essential for rearrangement [177,1781, in agreement with formation of the cyclopropyloxy intermediate. Wollowitz and Halpern [179] have compared the migratory aptitude of several functional groups (all with sp2 C) as substituents in radicals derived from dimethylmalonic acid in the absence of Co. They established the order olefin (modeling methyleneglutarate mutase) > ketone- 60-Me > -Ph > thiolester - 60-SEt (modeling MMCM) > ester-CQ-QEt. The rates fall from kl = 3 x lo5 s-l at 45°C for the olefin to .c; 10s-I at 113” Le., not detected) €or the ester. Extrapolation of the rates observed for the thiolester over the range 61-120°C gave a value of 2.5 s-’ for migration of the thiolester group at 20”; as they pointed out, this is considerably less than the value of about 1 0 ‘ observed ~ ~ ~ with the MMCM enzyme. Ester migration requires catalysis by Co(I1) (see, e.g. ~167,179,1801),whereas thiolester migration appears to be a borderline case, catalyzed by Co(I1) under some conditions but not others, in agreement with the above order of substituents. The order -CO-Me > -60-SEt > -CQ-OEt may reflect increasing destabilization of the three-membered cyclopropyloxy radical intermediate due to increasing repulsion between the lone pairs as the substituent is changed in the order Me < SEt < OEt. This, in turn, would explain the role of CotII) as beneficial in assisting thiolester migration and essential in assisting ester migration. The parallel operation of both the Co-dependent nonclassical path and a Co-assisted classical path (for glycyl and ester migration, respectively) in the protein-free model for GluM (see above) further suggests that participation by Co in a CT complex is a common link in the mechanism of’ all the mutases. We therefore suggest that the Co(I1) ion in the CT complex serves to remove electron density from the radical in the transition state, whether reaction occurs via a Co-dependent nonclassical mechanism (as in GluM) or a Co-assisted

652

PRATT

classical mechanism (asin MMCM). This would explain the common occurrence of CT complexes with their unusual ESR spectra throughout the mutases, and also the apparent lengthening of the axial Co-N bond length observed in dl of the mutases studied (Sec. 2.41, which could promote partial reduction of the five-coordinate Co(1I) to the normally four-coordinate Co(1) state. Further promotion or control by the protein through steric and/or coulombic effects would naturally be expected, but mechanistic proposals that completely ignore the possible role of charge transfer from radical to Co are probably unrealistic. The two other mechanisms that have recently been advocated are (1)rearrangement as the alkyl-Cbl, formed by reaction between Co(I1) and the substrate radical [1811, and for the C-skeleton mutases ( 2 ) fragmentation into acrylate and a radical (e.g., glycyl from Glu), followed by recombination at the vicinal C atom [1821. The first conflicts with the known inhibitory effect of Co-C bond formation in protein-free corrinoids (see above) and is effectively excluded by the distances observed in the three mutases (see Sections 2.4 and 4.1). The fact that acrylate and e.g. glycine together (but not alone) will cause Co-C bond fission in GluM was cited in support of (21, but other explanations are possible; the ability of component parts to act together as the whole is not unknown, even in the BI2 field [%I. It conflicts with the high yields (up to 97%) of rearranged product reported for some protein-free models [1811and, conversely, with failure to detect any crossover products in experiments designed to test the fragmentation-recombination mechanism in ketone or thiolester migration 1179,1831, failure to detect acrylate or glycine as products from the protein-free model for Glu rearrangement (see above), and failure to detect Glu as the reverse product of condensation from acrylate and Gly in the enzyme [561. Neither mechanism suggests any explanation for the general occurrence of CT complexes with their unusual ESR signature. Ideas and results on 1,Z-alkylshifts in radicals have been few and scattered and have never previously been brought together. Nevertheless, it was surprising to realize that €ew if any of those most closely interested in the B12-dependentmutases were aware of the work of Rooney [168-1701, let alone the work of Peisach [1531 and Walling [1581. The central mystery of BIZis not the mechanism of radical rewrangement but rather the failure of databases and intelligence gathering. B12has, in fact, made a substantial contribution to our understanding of rearrangements in general and to the emerging field of 1,Z-alkylshifts in radicals in particular. Such a shift may be made possible by some donation of electron density from the nonclassical cyclic transition state to a bonded (i.e., coordinated) transition metal ion, to a nonbonded organic acceptor or, as now exemplified by BIZ, to the nonbonded transition metal ion in the Co(I1)-radicalCT complex.

4.4. Summary of Main Points (1) A comparison of the properties of free and protein-bound AdoCbl (Secs. 4.1.2. and 4.1.3)shows that the mutases are capable of increasing the apparent equili-


653

brium constant for homolytic fission of the Co-C bond logK by a massive +18 from -18 in free AdoCbl to about 0 in the mutases when substrate is bound; this reflects a smaller increase in the forward rate constant logkf by 2 11from about -9 to about +2 and a decrease in the reverse logk, by -7 from 9 to 2 (all s-’). Since logk, falls, the role of the protein cannot be described as stabilization of the transition state, except indirectly as a consequence of stabilizing the products of the equilibrium, and changes involving the coenzyme cannot be treated in isolation from changes to the protein. (2) The overall increase in logR is achieved in two stages, i.e., when AdoCbl is bound by the apoenzyme (stage 1)and when the substrate is bound by the holoenzyme (stage 11).The proportion of the total increase in logK represented by stage I varies from about 75% in EAL to less than 50% in NPMCM (Sec. 4.1.4).Little is known about the changes involved in stage I, which is revealed as a slow protein-catalyzed cleavage of the Co-C bond by reagents such as oxygen. (3) Stage 11involves fission of the Co-C bond and a major conformation change. The steady-state UV-Vis spectra and the kinetics suggest the occurrence of‘ some spectroscopically distinct intermediate, which may be a highly strained, five-coordishow nate form of AdoCbl (Sec. 4.1.3).The X-ray structure determinations of that the binding of substrate is accompanied by a major conformation change and the loss of more than 30 molecules of water (already described in Sec. 2.4).Solution studies suggest that this step is associated with a significant increase in entropy (See. 4.1.3). (4) Protein-free corrinoidv have provided two examples of the 1,2 migration of an sp3-hybridizedC atom (including the glycyl group, as in GluM) under the influence of the nonbonded Go(l1) ion (Sec. 4.3.1) and also demonstrated that even the 1,2 migration of an sp2 C atom may require catalysis by the Co(I1) ion, e.g., where the migrating group is an ester (Sec. 4.3.2). The only mechanism that is not excluded by the experimental data involves rearrangement of the radical via either a nonclassical three-membered cyclic transition state (e.g., GluM) or a classical cyclopropyl intermediate (other C-skeleton mutases), together with removal of some electron density by the nonbonded Co(II) ion to give the radical some carbonium character. The mechanism also explains the occurrence of the Co(I1)-radical CT pair, with its distinctive ESR “signature”, as an intermediate. There appears to be no conformation change associated with the rearrangement. In retrospect, it would appear that the main points relating to both Co-C bond fission and radical rearrangement had been established in principle by 1985. ow ever, they remained controversial and had to wait until 1999 for adjudication by the structural data and the improved spectra, kinetic, and thermodynamic studies, which have now become possible thanks to the application of molecular biology.

5. P

CTlVE

Answers are rapidly being found for many of the questions about A ~ o and ~ the ~ l mutase reactions, which have been the focus of attention in the BIZfield for 40 years

PRATT

654

(Sec. 5.1). The spotlight will now almost certainly switch t o the ~ethyltra~s€erases and other reactions of the primitive anaerobic bacteria, which really form the B12 heartland, and to the information they may provide about evolution as well as enzymatic mechanisms.

5.1.

ependent Mutases: Precis of Present Results

Some of the challenging questions about enzymatic mechanisms that confronted BI2 researchers by the mid-1970s are listed in Sec. 1.1.Some 25 years later most of the questions relating to the mutases have probably been answered, at least in broad outline.

52.1. How Does th,e Protein Promote HowLolytic Fission of the Co-C Bond? (Secs. 2.4.2. and 4.1.2-3) Comparison of the properties of AdoCbl when free and protein-bound, both before and after adding substrate, shows that the protein can increase the apparent equilibrium constant for Co-C bond fission to give Co(1I) and a radical from logK = -18 in free AdoCbl to about 0 in the mutases, and that this massive increase of -k 18 in the value of logK is achieved in two stages: when AdoCbl is bound by the apoenzyme (stage I) and when substrate is bound (stage 11). Stage I1 is now well characterized in the case of at least one mutase (MMCNI) and shows the following features: a simple equilibrium between the Michaelis-Menten complex with substrate and intact Co-C bond and a second form with Co(II), AdoH, and the substrate-derived radical; a unique split TIM barrel that closes over the substrate, expelling more than 30 molecules of water; binding of substrate and Co-C bond fission probably associated with a large increase in entropy; the Co-C bond ruptured by a shear motion of the protein, linked to the hinge motion of the TIM barrel; evidence from the UV-Vis spectra and kinetics for some intermediate, probably a highly strained five-coordinate form of AdoCbl. This probably provides the first case in any protein where one can see how a conformation change is used to link together two equilibria (involving the substrate and the Co-6 bond) at separate sites. Discounting the honorary enzyme Hb, it also provides the best insight into protein machinery in any metalloenzyme. The changes involving enzyme-bound AdoCbl agree with those expected from studies of proteinfree corrinoids. The mutases show how the protein can use the egress of a large number of water molecules both to build up the required strain energy in small increments and to drive the mechanical hinge movement required to focus this energy at one point and rupture the Co-C bond-a reciprocating "molecular nutcracker or jaw-crusher'--with completely reuersibEe energy input/output!

655 5.1.2. How Do the Substrate-Derived Radicals tindergo Rearrangement?

(Secs. 4.2 and 4.3) Radicals, unlike carbonium ions, do not undergo 1,2-alkyl shifts unassisted. So how does the ~ 1 enzyme ~ M manage it? The 1,2 shifts both of alkyl groups with m sp3 C atom (as in the substrate glutamate) and of ester groups with an sp2 G atom have been successfully modeled in radicals derived from protein-free corrinoids, but a r e ~ u i r e ~ n efor n t the nonbonded Cof11) was also demonst~ate~. Taken together with the observation (from their distinctive ESR spectra) of enzyme-bound Co(~I}-radical GT pairs as reaction intermediates, this fairly conclusively establishes a mechanism ~ b ~ in~rmediate ~ ~ d or transiti~nstate, together with involving a t ~ r e e ~ m e cyclic paxtial removal of electron density by the nonbonded Co(II) ion to give the radical some carbonium ion character. 1,2-Alkylshifts in radicals may occur, it now appears, if the nonclassical cyclic transitio~state can be stabi~zedby transferring charge to (1) a coordinating (k7 bonded) transition metal, (2) some nonbonded (or indirectly bonded) nonmetallic acceptor such as a phenyl substituent or (3) a nonbonded transition metal ion in a 67:c o ~ p l e xas~ in the ~ ~ ~ - ~ e p e nmutases. dent

5.1.3.

? Why Doc?s Co(111) Form Such Stable Co-6 and Co(See. 1.5)

Go is a rare metal in the environment, so why the need for Co? The enzyme requires a metal ion that will form sufficiently strong M-C bonds and sufficiently accessible oxidatio~states to carry out the necessary catalytic cycles. Studies on simple M and other ions in the gas phase (M is a transition metal) have shorn that M-H and MC BDEs increase with increasing participation of the outer s orbital (4s in Co); the 3d orbitals are too ~ontractedto provide the overlap needed to form a strong semicovalent bond. They also provided an “intrinsic” M-H BDE of about 57 kcal/mol, similar to the Co-I3 EDE of about 58 kcaVrno1 determined for the fully ligated C o ~ I I I~entacyanide ~ ion; this provides direct experimental evidence for a high 4s character in Go-H and, by extension, Co-C bonds. It can also be shown that, among te low-spin complexes of metals of the first transition series, the most and M-C bonds are expected for the Co(II1) and CrCIll! ions, as is observed. The lower valent Cr(II) and CrU>required for enzymatic activity would, t . leaves Go as nature’s choice however, be inaccessible in a b i o ~ o ~ cea~l v i r o ~ e nThis for the widest rang?of o~~~anometallic chemistry in a m e t a l l o e n z ~ cBI2 . i s probably the first case where nature’s choice of metal can be explained and traced back to the electronic structure of the ion involved.

656

5.1.4.

Corrirt? (See. 1.6)

Co p o r p h ~ ~ occur n s in nature and one a s s o c i a t ~e n z ~ a t i reaction c is known f143,

so why the need for the much more complex corrinoids? The corrinoids have many e s are not e x h ~ b i by t ~ the unusual photo physic^ and photochemica~p ~ o p e ~ i that ~ ~ r p and h that ~ ~probably n ~ reflect the presence of one or more low-lying metastable electronic states. Therefore it is possible that the comin ring could offer the protein a ~ nas a much more readily ~ ~ i ~ ~or a‘~tunab~e’, t e d cis effect than the p o ~ h ring means to modifv and control the reactions o€ the Go; but this remains to be established. The corrin ring also dif€ers conside~ablyin steric ~roperties.There is no evidence that its unusual electronic structure plays any role in the mutases, but its steric p r o ~ ~ r tmay i ~ said Co-C fission by preventing u n p r ~ ~ ~ ~ ‘4sl~ppage” ctive as pressure is ap~lied(See. 2.4).Evidence for any ‘‘electronic’’role of the cosrin ring must be or even m o n g more primitive ~ * ~ - d e p e n ~ e ~ o~~~n~ ~ the ~methy~trans€er~se~ ~ t e n z ~ yet e ~to be discovered. ~ n d e r s t a n ~ the i n ~electronic structure of the corrin t ~most ~t ring and its mechanistic relevance remains one of the most i ~ p o ~ ”and neglected ~ ~ o b l e m insBlz chemistry.

Ado as Ligand? (Sec. 2.3) Any r ~ ~ ~bulky ~ alkyl a ~ ligand l y could ~ r e s u ~ a boffer l y the ~ o s s i ofb Co-C ~ ~ bond fission by steric distortion, as occurs in the mutases. Comparison of the structures of the m u t ~ s e sm d Me t r a n s f e r ~ e ssuggests that the former might have evolved from the ~ ~ ~and t that e r Lhe Ado ligand could have originated from the use of either ATP or A ~in the~‘ ~ ~ c~t i v a t i~~m~on d’ ’ut ~ eof~the methyltra~sferas~s.

As with all good detective stories, the final answers to each of the main questions in Secs. 1.5.1-1.5.4. include an unexpected twist-nothing really new, just the need to apply in practice what we probably know in theory and may even teach, but often forget or ignore. ider erst and in^ why Co forms such strong Co-C: bonds requires s ~ t c h i attention n~ from the popular 3d orbitals to the generally ignored 4s orbital. ~ n d e r ~ the t ~ ~~ ~gu es l u~ ca t ~r o ~s t~~cc t u r eof the corrin figand may require e ~ t e n d i nour ~ theoretical unders~~nding of the balance between one-electron resoc o u ~ o mrepu~sion ~ terms from the fairly r e ~ l a polyenes r to a very s ~ r a ~ ~ role of @o(II)in and b u c ~ ~ ecyanine, d such as corsin (Sec. 1.6.2).~ n d e r s t a n d i nthe the radical ~ e ~ r a n g e ~ emeans n t s ~ c c e p t i nthe ~ reality of the expo~entialdecay o f orbitals with distance, as taught to our u n d e r ~ a d u ~ t eand s , the possibility of transient and/or partial electron transfer between n o n b o n ~ epartners, ~ ~ n d e r s t a n d the ~ ~ role g of the protein in Co-C bond fission requires including the protein or “solvation” environment as an integral part of the Co-@bond. The mutases prov~dean object lesson in the dangers, firstly, of focus in^ exclusively on rate constants while ignoring equilibrium constants and, secondly, o f invoking the Hammond

COBALT IN V I T A ~ I NBj2 AND ITS ~

N

~

Y

M

~

~

657

postulate while ignoring the Hammond caveat that “the postulate deals directly with potential energy rather than free energy relationships.. .and entropy changes. . . are essentially uncontrolled by the postulate” El841. It is frequently assumed that the enzyme enhances r e ~ t ~ v iby t ys t a b ~ s i n some g t r ~ s i t i o nstate, In the ~~z-dependent mutases, however, the protein actually decreases the rate of the reverse reaction (required to regenerate the Co-C bond) by lo7 (See. 4.1.2);there is no seal evidence for the stabilization of any “transition state”. Co-C bond fission psobabl~proceeds via by e n t r ~ p y some d e t e c ~ b ~~e~ t e r m e ~ (See. a t e 4.1.3), appears to be do~n~nated changes, and reflects the role of the protein in changing the overaIl equilibrium constant which, by definition, also changes the ratio offorward to reverse rate constants. r~ Changes involving the Co-C bond in the enzyme cannot realistically be c o ~ s i d ein isolation from changes involving the protein environment. Perhaps most important of all, the Blz field so clearly demonstrates the importance of “model” studies. The ability to compare the structures and physical properties, e ~ u ~ l i b r iand u ~ rate , c~nstantsofthe cofactor in the enzymes directly with those of prote~n-freeCo corrinoids provides the means to p ~ p o i nand t quant~fythe role of the protein. Further comparison with noncorrinoid Co complexes provides the means to assess the separate contributions of the corrin ligand and the Co ion. 12 in the

~ e ~ oPast: t e l ~ s i ~ into h t Evotution ~

The recent structure d e t e r ~ i n a t ~ oof n sthe mutases and the ~ b l - b i n d ~ fn g~ a ~ eofn t MetS reveal a remarkably clear modular c o n s t ~ c t i o nin~which the corr~noidis held domain or module (See. 2,3); the same probably applies from below by a Cb~~binding to the Biz transport proteins (See. 3.2). This leaves the upper surface free to interact with other modules; in the Me t r ~ s f ~ r a s the e s upper face of the c o r r ~ ~ ~i si d s clearly exposed to diEerent modules in succession during the enzymatic reaction and for any additional “reactivation”. Sequence and module alignments between the mutases and the Me transferases suggest that the mutases may have evolved t r ~ f e ~ a s and e s that the latter may, in turn, have a c ~ u ~ some e d of their modules from n o n - ~ ~ ~ - d e p e n denzymes. ent One can virtually see exon shuffling in operation! So what was the role of eorrinoids before Me transfer? Leaving aside possible photochemical reactions, the linear relationship between rates of / C op ~o It ~e ~ (See. ~ i ~3.3) s u ~ e s that ~s met~ylationof Co(l1 and the ~ o ( ~ ~ ~redox any properties of the corrino~dthat are exploited for Me transfer ( e g , a tunable cis effect) could also have been exploited for electron transfer at low potential or for reactions involving the formation of complexes with other polwizable ligand atoms (eg., S or Se>. The additional parallels between AdoCbl and AdoMet as cofactors are instructive. A B12-independent lysine %,3-aminomutaseis known that requires AdoMet, a 4Fe-4S cluster, and a reductant but, like the Blz-depondent aminomut~es,also on is reversible f o r ~ a t i o n requi~esp ~ ~ i ~d h ~o xs ~[621. ~ ~The t ec ~ ~ m denominator of the Ado (or derived) radical, which may be achieved either from AdoCbl a ~ c o r d ~ n g

658

PRATT

to equation (1)or from the sulfonium cation AdoMet according to equation (4). Even Rs [63,185], which more surprising is the existence of three distinct families of share many features, including the use of a free radical, but differ in the nature of the radical and the means of producing it: 1. AdoMet is used to produce a glycyl radical, probably via the i n t e r m e ~ a t e formation of the Ado radical according to equation (4) (only in strict anaerobes). 2. AdoCbl is used to produce the Ado radical or, more correctly, a Cys thiyl radical (See. 2.1) (in both aerobes and anaerobes). 3. An Fe-0-Fe center is used to produce a tyrosyl radical (in aerobes, including humans).

The exchange and addition of regulatory domains to improve the specificity and control of a given catalytic domain is well known in, for example, the protein kinases, but the close functional parallel between AdoCbl and AdoMet as cofactors for both and aminomutases is probably unique. It demonstrates that the interchange of ns can include the interchange of cofactors and their associated cofactor binding domains. The above evidence suggests that the exchange of modules or domains may have occurred much more readily on a “pick-and-mix” basis in the distant past. The concept of a purely linear evolution of proteins, whether divergent or convergent, may therefore be too simplistic. Other unusual features shared by all of the RNRs include a single catalytic site reduction of all four substrates (ribonucleoside triphosphates in the case of the -dependent RNRs, diphosphates in the other groups) and an additional allosteric site for binding deo~ibonucleoside triphosphates, which act as allosteric effectors to control the substrate specificity of the catalytic site and maintain the required balance between the four products. Reichard has used the occurrence of this same allosteric control mechanism in all RNRs to argue for a common ancestor and, “if we accept divergent ev~lution’~, to argue that the AdoMet-dependent RNR is most closely related to the “Ur-reductase” [1851, However, the allosteric control mechanism bably developed and refined through a succession of production models. of the ready exchange of modules, each improved model could in turn have become associated with each of the available radical-generating modules to produce a multiv~iable“matrix”, not merely a linear process, for the evolution of the RNRs. have suggested that “the replacement of RNA by DNA,. .was probably the key step at or near the base of the whole evolutionary tree as we now know it” and speculated on the relative claims of AdoMet or AdoCbl as the main cofactor “before or after final consolidation of the DNA revolution” 11161. In the light of the evidence for a more ready exchange of modules in the earlier stages of evolution, we suggest that the base of the evolutionary tree may reflect the appearance of the present model of the allosteric control mechanism which (probably like its precursors) rapidly became associated with both the AdoMet- and AdoCbl-dependent radical-generating modules. It may be unrealistic to expect a simple divergent evolution of the RNR enzymes.

AdoMet and AdoCbl provide the main cofactors for enzymatic reactions involving sp3 C-centered radicals that until recently were considered too reactive to be used in a biological environment. Six different AdoMet-dependent reactions have now been discovered, all of which probably involve the reversible formation of free radicals by one-electron reduction of A ~ o ~ esee, t ; for example, l l 8 G l . Buckel and Golding have pointed out that radicals offer considerable advantages as intermedi? the r a t e ~ ~ m i t i n ~ ates in e n ~ ~reactions t ~ c because of their high r e a c t i v i ~while step may be formation of a radical from i t s neutral precursor; and that, since free radicals react rapidly with oxygen, such reactions are used mainly by anaerobic orga~isms/1873. One corollary is that for reactions of this type the role of the protein is to produce the initial radical from the stable precursor (cofactor) with an input of energy, whereas the subsequent reactions i ~ v o l ~ free-radical ng intermediates may be intrinsically fast with no need for further catalysis by the protein. In complete contravention of the biochemistry textbooks, therefore, the role is to activate the cofactor (not of the protein in these ~ a d ~ c ~ - g e n e r a tenzymes ing the substrate) and to increase the equilibrium constant for reversible radical formation through an input of energy (not merely to increase a rate constant by s ~ a b ~ l i an ~ i n~ntermediate}-for ~ which the ~ ~ 2 - d ~ p e n dmutases ent provide the paradigm. An add~tionallikely co~sequenceof using reactive and relatively unspecific radicals is that the radical-~eneratingmodules could be used as interchangeable “plug-in” modules wherever required-as demonstrated by the existence of six (so far) very d ~ f ~ e r eAdo~et-depend~nt nt enzymes IlSGl. Two of these (RNR and the inom om^^^ tase) also have AdoCbl-dependent analogues, whereas one (the so-called spore photoproduct lyase for the repair of UV-damaged DNA) 11881 has a light-d~pendentDNA photolyase analogue llSSf, which depends on a chromophore and FADE2;this further demonstrates the need for an input of energy, irreversible in the photolyase but reversible with A d o ~ e and t AdoCbl, to form the radical. Similar i n t e r ~ h a n ~ a ~ l e modules may be expected for the energy-dependent delivery of other fairly nonspecific reagents, eg., for ATP-dependent electron transfer. These patterns revealed by BIZ-and AdoMet-dependent enzymes suggest an ancient, anaerobic world of much more readily interchangeable modules, which include a variety of plug-in energy transducers for catalyzing or driving r e a c ~ i o ~ ~ and which do not always respect the teachings of modern b i o c h e ~ i s t 1textbooks. ~

roblerns and Prospects B12 research over the last 40 years or so since 1961, when Lenhert and showed the presence of a Co-C bond in AdoCbl, has (1)resulted in the build up of a solid foundat~onfor the coordination and organornetallicchemistry of protein~freeCo corrinoids; (2) helped to clarify the differenl roles and contributions of the Co, the ligands, and the protein to the reactivity of the rnutase enzymes; and, at the same time, (3) added to our understanding of chemical bonding and ~nteractionin general,

PRATT

660

Not s ~ r i s i n ~ lprogress y, on u ~ ~ d e r s t a n dthe i ~ gmore medical aspects has been less d~amatic. One can predict, without much difficulty, that the spotlight will now switch from the mutmes to the Me t~ansferases(See. 3.3) and to other enzymes of primitive anaerobic bacteria (? also in the communities around the black smokers) and will reveal many features of interest for both enzymatic mechanisms and evolution. One transferase~.The r e d ~ c t ~ ~ might even find the ~ I ~ ” d e p e n ~~ecnet s t o of r ~the dehdogenaBe$ (Sec. 3.4)could prove to be of considerable interest because of their pot en ti^ use in biore~ed~ation. ince the 19‘70s work on the protein-free Go corr~noidshas tended to focus, perhaps rather too narrowly, on corrinoids with a Co-C bond; one suspects bias and This has resulted lack o f ~ s i o non the part of the funding a g ~ n c and ~e~ their re~erees~ in the neglect of areas such as elucidating the electronic structure of the corrin ligand and exploring possible reactions involving bonds between Co and other polarizable l~ga~ atoms d such as S and Se; see, for example, [8,1901, ~nterestingand ~ n d ~ r ~ c t l y “ t h e r ~ p e ~ t i uses c ” of BIZare being developed, based on using the BIZabsorption and t r ~ s m~ e oc ~~ ~ i ~formdse l i v e ~ ndrugs ~ and d i a ~ o s t i creagents 11911, but the cause of the direct and rapid therapeutic effect of BIZ is still unclear. Lester Smith, head of the Glaxo team that isolated B12 in 1948, concluded his monograph in 1965 111923 by saying that “we still cannot explain the dramatic efXects ofa single smaff.dose upon a relapsed case; the patient feels better within hours, has a normal bone marrow w i ~ i days n and is almost f d y recoved in a few weeks”. It appears that the cause of this rapid response within hours is still unknown.

A

I sincerely acknowledge the help received in preparing this review (and a previous one) from more than 30 colleagues in the B12 fraternity around the world-providing and additional ~ackground~ o r ~ i a t i o en ;~ c h ~ n ~ p r e p ~ ~ n tusn, ~ u b l i ~ h eresults d views; making samples available; even carrying out further experiments or calculations. This has allowed €or certain aspects to be probed to greater depth and certain con~lusionsto be drawn with greater confidence than would otherwise have been possible. Even if colleagues may not agree with all of my find conclusions, I hope they wiIl agree that their aesktmce has enabled tbis review to draw ~ o g e t ~ ae r number of threads at a definite turning point in BI2 reBearch.

A@ Ado A~o~bl

allylcarbinyl 5 ’ ~ d e o ~ a d e n o (as ~ y Iligand and radical) 5 ’-deoxyadenosylcobalamin

COBALT IN TAMIN IN

AdoMet

AM ATP

Cbl CN-Cbl CoA ~ CPIM CT dGTP

~

EAL ESR Et FADIT2 GluM ITC IF

Me MetS

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nP Ph Pr

Q ~~

TC TIM ‘ ~

~

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66 1

AND ITS ~ N Z Y ~ ~ S

~-adenosylmet~ionine 8-aminolevulinic acid adenosine 5~-t~phosphate vitamin BIZ bond dissociation energy cobinamide co~al~in cyanocobal~in03121 coenzyme A CQ d~e h y d r o~~ ~ ~ a s e/ i - Cs pot A h aAs e ~ ~ cyclopropylmethyl charge transfer deoxyguanosine 5 ‘-triphosphate diol ~ ~ h ~ d r a t a ~ e 5,6-dimethylbenziminazole ethanolamine ammonia lyase electron spin re son^^^ ethyl flavin adenine dinucleotide glutamate mutase ~ap~o~orrin inirinsic factor International Union of Biochemistry ~ n t ~ ~ ~ t iUnion o n a of l Pure and Applied ~ h e ~ methy1 methionine synthase m e t ~ y l ~ ~ o n y lmutase -co~ ~ o l ~orbital ~ ~ a r reduced nicotinamide adenine dinucleotide (phosphate) neopentyl phenyl ProPYl queuosine ~ ~ o n u c l e o t i reductase de ~~~~~obalamin tri~s~phosphatc isomerase 2~~ 2 , ~H ,6-tetra~~thylcyclohe~l

~

~

t

662

PFIATT

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667

138. 'I?. Tomya, E. JKrodel, A. S. Mildvan, and R. H. Abeles, Biochemistry, l#,417 (19791, 139, M. R. Holloway, E.1. A. White, K. N. Joblin, A. W, Johnson, and 0. C. Wallis, Ear. J. Biochem., 82, 143 (1978). . P. Eay and R. G. Finke, Polyhedron, 7, 1469 (1988). . J. Mmkwell, J. M. Pratt, M. 8. Shaikjee, and J. 6.Toerien, J. Chem. Soc. ~ a l t o nTrans., 1987, 1349. , Hisaeda, T. Qhno, and T. Ozaki, Chem. Letts, 1985, 477. 142. Y. M u r a k a ~ iY. 143. K, L. Brown and 5. Li, J. Am. Chem. Soc., 120,9466 (1998). . Eabior, J . Biol. Ckem., 245, 1755 (1970). , Babior and T. K. Li, ~ ~ ~ c h e m 8,i154 s ~(1969). ~ , 146. IT. A. Lee and R. . Abeles, J. BioE. Clzem., 238, 2367 (1963). 147. 33. M. Babior, J. Bid. Chem., 245, 6125 (1970). 148. 0. W. Wagner, R. A. Lee, I". A. F'rey, and R. ET. Abeles, J. Biol. Chem., 241, 1751 (1966). 1-49? T. Toraya, Arch, Biochem. B~ophys.~ 242, 470 (1985). . Pilbrow, in [ Z ] , Vol. 1,p. 431 (1982). Gerfen, in [I], p. 165 (1999). 152. V. D. Gbanekar, R. J. Lin, R. E. Coffman, and R. L. BlaMey, Biochem. Bitrphys. Res. Commun,., 101, 215 (1981). iol. Chem., 256, 100 153. K, Lerch, W. 33. Mims, and J. Warncke, R, S. Farid, and 154. (a) C. 6. Moser, J. M. Kes ~ f f i i ~ 355, ~ e , 596 (199~);(b) 6. 6. Page, C. 6, Moser, X. Chen, and P. 1,. utton, Nature, 402, 47 (1999). 155, D, Zhou, P. Wilder, R. Scheffold, and L. Walder, Hdu. Chim. Acta, 75, 995 (1992). 156. H. R. Zimmerman and A. Zvveig, J. Am. Chem. Soc., $3, 1196 (1961). 15'1. H. E. Z i ~ ~ ~Acc.~ Cllem. ~ a Res., ~ i 5,, 393 (1972). 158. 6 . Walling and A. CioEari, J. Am, Chem. Soc., 94, 6064 (1972). , Collins, P. von urns, M. A, M ~ ~ ~ eJ.rJ.~ e ~ , , N. 6. S a m ~ a nJ. hleyer, and E, Osawa, Chem. 1977,95. Clarke, 0. E. Finlayson, a n d J. J. Rooney, Chem. Cowzm., 1982, 1277. cKervey, J. J. Rooney, and N. G. Samnmn, J. Caiiaf., 30, 338 (1973). isenstein, N.T. Anh, 1~~~~~ J. ~ ~ ~ 1 , r3 n ~ ~ , . Kmg, J. Am. Chem,. Soc., 97, 4754 (1975). Flohr, u. M. Kempe) T. Krebs, and J. R6teJ7, A ~ ., 14, 822 ~ 1 9 ~ 5 ~ . . Pratt, Chern. Commun., 1976, 988. 166. A. I, Scott and R.Kang, J. Am. Chem. Soe., 99,1997 ~1977j. a

f

~

668

PRATT

167 S, RQtey, in Vitamin BI2 (B. Zagalak and W. Friedrich, eds.), de Gruyter, , 1979, p. 439; M. ~ o u n t o u l ~and s J. Rbte-y, C ~ e Ber., ~ . 113, 650 168. R. ITa~ilton,T. R. B. Mitc~ell,E. A. M c ~ l g ~J. r ~J., RooIiey~and M. A. McKervey, Chem. Com~nun.,1981,686. 169. R. ~ a m i l t o n ,T. R. B. g itch el^, J. J. ~ o n e y and , M, A. M c ~ ~ r vChem, e~, Comrnun., 1979, 73s. 170. ~ i r ” ~ b a r a ~J.i m Grimshaw, , E, A. Mcllgoarm, T. R. B, Mitchell, and J.J. oney, s.Mol. Cat., 27, 337 (1984). 171. M. Marques, personal communication. 112. S. E m , IT. L. Chea, N. ~ ~ i l ~ ~ eand n ~J-e~ ~~ dp , e rJ.nAm. , Chem. Soc., 110, 3120 (1988). 173. Y. M ~ r ~Y. ~ Hisaeda, i , and T. Ohno, Chem. Letts, 1987, 1357 and Chenz. Commun., 1988, 856. 174. U. ~ u r a Y.~Hisaeda, , X. Song, and T. Ohno, Chem. C o m m u ~ 1992, .~ 1527. 175, Y. Hisaeda, persctnal communication. 176. 0,Zelder, €3. Beatrix, U. Leutbecher, and W, Buckel, Eur. J. Biochem., 226, 577 (1994). 177. Y. Zhao, A. Abe~d,M. Kunz, F. Such, and J. R & y , Bur. J. ~ i o ~ h e 225, m.~ 891 (1994). 178. A. Abend, V, Illicb, and J. Mtey, Eur. J. ~ ~ o ~ h e249, m . , 180 (199~)’ 179. S. Wollowitz and J. Halpern, J. Am. Chem. Soc., 110, 3112 (1988). 180. T. Darkwe, R. Keese, V. Siljegovic, Chem. ~ o ~ m u n1996, . , 1561. 181. M. Me and P. Dowd, J. Am. Chem. Soc., 120, 1133 (1998). 282. eatrix, 0. Zelder, F. K. Mro& G. Orlygssorr, B. T. Golding, and W. Buckel, Angew. Chem. Int. Ed. En&, 34, 2398 (1995). 183* A. I. Scott, P. Karuso, €3. J. Williams, d. Lally, J. Robinson, and 6.P. Nayar, J. Am. Chem. Soc., 116, 777 (1994). 184, 6. S. Hammond, J. Am. Chem. Soc., 77, 334 (1955). 185. P. Reichard, TJBS, 22, 81 (1997) 186. S. Ollagnier, C, Meier, E. Mulliez, J. Gaillard, V. Schuenemann, A. ~ a u t w e i n T. , M~~ti~ M.l iLutz, , armd M. ~ o n t e c ~ vJ.e ,Am. Chern. Soc., 121, 6344 (1999). ~ , ~ i c r o ~Reus., ~ o ~22, . 523 ~ 1 9 ~ 9 ) . 187. W mBuckel and B. T. ~ l d i n~E~~ 188. R. Rebeil, Y. Sun, L. Chooback, M. Pedraza-Reyes, C. Minsland, T. P. and W. L. Nicholson, J , Bacteriol., 180, 4879 (1998). 189. T. Carell, Angew. Chem. Ink Bd. En& 34, 2491 (1995). 290. J. I. Toohey, J. k o r g Biochem., 49,189 (1993). 191. . P. C. Hogenkarnp, D. A. Collins, 6 . B. Grissorn, and F. G. West, in [I], p. 385 (1999). ~ B I Z~3rd , ed., ~ ~ em ~ h u e~ London, n , ~ 1965. 192. E. 1,.Smith, V

1

~

~

p

~of ~t o~ - ~ e nn ~ ~r o nScience ~ e nand t ~~ ~ h n o l~on i~v e, r s iof t ~~ o l o ~ ~ a Vide Berti Pichat, 10, I 40127 Bologna, Itdy

e p a r t ~ e nof t Chemistry, University of Siena, Via Aldo Moro, I 53100 SBna, Italy

1. I N T R O ~ ~ ~ ~ ~ O ~ 1.1. Bioinorganic Role of Nickel

2.1.1. ~ c c u r ~ e and ~ c eBiological Role 2.1.2. Overdl Structural hchitecture .3. ~ ~ u c t uof r ethe Active Site 2.2.1. Occu~renceand Biological Role 2.2.2. Overall Structural Architecture 2.2.3. ~ t ~ c t ofu the r ~Active Site 2.3, Methyl-Coenzyme M Reductase 2.3.1. Occurence and Biological Role 2.3.2. Overall Structural Architecture 2.3.3. Structure of the Active Site

3. ~ ~ ~ E ~ ~ ~ OU~~~~ ~ E I~ N~ S' ~ U C T 3.1. Carbon ~ o n o ~Dehydrogenase/Acetyl-Coenz~e i d ~ A Synthase 3.1.1. Occu~rence,Biological Role, and Properties of the Protein 3.1.2. Structure of the Active Site ~~~

669

670

670 671 671 671 671 672 675 6'35

676 678 680

680 681 682 ~ 683 ~ ~ 683 683 684

ClURll AND ~ A N ~ A

670

3.2. Nickel Superoxide Dismutase 3.2.1. ~ c c u r r e n ~ c ~~ ~o l o ~Role, c a land ~ ~ o ~ rof tt'he ~ Protein e s 3.2.2. Structure of the Active Site 3.3. Nickel Chaperonins 3.3.1. Occurrence, ~ i o ~ o Role, ~ c aand ~ ~ r o ~ e ~of~the i e Nickel s Site 4. S ~ 4.1. 4.2. 4.3. 4.4. 4.5.

5.

~~~~~~

~ U C ~ ~ ~ ~ - F R ~ULNA ~T ITQIN~S N ~I~~ Urease [Ni~el"~ydrogenase ~ e t h y l ~ ~ o e n ~MyReductase me Carbon Monoxide Celrydrogenase/~~etyl~~oA Synthase Superoxide Di smutase

~

~

~

~

685 685 686 687 687 689 689 690 693 697 699 699 ~ 700

700

Nickel a ~ u n ~ a nin c etho Earth's crust is ~ b o ~Oi .t0 2 ~about ? 500 times less than Fe. Nickel bas been claimed to exist in biological systems in oxidation states ranging from Ni(O) to Ni(ITI), but it is mostly present as Ni(II) [ll. i(II) (d8) is ~redomina~itly found in a ~ a ~ a ~ a ~ h ie ~t ih c- is s ~ = 1) ~ ~ octaheic s q ~ a r e ~ ~ l ageometry. nar dral electronic configuration, or in a d i ~ a ~ e tlow-spin ~ o ~ ~ ~ v e - c o o ~complcxes ~ n a t ~ of Ni(II) are also very common, either in a t r ~ bipyramid& or s~uare-planargeometry. Low-spin our-coordi~ate s q u a r e - ~ l a n~~ i ~ possesses a filled dZA orbital and behaves as a strong Lewis base, while its oxidized ~ o u n t e r p a~~i t~I I ~is) ,a reactive free radical. In general, the ~ e ~ c ob ~ r~d ien a t ~ o ~ i m ~ t ~a l l oeb ~ o~~ o ~~eto c ube e~ e s~ geometry of nickel causes its coordination ~ r ~ in ~ dictated largely by the protein matrix. The attitude o f Ni(1I) to hi*mdinuclear complexes is e ~ l o i t e dby nature to construct the c a t a ~center ~ i ~ o f some N ~ - c o ~ enzymes. ~ ~ ~ complexes o r of ~ ~i~~~ ~ ~ ~ ci a r n do) ~ ~are ~ st L~a cb i ~ by i ~ ~ ~~ a c c e ~ . (paral i ~ a n d ssuch as carbon monoxide or cyanide in a t e t r a ~ e d gr e~o m e t ~ Niii) magnetic d9) is most likely in a high- or low-spin tetragonal (D4h) ligand field and is also o ~ t ~a?sn~ o ~ i a t ewith d ~ - a c c e p ligands. ~r On the other hand, ~~(~~~~~~~~~~~~netic d7, usualIy in a low-spin Letragonal config~ratio~~) 3s co~plexedwith ligands c o n t a ~ ~anionic ~ i n ~ oxygen, nitrogen, or sulfur donors [I]. Nickel i s generally toxic for living o r ~ a ~[a], i ~its~ t o~ ~sc i t ybeing probab~y to Ni binding to DNA, but nevertbe~~ss several b i o ~ o processes ~c~ require Mi for their function. The b i o l o ~ role c ~ of nickel in e n ~ y m a t ~c ca t ~ y s inow ~ ) eom-

N I C K ~ L - ~ Q N ~ING A I NENZYMES

671

prising several systems [3-9], was first established in 1975, when Zerner and coworkers reported the presence of this transition metal ion in urease [lo]. This discovery prompted efforts to shed light on the chemistry of nickel in other biological detected in CO dehydrogenase from photosystems. Its presence wits s~~bsequently synthetic bacteria, in methyl-coenzyme M reductas~-boundfactor Ni-I"4,0 from methanogenic bacteria, in bacterial [NiFel-hydrogenase from several m~cr~organisms, in acetyl-CoA synthase from methanugenic and acetogenic bacteria, and in superoxide dismutase from actinomycetes. Other enzymes, such as glyoxalase I and peptide d ~ ~ o r m y l amainly s e ~ isolated as Zn(I1) rl11 and Fe(I1) C121 derivatives, respectively, have also been reported to function in the presence of nickel IS].

2.1.1. Occurrence and Biological; Role

Urease catalyzes the h y ~ o l y s ~ ofsurea in plants, algae, fungi, and several microorganisms f2,13,141 in the final etep of organic nitrogen mineralizat~onto produce ammonia and carbamate:

This process occurs times faster than the noxicatalyzed reaction at pI4 7-8, with a half-thne of the order of microseconds. The carbaniate produced during this reaction spontaneously decomposes, at physiological pR,to give a second iiiolecule of ammonia and bicarbonate:

The products of this reaction undergo further hydrolysis that causes an ovendl pH increase. s related to a nitrogen assimilato~ The role of urease in ~ c r o o r g a n i sis~ mostly mineraiization process [ZI.The role of urease in plants is less clear. It has been shown that ~ l t ~ v a t of ~ oplants n in nic~el-deficientmedium leads to leaflet tip necrosis due to urea accumulation L 151.

Jack bean urease (JSU) was the first enzyme to be crystallized more than 70 years ago [16], In recent years, the X-ray crystal s t ~ c t u r e of s urease from ~ l e b s i ae7-oe~~~

€3 code 1WJ)CJ7-J 91 and Bacillus pasteurii (BPU, PD 120,211 were determined. The s t ~ c t ~ r of e sthese two microbial ureases are largely

~ ~ u i v a ~ eboth n t , being h e t e ~ o ~ o l ~ eproteins ric (ol&yts of about 250 kDa. On the other hand, plmt amases are hexameric proteins of about 600 kSla, each a-subunit being highly homologous to the (a&y)assembly of microbial ureases. The highly conserved m i n o acid sequences of all kno.viln ureases and the constant presence of two Ni ions and of their ligands in the active sites 114,2121 imply the presence of a common catalytic pathway. In the ~ s c o i d - s h a p( ~a p ~trirner )~ (Fig. 11, each a subunit packs between two s ~ ~ ~ e t r y - r e al asubunits t e ~ to form the sides of a triangle, whose vertices are topped by the three j3 sub~nits" Each y mhunit makes contacts wikh two CI subunits, f a ~ i l i ~ a t ~ ing trimer formation. The trimer shows a loss of about 5000 A2 of solvent-accessible o both n . KAU and BPU, each 01 subunit I-570 surface area upoil complex ~ ~ r ~ a t iIn barrel domain and a 0 type d o m ~ iwhile ~ , the f3 r e s ~ d ~ise made s ~ o f an el lip ti^ subunit (- 120 residues) and y subunit (- 100 residues) are mixed aj3 domains (Fig. 2). The nickel-contai~ingactive site is found in the CL subunit, at the bottom of a 15-A-deep - ~ e ~flap, which has chwinei c~aracterizedby the presence of a h e l ~ - t ~ r nflexible been ~ b ~ both ~ in~ aneopen d (BPU) arid in a closed (K4U) c o ~ f o ~ m a ~ i o ~ ,

~ t ~ c t u of~ native e s K. aercgenes urease 1171 and its several mutants I18,191 have initially ~ r o ~~ in f~~ ~~ m d aon ~ ithe o noverall h e t e r o p o ~ ~ e~r ~~c a t ~structure r n a of ~

FIG. 1, Structure of the ( ~ f i y heterotrimer )~ of B.pasteurii urease, represenhad schematicdy. The a,f3, and y subunits are colored black, gray, and white, respectively. Circles indicate the position of the active sites, where the Ni ions are shown as spheres. The arrangement of thc Nibound amino acid residues and water molecules in the active site of the native enzyme is shown in the ~ ~ l a ~ ~ ~ ~ e ~ t .

NTAlNING ENZYMES

673

the protein and revealed the ~ r ~ g e m eofn the t metal ions and amino acids in the urease in the native f211 and active site. ~ u b s e q u e n t ~the y ~structures of B, ~~~~~e~~~~ inhibited forms [21,23,241 confirmed several observations relative to further clarified the structure of the nickel-bound water molecules in the of the resting enzyme. New structural details on the binding of inhibitors such as J3~ ~ r ~ p t o~ e t ~h acetohydroxam~c ~ ~ ~ ~E )acid ~ ( ~ and pheny1phosphoro~a~~ , date (PPD) were also elucidated /21,23,241. In the active site of native BPU E211 (PDB code BURP, Fig. 3A), two nickel ions are b ~ d ~ by e dthe c a r ~ ~ m yLysa220' ~ a t ~and a h y ~ o x ~ d e with an Ni-Mi distance of3.7 A. One Ni ion (Ni-It) is fbrther bound to Hisaz4' and wy275,while Ni-2 is bound , and Asp"363.The coordination geometry of each Ni ion (pseudos q u a r e ~ p y ~ a mfor i d ~the pentacoordinate Ni-1 and pseudooctahedral for the hexac o o ~ ~ Ni-2) ~ ~ ias tc o~ m p l e ~by~ one water molecule. A fourth water m o l e c ~ eis bound to the N i - ~ o ~ ~solvent n d molecules by H-bonds and completes a tetrahedral molecules. ae Several structures o f U U , mutated at the cluster of w a t ~ r ~ y d r o ~ c ~ b a ~ y ~lysine a t ~residue d bridging the two Ni ions in the active site, have revealed that the ~ m p o ~ of ~ such c e a ligand does not rest in the peculiar electron donor irty of the c ~ b a m a functional ~e moiety; instead it simply serves to hold the ions close to each ather for optimal bimetallic catalysis C191. In BPU inhibited with BME 1231 (PDB code LUBP, Fig. 3B) the sulfur atom of ~~E s ~ ~ e t r~ ri i ca ~~the e s~~ i ~ u cNil center e ~ and further chelates N i - i using th Ni ions being pent its terminal OH, re : Nit11 is distorted s~u~re-pyra~id~~ rted trigonal~bip~amidal. er molecule af &ME forms a mixed disulfide n eadnei group of ~a""', ~ ~ ~ i t i o on g loop, fixing the flexible active

with AHA f24] (PDB code 4UBP, Fig. 36) shows etrically b r i ~ ~ the n gtwo Ni ions in the active u ~ ~ gen (at 2,O A) and chelating Ni-3 t h ~ o the ntacoordinate with a p s e u d ~ t r i g o n ~ " b i p ~ a ~ i d

674

Cn, FIG. 3. Stmcture of the dinuclear Ni center in R. pnsteeurii urease in the native (A), BME), A ~ - j s ~(C), ~ and i ~ ~~ A~ ~ e- ~i n ~ ~ ~ ~ i t e ~

geometry. The flexible flap flanking the active site cavity is in the open conformation. The structure of a KAU mutant, complexe(~with AHA (PDB code 1~~~ [MI, shows a molecule of the inhibitor bound to the Ni ions in a similar way as observed for inhibited BPU, but a pronounced asymmetry is observed for the Ni-bridging atom (Ni(1) - OB 2.6 Ni(2) - Op, = 1.8 This difference can be ascribed to the closed conformation of the active site flap that affects the properties of the active site cavity. A molecule of ~ i a m i ~ o p h o s p hacid o ~(DAP) ~ is found in the active site of urease crystallized in the presence of the slow, tight-binding inhibitor PPD [ Z l l (PDB code SUBP, Fig, 3Dt. DAP is the product of the enzymatic hydrolysis ofPPD 1251and binds to the bimetallic center using three of the four potentially coordinating atoms: one oxygen AP replaces the Ni-bridging hydroxide observed in native BPU, while one oxygen and one nitrogen atoms bind to Ni-l and Ni-2, respectively. The second nitrogen atom of DAP points to the cavity opening [211. In DAP-inhibited BPU the active site flap is found in a closed con€or~ation.Changes in the active site flap conformation, besides d e t e ~ i n i n gthe accessibility of the active site channel, directly affect the position of important active site residues, such as Hisu323and The former residue, known to be responsible for the lower pK, of the bell-shaped p~/activityprofile L26,271, is shifted away from the Ni ions when the flap is open. Similarly, points its carbonyl group to, or away from, the Ni ions when the flap is closed or open, respectiveiy. Only when the flap is closed can both and fSisu323 be involved in the H-bond network that stabilizes the binding of DAP to the binuclear Ni center [213. =I

A;

A).

NIC~~~-CONTAININ~ ENZYMES

75

Hydrogen gas (N2) is produced by bacteria both in anaerobic and aerobic environments [ZS]. The classes of anaerobic bacteria metabolizing H2 include methanogenic archaea, aceto~enicbacteria, sulfate- and nitrate~reducingbacteria, as wet1 as some h ~ e r t h e r ~ o p h ibacteria. ~ic In these cases, fermentatio~io f organic substrates (proteins, nucleic acids, sugars, and lipids) yields Ha. Reduction of H+ to used by ~ e ~ ~ m e nbacteria t ~ n g to dispose of excess reducing equivalents a suitable redox ~ o t e n t i i ~nl the cell, The H2 gas is s u b ~ e ~ u e n tutilized ly as a lowpotential reductant by bacteria living in the same enviro~ment,and its oxidation is usually coupled to the reduction of nitrate, sulfate, carbon dioxide, and fumarate, leading to ATP production. In aerobic condit~ons,H2 is produced by some p ~ ~ o t o s ~ t h ebacteria tic and algae as well as during the dinitrogen reduction process catalyzed by nitrogenase also in azototrophs [28l. is then used as a r&.xtant by the same species [281 as well as by the so-called ballgas bacteria, a class of ~ i c r o o r g ~ s m living s in aerobic environments and able to Eve on H2, O2 and COz E29f. All of the above species s hydrogenases, enzymes that catalyze the reversibte ~ ~ ~ e r c o n v ~ rofs iH+ ona [281:

Ea' = -0.414V

(3)

Hydrogenases are also able to catalyze prot~um-deuteriumand prot~um-trit~um in absence of elec~rondonoi*/~c~~ptor partners (r30 I and r e ~ e r e ~ c~e s~ e r e i ~ i ~ the ~ g type and predrogenases can he grouped in four classes, d e p ~ i ~ don sence of the metal cnfactors. Several hydr~genasesinvolved in E-I, uptake in aerobes, f~c~iltative a n a e ~ ~ band e s ~anaerobic bacteria contain an [NiFe1 binuclear center E311f. This class comprises both ~embraiie"bo~nd and soluble hydrogenases, [NiFeSelh y d r o g e ~ a ~isolated e~, from sulfate-reducing bacteria, contain Se in the form of selenocysteine [311. Another class consists o f the Fe-only hydrogenases from strictly anaerobic bacteria [32l. Fe-h~drogenaseshave been treated in Chapter 10 of this book, and this chapter will deal only with fNiFel- and [Ni~eSel-hydrogenases.The hydronewly discovered H2-formingmethy~e~e-tetrahydro~ethano~teri~-dependent genase IEC 1.12.99.-) appears to he a purely organic catalyst, and c ~ n s t ~ t u tae sclass on its own that does not contain metal cofactors t331. Several gene products (hup or hox genes) are involved in the hydrogenase expression and maturation r34,351. cently, the molecular biology o f l ~ i ~ e ~ - h y genases has been enri ed by the ~ n d that ~ ~ the s~1~~~ gene in ~ ~ ~ ~ pencodes ~ an ~ l Bensor ~ that ~ is~itselfs a hydrogenase that init~atesa chain of events leading to enzyme expression [36,3?1. An analogous route leads to hydrogenase e x ~ ~ e s in ~ iAlcaligenes ~n e ~ t r o ~initiated ~ ~ , s by the products of the hoxX in ~ o n ~ ~ nwith c ~ the ~ otrans ~i ivator kuxA I381 ~ h a ~ a c t c r i zo~f t ~ o activity studies showed that this (.

676

CIURLl AND ~ A N ~ A ~

hydroge~iasehas an active site constituted by an Ni-Fe cluster bound to one GN- and two CO molecules as found in "standard" I~iFeI-hydrogen~es [39]. However, this sensor, in c ~ n t r to ~ tstandard h y d r o ~ e n ~ is e , i n s e ~ i t ~ vtoe oxygen exposure 1391.

Several [NiFel- and [NiFeSel-hydrogenases from various sources have been sequenced [341, Many of these consist of two distinct subunits, 01 and p, of 46-72 a and 23-38 kDa, r e ~ ~ e c t i v L8,28,301. ~ly ~ e m b r a ~ e -m b uo l~t i ~~ u~b u ~ [NiFelt hydro~en~ses, dl of them also bearing similar a and @ subunits, have been characterized. They comprise hydrogenases 3 and 4 from Ea coli 140-423, the GO-induced ~ ~ G d ~ ~ ~ndvwn ~ ~ ~ hydr~genase l l u r n [43,441, E. C G Z ~ type 3 ~ y ~ o g from ~ n ~ e ~ ~ t ~ f f , n o barkeri s ~ r c t451, ~ ~ aand ~ e t ~ ~ ~ n ~ b a c ~t e r~ ~ ei ~ r nr n ~ ahydro~ t ~ t nase C461. The large a: subunit hosts an NiFe h e t e r o ~ i i u ~cluster l e ~ bound in the active are characterized by the site. The sequences of the a subunits in r~iFeJ-hydrogen~es presence of five conserved sequence motifs, two of which, the N-terminal R-X-G-X2-G and the ~ - t e ~ D-P-C-X2-G ~ a l peptide segments, provide the cysteine residues ~ e s p o ~ i for b ~ enickel and iron bind in^ l28l. The subunits show at least three characteristic amino acid sequence fragments that are conserved despite a large degree of sequence and structural variability, having the role to host one or more s s ~ ~by the redox FeS cluste~sr~sponsib~e for the electron transfer ~ ~ o c e needed reaction f281. The crystal structure of' six [ N i F ~ l ~ h ~ d r o ghas e ~ been ~ e s determined. Two of ~ ~r ~~~ , ~4 8(PBB iJ ~ codes ~ o1 Z ~ ~ oBe V ~from , them are from I ) e ~ ~ gigas ~ e s ~ l ~ o u ~f rbzr~i co~ o s o u ~1491 r a ~(PD s code l ~ two fro ~ ~ ~ ~ 1,vulgaris ~ Miyazaki [50,51] (PDB code 1H2A, 1 2R), and the last is the reduced form of the ~ ~ ~ u l ~ t [3l] f r u r(PDIE: n code 1GC1). The ~ ~ ~ e ~ e l - h ~ d r o gfrom e n a~e~ul~orn~crobiurn se codes IFRV, 2FRV, and IHZA refer to the inactive, oxidized form of the e n z ~ e , whereas lCG1 and 1H2R refer t o the active, reduced form of the enzyme. The resolution obtained for I). frfructosouoranshydrogcnase (JFRF) i s too low (2.7 to extract from it i n ~ o ~ a t i roeng a r d ~ nthe ~ mechanism. The c ~ s t d l o ~ a p hresults ic show that these [NiFeI~hydrogena~es are heterodimers having a very similar ajl quaternary structure. The molecule has an almost s an e x t ~ ~ d e d s ~ h e r i shape c ~ with a radius of about 25-28 A. The two s ~ b u n i t show contact surface (- 3600 li"> that stabilizes the dimeric structure without the intervention of covalent bonds (Fig. 4). The B subunits of the above enzymes (- SO kDa) are all character~ze~ by an Nterminal domain ~onsistingof a twisted p sheet surrounded by CI helices in a so-called a# twisted open-sheet structure (Fig. 5), similar to that adopted by Clostridium M.P. ~ a v o d o x ~1521. n The C-terminal domain is less structured, being cliaracterized by one l~ s t ~ c t (Fig~ r 5). ~ Tho B to threc 01 helices embedded in a ~ r e d o m i n a n trandom-coil subunit hosts three FeS clusters o f different types, and in all structures the M-term-

677

FIG. 8. 8tructure of the aB dimer o f [NiFeI-hydrogenasefrom D.gigus, represented schematically. The M. subunit is colored black and the j3 subunit white, The metal ions (NiFe center and F& clusters) are shown as spheres in the opposite color with respect to the ~ r ~ e ~ p o ~ d i subunit. The ~ r ~ ~ mof ethenI NLFe t I-bound ligmds in the reduced active form oF the enzyme is shown in the e ~ K ~ r ~ e ~ n ~ n t .

FIG. 5. Secondary and tertiary structure of the hydrogenase.

M

and f3 subunits of

a. gigas

[MiFel-

inal domain provides the ligands to only one of them, the proximal cluster, which is the closest to the NiFe active site located in the a subunit. All p subunits of hydrogenases host two Fe,S4 and one Fe8S4 cluster, with the exception of D. baeullatzcnz hydrogenase, wkich features three Fe4S4 clusters 1311, and of D. ~ ~ e ~ u ~ ~ u u r hydrogenase, for which the native Fe3S, cluster was converted to an Fe,S4 cluster by a Pro238Cys mutat,ion [49]. The FeS clusters are evenly separated by about 9-10 A and build up a chain of redox centers within the subunit, The proximal FeS cluster lies at about 10-13 from the Ni ion of the NiFe heterodin~clearactive site cluster, located in the rx subunit (Fig. 4). The secondary structure of the a siibunit {- 60 kna) consists of five different domains: three domains have a a@ structure, one is largely unstructured, and the third one, the largest, is characterized by a bundle of four long a helices (Fig. 5).

A

The crystal s t ~ c t u r e of s ~Ni~el-hydroge~ases have de~onstrated that the active site of the enzyme (Fig. 6) consists of an he1,erodin~clearNi-Fe cluster bound by thiolate sulfur atoms of cysteine residues [31,47-511. The coordination geometry of the two metal ions is completed by nonprotein ligands. However, the picture of the [NiFethydrogenase active site as emerging from these studies is not unique, and relevant di~f~rences have been reported concer~~ing the nature of the exogenous ligands to the heterodinucle~cluster. All crystal. ~ t ~ c t uof~ l~i~ei-hydrogenases es so far determined show the following common features of the active site: (lj The active site c~~iitaining the NiFe cluster is located in the a s ~ ~ ~ u at nit, the b o t t o of ~ a deep pocket situated almost at the center of the ap heterodimer, about 25 A from the surface (Fig. 4). It is ~ o m p ~ e t baried e ~ y and no water m o l e c ~ have ~e~ diffusion patterns into and been located bound t o it or in proximity to it. Possible t ~ the hydroplio~iccavities evidenced by out of the active site may bc c ~ n s t i t u by calculations involving mapping of the I). gigas molecule with a probe of 1.0 A radius ~~ran,~ 153,541, A xenon binding experiment conducted on the I). f r i ~ c ~ ~ ~ o zenzyme shows that the Xe binding sites are located in the hydro~hobicchannels described above [551. (2) Accurate metal analysis and e ~ ~ t ~ ~ ~a an op ~h ai l ~c~~) p~e~~ ~s data ion [48J have shown that the active site consists of a heterodin~clea~ metallic cluster e d 3 A. The protein ~ ~ o v ~four des made o f one Ni and one Fe atom s e ~ ~ ~byt about Cys ~ ~ i o l aligands te to the metals (Fig. 61-All o f these cysteines bind to Ni, and two of by three h ~ x o g ~ ~ o them also bridge to the Fe ion. The latter i s further coord~n~ted d i a t o ~ ~ligands. ic An additional monoatomic bridge exists between Ni and Fe in the o ~ ~ ~inactive ~ c dform , of the enzyme (Fig. 6A), leading to a f i ~ ~ ~ c o o ~ d idistorted nate te geometry for Fe. s q u ~ e . . p y r a m i geometry d~ for Ni and to a s ~ - ~ o o r d i n aoctahedral ( 3 ) An a d d ~ t i metal ~ n ~ binding site has been found in the ~ ~ tpart eof ~ IX subunit. The metal bound in this site has been refined as ~ ~in the( the ~~~~~~

cys

FIG. 6. Structure of the “334 cluster as determined from the crystal structure of oxidized D. $gas and D. vulgaris hydrogmases (A1 and reduced D.uulgaris and n. b u ~ ~ hydrogeW ~ ~ ~ u 1- The different attribution of the diatomic Fe ligands in the D. vulgaris enzyme i s shown in parenthesis. The bridging ligand X has been assigned as an oxygen species in D. gigas and as a sulfur species in D.vulgwis ~ y ~ # ~ ~ n ~ s e s .

structural model o f D. gigas [481 and D. vulgaris hydrogenases [SO]. In contrast, anomalous difference maps indicated the presence of an Fe atom in the same site of [ ~ i ~ e ~ e ~ - h y d r o g efrom n a s eD. bmulatum [3U1

In addition to these structural s ~ l a ~ t ~differences es, among [ ~ i F e l - h y ~ o g e nase structures have been reported that concern (1)the nature of the monoatomic ligand bridging Ni and Fe in the oxidized inactive state (Fig. 6A), and (2)the nature of the three exogenous ligmds to Fe. In D.vuZgaris hydrogenase, a sulfur atom was proposed to bridge Ni and Fe on the basis of the crystallographic refinement [501, while in the LI. gigas enzyme an oxygen species was proposed to occupy that site [481. The nature of the nonprotein ligands, as well as the identity of the metal ion close to the nickel center, remained undetermined in the first crystal structure of the 13. gigas hydrogenase [47J. The presence of new characteristic absorption bands in the IR spectrum of hydrogenase from C. vinosurn was reported 1561 even before the publication of the first crystal structure, and their presence later confirmed in the structural refinement of the D. gigas enzyme [481. The use of I5Naand 13C-enrichedhydrogenase pe~mittedthe attribution of the three bands to stretching vibrations of two CN- ions and one CO m o l e d e 1571. In contra& the crystal stnrcture analysis of D.vulgaris hydrogen~e,together with pyrolytic mass s p ~ c t r o ~ e t rdi ce t e ~ i n a t ~suggested ~~s, the presence of one SO molecule replacing one of the diatomic CO/CN- ligands found in D. gigas, D. baculatum, and D. fructosovorans enzymes L501. Two c ~ s t a l l o ~ a p hdeterIninations ie of the reduced form of the enzyme have recently been reported. One involves the D. vulgaris (Miyazaki) enzyme [51j, and one a M ~ i~ l e the i ~overall s~ruetureof the the [ ~ ~ e ~ e l - l ~ y ~ o gfrom e n af). s~ e c u ~ t311. molecule is unaffected by the reduction, the active site undergoes significant changes. In both reduced hydrogenase structures the monoatomic ligand bridging Ni and Fe has disappeared, leaving the nickel four-coordinate and the iron five-coordinate (Fig. 6s). All metal-ligand distances are not significantly different with respect to the oxidized forms. Ni-Fe distances of 2.5 A and 2.6 A were observed in I). b a c ~ Z a t ~ ~

and D. vulgaris hydrogenases, respectively. These values arc essentially unchanged with respect to the 2.6-k distance determined in the oxidized D. vulgaris hydrogenase. On the other hand, an Ni-Fe distance of 2.9 was reported for the inactive, t l y than the Ni-Fe oxidized D.gigas hydrogenase [48J, a value that is ~ i ~ ~ i f i c a nlarger for the reduced form of various h ~ ~ o g e distance of 2.65 d e t e r ~ i n e dby nases [581. This latter study revealed a substantial similarity of the nickel ~ n ~ i ~ o n t ~ Qthe n among the various h y d r o g e n ~ eand ~ made the interest in^ o ~ s e ~ ~ a that reduction of the Ni ion from the inactive oxidized enzyme to the reduced forms is cu~sistentonly with the one-electron reduction of the metal, ~~~

nt of m i n e ~ a ~ i z a t ~ o ~ ~ i o ~ m~e t~~ c~ oa ~ ~e ni se one ~ i sof the most i m ~ o ~ a routes of organic matter, occurring in environments such as fres~wate~. and marine s e ~ ~ m e ~swamps t s , and marshes, rice fields and landfills, as well as in the ~ ~ t e s ~ i ~ of ~ ~t591, In isuch anaerobic ~ b ~~o t ~ b~ ~e tos ~~ o ~ y w ~~ ee rt ~s a n ~ f o r ~toe d ~ r i ~ . hydrogen, carbon di~xide,formate, and acetate by fermentative ~ a c ~ Th cules w e then utilized by methanogenic Archaea as their sole ~ n e s r ~ ~ ~ o that ~ ~leads s sto f o r ~ a t i o nof GH, via c o ~ ~ l eroukes x ~ n ~ ~ ~ several ving e ~ ~ y ~ e ~reactions ~ ~ t ~~ r y z~ and e9 ~references , ~ ~ ~ therein). It has been estimated that ~ p ~ r ~ x ~ m10’ ~ t tons e l y of methane is produced every year by the microbial of o r ~ a nsubstances. i~ decQmp~sit~on (MCR) is the enzyme that catalyzes the final step ~ e t h y l ~ ~ o e n z yMmreductase e of ethane biosynthesis in ~ e t ~ ~ o ~byc converting h a e ~ 2-(rrrethy’lthio)ethanesulfonate ~ ~ e t h y ~ - c o eM n zor~~e ~ ~ in - the ~ presence ~ ~ of o7-tbio ~ ) , COMn ~ n e p h o s p ~(coenzyme ~~te B or CoB-SH), to methane and the disulfi ~~~~~~:

4

The reaction proceeds with a net energy gain of 45 kJ.mo1-l but no coupling to A'I'P synthesis has been proven. The subsequent reduction of the h e t e r o d ~ s ~ to ~ ~regende erate the free coenzymes M and B i s a reaction catalyzed by the enzyme heterodiaulfide reductase. enzyme is unique to all methanogenic bacteria [59], even though genes encoding a ~utativeMGR have recently been found in several n o n m e ~ h a ~ ~ g e n i c bacteria [611, indicating that these enzymes may not be an exclusive feature of kcbaea. The ~ c c ~ r r e n of c eMGR ~ s o e n z . ~ has e s been demonstrated in M. thermaaz~trophieu~n C621, M. fervidus L631, and M. jannaschii [641, and their gene expression is differently re~ulatedby the growth conditions [591. 2.3.2. Overall $ ~ r u ~ t u r Architecture ul

MGR from M e t h u n o b ~ e ~ ~ r ther~oautotroph~cum ium i s the most thoroughly character~c ized enzyme of this class. This microorganism,like most of the ~ e t h a n o g ekchaea, can live and CQ2 as the sole electron and carbon sources [651. In the course of rnothfur cmried out by M. t~ermouutotrophicum,the 602 is first reduced to the methyl o x ~ ~ a t i oLevel n via i n t e ~ m ~ ~bound t e s to methfur~furanand tetrahydrom e t h ~ o ~ t e r iThe n . methyl group is then transferred onto coenzyme M, which is the .The latter is c o n s t i t ~byt ~a dimer o f h e t e r o t r i ~with ~ ~ san ~ a2@2y2 quaternaiy a t ~ ~ c t uand r e a molecular weight of about 300 kDa. The same structural o r g ~ n i ~ a t i oisnbelieved to exist in all MCR enzymes, even though a putative monos contain ~ a single active meric MGR, having a x@yquaternary structure a i d p r o ~ o to site, has been purXkd from ~ethunosareinathermophila C661. M. t ~ , ~ r m o u u ~ o ~ r ~ cum M~~ contains two sets of COBand CoM in two active sites p ~ e s ~in?the ~ t holoenzyme. Each active site lies at the end o f a 30-hong and 10-A-widecrevice created at the ~ ~ t e r f between ~ c e the (a.a',@, and y) and (a', a,@', and y') subunit ensemb~es.The need of residues from subunits belonging to different xPy heterotrimers in order to build the active site cavity explains the quaternary structure of the enzyme. ) from M. t h e r m a p h ~ [SSI ~~ the reported p ~ r i ~ ~ a tofi othe n active monome~c( x f ~ fNCR may indicate the existence of a possible different buildup of the active site. The crystal structwe o f M. t h e ~ ~ o u u ~ r oMCR ~h~ 167,681 e~~~ entry 0 )has provided the first step toward the structural understanding of the bioc ~ e ~of imethane ~ t ~bios~thesis.The structure reveaIs>hat the protein has an e l l i p ~ o ~shape d ~ with main axes of about 80 x 85 x 120 A, with the six subunits )~ structure (Fig. 7). The two a P y that are tightly asRociated in the ( ~ p y quaternary trimers conatit~tingthe whole molecule are related by a ~ o n ~ ~ ~ t atwofold ~ ~ o ~ axis, and the area excluded from the solvent upon formation of the hexarner amounts to about 8 3 0 A2. ~ The large distance s e p ~ a t i n gthe two active sites f- 50 excludes any cooperativity in the process of methane formation. The ~ t ~ cof tthe~ M,esubunit (548 residues) can be divided into four domains e (Fig. 8): (1) the N-terminal domain (residues 3-101) along the p o l ~ e p t i ~chain characterized by three a helices and two short @ strands; (2) an ap domain (residues ~ 0 2 - 2 wherein ~ ~ ) a f o u ~ - s t r ~ d eP dsheet is surrounded by helices in an @c s a n d w i c ~ ~~~~

682

FIG. 7. 8tmcture of ( c ~ p y )heterodimer ~ of M . t h e r n o a u ~ o o h . ~ ~ hMCR, i o u ~ ~represented schematically and viewed a p ~ ~ o x ~ m a talong e l y the n o n ~ ~ s t ~ ~ otwofold ~ a p havis ~ crelating the two crpy fragments constituting the enzyme. The arrangement of the Ni-bound Faso and protein residues in the active site of the enzyme is shown in the enlargement.

(3) an a-helix domain (residues 277-506) composed of' eight u. helices arranged in a helix s ~ d w ~ and c h (4) ~ the C-terminal domain (residues 506-549) wherein two short helices are separated by unstructured polypeptide. Subunit p (442 residues) has an Nterminal, u./B type domain and a C-terminal, a-ty-pe domain. Fintally, subunit y (247 residues) is constituted mainly by an @3 sandwich. 2.3.3. Structure of the Active Site

Each catalytic center or MCR contains Ni in a yellow chromophore named factor F4s0 169-721. The name of this cofactor is the result of ita absorption maximum at 430 nm f721, Figure 9 reports the stmco b s e in~ cell ~ extracts of X . ~~ermaauLoLro~hicLlnz ture of Ni-F4so determined by the crystallographic analysis of MCR 167,681, This cofactor is an u n ~ o ~ ~highly o n , ~ a t ~ ~ . a ~t ei -dc o ~ t a i n i pn og r p h i n o ~system ~ termed

~

-

S

~

~

U

~

~

~

FIG, 8. Secondary and tertiary structure of the a,0, and y subunits of M.therm,oautotrop?zicz&mMCR.

683

___

FIG. 9. Structure of the “corphinoid” Ie‘cs~of MCR.

“corphin” meaning a hybrid between a cominoid and a porphinoid tetrapyrro~c macrocycle. F430is the most reduced tetrapyrrole system found in biology, having only five double bonds within the cycle. This property i s probably related to the fact that this cofactor has been found so far only in m e t h ~ o g e n i cArchaea and hence it has evolved in anaerobic environments [73,741. When released from MCR, 3’430 contains Ni in the + 2 o ~ d a t i o nstate. The reduction potential of the Ni(IIYNi(1) couple in the soluble factor FqgOi s very close to that of the cob~~~)/cob{1~alamin couple (-0.65 V vs. -0.64 V) 175,763. Free N i W reacts with methyl iodide to yield methylated ~ i ~ I I ~ - Fwhich 4 ~ o ,spontaneously undergoes protanolysis t o yield methane and Ni(II)-F430 1771. These properties sup gest that factor can catalyze the reduction of methyl groups to methane, in contrast to cobalamin, which is a good methyl transfer catalyst. The crevice host~ngthe cofactors is very narrow and its d i ~ e t ei rs too small to allow an Fasomolecule to enter it. This observation suggests that the assembly of the hexameric enzyme occurs after coenzyme association with one of the subunits. The cofactnr is tightly bound to the enzyme through a series of non-covalent interactions among which 21 hydrogen bonds.

Carbon monoxide ~e~ydroge~ase/acetyl-CoA synthase ( C ~ ~ f 781 ~ i s/a multi~ C functional, sometimes mdticomponent enzyme capable of catalyzing the interconverC activity): O ~ ~ sion OF CO and COz ~

~

684

60 I- H,O

COZ + 2H++ 2c -

(5)

as well as the synthesis ofacetyl-CoA from CO, c o e n z ~ A, e and a methyl group ~ACS activity): CH;

+ CO -tCOA-S- + G

~ ~ - ~ O - ~ ~ ~ O A

(61

The CO required for the formation of acetyl-CoA catalyzed by AGS is generated by the CQDH activity W9]. The methyl group required in the second process origi~ His trans€erred ~ j to AGS through a nates from ~ethyltetraliydro€olate( G ~ ~ - and me~hy~transferase. The latter is a heterodimeric protein composed of a larger r subunit (55 kDa) hosting an [Fe4S41cluster, and a smaller p subunit (33 kDa) containing a C o ~ c ~ r cofactor ~ ~ ~ o (a~ vitamin d B12 derivative). The methyl t r ~ ~ ~from € e rCNSTHF to the cobalt center involves a nucleophilic attack o f a CofXj species onto CR3, to form a CM3-Co(III) moiety, In a subsequent reaction, the methyl group mally as CI-r.;> i s transferred to A C ~ / C rOe ~g ~~ n~~ r athe t ~ n&(I) ~ center [SO]. Methanogenic Archaea and acetogenic bacteria contain the bifunctional two~ A These ~ organisms S use the ~ o ~ ~ ~ jpathway u n ~to d ~ subunit ~ ~ D enzyme. grow autotrophically on CO, and R,; carbon dioxide i s k e d by the @O reduced to CO, which is utilized for acetyl-CoAsynthesis and subsequ organic matter, in the reaction catalyzed by ACS. In contrast, some theti ti^ bacteria, such as ~ ~ o d o ~ pr ~~ r ~i ~contain ~- uu ~a~ sing1 , that lacks ACS activity 178,811. The assembly, as well as the genetic o r ~ a n i ~ a and t ~ oregulation ~ of the CO o ~ d a t i o n(Coo) system in R, ~ ~ ~entails r ~a m coo gene , cluster c o n t ~ ~ genes in~ for a membrane-bound Fe-S electron transfer protein coding for CODH (COOS), (cookj), for a m e n ~ b r a n e - ~ o uhydrogenase ~d (cooH), and for metd cluster assembly y ns t ~ ~ p ~ o t ~ i and n s a CO-induced hydrogenase coo^^^ 143,4482 1. The e ~ ~ r ~ s ssi o dsa includes a he~e-contain in^ CQ-sensing ti~anscriptionregulatory protein (CooA) that, binds to the coo promoter region when the heme group binds C 8 183-851. ACS from ~ ~ ~t ~ et r ~~ o ~ ~~the e most td ~ cstudied ~u ~ u~ system ~ of this type, is an izzpz tetramer [86], with each ap dimer housing three ~ o ~ y m ecenters t ~ ~ cnamed cluster A, cluster B, and cluster C. Cluster A is the active site for ACS activity and is located in the Q subunit I87-911. Cluster C is the active site for CO H activity 192,931 and is located in the p subunit in close proximity to cluster J3, a conventional EFe,&&I C and e x t e ~ aredox l partners cluster. The latter t r ~ n s ~electrons e~s b ~ t ~ e cluster en [94,951. In the photos~nth~tic bacterium R. rubrum a simpler COD^ enzyme, constituted by a single a subunit of 62 kDa, catalyzes only the ~ Q / Ci ~ tze r c o n ~ ~ ~ s i o using cluster C. 3.1.2. Structwe of the Active Site

A large combinat~onof spectroscopic studies indicate that cluster A, the site of ACS activity, consists o f an [I"e4S47 cluster covalently linked, through a cysteine or a sulfide, to an Ni ion residing in a distorted square planar, S , ( ~ / O j zcoordinat~o~

685

FIG. 10. Putative?structure ofthe catalytic cluster C of CODH (A) and cluster A of' ACS

e n v i r o n ~ e n{Fig, ~ IDA) 2941. S i ~ ~ a rthe l y spectroscopic ~ properties of cluster C have been interpreted in terms of the Ni-X-[Fe4S4]center displayed in Fig. IOB [96], where l y i ~ a g n e t i c coupled ~ ~ y to an Ni center t h r o u g ~an the fFe4S41cluster i s c h e m ~ c ~and unknown ligand (possibly a cysteine thiolate, an histidine, or a sulfide), The nickel atom resides in a mixed ~ S ~ 3 ( coordination ~ / 0 ~ in distorted pentacoordinate geom~t~.

3.2.

Nickel ~ ~ ~ ~ r o x i ~ ~

3.21. Occurrence, ~ ~ ~ ~ lBole, o ~ and ~ cProperties ~ a l of the Protein

Activated oxygen species, such as the hydroxyl radical, l i y d r o ~ e ~ ~ x i and des~ superoxide anions, are physiological products of cellular O2 metabolism, and their intracellular levels are subjected to increase when the cells are exposed to oxidative stress by pathalogical events 1971. Aei*obic organisms have evolved a complex defence ~ a ~ h i to n eprotect ~ themselves from such highly reactive oxygen species. Superoxide dismutases (SODS) are i ~ ~ o r tcao ~~ pt ~ n e noft ssuch defense systems i othe n superoxide anion to because their function is to catalyze the d ~ s p r o p o ~ i o ~ a tof hydrogen p e r o ~ d eand dioxygen: 20,

$. 2H'

+ 0 2 4- H,O,

(7)

The p o t e n t i ~ ~hy ~ fhydrogen. ~ l peroxide is then scavenged by catdase, an e n z ~ m e capable of ~sproportionatinghydrogen peroxide into water and 02. All SODS are ~ e t a ~ l o e n ~ ~and n e catalysis s, occurs by taking a d ~ a n t a of ~ ethe redox properties of the metal, which should possess a reduction potential intermediate between the Q 2 / O i (E'' = -0.33V) and OY/H2Qz (E*' = +0.89V) couples. De~ndi~ ong the metal cofactor, four types of SODS are known: copper-zinc SOD (~u,ZnSOD),~ ~ n ~ a SOD n e (s~ n ~ S O D )iron , SOD (FeSOD), and the recently disic covered [98] nickel SOD (NiSOD). Mn and Fe SODS are bacterial c ~ o s o ~enzymes, which, despite different quaternary structures, share nearly identical sequences and tertiary structures indicative of evolution from cominan ancestors. It is interestii~g that some bacteria, such as P r o p ~ o n ~ b ~ tse r~~ ue ~ r and ~ ~ t nr e~ ~ ~~ omutans ~ ~ c ~ u s

produce an SOD, called “ c a m b i ~ s t i cSOD”, which can bind either Fe or Mn giving rise to active FeSOD and MnSOD [991. SODSare widely spread enzymes, found in bacteria and e u k ~ o t eas s well as in plants and higher organisms [971. In contrast, NiSOD has been found so far only in ~ ~ r e ~ tspecies ~ ~ ~[98,1001 c e s from which it has been c h ~ ~ c t e r i z easd a homotetramer of‘about 13 kDa subunits with no sequence similarity to any other kn0~7nSOD. EPR and XAS skudies have established that nickel is bound to the o~idizedenzyme as NiUII) ion [98,lOll. Therefore, the N~(IIl)/Ni(II)couple has been proposed to be responsible for s u ~ r o ~ ~isproportion~t~on, de even though its reduction potential lies outside the region of the couples involved in superoxide disrnutation [98,1011. ~ S. see o ~r ~ e ~ The genes encoding N~SOD~ s o d Nin~ S. c @ e ~ ~ c~o ~ ~~rand in have been cloned and sequenced, showing 91% homology [lo1I. Ni(I1) ions have been found to r ~ ~ l athe t eexpression of N i in S.~c o ~~l ~ o[1021. ~l o ~ ~ ~ h e ~ ~Ni(II? o r e , promotes sodN transcription by increasing the level of sodN transcript by about one order of ~ a ~ ~ t with u d respect e to controls l.1031. Ni(II) ions also regulate the posttranslational processing o f the enzyme necessary to obtain the active enzyme. One oe r~ gene ~ ~cloned in S. Lividam, exam~leof such a process is the S. ~ @ e ~~ ~ lc lsodN expressing a functional NiSOD only in the presence of Ni(l1) 11031. En contrast, the O the ~ presence of Ni~Il? same sodM gene expressed in E. coli prduced active N i ~ in only after deletion of the nucleotides encoding for the N-terminal 14 amino acids, thus s u g g e s t ~ gthe need of accessory rotei ins^ absent in E. coli, to properly fold and process the NiSOD polypeptide [1031/. Therefore, nickel appears to play a unique role in this system, being involved in enzyme expression, maturation^ and active site assembly. 3.2.2. Structure of the Active Site

The structure of the nickel site(s) in NiSOD has been investigated only by X-ray abso~ptions~ectroscopy[loll. Nickel Kedge spectra of the oxidized and reduced enzymes confirm that reduction occurs at the nickel center, although the observed I) The edge shift (1.2 eV) is smaller than expected for the N i t I I I ~ ~ i { Ireductio~. analysis of the EXAPS spectrum shows that nickel in NiSOD has a coordination sphere d o n i i ~ a t ~byd sulfur donors. The Ni ion is in fact bound to three S ligands on average, both in the oxidized and reduced forms. Two N/O ligands complete a fivecoordinate envi~onmentin the oxidized form (Fig. IlA), while reduction is proposed to cause the loss of one of such light ligands to yield a square planar coordination (Fig. 1. The presence of a binuclear Ni site {Fig. UC, D) was suggested tl0ll by the analysis of the amino acid sequence, featuring a Cys2-XXX-CysG motif that contains the only Cys residues found in the protein. The EXaFS data do not exclude this possibility, indicating the presence of an Mi-Ni interaction at 2.88(6) A Cl0l.l. In the case of a binuclear active site, the ligands to the two Ni ions would be provided by two different subunits in a hornodiineric enzyme. Indeed, sharing two Cys ligands in a double bridge between the Ni ions would account for the presence of three S ligxnds per nickel found by EXAFS. On the contrary, a mononuclear Ni site should invoke the

I D

~ )reduced ( FIG. 11. Putative structure of the oxidized ( ~ , and

cvs

1 Ni center of NiSOD.

p a ~ i c i ~ a t i oofn the o d y methionine residue present in the enzyme (Met28) in order to have the nickel ion bound to three sulfur donors.

Several proteins are involved in Mi uptake and tramport as well as apo-to-bolo metalloprotein conversion in organisms that rcquire nickel for essential cellular functions t35,1041. ~ ~ ~ " d e ~ eNin uptake ~ e n tin E. coli is regulated by expression of the five-gene tzik operon ~ n ElQsl.On ~ the other~ hand, A A ~ ~ - ~ n d ~e ~ ~~n ~d e~n~ t b r a ~ ~ bound Ni permeases have been discovered in AZcaZigenes eutrophus ~ H o x Nt 1061, B r u ~ ~ r h i ~ ~o bu ~p~ oi , ~~me , up^^ i ~ [106-1Q81, ~ ~ ~ ~ e ~ ~ c o b upylori c t ~ rfNixA) ~ 1 0 ~ , ~ 1 0 and Bacillus TI390 (UreEI) [llll. E. coli nihA, the first gene of the nih gene cluster, encodes NikA, a 56-kDa periplasmic protein that specifically binds a single Ni(I1) ion with high affinity [105,112,1131. According to X-ray absorption spectroscopic studies, the N i ~ - b ~ u n d Ni ion is six- or seven-coordinate,and its coordination sphere includes five or six O/N donor atoms (probably coining from aspartic or glutamic acid residues) with bonds averaging 2.06{2~A, typical for high-spin six-coordinate divabnt Ni ion [1141. An additional heavier scatterer atom, probably the sulfur of a methionhe residue, was also detected at 2 . ~ 7 ~ 2from ~ Ni.

LI AND ~ ~ N ~ A N In general, membrane-bound Ni permeases feature eight transmembrane helices with conserved (G1y-X5-Glu- is-Ser"Ser-Val-Val) and (His-X~-~p-His) sequences [115,116]. The latter motif is also found in E. coli NikC, where it has wn to be associated with Ni binding [loti]. ~utagenesisstudies of A. eutroxN have shown that the His and Asp residues of the second conserved sequence motif are responsible for Ni binding, whereas no cysteine is involved [1151. ~ a l o g o studies ~s on H. pylori NixA have confirmed these results [1161 and have extended the known essential residues to the His found in the first conserved sequence motif, as well as several additional Asp and Glu residues located on adjacent trans ane helices. ions that have crossed the cytoplasmic membrane through the systems described above are required for nickel metallocenter assembly. This process needs ~. ancillary complexes always include an Niseveral ac~essoryproteins [ 3 5 , 1 0 ~These and a nucleotide-binding protein. The best known systems are (1)UreE from bacteria [1171, involved in the assembly of the urease active site, (2) Hyp from bacteria pro cing NiFe hydrogenases 191, and (3) CooC and CooJ, proo ~ o ~ p i ~r ~u bl rl uu~C~ biosynthesis tl201. though all teins involved in ature histidine-rich r their overall sequence similarity three proteins o is very low. UreE are Ni-binding homodimeric proteins of about 34 a and are expressed by the ureE gene belonging t o the more complex urease operon. The latter also encodes additional proteins that include UreABC (the three subunits of the urease protein itself),the GT~-bindingUre~, as well as UreF, UreD, and UreH. The detailed function of the latter four proteins is poorly understood. UreD, UreF, and UreG have been G supercomplex, putashown to associate with the UreABC proteins in a Ur Lively essential for metallocenter assembly [14,1041. often, but not always, found to contain a histidine-rich carboxy terminal tail, and their sequence similarity is not very high 11211. The protein from X. aerogenes, the most studied system of this e of binding approx~matelyfive or six Ni(1I) ions with high affinity 221, while the t ~ n c a t e d the His tail, retains two highD studies have shown that, on g sites per dimer E1231. average, the Ni ions in the two sites are both pseudo-oct~edraland six- or sevencoordinate by N/O donor ligands. The two sites differ in the number of histidine ligands: three or four His for the higher affinity site, one or two His for the other [1241. It has been proposed that the Ni ions are bound at the interface of the dixneric UreE protein through related amino acid residues belonging to the two subunits. The ~NiFel-hydrogenase assembly protein HypB performs both Ni-binding and nucleotide-binding functions during the bios~theticincorporation of nickel in the protein [118,119]. H~~ features an N-terminal histidine-rich region, and is capable with an average Kd of 2.3 pM [115,125,1261.The assembly of H requires both the CooC protein, pre~ictedto contain the nucleotide binding domain, and the COOSprotein9 which binds four ~i/monomer = 4.3 pM) and is characterized by a histidine-rich C-terminal region [1201.

The i ~ o ~ofd~~idophosphoric e ~ ~ e acid found in the active site of^^^ ~ ~ t ~in l ~ the presence of p h e n y l ~ h o s ~ h o r o d i ~ i d(Fig. a t e 3D) is a transition state analogue, and its mode of binding to the Ni ions suggests a mechanism for enzymatic urea hydrolysis that allows a reconciliation between the availabk biochemical data and of the urease active site 19,211.This mechanism involves a direct s t ~ c t uproperties r~ ~n~ as the nuclcophilc in the process of urea hydrolysis, role of the ~ i - b r i d hydroxide as shown in Fig. 12A. According to this mechanism, urea replaces the three water molecules in the active site and undergoes a nucloophilic attack by the ~ i - b ~ d ~ n hydroxide concomitantly with the closure of the flexible flap, which adopts a conformation capable of stabilizing the catalytic intermed~ate,thereby lowering the activation energy for the reaction The steric constraints as well as the a s y m m ~ t ~ i c ~ s t ~ c t features u ~ ~ of the active site, poked to donate H bonds in the vicinity of Nil and receive IE bonds in the vicinity of Ni-2, induce the position and orientation of the transitian state. The structure of the tetrahedral transitian state in the active site, generated upon bond formation between the bridging hydroxide and the urea "inhibited BPU. The proton needed by carbon, is mimicked by the structure of the distal urea WR2 group in order to form ammonia by cleavage o f the C-N bond ~e itself. The latter would then also act as could be provided by the h y d r o x ~ nucleophile

FIG. 12. Alternative proposed mechanisms for urease-catalyhed urea hydrolysk. (A) Bridging hydroxide as nucleophile; ( Terminal hydroxide as n ~ c l e o ~ h ~ l e .

general acid, through a proton transfer assisted by Aspz365062, a residue shown to be capable of adopting multiple conformations while maintaining the coordination to Ni2. His”1323 would then act as a general base, by stabilizing the positive charge that develops on the distal ~ t r o g e natom during formation of nascent a ~ m o n i a This . mechanistic model, whose main feature is the role of the metal-b&iging hydroxide acting as the nucleophile, may well constitute a general rule for all hydrolytic enzymes containing bimetallic catalytic sites 1127I. A31 alternative mechanistic proposal is based on different roles played by the two Ni ions: Ni-l binding and activating urea, Ni-2 binding and activating the n ~ ~ c l e o p ~ l e hydroxide [17,128-1301 (Fig. 12B). However, this mechanism raises two problems E1301: one about the missing general base, which would deprotonate the Ni-2-bound water molecule at the optimum pH for enzyme activity (pH 81, and the second about as general acid, which must be protonated at p 8 even though it the role o f has a pKa of 6.5 f271. Therefore, according to this mechanis~,only 0.3%of all urease molecules would be in the optimal protonation state for catalysis, an inconsistency justi~edby the “reverse protonation hypothesis” 1130J. ~onsensuson which mechanism is followed in urease is not yet reached, and the matter is still under debate. A major step to understanding the urease mechanism has recently come from kinetic studies of the inhibition with fhoride 1131al; this anion slowly inhibits urease both in the absence of and, preferentially, in the presence of substrate. Evidence has been obtained to suggest that fluoride binds by bridging the two Mi ions, replacing the bridging hydroxide found in the native enzyme. In this state, urea can still bind to the Ni ions in the active site, but it, is not hydrolyzed, g during catalysis, and therefore suggesting a major role of the N ~ - b ~ d g i nhydroxide supporting the mechanism shown in Fig. 1%. The identity of the general acid capable of delivering a proton to the -NH2 group of urea to form ammonia is still not firmly esta b ~ ~ s h ~ d .

The mechanism of dihydrogen binding and activation by 1NiFeI-hydrogenases has still to be fully understood. However, the recent acquisition of structural knowledge of the oxidized and reduced forms of the enzyme, coupled to the available spectroscopic and biochemical data, has allowed unprecedented insights into the enzyme fir nction. s e been The existence of different nickel redox states in l ~ i ~ ~ l ” h y * o g e n ahas determined by EPR at low temperature [131b,132f. Subsequent IR studies have established that a ~ifferent1R spectrum is assocjated vith each redox state of nickel and related to changes in enzyme activity [48,56,13$J. The scheme reported in Fig. 13 11331 shows a summary of such enzyme states. D.agigus [ ~ i ~ e ~ - h ~ d rfrom o~~ s e contains mixtures of diEerent nickel states when prepared in aerobic conditions. One component is the so-called Ni-A state (see Fig. 131, which is EPR-active, enzymatically inactive, and inert toward activa-

691

N I ~ ~ ~ L - C O N T A I N IENZYMES NG

fully reduced cat. active

cat. active

unready cat. inactive

",..

ready cat. inactive

unready cat. inactive

FIG. 13. Scheme for the interconversion of the different states of CNiFel-~~y~ydrogenases, showing the relative catalytic, spectroscopic, and electrochemical properties,

tion by reducing agents (unready). A second state is the Ni-B form, which is EPRactive and enzymatically inactive, but can be readily converted to the enzymatically active SI state by one-electron reduction with Hz or another reducin~agent. The act~vat~on pathway of Ni-A involves its initial one-electron reduction to the and unready, NGSU state, which i s slowly converted to the E P ~ - s i l e n tinactive ~ active Ni-SI state. Ni-SU and Ni-ST have different IR absorption bands and reduction potenti~s.The o b s e ~ a t i o nof a significant shift to lower energy in the Ni-Kx X-ray a ~ s o r ~ t j oedge n in going from Ni-A to Ni-SI suggests that the reduction process involves NE (or its S ligands) but not Fe [581. The energetic harrier between Ni-SU and Ni-SI was suggested to involve loss of the bridging ligand s ~ later s found strong support in the between Ni and Fe ji13.41. This ~ ~ o t h e has structure of the active site of reduced [NiFel- and [Ni~eSel-hydroge~ases where the bridging ligand i s missing [31,511 (Fig. 6B). According to this hypothesis, the enzyme in the Ni-SU state can be activated only after the removal of this ligand, l e ~ ~ to n the g Ni-SI state, which is converted back to the Ni-A state upon exposure to dioxygen (Fig. 13). Two additional active forms of the enzyme have been found: reduction of Ni-S1 gives the EPR-active Ni-6, which can be furkher reduced to NCR (EPR-silent). ~nterconversionbetween the N i - ~ / ~ I states / C ~ is fully reversible C1331. All of these redox steps are associated with proton uptake and release (Fig. 13). The existence of two SI forrns (Ni-SI and Ni-SI-H) in a pH-dependent equilibrium (pK, = 8.2) has been established by IR spectroscopy [ 1331. The equilibrium was proposed to involve protonation o f a Cys thiolate in the active site.

692

CIURLl AND ~

A

~

~

Several schemes concerning the possible redox states of nickel and iron ions during the catalytic cycle have been suggested (see E31 and referenc~stherein). A recent study indicated that the iron ion is diamagnetic and low-spin FeCII) in all redox states of the enzyme [134], and the authors proposed that the nickel o x i ~ t i o ~ states are Ni(I1I) for Ni-A and Ni-ByNi(I1) for Ni-SI, Ni(1) for Ni-C, and Ni(O) (or -1 for Ni-R. This proposal is not consistent with data obtai~edby , which show small Ni-Kct edge shifts, compatible only with o reduction in going from the inactive oxidized form Ni-A (or Ni-B) to Ni-SI, as well as no edge v ~ a t i o n supon further reduction of Ni-SX to Ni-C and Mi-R forms E58,1351. On the other hand, it has been shown that Ni-R is the product of oneb e t ~ e e nthese e x ~ e r ~ m e ~obsertal electron ~ ~ u ~ofiNi-C o nt1361. The di~~repancy vations could be reconciled by assuming that redox chemistry occurs at ligand cysteines [3,134,1371. There is no conclusive evidence about the binding site and mode of Nz to the active site. The possible role of nickel in binding the substrate is suggested by an EPR study of the binding of the com~et~tive ~ n h ~ b iCO t o ~on the ~ ~ ~ ~ active i Ni-C c a ~ form, which showed spin coupling between "Ni and "CO, indicating the nickel as the 60 and thus Hz [138f. Further support for Wz ~ ~ to nickel n is~ ~ i ~ ~ site d ~for n g provided by the X-ray analysis o f the Xe derivative OED.fruetasouorans [NiFel-hydrogenase, which finds that the ~ydrophobicchannel closest to the active site points to the empty coordination site of nickel [553. ~pposi~ evidence g favoring Fe as the €Iz binding site is indirectly provided by the fact that CO ligation to Ni was not observed in EXAFS spectra of a ~ ~ " i n ~ i b ~ t enzyme form, which, however, featured CO stretching frequencies characteristic of char eta^ binding 133, ~ u p to~this o aIternat~ve ~ h ~ o t h e s i iss p r a ~ d by ~ da theoretical study indicating that Hz binds to the Fe ion of the NiFe cluster C1391. Several different hydragenase mechanisms have been proposed t o account for all of the experimental evidence on the enzyme knction ~ 3 , 3 ~ y 5 ~ , 1A4recent ~1. proposal assumes that the iron ion remains low-spin lFe(II) throughout all of the catillytic steps and that only N i ~ I ~ I ) / ~redox i ~ I changes I~ occur 11341, ~ c c o r dtoi ~ ~ this mechanism (Fig. 141, Ni(E1E) is assigned to the Ni-A or Ni-B state, and the first reductive step yields N i ~ ~ E )which - ~ ~ ,is the species c 5 m ~ e t e for ~ t Rz bin and activation. The reaction of Izz with Ni(IIC)-SE, coupled to the uptake of on of Wz, with subseque~tprotonation of the ion, causes the h e t e r o l ~ ~cleavage c terminal, Ni-bound, steina ate ligands { s e l e n o c y s ~ i n ~in~ ethe NiFeSe enzyme) as well as the binding of a bridging hydride anion between Ni(I1) and FeOI), giving rise to the fully reduced Ni(I1)-R state (Fig. 14). The N ~ { I ~ ) - H - ~ ~ e ~ centcr thus formed may facilitate the conversion of the hydride to a proton, ~ -Lo~N~i ((~ ~~ -~~ ~ " ~ O e ( ~I E~} ~. ~ ~ox~dation ~ c t r o n being i s ~ e l e c ~ to ~ oN~ c~ ( E ~ - and of Ni-R would then lead to Ni-C, best described as a ~ i ( I I ~ - t h iradical yl complex. Finally, release of two protons and further one-electron o ~ ~ d a t i o~n~ g e ~ e r athe tes Ni-SI state (Fig. 3.4).The two electrons released during the catalytic Hz o ~ ~ a ~ , i o n must be transferred one by one to an external oxidant through the chain of FeS clusters located in the small fl subunit. A possible pathway for electron Lra~sfer,

93

FIG. 14, Proposed catalytic mechanism for [ ~ i F ~ ] - ~ ~ ~ ~ g ~ ~ a s ~

through~bondsand/or through-space, from the active site to the redox partner is shown in Pig. 15 1301. A Werent ~eclianisK~ has been proposed that does not assign formal oxidation states to the metal ions. This mechanism does not invoke the formation of the hydride bridge but suggests the protonation of one of the cyskinate bridges in Ni-R and Ni-6 forms 131.

M. t h ~ r ~ ~ a ~ t u tcells r u grown ~ ~ ~ inc the u ~presence of an 80%:20% H&D2 mixture that can exist in different states, depending on different harvesting and ~ ~ o ~ cao ~ d~i t oi ~nn[741. s ~ a e i ~~ ~ ol afa ~ MCR ~ ~~leads ~ ~n to the c a t ~ ~ i c ~ ~ ~ ~ ~Ni(I1). , Treating the cells with d e n t form named M 6 ~ icontaining 100%Hz prior to harvesting produces a catalytically competent and E ~ R ~ a cform t~v~ named Meltredl, which displays an EPR spectrum characteristic o f Ni(I)-F*so (1411, n the other ~ ~~ r de ~the , t ~cells ~ gwith an $ ~ ~~ : ~2 m0~ /x~t p~ior u ~~ ~to ~ ~ collection produces the catalytically inactive, EPR-active, form named ~ ~ R has been proposed that the MCROxlform contains Ni(II1) bound to CRoxl is also obtained by incubating the cells prior to harvesting in the ~~~s~~~~ of Na2S, while incubat~onwith Na2S03 yields ~ C a d i ~ e r ~ n~t EPR-active and catalytically competent form [1441. Reduction of MCROxIwith a strong reductant (Ti(II1) citrate at pH 10) generates MCRredl [141].The MeltOxl,and ~ e R oforms x ~ are t r ~ s € o ~into e d EPR-silent and catalytically inactive ~ ~ ~ ~xR , o lxupon 2 ~ ~ expo~ ~~ ~ ~ forms named, respectively, ~ ~ ~ ~ ~ - ~s i ~ l and~ M sure to oxygen “741. M6Rl.edl is particularly sensitive to oxygen, and its activity disappears rapidly even in strictly anaerobic conditions due to its conversion to the

694

ClURlI AND M A ~ G A N I

PIG. 15. Proposed pathway for electron transfer connectingthe [NiFe] center in the R subunit and the FeS clusters o f subunit @ o f [NiFel-hydrogenases.

N i ~ ~ I ) - c o n t ~MCR,edl-,,lent nin~ form 1141 I. On the other hand, MGR,,, is relatively stable in the presence of oxygen I1451. The published crystal structures of MCR refer to the N I C R and ~ ~ ~~C~R~~~, x ~ ~ s i , states [67,681. Only the stmcture of the MCR,,x,,,l,,t form has been deposited in the PDB (code 1 ~ ~ These ~ structures ) . have allowed unprecedented insights into the enzyme niechanism despite the fact that they describe two inactive forms of the enzyme. In both MCR crystal structures, the Ni(I1) ion is equatorially bound to four t ~ oxygen ~ nitrogen atoms of the pymofe rings of and is axially c o ~ r ~ nbya the (Fig. 7). The structure o f the cofactor isolated atom of the side chain of GlyC"lh7 in the absence of the protein matrix i s identical to that determined for MCR-bound F430 1711, ~ i f f e ~ nonly g for the fact that in the enzyme the tetrapyrro~ering is almost planar, possibly because of the hexacoordinate state of the Ni ions. The identity of the second axial ligand completing the octahedral coordination geometry o f Ni(II) in the e~~yme-bound F40 depends on the MCR form. In the s ~ r u c ~ of u~e the M C ~ " form, ~ ~the-Ni~ion~i s bound ~ ~ to ~ the ~ sulfur atom o f CoM (Fig. 16A). The latter receives two HI-bonds from Tyr"333and TyrP3679 residues located in the active ~ site cavity. The structure of the NICR,lent state differs from that of M ~ d e of COBbecause it binds a sulfonate oxygen atom of the oxidized h e ~ e r o ~ s u l adduct SS-CoM (Fig. 1613) 167,681. In forming the heterodisulfide in MCR,,le,t, CoM rotates away from the position occupied in MCR,,xr-s,lent.The Ni-bound oxygen atom is also Hbonded to Tyrc/333, while a second sulfonate oxygen atom is ~ " ~ o nto~ Tyre367. e d Five chemically modified amino acid side chains, belonging to both the c1, and a' subunits, have also been identified in the active site. Four of these modifications of the side chains of N;isu257,Argm271,~ l ~ ~ *and *', involve methy~~tion appears to have its carbonyl oxygen substituted by sulfur [67,6$f. whereas GlyC"445

695

FIG. 16. Structure of the MCE active site as determined in the MCRox1-sllent(A) and

Analysis of the molecular structure reveals that the F430 binding pocket is mainly lined by hydrophobic residues. The small diameter of the active site channel implies that only small molecules can enter it, and that COB and methyl-CoM cannot simultaneou~lyoccupy the channel, but should enter it in an orderly way. In particular, met~yl-CoMshould enter first, followed by a molecule of COB, coxisistently with the ordered formation of a C ~ 3 ~ S ~ C o M / C oternary ~ - S ~complex ~C~ shows occurring during catalysis [1461. Furthermore, the structure of MCRoxr..sdellc that COB cannot penetrate the channel deep enough to reach the available &al nickel coo~dinationsite (Fig. lGiA), with its thiol group lying at about 9.0 from the Ni(I1) ion. The above structural features, coupled with kinetic and sp~troscopicdata for the active forms of the enzyme, have resulted in a proposal for the catalytic mechanism of MCR, which accounts for most of the available e ~ ~ r i m e n t evial dence 167,741. This hypothesis assumes that a molecule of methyl-CoM enters the active site channel of the enzyme in the N ~ ( ~ ~oxidation - ~ 4 ~ 0state (Fig. 17A). The binding mode of the CoM in the MCRoxl-s,lentstructure (Fig. 16A) suggests that ~ ~ ~ - S - Cmay o M bind at the same position in the cavity. The ~ C ~ - s ~ ~ s complex is then proposed to be the adduct with CH3-S-CoM,in which the methyl goup is close to the empty axial position of N i ( ~ ) - F ~as o ,shown in Fig. 17A. The productive Ni(II)-CN3 interinediatc can be formed by heterolytic cleavage of the ,-S-CoM bond upon nucleophilic attack by the electron-rich Ni(I)-F430 (Fig. 17B)

*

C ~ ~ ~+- H" ~ + - Ni(I~-~430 C o ~ C ~ 3 - N ~ ( I ~ I )+- F~~o~M o -~H

(8)

696

SH

I

FIG, 17. Proposed catalytic mechanism for MCR.

followed by one-electron transfer from CoM-SH (Fig. 27C):

~ - ~ i { ~ +I CoM-SH I ) ~ ~+ * ~~ ~~

~

-

-k ~ CoM-5 i (

~

I

9

~

~

~

The proton needed in the first reaction could be provided by a nearby Tyr residue (Fig. 16), which then would be repr~tonatedby dissociation of Co

~

alternative route to N ~ t 1 I ) " C format~on ~~ can be provided by the honiolytic cleavage of the m e t h y ~ - C obond. ~ This route requires the formation of a thiyl radical on CoB (COB-S') by the action of an unknown oxidant group present in the cavity and proposed to be the modified thioglycine ~ l y ~ * ~This ~ ~radical [ ~ 0would ~ . react with C CoM to yield C o M - S - ( C H ~ ) ~ ~ 'The ~ C olatter ~ . would transfer a CH, radical to NilIIg ~ n e r a t i nthe ~ ~ e t e r o d i s u ~ dand e C ~ ~ - N if601. ~ ~ ~ ~ - ~ ~ ~ * Whatever route is chosen, the N i ( I I ) ~ ~ Eintermediate 3 would spontaneously - Fn ~~ 3c ~e , s ~s ~~ o t being on undergo ~rotonolysisto yield methane and N i ~ ~ ~ ~the provided by CoM-SH* ', The subsequent step should be the reaction of the ' thiyl radical with the anionic COB to generate the h e t e r o ~ s ~ l fradical i~e anion (Fig. 17D). The latter species has a reduction potmtial negative enough to reduce Ni(II1 to Ni(T) via electron transfer through the residues present in the active site (possibly again thioglycine 61y"**5),reconstituting the active form of the enzyme.

ehydl.ogenase/AcetyI-CoA § y n t h a ~ ~ The catalytic center of carbon monoxide dehydrogeriase activity, cluster C (Fig.

Ion) can assume four redox slates, named Cox,CreclllCkk, and Cia. A one-electron reduction (En'= -0.15V) o f the diamagnetic state C, (Ni(~1)-~-[Fe4S*l2~ [96, 961) yields the Credl state (Ni(~I~~X"[Fe*S~]") 11471. This S = 1j2 state is EPR active, and the Ni(I1) ion is at most weakly coupled to the FeS cluster 194~96,14~]. At lower potentials (E"' = -0.39V) another S = 1/2 state i s obtained, named CWd2j91,941, This state has been prop@sedto be two electrons more reduced than Credl and to possess an IJ,d-Ni(l)-X-[Fe*S*]' *) configuration, with L being a metal-bound ligand silent state named Cint,~ n t ~ r ~ e d ibetween ate Credland Grea, ha;., also been detected [149,1503.A putative mechanism for CODE activity 1151,1521(Fig, 3 8 ) involve8 the binding and d e ~ ~ ~ t o n a tof i oanwater molecule at the N i ~ ~site I ) in the 6, form of cluster 6 , generating a nucleophilic hydroxide anion. A subsequent oneelectron reduction would produce Credl,the form of cluster C that is proposed to bind CO at the proximal Fe site. This molecule of CO would then undergo a nuc~eQphilic attack by the hydroxyl group, producing an Fe-bound COOH intermediate. The latter can be deprotonated, yielding C02 and a cluster C reduced by two electrons tCrd2). The regeneration of the C,,, state would proceed through a two-electron oxidation via cluster €3 to an external acceptor, i d e n t i f i ~in an H2-evolving hydrogenase in the case of isolated CODH activity (as in R, rubrum), or in ACS in the caBe of b ~ f ~ c t i o n a l ~ ~ ~ e ~~ ~/(as~in A C. ~ ~~e s~S ~ ~An ~mt e r n~a t i v em ~ ~ ~ h ~hasi &so es ~u been proposed that entails a role reversal between Ni and Fe in binding CO and would i b ~ hold i t y to describe the o ~ ~ o s h y ~ ~ ~f1481. x i ~Ine any case, ~ i ~ r o r ~ v e ~ ~ process of CO2 reduction. A possible m e c h a ~ ifor s ~ACS activity (Fig. 19) E5l involves the direct role of the Ni ion in cluster A (Fig. 1DA). According to this proposal, a o n e ~ ~ l e ~ treduction ron of the djamag~etic e oxidized state of cluster A (Ni(lI), &,I, using electrons ~ r o ~ during c e ~ -catalyzed oxidation of CO to GOz, would yield the param~g-

f

Y-

J

FIG. 18. Proposed catalytic mechanism for CODH activity.

1 L

J

I

FIG. 19. Proposed catalytic mechanism for ACE activity.

netic reduced state (NitI), AT&).This reduced state of cluster A would bind a molect~le of CQ (possibly coming from cluster C through an intramolecular tunnel), yielding the A,,d-CO state. The latter displays a typical S = 1/2 EPR signal named “NiFeC signal”, i n d i ~ t i n the g presence of the putative chemical moiety [ ~ e * S * ~ - ~ ~ N i ~The I~-CO Ni center of cluster A, believed to possess two cis open coordination sites, would then enzyme, u ~ d e ~ ~ oai formal ng bind a CH: group, coming from the ~ethy~transferase two-electron oxidation to yield an unstable NiflI1)-containing intermediate. A oneI) electron reduction of this state, followed by insertion of CO in the C ~ ~ - N i ( Ibond, fragment. N i ( I h~subsequent ) nucleophilic attack by the thiowould yield a C ~ ~ - ~ ~ ~ late group of CoA on this fragment would yield acetyl-CoA and regenerate the active Ni(1) form following release of one electron (Fig. 19).

The current knowledge on the chemistry of NiSO prevents the formulation of detailed mechanisms for the catalyzed reaction. As pointed out earlier, the reduction I ~ ~ + 1.0 V) potentials of the possible one-electron reduction couples, N i ( l l l ~ / N i ((about and N i ( ~ I ) ~ i ((aboul I) -1.0 V) L1311 have potentials lying outside the possible range for supe~oxid~ ~ i ~ m u t a ~(from i o n -0.33 to +0.89 V). However, it has been establis~ed i~II) that thiolate ligands should lowar the reduction potential of the ~ i ( 1 ~ 1 ) ~couple to app~opr~ate values 113~~1~31. The dismutase activity of NiSO has been measured and found to be similar Lo that of C u ~ ~ n S and O MnS ~ (kWt= 1.3 x M-’ s-‘/Ni ion) [l01]. The a c t i ~ ~ist y constant over the pW ran 8 and it has little dependence on ionic strength with Q ~D n S tl0ll. ~ D NiSQD appears to be inhibited by cyanide, respect to ~ u , ~ n ~and but very little inhibitory effect has been observed with azide, which is instead a competitive inhibitor of Cu,ZnSQ 1981. The lower affinity for anions, and the little eEect of ionic strength on activity of N ~ Slead ~ to ~ the , proposd of an outer sphere mechanism for superoxide dismutation in NiSQD operating for both redox reactions g the ~ o s s ~ b ~ lof i t yo ~ e ~ a t i nwith g an outer sphere fl0lj. It is worth ~ o t ~ nthat mechanism was also recently sugge;estedfor Cu,ZnSOD [1541.

5.

IVE

~ o w l e d of ~ ethe b i o i ~ o r ~ a role ~ i c of nickel is a relatively new field of research, promising to reveal more surprises and discoveries in the years to come. In particular, the ~ ~ s t e r i of n gm o l ~ cbiology ~ l ~ techniques to overexpress ~ i - ~ o ~ ~t ~~ io ~t i en will allow the obtaining of large q u a ~ t ~ tof ~ eprotein s necessary to facilitate mechanistic and s ~ ~ c t~ ~t ~r da~The ~e s latter . will certainly invalve the d ~ t e r ~ i ~of~the ti~n structures of known Ni-containing systems, such as those related to Ni t r a ~ f i c ~ i ~ g , r as well as possibly afford s t r u c t u r ~ gene regulation, and m e ~ l l o c ~ n t eassembly,

informat~onon enzymatic ~ t e x ~ e ~ aStudies ~ e s . of the bio~ogica~ i ~ p l ~ ~ a t i of ons nickel toxicity, and in particular the elucidation of Ni-nucleic acid interactions, will also possibly shed light on the ~ e ~ t h - t h r e a t e ~mechanisms ing for this important y ~ l i ~ d u s t ~~~oal ll ~ t aand n t h o p e ~ l l yhelp in the designing of e n ~ r o n ~ e fn ~~ ~~ n Ni-bas4 catalysts.

A

BME BPU COB COT3 CoM DAP

EPR IR JBU

mu MCD MCR PDB PPD SOD

XAS

R

acetohydroxamic acid acetyl-CoA synthase ~-~~ercaptoethanol us ~ ~ s t e urease L ~ ~ i coenzyme R carbon monoxide dehydrogenase coenzyme M d i ~ ~ i ~ o p h o s p h oacid ric electron p a r ~ a ~ e tr i ~c s o ~ ~ n ~ e extended X-ray absorption fine structure Fourier transform infrared spectroscopy infrared jack bean urease ~ ~ e b saerogenes i e ~ ~ urease ~ magnetic circular dichroism methyl-coenzyme M reductase Protein Data Bank phe~ylphosphorodiamidate superoxide dismutase tetrahydro~olate X-ray absorption spe~roscopy

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'School of Chemistry, University of Leeds, Leeds LS2 9JT, UK 2School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK

710 710 711 713

1. ~ N T ~ ~ ~ U C T I O N 1.1. ~ o o r ~ n a ~Chemistry ion of Copper 1.2. Bioinorganic Role of Copper 1.3. Homeostasis and Metabolism

2. E ~ 2.1. 2.2. 2.3. 2.4. 2.5. 2.6.

~ E S ~ ~ O T WITH E I N~0~ S S T ~ U ~ T ~ ~ E Galactose Oxidase b i n e Oxidases Peptidylglycine a-Hydroxylating Monooxygenase Ilemocyanin Catechol Oxidase Cytochrome c Oxidase

3. S T ~ U C FT~ ~~ T ~ OF R ECOPPER S O ~ D A T EI ~ ~ ~~~0~ 3-D ~ T ~ U C T U R E 3.1. Lysyl Oxidase 3.2. Doparnine p-Monooxygenase 3.3. Tyrosinase onooxygenase and Ammonia Monooxygenase 3.5. Nitrous Oxide Reductasc

4, S T R U C T ~ ~ ~ ~ ~~ ~E C~ T I~ OO NNS ~ ~ P S 4.1. Catalytic Mechanisms 4.1.1. Galactose Oxidase

709

~ OFE

715 720 721 723 725 726

727

S 730 730 730 735. 732 733

734 734 734

4.1.2. Amine Oxidase 4.1.3. Dopamine p-Monooxygenase and Peptidylglycine a-Hydroxylating Monooxygenase 4.1.4. Tyrosinase and Catechol Oxidase 4.1.5. Methane Monooxygenase and Ammonia Monooxygenase 4.1.6. Cytochrome c Oxidase 4.2. Structure-Function Comparisons Retween Enzymes 4.2.1. Galactose Oxidase and h i n e Oxidases 4.2.2. Dioxygen Binding by Hemocyanin, Tyrosinase, and Catechol Oxidase

~ S ~ ~ C T AND I V ~OUTLOOK S

736 738 741 744: 746 746 746 748 749 751

751

emistry of Copper

Although isolated examples of zero-valent and trivalent copper compounds are known [I],the chemistry of copper with biologically relevant ligands is exclusively that of the +I and +2 oxidation states I2j. The copper(1) ion is the only biologically relevant monoval~ntion, which prefers “soft9’ S ligation and coordination numbers of two, three, or four [ll; four-coordinate copper(1) is electronically saturated, and cannot se it is a closed-shell ion, compounds of copper bind a substrate. characteristic spec pic signatures in the optical spectrum and are EP Its diamagnetic nature also means that the NMR spectra of c~pper(1~”contain~ng n~te~ proteins contain no contact-shifted sigsials diagnostic of m e t ~ - c o o r ~ ~residues. This means that information regarding the structures of‘ copper(%)sites in proteins can be gleaned only from site-directed mutagenesis experiments, fi-oni their luminesS and single-crystal X-ray cent p r ( ~ ~ e r t i[3], e s or from the stntetural techniques E diffraction. Copper(II), by contrast, favors “harder” N-donor ligands and higher coordination numbers. ~ i v e - c o o r d i ~ aist ~most o ~ common for Cu(II) complexes, although fourand six-coordinate complexes are also well known. Thanks to their d9 c o ~ i ~ ~ r a t i o n complexes of Cu(II) have very plastic coordination spheres, giving rise Lo rather dist o r t ~ dgeometries compared to the “ideal” polyhedra found for many other metal ions. Tetragonal [4+ XI (x = 0-2) structures ~ r e d o m ~ n aderived te~ from a [CuL412+ square plane with zero, one or two apical donors, the latter often lying at, long distances of up to 3 A or more from the metal ion [ll. In contrast to copper(I1 compounds, the optical and EPR spectra of copper(TT)complexes are very informative [I], so that much more is known about copper(1~~ sites in proteins. An EPR spectrum will

Cu PROTElNS IN T R A N S P ~ ~AND T ACTIVATION

711

identify the highest lying d orbital of the copper(1I) ion, which can be used to cfistinguish between tetragonal, tetrahedral, or trigonal bipyramidal Cu(II) centers 141. It can also detect the presence of S or N ligation to copper using samples enriched in 33S or, if necessary, 15N, although the use of double-resonance methods may be required to observe couplingp to these nuclei 151. W i l e a UV-Vis spcctrum o f a copper(I1) complex will contain one or more d-d absorptions, these are often broad and overlapping. However, a simple optical absorption spectrum can still be diagnostic for tetrahedral or six-coordinate copper(I1) centers in particular 161. If necessary, d-d spectra can be deconvoluted and assigned using single-crystal spectroscopy or, in solution, circular dichroism methods. The cupric ion is the smallest, and hence the most Lewis acidic, of the divalent ions of the first-row transition series, so that many copper(I1) complexes are highly effective Lewis acid catalysts [7$1. However, there is no known example of copper being used for this a~plicationin biology, possibly because of the very high metalligand bond strengths of copper(lc1) complexes, which would make product release during turnover rather difficult. There are also no known examples of structural copper sites in proteins. Instead, almost; all known biological applicati~nsof copper 1 ~ either ) to carry out a simple involve redox reactions utilizing the ~ ~ 1couple, electron transfer event or to effect a redox transformation at a substrate (Sec. 1.2). The very different coordination preferences for copper(1) and copper(II), coupled with their d ~ ~ e r eionic n t radii (0.96 m d 0.72 respectively-a d i ~ ~ r e n c e o f 25%),means that Cu(1III) redox reactions almost always involve large geometrical changes at copper. This contrasts with the other metals used in biology for redox or atom transfer applications, namely, manganese, iron, cobalt, nickel, molybdenu~, tungsten, and (possibly) vanadium, which all form compounds that can undergo one-electron reactions with small to minimal changes in niolecular structure [9]. The large structural reorganizations associated with copper redox are useful €or catalysis in that they facilitate the oxidative substrate-binding reactions found in copperldioxygen chemistry DO], for example. However, a large reorganization energy is disadvantageous €or applications requiring rapid electron transfer to or from copper ll1L The type 1copper site is art ingenious evolutionary solution to this problem (Sec. 1.2).

The majority of copper sites in proteins fulFill one of two functions, namely, electron transfer or the binding and activation of small molecules 1121, .Almost ail electron transfer copper centers are based around the type t or “blue” site, whose structure is derived from a trigonal pyramidal [Cu(Cys)(I-Iis)Z(L)l*’+(L = methionine or a main chain carbonyl group) motif, with a long CUL bond of close to 3 A (Fig. 1A) [13,141 (see se the also Chapter 17). This structure can be thought of as a ~ o ~ p r o m i between stereochemical and electronic requirements of copper(1) (soft S-donor ligands, three-coo~dination)and copper(I.1) (hard N-donor ligands, four-coordination). As a

712

A

FIG, 1, ~ o l estructures c ~ of known electron transfer copper centers in prokina. (A>'Type 1 copper site. (Bj CuA site from cytocbrome oxidase and ~itrousoxide reductase.

result, the geometries of the oxi&zed and reduced type 1copper sites a identical 1141, and nature has suecessfully evolved a Cu(lZ/I) couple wi range c250 to 4-775 s. NEE, oxidized type 1copper centers scopie finge~rints: 1 A,,{63365Cu} coupling constant in th jess tbm 70 10-4 and an S-Cu charge transfer band lo3 cm-'(~= % 5,000 M-' em-') which gives rise to the ch t known f131. Type 1 copper proteins m e discussed snore fully in transfer site that is closely related to the type 1 c d GuA "El, has been c ~ s t ~ o ~ a p h icharacterized c~ly in cytochrorne oxidase s oxide ~ ~ u c t ~N~~~~ ~ s e See. 3.6)

group>complex (Fig. JB). In een the [Cu2'Gu+I3+ and t C u l ] sed to donate two electr Lo be C O ~ The~ ~ . form of this site shows a seven-line sigaal, indicat to both copper ions [MI.The optical spectrum 12.0, 19.0, and 21.5 x 10 1' rhomb and giving a nusual electronic s electron i s de$ocaliz 1.5-t 31. 8 rcuz 1 species ~191.

713

~ r o a s~p ye a ~ n there g ~ are two types of copper biosite that are known $0 take inding and activation. The “type 2’, copper centers are mononucdo not contain thiolate ligation. A variety of structures fall into ( ~=~1,2) z ) ion ~ J in~ amine ~ ~ ~ n [205, including the [ C u ~ H i s ~ : ~ (x complex in galactose oxidase oxidases (Sec. 2.2 and 4.1.2), the [Gut s)a(Tyr)z(OH~)] t ) ( O center ~ z ~ ~found in p e ~ t i d y ~ (Secs. 2.1 and 4.1.1)9and the l ~ u ( ~ i s ) ~ ( ~ e ‘‘Cug’’ o n the glycine ~-hydroxylatingm o n o o ~ ~ e n a (Secs. s e 2.3 and 4.1.3). By c o m p ~ ~ s with type 1 sites, oxidized type 2 centers exhibit EPR spectra that are more typical of t e t r ~ ~n ~ u species, ~ Iwith~A,,(“”,’cu) ~ > 120 x cm-’, and only wedc d-d < 200 M-’cm-lf lZO1.‘Type 3” coppet visible ~~a below 22 x 103 em-’ are dinuclear complexes whose reduced and oxidized forms have the struc ~ s ~ ~~) ~ t ~s i iL - L= I x *~ x = ~4 or ~L = 0 r ~ ~is+ u ~ ~~ OHz, per(I1) forms their optical spectra rese respectively [all. type 2, centers, but they are E ~ R - s ~ l e nwhich t, reflects the presence derived Egand b ~ ~ d two ~ copper n ~ ions [all. With the possible exc dopamine [hnonooxygenase and peptidy~glycinea-hydroxylating m o n o o x y ~ e n ~ ~ e (Secs. 2.3, 3.2, and 4.1.3) [ZO], dl known type 2 and type 3 copper centers form the active sites of their proteins, In addilion, in all examples whose ~echanismsare we11 understood, the type 2 or type 3 copper center binds a small molecule during catalysis, of an organic s ~ b ~ ~ rmaot lee c ~ eto these cap^^ ~lthou~ direct h ~ a ~ p forma~ion ~ e x biosites does not always take place. In addition to the above catalytic and transport applications, other roles for copper in biology are just b ~ ~ n to~ be ~ elL~c~dated. n g For example, a ~ ~ e p t for or the plant hormone ethene has recently been isolated, whose ethene binding site is a cop~er(1)ion of u n c e ~ a i nstructure that contains both cysteirze and histidine ligation f221. It should also be noted that three important classes of copper enzyme that carry out redox chemistry on small molecules are discussed in other chapters of this book. These are the blue sup ti copper oxidases, which possess a trinuclear active site that can be thought of as a combi~ationof one type 3 and one type 2 (Chapter 16); and the type 2 copper proteins superoxide dismutase ( ~ ~ ~nitrite a r et d ~oc t ~~ e~ ~ a p18). ter

A recent study of SOD activat~onin the yeast ~ a c c ~ r o ~ ycerevisiae ces c of free (i.e,, n o n p r ~ t e i ~ a ccopper ~ o ~ s is ~ that the c ~ o p l a ~ ~concentration iic 10-Is NI,or less than m e atom per cell [23I. Regardless of whether this result can be ized to other organisms~it is clear that cellular copper is very strictly ~ ~ l and stored. This is necessary because ofthe ability o f copperf111to generate extremely toxic hydroxyl radicals from superoxide or peroxide species, which are trace byproducts of or^^ r n e ~ ~ b o While l i ~ ~ the . protein m e ~ ~ l o t ~ o acts n e ~asn a sequestrating agent and storage medium far cytoplasmic copper, it does not appear to be involved in the transport o f copper to specific parts of the cell or the insertion of

~

~ A ~ C ~ O NW OWL^^, , AND PHILLIPS

714

copper into freshly prepared apoproteins. Rather, specific mechanisms for the donation of copper to each individual enzyme appear to have evolved that are only beginning to be elucidated. The systems for copper uptake, delivery and regulation from S. cereuisiae are the most fully understood to date (Fig. 21, and are briefly described below. ExtracelluIar copper, which under ambient aqueous conditions will be mostly in the 4-2 oxidation state, is reduced to copper(1) by the protein FREl, or one o f several hornologues, at the plasma membrane. Three copper uptake proteins, GTR13, have been identified, which import copper(1) across the plasma membrane and transfer it to one of several copper-sensing or chaperone proteins, by a mechanism that is currently unknown [24-261. Two copper-sensing proteins are well understood, ACEl and MAC1, which operate in different ways [261. ACEl is a copperbinding protein, which coordinates four Cu(1) ions in a thiolate-rich cluster when the cellular concentration of copper is high r261. This activates the protein to bind a specific DNA sequence, which triggers the transcription o f two proteins: metal-

0

ATXI

FET3

COX17

FIG. 2. Ehown copper uptake and chaperone proteins in S. cereuisiae.

r"""T" S-.C""S

?

u

T^"T" s-cu-s I

*

r" "^fSN" s cuI

I

-

u u

T""r" s I

?

s-.Cu,.

SH

d

"T""T" sn SB _a_

s-cu-s

FIG. 3. Mechanism for the transfer of' copper between a chapcrone and target protein, as proposed by Puf'ah€et al. 1373.

lot~onein,to sequester the excess cellular copper t27); and SOD, to mop up cytoplasmic superoxide that might react with this free copper 1281. MAC1 is required for the expression of the FRE and CTR genes; while it does not require copper for binding, W C 1 does contain a potentially copper-binding amino acid motif 1291 and is rapidly degraded in the presence of excess copper 1301. This represses the expression of the cellular copper uptake system and prevents the introduction of even more copper into an overloaded cell. Three copper chaperone proteins have been identified in S. cereuisine (Fig. 2) 1241. CCS (a.k.a. Lys'i) is responsib~efor loading copper into the enzyme SOD (Chapter 18) [311. ATXl, which also has antioxidant activity 1321, delivers copper to thc vesicular transport ATPase CCC2, which in turn passes it on to the ~ ~ u l t ~ ~ o p p e oxidase FET3 (a ce~loplasminanalogue, Chapter 16) inside post-Golgi vesicles t331. ina ally^ COX17 assembles the CuA site of ~ p o c ~ o c h r o oxidase ~ e (Sec. 2.6) at the inner mitochondria1 membrane [341. Analogues for all o f these proteins have also been isolated from Homo sapiem. A ~~~C metal~bindi~g motif and "open psandwich" ferredoxin-like domain are common to the sequences of most of these chaperone proteins, with the two conserved cysteine residues acting as ligands to a two- or three-coordinate copper ion [24,35,361.A mechanism has been proposed for the exchange of copper ions between two proteins of this type, involving two- and three-coordinate copper(I) intermediates (Fig. 3 ) [37].Transfer of copper from CCS to SOD cannot take place in this way; however, the sequence and crystal structure of CCS show an obvious docking site for m apo-SOD subunit [31,361, which suggests an alternative mechanism 138I. Inte~estingly,60x17, which does not share the above homologies, forms a dinucleai-copperlthiolate center 1391. Given that its function is to assemble the dicopper CU, site of cytochrome oxidase (See. 1.21, it is tempting to postulate that both copper ions are transferred from COX17 to apo-COX simriltaneously.

Crystal s t ~ c t u r eare s now available for all of the major classes of dioxy~en-b~n~ing or dioxygen-activating copper protein. These are listed in Table 1,while relevant views of the copper centers in these proteins are shown in Fig. 4.

746

semi, pW 4.5 semi, pW 7.0 w o semi s semi, T semi, Cys22$G~~

1.2 1.02 1.8

35 35 36

1.7 1.9 2.2 2.0 2.1

43 43 43 230 230 230

2.6

2-0 2. 2.0 2.4 2.4 2.1 2.2 2.0 2.2 2.2

67 57

68 183. 181 181 67 67 61 58

717

sweet potato Sweet potato Sweet potato

2.8 2.2 2.4

59 59 60

2.15 2.1 2.1

13. 72 71,72

3.2 2.18 2-4 2.4 2.3

-

79 81 222 85

2.5 2.7 2.7

1BT3 1BT2 1BUG

2.7 2.3 2.35 223 2.9

86

92 92 92

96 98,99

99 99 99

718

Cu ~ R O T ~ ~INNTS~ A N S P ~ AND ~ TACTIVATIO~

719

I FIG. 4. Active sites in the crystal structures of the copper proteins described in %. 2. (A) Galactose oxidase grown a t pR 4.5 142,431, (B) Active form o f E. colt amine oxidme [ST]; (We, Wa and W2 are water molecules, see Section 2.2); (C) 2-phenyleth3.lamine-red~ced E"eoEi amine oxidase, following exposure to air TlSll; (the cofactor "TPR' is in either its aininoquinol or ~ m form); iD)~ The oxidized ~ catalytic ~core of rat ~~ p t i d y ~~g l y 2-hydroglating ~j~e~ ~ monooxygenase [7U; (E) The oxidizod catalytic core of sat peptidylglyci~ea - h y ~ o q y l a t i ~ g monooqygenase, incubated in the presence of N-a-acetyI-3,5-diiodotyrosylglycine[721. (F) P. ~ ~ ~ e~ ~r# . ~ r ~ ie m~o c~y1791. a~ n i (GI n~ L. ~ # ~ yo~yhemocya~il~ p ~ ~ ~ 12221. ~ §(€3)Sweet potato met-catechol oxidase 1921. (I) The heme-cc$CuB site of oxidized beef heart cytochrome c oxidase 1991. This figure was produced using Midasplus software 12311. See Figure 15.4 in the color insert.

720

Galactose oxidase (GOase, EC 1.1.3.9)is an extracellular enzyme produced by fungi of 0t ~catalyzes . the o ~ d ~ t i oofnp r i a~coho~s ~ ~ to aldethe ~~$~~~~~~ genera [ 2 ~ ~ 4 X hydes by molecular oxygen, producing hydrogen peroxide as a byproduct (equation 1):

ase is a single polypeptide chain of 639 amino acids [4U, whose structure has been d e t e ~ ~ i n etod 1.7 A resolution [42,431. The p o l ~ e p t ~ dchain e is divided into three ~ r e d o ~ i ~~~- sn tt~~c yt u rdomains. al Domain 1 (residues 1-155) has a @-sand, which is Linked to domain 2 by a well-ordered stretch of main 2 (residues 156-5324) has p s e ~ ~ o - s e v e n fsymme o~~ nee of a seven-bladed propeller wherein each blade consists of a a ~ ~ i ~fi asheet. ~ ~The ~ copper e l ion, which is part of the active site, solvent-accessible s e of this domain close to the sevenfold axis. ~ o m a 3~ {residues n ~ 3 ~ "lies ~ 3on~the 1 opposite side of d o ~ 2~from n the coppe~ center. Two of the seven B strands in domain 3 form a hairpin bop that extends i ~ e (NisEi8P) t h r o u g ~the pore in the middle o f domain 2, p r o v ~ d i ~ag~ i s t ~ dBgand for the c o ~ ~ion. e r The space be~weenthe h ~ r and ~ the i pore ~ walls in d o m ~ n 3 is filled with well-ord~r~d water molecules. The c ~ ~used t to ~ d se t e r ~ the ~ ~ se t ~ c ~of~galactose r e o ~ d a ~~ e4 2 were ~ 4 ~ ~ grown In acetate buffer at pH 4.5, a pH at which galactose oxidase is inactive. The ~0~~~~site is seen (Fig. 4%) to have a ~ ~ r o x i ~ asquare t~Iyp ~ coordinat~on ~ ~ ~581, and an acetate as the in-plane ligands m d Tyr495 as a I ligmd. A striking feature of the active site structure e+ dent in the electron density map is the presence of a thioether bond between Tyr272 (one of tchecopper ligands) and Cys228, ortho to the tyrosine hydroxy~s~bstituent. ~ n t a variety of i n d e p e n ~ eevidence ~t fro This ding is c ~ n s ~ s twith istry [41,42,44], resonance Raman spectroscopy 145I, and [46,47J.The ~ p ~ r o s c o pdata, ic consider^ in c o n j u n ~ t ~with o ~ the X-ray crystallographic data, indicate that a €ree radical, generated by oxidative activation of the resting enzyme (See, 4.1.11, is located on Ty1-2'62.The thioether c r ~ s s ~cl~~~~t k~ ~ b u t to lowering the oxidation potential of this phenoxide ring [481, and possibly also to its kinetic stability. Another striking feature of the active site is that the indole Sing of T~~~~ lies parallel to the plane of Tyr272 at a distmce of about 4 A and is stacked ~ of~Trp ~ side e rchain e dis over the T ~ 2 ~ 2 ~ ~ bond y ~ 2such 2 8that the s ~ ~ ~ ~ring directly ovttr the Gys s u l h atom. The other face of the indole ring is exposed to solvent, so that Trp290 acts to protect the radical center from attack by solvent. * radical is c o n s t ~ ~ n e within d an a r o ~ a t box, ~ c which both ~ ~ ~ c t ~ vtho e l~~2~~ y, stabilizes the radical and directs its reactivity to achieve catalysis. Elegant spectroscopic studies, p ~ t i c u by ~ ~ ~i yt t ~ and e rco-workers, have the~ s ~ ~ u c tand ~ r chemistry e of the active site in been crucial to our ~ n d e r s t a of n~ GCbase 1491. Circular dichroism studies C501 indicate these is a change in the geometry ~~~~~~

of the active site between the resting (inactive) form and the oxidatively activated form; however, the E M S for the inactive and activated f o m s are virtually ~ ichange ~ g in copp~r-to-li~an~ d ~ s t a n ~ eIts *is ~ ~ p oto r ~ identical €513,i ~ ~ i e ano addres~the question of whether the X-ray crystal structure determined at, pfz 4.5, where GOase is catalytically inactive, could mislead our ~ n d ~ r s t a n of ~ nthe g chemr ~cryst& t i o ~ tive site during c~talysisr4.91. A direct d ~ ~ o ~ s ~that .5and then t ~ ~ s f e r to r ~pH d 7.0 catalyze substrate t ~ r n o v e rhas not been possible. This is because, in this crystal form, the active site of very close to a s ~ m ~ e t r i c a lrelated ly molecule in the crystal lattice and there is no spaee for s u ~ s t ~ - a t(such e s as D-galactosef to entw the active site. A c q s t i i ~ ~ ~ c of activated galactose oxidase at pH 7.0 in a new crystal form allowing substrate to be ofgthe m o l e ~ ~ l dfised into the active site is needed to advance our ~ n d e ~ s t a n d i n basis for catalysis. The copper e n q m e glyoxal oxidase from ~ ~ ~ ~c ~~ ~ r s o~ has $c spec~ ~ ~ zr troscopic and redox properties similar Lo those of GQase, suggesting that it also possesses a t ~ o ~ ~ n e - f ' a c y ~ f ~t eei n- r~a d i cofactor c~ and a catalytic m e c h ~s i ~ ~~ to GQase [52]. This suggestion was confirmed by the recent identi~cat~on by mutax~ genesis of the residues responsible for forming the Tyr-Cys cross-link in g ~ y o ox%dnse [531. The close s i ~ l a rbetween ~ t ~ these enqmes is ~ e r h a s~us ~ r ~ sbeca~se i~g there is only 28% protein sequence ~dentitybetween them t531.

~ o p p e r ~ e o n t ~m~ innieno~x i d ~ e s~ ~ EC A 1.4.3.6) ~ catdyze s the ~ aewbic ~ ~ ~ d a t ~ o f primary amines to aldehydes (equation 2) [20,541.

They are ubiquitous in nature, examples having been isolated from bacteria, yeasts, plants, and animals (including humans). All w e ~ ~ c h a r a c t e r iCAQs z ~ are h o ~ o d i m e proteins ~c with subunit molecular masses in the range 70-95 k taining one copper ion and one carbonyl eofactor per subunit. The cofaf'actorin most (if not all) CAQs has been shown convincingly to be the quinone derived from 2,4,5t r i h y d o ~ h ~ n y l ~ ~ i n ef551,which results from a c o p p ~ r - ~ e p e n~~ ~ n~t t t ~ a n lational modification of a tyrosine side chain (Sec. 5) [5&l. ~~~~~

HALCROW, KNOWLES, AND PHILLIPS

722

T The first CAO crystal structure, of the Escheriehia coli enzyme, was reported to in 1995 t571. Su~se~uently, the structure8 of GAOs from pea seedling 2 A reso~utio~i 1581, Arthrobarter ~ l o b i f o r ~f591, i s and Nansemda p o ~ y ~ o r ~1601 h ahave also been published. Since all these structures are similar, a description of the CAO from E. coEi will be given here. The E. coli CAQ dimer is mus~oom-shaped,with the first 85 residues forming the “staW9 (domain 1) and the remaining 640 residues the “cap”, Domain 1 consists of a five-stranded antipmallel f3 sheet twisted around an cl-helix; the p sheets from this domain on the two inonomers face esch other but are not tightly packed. The other amine oxidases whose structures have been reported lack this stalk domain, whose function is currently unknown. The 440 ~ - t ~ r m i nresidues al of each monomer form the core of‘ the molecule (domain 41, which contains the active site and comprises a pair of twisted 1-3 sheets that contribute to the intersubunit interaction. The area of the inter-subunit inter€ace is very large at 7250 A2, which con~~ibutes to the high stability of the dimer. In ; arm from each addition, the dimer is held together by two pairs of p-hairpin w m ~one subunit lies across the top of domain 4, while the other arm from each subunit lies toward the bottom of the cap and penetrates deep into the other subunit. The latter provides what appears to be a structural link between the active sites in the subunits. Finally, in terms of the overall structure, each subunit has a pair of small domains ~ ~ o m a i n2 sand 3 ) on the niolecular surface, far removed from the active site for catalysis. These domains are closely similar in terms of both their primary and tertiary Rtmctures, suggesting a gene duplication event. Amino acid sequence compar1611 isons between amine oxidases from m e r e n t o ~ g a ~ i s m s show that domains 2 and 3 have been conserved over a long span of evolutionary time, suggesting an important biological role. This is presently unknown but may be associated with adhesion [621. The crystal structure of c a t ~ ~ i c active ~ l y amine oxidase from E. coli 1571 reveals that the active site copper ion is pentacoordinate in an approximately square pyramidal configuration by four equatorial ligands tHis524, His526, His689, and a water ligand) and an apical water molecule (Fig. 4B). The presence of basal and apied water ligands had been deduced earlier from EPR, water proton relaxation, and kinetic studies on pig plasma CAO [631, whereas the overall configuration of the S f641. The basal water ligand is copper site had been predicted from E ~ F studies

labile and appears not to be present in all m i n e oxidases. Hence, the X-ray structure of H.~ o ~ ~ ~amine a r oxidase p ~ u [GO] reveals the basal water molecule in some but not all of the six independent subunits in the asymmetrical unit. In addition, proton relaxation studies indicate that the basal water molecule is present in pig plasma o but ~ not ~ inc the~ enzymes e ~ from bovine plasma or pig kidney and ~ r ~ ~ PI ~CAOs, C6SJ. The spectroscopic properties of the copper site in CAOs has been reviewed by Knowles and Dooley 1661. The TPQ cofactor is not coordinated to the copper ion in the active, resting form of E. colt CAO but is located close by 1571. The overall position of the TPQ side chain is clear from the electron density, although the orientation and c a n f o ~ ~ t i of o nthe sixmembered ring can only be judged approximately owing to its high mobility. Closer e x ~ i n a t ~ of o nthe E. colt CAO structure reveals that the TPQ residue is located in a wedge-shaped pocket, which allows freedom of pivotal motion at the substrate binding position (C5 of the TPQ ring), which is essential for optimal catalytic activity 1671. In the H. ~ ~C A 8 structure l [so],the ~ electron density ~ for the o TPQ ring~is better ~ resolved than for the E. coli enzyme, implying variation between enzymes in the cofactor mobility required for catalysis. The structure of the complex of Eo colt m i n e oxidase with the substrate-~ikeinhibitor 2"hydrazinop~idinehas 'been determined and now reveals clear electron density for the TPQ ring l'681. A short hydrogen bond is evident between the 0 4 of the TPQ ring and the conserved residue ' l ~ ~ G 9 , while there is also a hydrogen bond between 0 2 of TPQ and the apical water ligand of the copper ion. These results indicate the importance of hydrogen bonding in stabilizing the ~ r i e n t a t ~ oofn the TPQ ring during catalysis.

(PAM, EC 1.14.17.3) is a u b i q ~ t o u senzyme ~ e ~ t ~ d y l g l y c~i n-e~ ~ i d a tenzyme ing from the nervous systems of higher organisms, which catalyzes the cleavage of the terminal acetate group from C-terminal glycyl peptides E20l~This conversion takes place in two stages, involving two active sites within the protein. First, the acetate group is monooxygenated at the methylene carbon atom (equation 3, to yield a carbinola~idethat is subse~uentlycleaved to give the finalproduct ~ e ~ ~ a4)t:~ o n ~ C { Q } ~ H C ~ 2 C+0Clz 2H -+ 233' ~ C ~ C l ~ ~ H

+ 2[ascorbate] + C+ H ~ 2(0-+0 2[dehydrose~ia~corbate] ~ ~ ~ C l ~ ~

(3)

(4) Multiple forms of PAM are produced in vivo, with M between 40 and 100 Wa. The soluble form ofthe enzyme, which has M 75 kDa, has been the best studied. This i s available by isolation from a variety of or~anismsor by recom~inantmethod^. In addition, the peptidylglycine a-hydroxylating monooxygenase (PHM, EC 1.4.17.3) and pe~tidyl~idoglycolate lyase (PAL, EC 4.3.2.5) domains of PAM can be produced cDNA separately, either by proteolysis of intact PAM or the use of clones containi~i~ I -

c o ~ i n gfor the relevant half of the p o l ~ e p t i d eonly [69,701 and FAL produced in this way retain their catalytic activity. W i l e P r enzyme, the active site of PAL contains zinc, Therefore, only the former protein will be described here. A crystal structure of the “ c a t a ~ ~COE” i c (residues 42-356) of oxidized rat PI334 at 2.3 resolution has been published 1711, The peptide contains two distinct ninestranded ~ ~ s a n d domains ~ c h of about 150residues each. These domains have similar t o p o l o ~ e seach ~ being based around an eight-strand jellyroll motif. There is only one small region o f ~ ~ t e r ~ ~ n e t r abetween t i o n the two domains, which are otherwise separated by a solvent-fi~edcleft whose width averages 8 A. The two copper ions lie on either side of this cleft, one in each domain, and are separated by 11 A (Fig. 4D). One of these, termed CUA,forms a near-planar T-shaped complex vith three histidine side chains. The other copper ion, CUB, has a trigonal pyramidal structure with two histidine and one water ligands, and a long bond of 2.68 A to an apical ~ e t ~ o ~Si n e donor. These geometries are consistent with 1EXAFS measurements, which show 2-3 histidine i1-2 O/N ligands per copper center, all at 1.97 A (no Cu-S i ~ ~ r a c was ~~on detected in this state) [TO]. Reduction of both copper ions to copper(1) in the crystal cattees na interdonai~notion and only minor ~ o ~ e m eofn tthe co~rdinatedligmds, the main difference being that Cu, becomes slightly more planar; there is no change B L72l. This contrasts with Cu ~~S m ~ a ~ u ~ e ~on ents in the C U ~ - C ~distance ed P H ~which , showed the loss of one OiN scatterer compared with oxidized ,together with 0.5 of a Sic1 scatterer at 2.27 [701. This s u ~ ~ ~a s ut sb s t ~ t i ~ s h o ~ e n i n gof the CuB-Met314bond following reduction, which is not observed in the crystal. These differing conclusions from solid and solution phase ~easurements have not yet been reconciled. Both copper sites in P states are redox-active [733, and in their coppcr(1~~ type n ~2 exhibit almost identical EPR spectra that are typical of isolated, n o n i n t e r ~ c t ~ ing only the CUB site i s capable of copper sites (See. 1.2) [69,701. ~ o ~ l o ~reduction, b i ~ the ~ competiti~e g inhibitor carbon monoxide, implying that only this copper ion is accessible to small molecules [?a]. This led to the suggestion that CUBforms the c a t a ~ ~site i c of PHM, because only this site should be capable of binding dioxygen. A czystal structure of oxidized PHM incubated with ~ ~ ~ ~ a c e t y l - ~ 9 ~ * d i i o d o t ~ o s y ~ substrate at 2.1 A confimed this proposal, with the substrate molecule being bound within a large hydr~phobiccleft close to CUB 171,721. The reactive glycyl methylene carbon atom is 4.3 from CUB sand is oriented to the CUBsolveiit ligand, This is a suitable distance to allow the reactive C-H bond to interact with an Oz-derivedligand c o o r ~ i n a t to e ~CuB. while the substrate is held in place by wveral hydro~enbonds to amino add side chains, one particularly significant interaction is a three-center h y ~ o bond ~ ~ chain n between the substrate c ~ b o ~ ~group, a t e a waker ~ o l e c ~ ~ e and ~ l n ~ on 7 0the opposite face of the solvent-filled cleft (Fig. 4E) [721. This providw ~by b~~~~ ~~ ~ i bso u~ a clear ~ a t h w for ~ yelectron transfer from CuA to CUB~ that Cu, c a n act as an electron transfer site during catalysis. Since the shortest ~easibleelectron transfer pathway between the two copper sites in the absence of

A

A

substrate i s ~rohibitive~y long at 24 residues, the substrate-mediated pathway offers an attractive rationale in the catalytic mechanism.

emocyanin (Hc) is the vascular respiratory dioxygen transport protein employed by It is a large, ~ u l t i - s u b u ~protein it whose various m o ~ u s arid ~ s a ~ h r o ~ o [74,751. ds primary and quaternary structure varies strongly depending on the source of the protein. Arthropod Pxc subunits have M 75 kDa and are globular. These associate in vivo into oligomers of hexmers, whose precise aggregation state depends on the a n i involved; ~ ~ e x ~ ~ lc eo nst ~ n i n up g to 48 subunit^ per molecule (i.e-,an octamer ~ and ~ of h e x ~ e r are s ~known 175,761.In contrast, molluskan He subunits are t associate linearly in multiples of 10 to yield cylindrical molecules [?5,741. Each subthe widely unit contains seven copper~bindingsites and has M * 400 kDa. ~eflecting varying ~ ~ a t e r n a sr yt ~ c ~ u roef sdiEerent Hc’s, there is no s t ~ n d a r ~ nQni~nc~a~ed ow substantial homologies ture for their subunits. Although Hcs within each genu with each other, except for one copper binding region from an arthropod and a mollusk exhibit very little sequence similarity 1783, In this section we will describe the ~ sHc will be ~ s € ~ sins ~ d s t ~ c tproperties ~ ~ r ~ of EIc; the d ~ o ~ ~ e n - b i npdr~onpge ~ i of Sec. 4.2.2. ed form are availCrystal structures of two arthropod Hc’s in their dea able 1751. The structure of a hexameric unit from the spiny lobster ~~~u~~~~~interi ~ contains ~ ~ u $ three ~ ~ haspbeen~ refined u to~ 3.2 resolut~on1791. P. i r 2 ~ ~ r ~Hc distinct subunits a-c; the c r y s t a l l ~ ~ a p h i ccharacterized ~ly h er is composed of subunits a and b only. The a and b subunits differ in only 2.7% of their amino acid residues, whereas the c subunit is 59%identical to subunit a E801, All three subunits contain one ~ c a p p binding e~ site, and the existence of three distincr, subunits is therefore thought to reflect an evolutionary gene duplication event I801 The stmcture of 8 hexamer formed from a purified preparation o f subunit I1 from the horseshoe crab L i r n ~ ~ ~has ~ been ~ ~ refined y ~to 2.18 ~ ~A resolution r n ~ ~[MI. This Hc is a 48mer with eight differ en^ subunit types in its native form. For both Hc structures, each subunit has three domains. Domain 1is dominated le, with a protruding “ endix“ consisting of one cc helix and by a five-ccone @ sheet that is involved in i n t ~ r ~ s u b interactions. u~t Domain 2 c o n ~ ~ the ns dicopper binding site, which lies huried within an antipar~lelfive-a-helix bundle. Four of these five helices contribute Zigands to the copper ions. most exposed to the surface of the hexamer, and consists of a seve with several large loops p r o t from ~ ~it, some ~ ~ of which penetrate into domains 1 and 2. Overall, the hexamer has effective 32-point symmetry and can be thought of as a “trrimer of tight ers”. While all three domains are involved in inter-subunit that domain 2 is the most important in maintaining the Hc interactions, it is c ~ u a t e r stm~ture. n~ This also su ts a role for domain 2 in ~ n t e r - s ~ ~ u ~ i t t ~ e~ ~ c~ erativity (see below). In addition to the copper site, the L. ~ o ~ y Pzcp s ~

A

I

726

HALGROW, DO OWL^§, AND ~ ~ I L L I ~ S

also contains a calcium binding site on the exterior of the hexamer, which may play a structural role and/or mediate i n t e r h e x ~ e contacts r in vivo. Each Hc subunit contains one dicopper center at its active site. The copper ions are each bound by three histidine side chains (Fig. 4F). In dmxy-Hc, both copper ions r ~ ~ all t ~sixs have approxi~atelytrigonal planar geomet~es.In the P. ~ n ~ ~ rstructure dicopper pairs are separated by 3.5-3.6 A, a distance in accord with EXAFS studies e ~ u ~ the intercopper distances surprisowever, in the L. ~ o Z y p ~ structure ingly refined to 4.6 A. It was suggested that these two active site c o n ~ o ~ ~ a t i o n respectively, represent “relaxed” (high oxygen affinity) and “tense” (low oxygen affinity) skates of the protein [sl]. Comparison of the two structures showed some confor~nationa~ differencesresulting in a 7.5”rotation of domain 1relative to domains 2 m d 3, sug~estingthat movement of the copper ions upon &oxygen binding might form a basis for cooperativity between He subunits L811. This cooperativity would appear to be mediated by a chloride binding site situated spanning doinains 1and 2 [84,851. ‘In addition to the above arthropod Hc structures, the crystal structure of a single 47-kDa “functional unit” o f a molluskan oxy-Hc from Octopus dofleini has ~ ~ t two d o ~ ~ nas largely : Ebeen ~.eported1861. This p o l ~ e p t i d ef r a ~ e contains helical copper binding domain, which shows substantial differences Lo domain 2 of the arthropod structures; and a “jelly~roll”six-strand p-sandwich domain. In the is formed largely crystal, the ~ o l y ~ e p t i associates de into ~ o ~ ~ d i whose m e ~interface s by the fi-sandwich domain. This was proposed to reflect the disposition of isolated functional units within the tertiary structure of intact molluskan He. In agreement with spec~ro$copicdata 1871, the molecular structure of the 0. dofleind Hc active site is generally similar to those described above, with two trigond copper ions separated by 3.5 A, each coordinated by three histidine ligands. Interestingly, however, one of these histidine sidechains has been chemically modified by an oxidative ci-oss-link to a cy&eine residue (cf. catechol oxidase, Sec. 2.5, Fig, 433). The function ofthis cross-link is unknown, although it appears to be a feature common to other molluskan hemocyanins 1881. The dioxygen binding mode in this my-f-lc structure will be discussed in Sec. 4.2.2.

Catechol oxidase (COase, EC 1.10.3.1) is a plant enzyme, which uses dioxygen to (5) [891. oxidize ~ ~ ~ z ~ -todquinones ~ ~ ~according e ~ o to~ equation s

The copper requirement, redox activity, and spectroscopic properties of oxidized and reduced COase closely resemble those of the animal and bacterial enzyme tyrosinase, which catalyzes both phenol m o n o ~ ~ g e n a t i oand n catechol o ~ d a t (Sec. ~o~

Cu PROTEINS IN TRANSPORT AND ACTIVATION

727

3.3) [90,911. COase is therefore best thought of as a tyrosinase variant that lacks phenol monooxygenase activity. There is 26% identity between the sequences of sweet potato catechol oxidase and human tyrosinase [92J. The crystal structure o f sweet potato COase has been published to 2.7 resolution [92]. It i s a monomeric protein of mass 39 kDa with an overall elliptical shape, s which is composedl p r i r n d y of CI helices. The core o f the protein, which c o n t ~ n the active Bite, is composed of a four-a helix bundle that i s in turn surrounded by two further whelices and some short p strands. All four helices of the central bundle contribute ligands to the dicopper site. The two copper ions, termed CuA and CUB, are each bound by three histicline side chains (Fig. 4H). The ixnidazole ring of one of the ligands to CuA, HislOQ, is covalently cross-linked at the 2-position to Gys92, forming a thioether linkage (Fig. 4H). A similar His-Cys cross-link is known to occur in some ~ o s i n a s e s(Sec. 3 . 3 , and in mollusk hemocyani~s(Sec. 2.41, Overall, the structure of this catalytic core i s very similar to that of the copper-bjnding region of hemocyanin (Sec. 2.41, despite the lack of any significant sequence i d e ~ ~ ibetween ty the two proteins. In resting, oxidized COase (i.e., met-COase, Sec, 3.31, the copper ions lie 2.9 A apart and are bridged by an exogenous solvent-derived ligand, which on chemical grounds is most likely a hydroxide ion (Fig. 4H). Both copper ions are trigonal pyramidal in this state, with a His ligand in the apical position. Upon reduction to its dicopper(I1deoxy statc (Sec. 3.31, the bridging ligand is lost and the copper ion separation increases to 4.4 A, which is acco~plishedwith only a small motion of the coordinated histidine side chains and no obvious conformational change of the rest of the protein (cf*deoxy-hemocyanin, Fig. 4F) W21. In this form the copper ions have noticeably different geometries, so that GuA has an essentially trigonal planar geometry, ~ s two whereas CuB is T-shaped. It was suggested that the different s t ~ c t ~ofr the reduced copper ions might reflect the presence of the His-Cys cross-link at GuA, which should constrain its coordination geometry by reducing the mobility o f this ligand.

2.6. Cytochrorne c

ASE

Gytochrome oxidase (COX, EC 1.9.3.1) and the closely related ubiquinol oxidases are the terminal oxidases in the electron transport chains of aerobic bacteria or the mitochon~riaof e ~ a r y o t e s[93,94J. They are membrane"~oundproteins, which carry out thc? reduction of dioxygen to water concomitant with the oxidation o f four equivalents of cytochrame or quinol electron carriers. The free energy generated by this process is used to pump protons through the protein molecule and across the membrane, so that the overall COX reaction is expressed in equation (6) (8= cytochrome, quinol):

W i l e differing substantially in their quabrnary structures, the metal and cofacx tor invento~esof most of these terminal oxidases are the same. Each mol contains the following metal sites: a dinuclear CUA ele n transfer site; an isolated is usually a heme-a group, and also fulfills an electron tran~fer heme c o f & c ~which r function; and a second heme cofactor (generally a hememagtype) and a typ ion I“CUB”)in close proximity. This latter ironicopper pair is the site of reduction by COX. Since of all the terminal oxidases structural data are thu available for cytochrome e oxidase (CcO), we will confine our discussions here and in Set. 4.1.6 to this enzyme. Crystal structure analyses for two fully oxidized CcQ’s have been reported, from ~ sAr [95,96] ~ ~resolution ~ a and ~ from s bovine the b ~ c ~ ~e a~ ~ uu ~~~~~ ~~ }~ ~ ieat ~2.7 heart at 2.3 A [9?-991. Bacterial CcO’s contain 3-5 subunits, of which subunits 14x1 are present in a31 enzymes with a high degree of sequence conservat~o~ I100 I. The P. ~e~~~~~~~~~~ CcO is a four-subunit enzyme, subunits I-N possessing 564, 252, 273 and 56 amino acid residues, respectively /95,963. Subunit I lies almost entirely within ~~ the ~ e m ~ r a nand o consists of 12 membrane-s~anninga helices a r r a n ~ ewith appro xi mat^ overall three-fold symmetry, This subunit contains the dioxygen reduct contains two tion activo site (see below) as well as an isolated heme moiety. ~ u b u n i11 t r a n s ~ e m b r pxa h ~ ~ ces with a short N-terminal loop, linked to a 10-strand P-barrel 52) that ~ r o t r ~ don e s the ~ e r i p l a s ~side i c of the m e ~ b r ~ n e . dom~n (residues 1 This barrel has obvious s i ~ i l a ~ i t to ~ etype s 1 copper protein folds and contains a dinuclear GuAelectron transfer center (Sec. 1.2).Subunit 111 consists of seven meme-spanning px helices arranged in two bundles, which are sepa~atedby a cleft that be a site for the docking of the physioIogica1electron donor, reduced cytochrome e. ~ u b u n i t sI1 both interact extensively with s u b u ~ i It but have no contact with each othe unit rV is a fragment from a larger procursor protein, which of one t r ~ s ~ e ~ b r r helix ~ n and e is in c o n t a ~with ~ all other subc o n s ~ s ~~~1~ t~ units, The function of subunit IV is unknown. Impo~tant~y, a protein composed of subunits I and II only retains activity for d n reduction and proton p ~ p i n g 11011, ~ ~ o that w ~ subunits n ~ T I 1 and IV are not required for this chemistry. e ~thec crystal, with each The bovine head CcO, in contmst, is h o m ~ d ~ in m o n o ~ c~ ro ~ ~ ~ 13 ~ different n i n ~ subunits with a total mass 204 kDa L97-991. Subunits 1-111 have essentially similar tertiary structures, and identical metal and cofacto~~ n v e n t ~ r ito ~ sthe , correspond~ngs u ~ u n i t sin the b rial Cccb, Since o d y these three s u b ~ n i t are s encoded by m i t o c h o ~ d r rather i~ t uclear genes, this is ~ e r ~ a up s~ ~ r ~The s other ~ n 10~subunits . are much s han subunits 1-111, and ail except three contain at least one l r a n s ~ e m b r a n ~ n e ~ a t i v e ~charged y cytosolic surface of the protein, formed by three subhaped region on as identified as a potential ~ ~ o c hc rdc&ing o ~ ~site. tuding subunit 1 Otherwise, the individual functions of the extra subunits present in eukaryolic CcOs are not known. on by CcO takes place at a dimetallic Ireme-copper site that is well buried ~ i t ~si~ nb u n i It (Fig, 4.X). The heme cofactor is a typical heme-ug ring with one apical hi ine ligand, whereas the copper ion, CUR,is a type 2 copper site


729

with three histidine ligands disposed in a T shape. Interestingly, in both CcO structures the pyrrolyl N atom of one of these histidine residues is cross-linked to the 2position of a t.yrosine side chain [96,99,102]. The resultant 2-(imidazol-l-yl)phenolis oxidizable and appears to act as an electron and proton shuttle during catalysis (Sec. 4.1.6). The two metal ions are separated by about 5 with electron density between the two atoms indicating the presence of exogenous ligandt s) wit.hin the intermetallic space. In the P. denitrificans structure, this was interpreted on the basis of one water ligand to each metal ion, affording octahedral iron and square planar copper centers f961. However, in the (slightly higher resolution) beef heart CcO structure, an “endon” r\l,q’-peroxide bridging ligand was modeled between the two metal ions 1991. While the existence of this bridging peroxide species in resting CcO has not yet been definitively proven, it is supported by E M S [103,104] and magnetochemical I1051 data suggesting the presence of a bridging Ligand between the metal ions in this state. Upon reduction of ciystals of fully oxidized bovine heart CcO the putative peroxide bridging ligand disappears, although the metal-metal distance remains close to 5 1991. Reduction is also accompanied by small changes in the conformation and hydrogen bonding within the polypeptide, which are suggestive of a rcdox-induced proton pumping pathway (see below). Crystallogaphic analyses of azide- and carbon monoxide-inhibited CcO also show minimal structural changws in the protein, and confirm the space between henie-a3 and CUB as the site of small-molecule binding [991. There is no obvious channel in either CcO structure to allow access of small molecules to the heme-a3/CuB center 196,981. This suggests that a conformational change is necessary to allow dioxygen to diffuse into the active site. However, in the P. denitrificans CcO two potential proton transfer pathways were detected [95, 961. The K pathway is composed purely of residues from subunit 1 and leads from the surface of the protein to the cross-linked Tyr-His residue at the heme-copper site. The other channel, the L)pathway, utilizes residues from subunits I and 11, and spans the protein molccule perpendicular to the membrane via a solvent-filled cavity within subunit I. Three similar potential proton channels were also located in the bovine CcO structure [%I. The existence of at least two proton channels in the protein is consistent with earlier conclusions that separate mechanisms existed Sor proton transfer to the dioxygen substrate, and for the generation of the transmembrane proton gradient 1106,1071, Mutagenesis data have suggested that the K pathway delivers protons required for the initial reduction of the heme-a3/CuBsite prior to substrate binding, whereas the Il pathway is utilized both for proton pumping and for proton delivery to bound dioxygen [1081.

A,

A

FEATURES OF COPP 3-0 S ~

~

~

~

3.1. Lysyl Oxidase

Lysyl oxidase (EC 1.4.3.13)is an extra&ellularenzyme that catalyzes the o ~ d a t i o nof lysyl side chains in structural proteins such as collagen and elastin9as the initial step in formation of cross-linkages. Purification of lysyl oxidase from. mammalian tissue l t and requires an initial urea extraction step. has proved t o be very d i ~ ~ u[I091 active, y it is uncertain whether Although the refolded purified protein i s ~ t a l y t i c ~ this corresponds to the full in vivo activity. The purified protein has a moiiomer molecular mass of 32 kDa and contains one mole-equivalent of copper [ l l O l . The carbonyl cofactor has been shown to be a novel quinone, termed lysine tyrosyl quinone (LTQ), involving post-translational cross-linking between a lysine and a modified tyrosine residue [lllJ.Lysyl oxidase cDNA has been sequenced to reveal a 1233bp region coding for a 4 l l - a m i ~ o - ~ ~46.6-kDa id, protein Ill21 that is further processed to give the 32-kDa secreted protein. Attempts to express the lo gene to givc a soluble form of the protein, suitable for crystallization have not been successful. We have no i ~ o r m a t i o non the 3-33 structure of lysyl oxidase. Since it is a c o ~ p l e ~ e l y different protein in kerns of i t s primary structure and molecular weight from the amine oxidases whose structures have been discussed in Sec. 2, homology modeling of the lysyl oxidase 3-D structure is not possible.

LT

Dopamine p-monooxygenase (DPM, EC 1.14.17.1) i s an enzyme found in mammalian brains that catalyzes the conversion of dopamine to norepinephrine [equation (7) 1 [20,113].

731

This reaction shows substantial similarities to that catalyzed by peptidylglycine 2.3). Given that both e n ~ are ~ copper~ s ~ ~ h y d r o x y l a t i~no~n o o x y g e ~ a s(Sec. e dependent, it is not surprising that the PHM domain in peptidylglycine ix-amidating enzyme shows 30% sequence identity to amino acids 190-490 in DPM 1114,1151. While the active site structure of DfiM is assumed to be highly homo15~ousto that; of the crystallo~a~hically characterized PHM (Sec. 2.31, little information is available about the structure (or role) o f the remaining 50% of DPM. It should also be noted 'and PAM differ in their oligomeric s t ~ c t u r e s : is a dimer of dirners that D ~ M 11131, whereas PAM is a bifunctional monomer. Both mernbrane-associatedand solubIe forms of DPM are found in vivo, primarily within the neurosecretory vesicles of the adrenal gland 1113,1161. It has been known for many years that DPM contains copper and that this is a requirement for catalytic activity 11161. Due to the facts that DPM binds copper somewhat weakly and that trace amounts of copper can reconstit~tethe small ~ o u n t of s DPM present in t~ steady-state kinetic experiments, it proved difRcult to establish the s ~ i c h i o r n e of ~ o p p e ~ / p r o t required ei~ for activity. When fast-reaction kinetic experiments using higher conceiitrations of protein were performed, it was demons~atedthat catalytic activity corresponds to two coppers per protein polypeptide chain monomer of about 73 kDa f1171. The ~pectroscopicproperties of these copper ions strongly mirror those shorn by PHM (Fig. 4D, Sec 2.3). Hence, in their resting state, the DPM copper sites (termed CUAand CUB)are difficult to distinguish by EPR or by EXAFS Tlli3t; both are m o n ~ n ~ c l and e a ~have two or three histidine ligands and one or two oxygen ligands coordinated to the copper with a copper-ligand distance of 1.97 A, giving a site with apparently tetragonal symmetry. Reduction o f DPM converts both copper ions in each monomer to the crxprous state [113,11~1" This leads to a dramatic change in the toordination at one of the copper sites (CUB)with a Coordinated sulfur atom at 2.25 A from the copper replacing one of the oxygen or nitrogen ligands [1201. Carbon m o ~ o x i dis~a competitive i ~ ~ bagainst i ~ dioxygen ~ r b ~ ~ d i to n gred~ced DpM and has been shown to bind exclusively to the CUBcenter ClZll. This is strong and direct evidence for a distinct role for CUB in the binding of dioxygen. The exact substrate g ~ d s c o ~ b abt ei n~ d ~ to n ~D~~ is unclear site of atnine substrate a d r e d ~ c i ~ at present, though it has been proposed that the CUAsite is involved in mediating electron transfer from ascorbate to the site of oxygen cleavage. D i s ~ ~ s s i oofn these r n e c h ~ ~ sissues t ~ c will be resumed in Sec. 4.1.3. ~~~

Tyrosinase (EC 1.14.18.I) is a copper-conta~ni~~ enzyme that catalyzes two differe~t reactions: the hydroxylation of monophenols to ortho-diphenols [phenol rn~nooxy~

~ e n a t ~ oactivity, n equation (8)J; and the o ~ d a t i o nof o r ~ ~ o ~ d i p h e noro ~catechols, s, to o r ~ ~ o ~ q u i n oXcatechol nes oxidation, equation GI1 [211:

The enzyme is widely distributed, and the primary stmctures of c tyrosinases from Streptomyces [1221, Neurospora crassa [1231, Rana nigro Lf241, ~u~ r n ~ s e ~ l u11251, s and Homa s a ~ ~ [f26f ~ n s have been r e ~ ~ The r ~frog d ~ C1241, mouse [1251, and human I1261 enzymes show 81% identity and are roughly the ~ dasrotei in same size at 532,533, and 548 amino acids, respect~vely.This c o ~ e s p o ~to molecular weight of about 61 kDa. However, these are quite distinct from ora t1231 which have 273 and 407 ~ ~ ~ e p ~ o l122l ~ y c and e s ~ e ~ r o s ~ tyrosinases amino acids (correspondi~~ to M = 30.9 and 46 kDa1, respectively, with only 23% identity between the Strepdomyces and ~ e ~ r o s p oproteins. ~a These diEerences between tyrosinases extend to higher levels o f structure; thus, tyrosinase from the fungus Agricara bisporus is an azPz tetranier with subunits of 43 and 67 W)a, human t ~ o s i n is~ as x~n e ~ b r a n e ~ b omo~omer u~~ [12?], rosinase has proved difficult to characterize due to pigment Contamination c e~ u l t i p l eforms [1281. ~ ~ ~ “ cor y s~t ~ g n e ~~ho ’ s ~ n afrom se and the o c c ~ ~ r e n of Neurospora was reported in 1963 [129], attempts to obtain diiyraction quality crystals have been frustr~ting.Wowever, stmctural furm mat ion has come from s~ectroscopic studies and from co~parisonswith the related proteins cateckol osidase and hemrrcyanin. Tyrosinases from all sources possess a type 3 binuclear copper center with c o o ~ i ~ ~ a t to i o nsix histidine residues [1301 that is directly ~nalogousto those in hexnocyanin and catechol oxidase (Fig. 4F, IT, Secs. 2.4 and 2.5) [2l]. Sequencing e s of these histidine ligands has been studies have shown that in some t ~ o s i ~ a sone po~~-translationally modified to form a His-Cys cross-link [1311, as is shown by cate2.4 and 2.5). The protein as isolated is chol oxidase and some ~ ~ m o c y a n i n(Secs, s mostly in an EPR silent met state with the two copper(I1) centers bridged by a s o l ~ e n t ~ d e ~ ~ligand v e d [1321. Two-electron reduction of the met state gives the deoxy state, which reacts with dioxygen to form oxy-tyrosinme. isc cuss ion of the properties of these different states of tyrosinase will be resumed in Sec. 4.2.2.

The routes devised in nature to activate C-W bonds and to hyd~oxyla~e hydrocarbo~~~ continue to attract both academic and technolo~calinterest CX331. Methane monooxygenase (EC 1.14.13.25) catalyzes the conversion of methane to methanol using d i o ~ g ns e ~the cosubstrate [equation (911:

There are two methane m o n o o ~ g e n ~found e s in methanotrop~cbacteria: a soluble form tsMM0) 1.1341located in the c ~ o p l a s mthat comists of three component e, and a small regulatory protein^ and a memproteins (a h y ~ ~ ~ l aasreductase, ~t211. The ~ soluble ~ form0 has been ~studied extenbran~~bound particulate form ( sively 11343 and the 3-D structure of the hydroxylase compone~tsolved 11351; this showed that sMMO has a nonheme binuclear iron cluster as its metal cofactor and is discussed further in Chapter 11. Studies on p~~ have been made difficult by the lability of the activity when oxygen is present. However, it seems establi~hedthat the protein, which has a total Mr of 94 kDa, consists of three subunits of MF45, 26, and 23 a, ~ a l o g o u to s the three subunits of s ~ QCurrent . analyses 11361 indicate 12oppers per 94-kDa Q unit, which axe organized into distinct copper sites performing both electron r aid catalytic functio~is[1371, The number and structure of these sites are uncertain but appear to include at least one mononuclear type 2 center [1381, whereas the catalytic site may be a trinuclear copper cluster /1391 with at least one ~ s t i ~ n e ~ with~ nitric 0 oxide affords a g = 4 EPR ligand [1403. Inc~bationof reduced p resonance similar to that shown by nonheme iron(^^)^^ a d ~ u c t a~ ;weak g = 6 ferrous signal can also be detected in some pMM0 preparation presently unclear whether this reflects an iron content for the 11421 or the presence o f copurifying cytochrome species i1361. Ammonia monooxy~e~ase ~~0~is an evolutionarily related membrane protein from ammonia-oxidizing bacteria that catalyzes the oxidation of ~ ~ nto hydroi a xyiamine by dioxygen. Purification of an AMQ has only recently been achieved, from Parncaccus denitrificuns, showing it to contain two s u b u ~ t ofMr s 46 and 38 kDa 61433. There is high sequence identity between AM0 and pMMO El441 and, a l t h o u ~ ~ g with few spectroscopic data are available, incubation of ~ Q - c o n t a i n i n membranes nitric oxide generates a g = 6 ferrous signal identical to that observed for 11451. Therefore, it is likely that the metal ion inventory and active site structures of the two enzyxies are essentially the same.

Nitrous oxide reductase CN20R) catalyzes the two-clectron ~ e d u ~ tofi oNZO ~ to Nzas one component in the pathway of reduction of nitrate to NZby denitrifying bacteria whose , physiolo~calelectron t1461. NzQR is a soluble enzyme found in the ~ e r ~ p l a s m donor is probably either a c-type cytochrome or pseudoazurin 11461. The NzQR from ~ s e ~ stutzeri ~ o has~ been ~ studied ~ s extensively 11471 and shown to have four copper ions per monomer in an 0% dimeric protein. The copper ions occur in pairs: one pair (Cu,) is an electron transfer site in the C-terminal domain that is strongly ~ ~ p a ~toa the b lCuA ~ site in COX, as indicated by EPR spectroscopy and MCD [17,148]. An additional EPR signal, termed “Cuz”, that was for a long t h e assigned to the active site copper pair [1481 is now known to derive from a different conformational form of the CuA site 1171. The second, active site copper pair is located in the

central protein domain and is EPR-silent in its oxidized form [171. This has led to the suggestion that this is a type 3 ~{Cu(Eliis>,),(~-OI)~~~ center, although the M20R protein sequence does not contain a recognized type 3 binding motif l.171.

The weight of evidence suggests that galactose oxidase catalysis involves a “pingpong’’ mechanism as first proposed by Hamilton et al. t1497. There have been proposals for an ordered mechanism [1501 and for a random order mechanism I1511, but recent work 11521 strongly favors a ping-pong mechanism as shown in Fig. 5. We can consider the elementary steps of this mechanism in turn. Although there is no crystallographic evidence that substrate sugars and other alcohols bind to the copper ion by displacing the equatorial water ligand (Fig. 5A), this is consistent with graphics modeling based on the 1.7-kstructure (Sec, 2.1)[431 and with water proton relaxation data 11531. The computer graphics studies offer a rationalization for the specificity of GOase for primary alcohol substrates that is broad yet stereospecific I1541 and includes oxidation of the terminal D-galactose residue in oligosaccharides. Activation of‘ this bound substrate has been suggested to involve transfer of the hydroxyl proton from the primary alcohol group of the substrate to the axial copper ligand Tyr495 (Fig. 5B).This suggestion originated from the observation that azide, like alcohol substrate, binds to the copper in GOase with concomitant uptake of a proton from solution, probably by Tyr495 [49?. Support for this proposal has also come from studies with the mutational variant Tyr495Phe, where there is no proton uptake follo~~ing addition of azide [1551. The protonation of 33-495 by the substrate would weaken its axial coordination to the copper, leaving it eff‘ectively four-coordinate. The rate-determining step in the reductive half cycle is hydrogen atom transfer from the methylene group of the primary alcohol substrate to the radical site located on Tyr272 (Fig. 56). This abstraction step is stereospecific for the pro-$ hydrogen of the methy~enegroup [1561 and involves a kinetic isotope effect ( k ~ / kthat ~ ) can be as high as 22 [1Fi23; this is suggestive of a tunneling process. Experiments with mechanism-based inhi~itorsprovide evidence for the proposed substrate-derived ketyl radical In addition to this atom transfer step, the visible spectrum i i i t e r ~ i ~ j a[157,1581. t~s NES spectrum [1601 of activated GOase following substrate reduction for reduction of Cu(II) to GuiI) conditions provide strong sup ver, it is not known whether om transfer from substrate to Tyr272 is preceded or followed by single-electron transfer to CulII). The four-cooidinate geometry of the Gu(IX)site created by breaking the axial bond to ‘Fyx-495 would faci~~tate fast reduction to Cu( ), The ping-pong mechanism suggests that product aldehyde is liberated following formation of Ertdralthough %hishas not been tested.

735


Q a

.I

FIG. 5. Currently preferred mechanism for deohol oxidation by galactose oxidase [I491

f

It has been proposed that the t ~ o - ~ ~ e~ ~e tor xo ~~ ~of ~ t i (Fig. ~ n 5E) involves CJ521, though there iL;. no pathways in the protein tertiary structure for electron and proton Ered to reduce dioxygen to hydrogen peroxide are also not known,

The cat^^^ ~ n ~ of c~ o p~ ~ ae ~ ~- asnine ~ s o ~~ t ~ ~ ~ ~ divided into reductive [equation (1011 and oxidative Ee

(lo1 (111 This s u g ~ s tthat s amine oxidases would exhibit ping-pong steady

r in the aminoi s derived from

e of Schiff base d

is the ~ u ~ s t rit ~istalways ~ , the pro-S

d~pendingon the source of the m i n e oxi

737


2

7)

N

R I

FIG. 6. ~ u ~ e n tpl rye f e ~ mechanism e~ for amine oxidation by copper arnine oxidases [ Z O ] .

~ O ~ - f odecrease ld in kcat. This is proposed to reflect disruption of the hydrogen bonding network holding the TPQ ring in place, allowing it to slip into a less accessible i c show that the TPQ is located in location t176J.For E. coli CAO, c ~ s t a u o ~ a p hdata a wedge-shaped cleft in the wild-type enzyme, allowing freedom of pivotal movement at the substrate binding position, which is suggested to be essential for optimal activity t671. Ifn the hp383Glu variant, the extra methylene group restricts the movcment of the TPQ residue, as is evident from its clear electron density by crystallography, which correlates with the very low catalytic activity of this mutant. Hydrolysis of the product Schiff base liberates the ddehyde product and generates the a m i ~ i o ~ u iform n ~ l of the cofactor (Fig. 6C), which must then be reoxidized ent (Fig. 6D). Dooley and co-workers 11771 demonstrated a ~ ~ p e r a t u r e ~ d e p e n dequilibrium between the ~ ~ I ~ ) / a m i n o q u i and n o l ~~~1~~~~ semiquinone forms of the resultant species, whose forward rate constant has n measured to be 75 and ZO,OOO s for two different CAOs [178,179]. These rates are much greater than the catalytic rate-limiting step, suggesting, but not proving, that the Cu~I)/semiquino~e form could bc an intermediate in catalysis. In chemical terns, Cu(lf would be a site for binding mol~cularoxygen as the first step in the oxidative half we-vw, Su md Klirman [I801 present evidence that the rate-~imitingstep in the oxidative half cycle is the initial electron transfer to dioxygen, which chemical precedent would suggest to be fast if binding of dioxygen was to Cu(B).It i s concluded that the rate-limiting electron transfer step is directly from the aminoquinol form of' the cofactor to dioxygen, with the copper remaining divalent. Su and Ulinman [lSOl further discuss the nature of subsequent electron transfer and proton transfer steps in the oxidative half cycle which lead to generation of the hydrogen peroxide product. They envisage that the a ~ i n o q ~ i ~form i o l o f the cofactor acts as a transducer storing two electrons and two protons for later delivery to dioxygen. crystal ~ t ~ c t u r of e$ ~ i ~et oal, [t 1811 have recently reported ~~i~~i-resolutioii species relevant to understanding the chemistry of the oxidative half cycle. The most interesting structure is that of a turnover complex formed when E. eoli CAO is reacted aerobically with substrate (Fig. 4 0 This structure allows the proton transfer pathways to the dioxygen molecule proposed by Su and K.hman [1801 to be visualized. It is suggested [I811 that the dioxygen-derived species in this crystal structure is proba~lyh ~ d r o ~ peroxide, en i m p ~ ~ that n g elcctron and proton transfer to dioxygen have already taken place. The reason why this species accumulates in the crystal state is because thc ~~nzaldehyde product from the r e d u ~ t ~ vhalf e cycle remains bound at the back of the substrate-binding pocket. The key roles played by Tyr369 and Asp383 in maintaining the correct location of the TPQ and as p ~ t i c i p a n t sin the proton transfer pathways emerges convincingly from these structural studies.

Sec. 3.2 of this chapter summarizes the &ructurtil and chemical evidence supporting two distinct mononztclear copper centers (Cu, and CUB)in dopamine ~ " ~ o n o o x y ~ e


739

ase, suggesting that CUBis the catalytic site of the enzyme. There Stre several experimental results implying that oxygenation of the substrate during D[3M catalysis is partially rate-limiting under physiolo@calconditions, and occurs by a radical mechanism I113I. An intrinsic 'I3 isotope effect of 10.9 was determined for dopamine hydroxylation 11821, which is at the high limit for a classical hydrogen atom abstraction reaction and implies a symmetrical transition state for the rate-determining step. H~droxylationof a series of substituted phenylethylamine substrates by DPM gives a linear free-energy relationship with p = - 1.5 [1831, a typical value for a radicalgenerating mechanism. A detailed free-energy analysis o f individual steps of these same reactions concluded that the fitial step of the catalytic cycle is product dissociation from an inner sphere complex to copper, These early results led to a proposition that binding of oxygen to fully reduced enzyme affords a copper(I1)ihydroperoxo species, whose close proximity to the dopamine substrate initiates hydrogen atom a b s t r a ~ t ~ o[1931. n More recently, an intrinsic "0 isotope effect of 1.025 was demons~ratedfor monooxygenation of dopamine by DpM F1841. Importantly, the same study showed that the 180kinetic isotope effect for DPM catalysis decreases as the reactivity of' a series of phenylethy~aminesubstrates increases. This important result suggests that 0-0 bond cleavage occurs before oxygen atom insertion into the substrate. This conclusion is contained within the revised m e c h a ~ ~ sshown m in Fig. 7, where an amino acid residue acts as electron donor Lo complete 0-0bond cleavage and reduction to water before the homolytic substrate C- bond cleavage. The oxidizable amino acid was proposed to be a tyrosine side chain, although at present there is no evidence to support such an ~ n v o l ~ ~ e r nofe an n ~ active site tyrosine residue in ~P~ catalysis. If this mechanism for DPM. is correct, a similar mechanism should apply for peptidylglyciae r-hydroxylating monooxygenase which, importantly, shows almost I 3 isotope effects [185,1861. Thc initial steps of PEIM catalysis identical intrinsic ' involve reduction of both CQ and CUBby ascorbate, which display ping-pong kinetics [18Sl and must take place before the peptide substrate can bind ilSS]. Steady-state lunetic data using reduced enzyme indicate an equilibri~~-ordered mechanism with peptide and oxygen binding sequentially to the active site [1851. This contrasts with DPM, where a rsendom order of binding of these two substrates was found under ~ h y s i o l o ~ ccond~tions al E1871. The o b s e ~ a t i o nof m e c ~ a ~ i s m - ibn~aec~~ ~ ~ aoft i o ~ by peptides contain in^ subs~i~uted glycines [1881 and by unsat~ira~ed thioacetic acids ClS91 supports involvement of a radical intermediate in the hydroxylation of the glycyl pro43 a-hydrogen, as proposed in the ICXinman mechanism for The c r y s t ~ l o ~ a pstudies ~ i c on PHM by Amzel and co-workers [711 indicate the i n e possibility of an i ~ t e ~ a cbetween ~ i o ~ the peptide sLI~strateand a ~ ~ o ~re~idue ~ ~ y r and 3 ~also ~ }show that the pro-S a-hydrogen of glycine points toward GuR, the site of oxygen binding in PHM. This is consistent with removal of the a-hydrogen atom by a eopper-oxygen species. ~ ~ r n a t im v eec h ~ i s m sfor PHM: have recently proposed. On the basis of their c ~ s t ~ oresults ~ a (9ec. ~ ~2,3), i h~z e l and co-workers proposed that it is a C ~ ~ ( superoxide species that abstracts a hydrogen atom from the substrate to generate

740

3*

FIG. 7. ~~~~~s~ for DPM and

catalysis proposed by KZinman and co-workers [l841.

C u ~ ~ I ~ ~ y d r o p e rwith o ~ dthe e , resultant substrate radical then promoting heterolytic cleavage of the dioxygen bond to rorm the peptidyl ~ " ~ y d r o ~ g l y cproduct ine (Fig. $1 1721. Jaron and Blackburn [1901 also observed differences in the properties of PHM following binding of the pe~tidylglycine substrate. Before substrate binding, carbon monoxide binds only to CuB; however, binding of the peptide opens up binding of carbon ~ o n o x i at ~ ea second site, p r o ~ a Cu,. b ~ ~This ~ implies that substrate b~nding may activate the Cu, center toward dioxygen binding. It was hence proposed that superoxide formed by reduction of dioxygen at Cu, difluses across the 11-A channel to relocate at the CUB site, where the subsequent events in the catalytic mechanism would be similar to those proposed by h z e l (Fig. 8) C721. 4.1.4. T'yrminase and Catechol Oxidase

The currently preferred mechanism for tyrosinase and catechol oxidase catalysis 121,1911 is shown in Fig. 9, This scheme was originally derived from studies of the binding of inhibitors and substrate analogues to tyrosinase [1921. Labeling studies of phenol hydroxylation using have demonstrated that the oxygen atom incorporated into the product derives from dioxygen rather than from water [1931. It is also known that the phenol monooxygenase and catechol oxidase cycles in Fig. 9 turn over with rates o f approxi~atelylo3 s-' and 107 s-I, r~spectively[194]. ~ n f o ~ u n a t e l y ~ phenol monooxygenation by tyrosinase in vitro exhibits complex kinetics, including a lag phase whose duration is dependent on substrate concentration. This arises because resting tyrosina~eis predominantKy in its met form (Sec. 3.3) [I321, and i s a consequence of the fact that monophenols can bind nonproductivdy to met-tyrosinase (Fig. 9). This leads to substrate i~hibitionof the reduction of ~ ~ ~ - t ~ o s iton a s the active deoxy form t1951. Hence, several details of the monooxygenase cycle in particular remain ~ c ~ r tCatechol ~ n , oxidation by tyrosinase appears to be a concerted two-electron transfer reaction, since no serniquinone intermediates can be detected during turnover 11961. While the binding mode of dioxygen to the type 3 dicopper site i s well established (Sec. 4.2.21, the mode of binding o f mono- and &phenolic substrates to the active site is less certain. It is known that the rate of phenol monooxygenation is ~ r a i n a t i c a ~ y decreased using substrates bearing bulky substituents, whereas the rate of catechol t e 1.s This suggests either that khe two oxidatian is barely affected by such s ~ ~ ~ t r a1192 types of substrate coordinate to tyrosinase in different ways, or that phenol monooxygenation is dependent on substrate mobility within the active site. ~pectroscopic analyses of half-met (i.e.$Cu(11)Cu~I~j ~osinase-substrate complexes have suggested that monophenols coordinate axially to a single copper ion in this form r1921. If this is also tme of ~~y-ty~osinase, then moleculai* models suggest that n i i ~ a t i o nof the substrate from an axial to an equatorial coordination site of the copper ion i s necesligand. It was thought for sary for attack at an ortho-C-1i-Xbond by a ~tz:~i2,ii~"peroxo some years that diphenols must coordinate in an ~ , ~ ' ~ b ~mode i d ~between n g the two copper ions 11921, in order to explain why tyrosinase does not exhibit 1,4-dipbenol oxidase activity r2ll. However, a recent crystal structure of a C O a s e ~ ~ ~ p h e -

742

3"

a

a FIG. 8. ~ ~ e r ~ ~e ~ h~ afor ~~ vi s eand ~ PHM catalysis ~ ~ ~ o bys hez de l and co-workers [721. ~~~

OTEINS IN T

~

A

~ AND ~ ~ ACTIVATION O ~ T

743

w deoxy

EI+

Q,,

n ~ l t ~ o i~n ~e bai t o rcomplex shows that the t l ~ i o u ~phenyl a ring iias within a hydrophobic pocket of the active site [92]. It€ diphenol substrates were also to bind within this pocket, this would also be consistent with monodentate ~ o o r d ~ n ~ tto i oan single copper ion. Although there are limited data pertinent to the details of arene h y ~ o ~ l a t i o n by tyrosinase, substantial mechanistic advances have been made in alkane and arene m o n o o ~ ~ e n a t i oreactions n by synthetic model dicopper(II)/peroxo cornpounds 110,1971. From this work, it is probabje that phenol monooxy~enatio1~ in tvosinase proceeds by an electrophilic attack of the active copper/peroxo oxidant at the substrate 6-I3:bond. This is consistent with the e~ectron-de~c~ent nature of tyrosinasebound dioxygen, as inferred from the spectroscopic properties of nxy-tyrosinase [1921 mat i s unclear is ~ h ~ t h0-0 e r bond cleavage takes place before or after this electrophilic attack, since examples of both scenarios are known in copper chemistry. This is ~ x e ~ ~ l i fby i e the d reaction in Fig. 10, whereby low-tem~er~ture o ~ g e n a t i o nof the ~ c o p p ~ r (precursor 1) affords a concentration-dependent mixture of [Cu&-02)12' and n I C u ~ ( p 0 ) ~ lspecies, ~' both of which undergo intra~olecularh ~ d r o ~ l a t i oupon warming [1981. These two different hydroxylation reactions give rise t o very dif€erent so that if these data were kinetic isotope effects and Hamrnett c o n s t ~ t I197,J981, s determined €or tyrosinase it would be possible to distinguish between these two ~ec~anis~s. ).

Both p ~ t i ~methane l a ~ monooxygenase ~ and ammonia monooxygenase hydroxylate or oxidize a wide range of compounds in addition to their natural substrates, including akenes, arenes, and sulfides 1199-2011. In addition, alkanes up to a CS chain at0the Ca, position for C3--C5 alkanes length are h ~ ~ o ~by~~ ~ t ~espec&cdly d ~ 12021. Thus far, there have been only two reported studies pertinent to the mechanand M0. First, alkane hydroxylation and &ene e ~ o ~ i d a t i oby n ism of p~~ 0 using isotopically labeled substrates has been used to show that oxygen i~sertioni n k the substrate 6-€3bonds occurs wikh complete r~tentionof configuration, by a concerted pathway that does not involve free radicals [202,2031. This is cons~sten~ with the observation that arene h y d ~ o ~ l a t i obyn occurs c o n c o ~ i ~ tantly with an "'NIH" heteroatom 1,2 shift l2041, which is strongly indicat t aC e ~ e c t r o ~ h i~l ~ec c h ~ i sfor m addition of an oxygen atom e q u i v ~ e n to This ties in with model studies, in that intramolecular ligand hydro~lationby Cu(II)/ peroxo or CU(~~I~)/QXO species commonly occurs by an electrophi~icattack of the ligated oxygen species at the relevant C-H bond (Scc. 4.1.3) [10,1971. However, given the uncertain nature of the active sites of pMMO and Al\/f0, the identity of the active oxidant in these enzymes is unknown. ~~

FIG. 10. Aerobic ~ t r a r n o ~ arene e c ~m~d alkene monoo~genationby a model dicoppr c o r n p o ~ d[1981. The p ~ o ~ ~€ram c t sthe ~ g h t " h ~ d reaction are d e ~ a d a t ~ oproducts n resulting from ~ y ~ o ~ 7 l aof~ 5gaxd i o n xylyl GHZ or isopropyf CH moieties. The isolation of about 50% of unchanged kigand from this reaction shows that only one ~ y ~ Q ~ ~ step a per t ~ &copper Q n unit takes place.

746

4.1.6. Cytochrorne c Oxidase

The active site in resting cytochrome c oxidase lies at the ~on(I~I)/copper(I~) oxidation level. Incubation of CcO with carbon monoxide allows the active site of the enzyme to be clamped in its fully reduced iron(I~~/cop~er(I) state. ~ h o t o l ~ ~ofs ithis s inhibited enzyme ejects the CO ligand from the active site, transiently generating fully reduced CcO, whose reaction with dioxygen has been extensively probed using Raman C20Sl and W - V k spectroscopies [$13],E~posureof ~ i m e ~ ~ e s o ~resonance ved hlly reduced CcO to dioxygen first generates “A-CcO”, which is believed to contain a simple iroddioxygen adduct analogous to that in ~ ~ ~ - h e ~ o g lCUB o b is ~ still n ; reduced in this f o m . A-CcO then transforms into the “P”-state, which in turn decays first to the “F” state, then to a final “H”-state (Fig. 11).The P and F states of the active site can also be produced by treatment of fully oxidized CcO with hydrogen peroxide or of partially oxidized enzyme with diosygen [206-208]. The currently preferred hypothesis is that the complete four-electron reduction of bound O2 occurs during the A+P step of the catalytic cycle (fig. 11).This mechan- t y r o s and y l is supism formulates P-CcO as an ~ i r o n ( ~ ) / o x o l ~ ~ p p e ~ ( ~ I ~complex ported by several recent observations. First, it is known that the active site heme center lies at the same oxidation level in both P- and F-CcO, which are both one electron higher than the resting enzyme l209I. Second, the A-tP transition is irreversible, suggesting that it involves cleavage o f the 0-0 bond [210,2111. Third, tlie cross-linked tyrosine residue can be oxidized to a radical by the incu~ationof resting CcO with hydrogen peroxide [2X21. Fourth, CUBin P-CcO is E ~ ~ ~ $ i l ewhich n t , can be explained by superexchange between the copper(1I) and phenoxyl. unpaired spins E2131. Finally, the P+F transition is a one-electron reduction, probab~yof the tyyrosyl radical [211]. Rate consta~tsfor most of the steps in Fig. 11are available. 0th the A -+P and P -+I? t r ~ i s ~ t ~occur o n s with rate constants of the order of 10*s-I r214,21519~ t ~ o u g h the latter step involves distinct electron and proton transfer events. The rate-determining step o f the cycle appears to be the F -+H reduction, which has a rate constant of 740 s-’ 1214,2151. The rates of the P -+F and F -+H t r a n s i t ~ o are ~s p~-dep~ndent, so that t r a n s ~ m ~ ~ bpumping r a ~ e of one proton is proposed to be coupled to each of these steps.

Both galactose oxidase and copper-containing anline oxidases catalyze similar reactions, namely, the activation of a methylene group of a primary alcohol or primary amine to yield a product aldehyde. Although there is no similarity in the overall structures o f the two classes of enzyme, both contain a monocopper center and it would be a reasonable first speculat~onthat the role(s1 played by copper in the two

Gu PROTEINS IN TRANSPORT AND ACTIVATION

747

A

fs

proteins would be similar. In both oxidases, copper is also essential for post-trans~ational processing of tyrosine to yield novel cofactors (Sec. 5). However, the role of copper in the catalytic t ~ a n s f o r r n ~of t ~~o~~b s t r a t seems es to be different in the two enzymes. Most obviously, in GOase there is evidence that the akohol substrate binds t ~ the active site copper center, whereas for amine oxidases the to, and is ~ c t i v a by, substrate binding site is remote from the copper ion. In the latter case, copper appears not to be directly involved in substrate oxidation, other than by ~ e ~ topcorrectly ~ g position the TPQ cofactor through hydrogen bonding. In both enzymes, the abstraction of hydrogen from the substrate molecule is stereospecific and is eEected by the organic cofactor. In GQase this step appears to proceed by hydrogen atom abstraction by the modified Tyr272 free-radical site. However, in CAO the TPQ cofactor activates the amine substrate toward deprotonag disparity, the active sites of tion by an active site aspartate base. ~ e ~ e c t i nthis galactose oxidase and a d n e oxidases are strikingly different, The GQase active site is nonpolar in character, which favors radical chemistry, and very compact, suggesting that the catalytic events do not involve large movements in active site residues. By contr~st,the CAO active site contains many polar residues, and movement in the TPQ cofactor is an essential feature of catalysis.

two a ncuprous i n , ions at an interIt has been seen in See. 2.4 that in ~ e ~ x ~ h e ~ o ~ , are each coordinaked by three histidine residues. This nuclear separation of 3.5-4.6 & provides an empty cavity for dioxygen binding between the copper ions, to yield oxyHe, Blue oxy-He is ~ ~ R - s i l eand n t essentially d i ~ m a ~ n e[2161, t ~ c and has a characteristic optical absorption centered at 345 nm, assigned to an O24 C u charge transfer transition E217l. It also exhibits a 750 cm-l 0-0vibration band by resonance Raman, consistent with a t~~peroxo)dicopper(I~~ center [218]. Isotopic labeling experiments proved that the peroxo ligand bridged between the copper ions rather than being atta~hedterminally to one of them F219l. Comparison of the spectroscopic properties of oxy-Hc and a model c o ~ p o u n din which two cupric ions were €inkedby a ila,qzperoxide [220] showed close similarity. In contrast, an alternative dicopper model r o ~ dexhibited e a very ~ € e r e nCu-Cu t disc o ~ ~ o c~onn d~ ~ n a~ ~n g‘ , ~ ~ ~ p ebridge tance and 0-0 stretching frequency [2211. Hence, it was concluded that oxytains a ~ ~ m e“side-on” t ~ ~ ~c- ? ~ z , ~ 2 - p e r o&and. ~ide The existence of this hitherto unusual peroxide bridge was confirmed by crystal e ~The ~ dioxyge~ ~ s t ~ ~ ~ofuoxy-He r e ~ from L. ~~~~~~~~s f2221 and 0. d o ~ I86l. moiety lies equidistant between the two copper ions, which in both structures are n t ~~~S ~ ~ s u r e m e n1831 t s (Fig. 4G). The ~ e p ~ by a 3.6& ~ e ~in a ~ ~ m e with copper centers have a distorted square pyramidal geometry, each having both oxygen and atoms and two histidine ligands lying in the basal plane at &stances of 1.7-2.2 one lengthened apical Cu-N bond of 2.4 The crystallographic tertiary and quaternary structures of the deoxy and oxy forms of this protein show only small differences.

A.

A,

The &copper sites in Wc a i d tyrosinase have similar histidine ligand cnvironrnents [211, although in tyrosinase the site is more open to exogenous ligands 1132,2231. It is plausible to suggest that dioxygen bridges in side-on fashion between the copper ions in o~y-tyrosinase.This s ~ ~ g g e s t ~isosupported n by the close resemblance o f the oxy-tyrosinase resonance Raman (v = 755 cm ‘1 E2241 and th,, = 345 nm, c, = 18,000 M-’em-’) 11321 spectra to those of oxy-He. 47xy-tyrosinass also has a Cu-Cu distance of 3.6 A by EX.AFS [2251, identical to that shown by 0qv-H~[83,222 I. The structural features that cause the additional activation of dioxygen upon binding to ozy-tyrosinase are uncertain, particularly given the essentially identical uo-0 vibration shown by the two proteins, iurd must probably await the c ~ s ~ ~ l o ~structure a p h ~ deter~ination c of this enzyme. C o ~ p a ~ i s obetween n catechol oxidase and tyrosinase is interesting. COase lacks the catalytic activity of tyrosinases for hydroxylating phenol derivatives to 1,%diphenols but shares with tyrosinases the catalytic activity for oxidizing a range o f 1,2dih~dricphenols to the corres~ndingquinones. The EXAFS, W-Vis, and resonance R m a n properties of the two enzymes in their deoxy, met, and oxy forms are essentially identical, strongly suggesting that their copper sites have identical structures and redox c h e ~ i s t r ybut differ in their mode of substrate binding t89-911.

It is interesting to consider tyros~ase,d o p ~ i n ef3-monooxygenas@,and t ethane monooxygenase as a group. These three proteins each perform essentially the same reaction, but on sequentially more demanding substrates (aryl Cweaker than alkyl C-H bonds, wMe methane is the hardest hydrocarbon substrate of all to functionalize [133,226]). Clearly, nature has evolved three differ en^ copperbamd oxidants, each tailored to the thermodynamic requirements of their substrates. Although no crystal structure for tyrosinase i s available, from a combination of spectroscopic and model data the identity of the active oxidant in tyrosinase catalysis has center for its resonance form. been clearly assigned as a 1(Cu(~~i~)3)~(l.l-O2)1”’ f(Cu(I-ris),(l.l-O)>,l’~,Sec. 4.1.4). What remains unclear about the tyrosinase mechanism is the mode of substrate binding at different stages of the catalytic cycle. For Df3M and PHI% the converse i s true in that, although a crystal structure is availa~lethat clearly demo~stratesthe substrate binding site, the mechanism of &oxygen activation by this class o f enzyme is still uncertain. For pMMO, both the structure and mode of action of the active oxidant are unknown. Further characterization of all these enzymes will allow deli~ea~ion of their st~cture-function relationships, ~ h i c hhave allowed nature to tune the oxidizing power of the copperidioxygen couple so precisely, The presence of cross-linked amino acid residues in several of the proteins discussed in this chapter is intriguing. For the Tyr-Cys cross-link in galactose oxidase (Sec. 2.11, it is well established that the thioether substituent formed by the cross-fink to cysteine serves to lower the oxidation potential of the tyrosyl side chain by about

0.5 V, and may also kinetically stabilize the resultant tyrosyl radical. However, this is the only example for which the function of such a cross-link is reasonably well understood. The EI-is-Cysresidues in some hemocyanins, tyrosinases, and catechol oxidases (Secs. 2.4, 2.5, and 3.3) are not redox-active, and their presence is particularly intriguing given that there are many h e m o ~ a n i n sknown in which this cross-link is not present. In the absence of any obvious electronic benefit, it has been suggested that the His-Cys cross-link plays a purely st,mctural role [SCl, although this remains to be confirnied. The functional role of the Tyr-His cross-link in cytochrorne oxidase (Sec. 2.6) is unknown. Mong with their function in active enzyme, the mechanism of formation of these cross-linked amino acids, and of the chemically modified tyrosine residue in GAQ, needs to be investigated. For GAO, amino acid modification is known to be a pusttranslational ~el~-processing event that i s dependent on both copper and oxygen [56]. For CAQ TPQ biogenesis is effected by the active site copper ion only (i.e., external copper is not involved) and occurs with apparent first-order kinetics [227]. However, i sf m o r ~ a t i o nof this or any of the other little else is known about the ~ e ~ h a ~ of modified amino acid side chains in this chapter which, assuming they are also oxidative reactions, involve transibrmations that have little precedent in organic chemistry. This clearly demands further study. In view of the extremely low concentration of free copper in the cytoplasm of eukaryotic cells [23],it is probable that incorporation of copper into all intracellular es ~ o p p e r ~ c o n ~proteins ~ n i n ~ involves specific copper chaperones. Of the e n ~ y ~covered in this chapter, it i s only for the chaperone-mediated route for copper incorporation into the multi-copper oxidase FET3 that significant structural and mechanistic e also been data are available [24,25]. However, a bacterial chaperone for t ~ o s i n a s has identified [228], and the search for copper chaperones for other proteins involved in oxygen transport and activation should be fruitful. Structural studies of how these chaperones mediate transfer of copper to the target protein will reveal whether the model suggested for copper incorporation into SOD [381 involving separate domains in the chaperone for copper binding and docking to the target apoprotein is generally applicable. For extracellular oxidative proteins (such as galactose oxidase and serum amine oxidases), there is currently no information on whether in vivo copper incor~ n t r a ~ l l ~ aor r l extrace~lu~arly y , and whether e x t r ~ ~ e ~copper lul~~ p o r a t ~ ooccurs ~ incorp~ration(if it occurs) i s regulated. Finally, the biological role of many of the proteins covered in this chapter r e m ~ to n ~be clarified. In some cases, this is clear; for example, the oxygen binding role of hernocyanin in mollusk or arthropod metabolism is u n ~ b i ~ o uass ,are the biological substrates stated to define enzymes such as cytochrome oxidase and dopamine ~“rnonoo~genase. However, in other cases, the b i o l ~ ~ csubstrate al for a wellcharacterized enzyme is not known, nor indeed are the full biological roles of the protein understood. Thus, for “galactose oxidase”, it is not known whether D-galac~ c ~ substrate, nor is the b ~ o l o ~ crole a l of the enzyme tose is the p h ~ s i o l o enzyme evident. Arnine oxidases are even more intriguing. Whereas for prokaryotic amine oxidases the role of the aniine oxidase would plausibly be to enable the organism to

grow on a p a ~ i c u ~m a ri n e s u b s t ~ a t efor ~ no e ~ a r y o t amine ~ c oxidase do we know the specific substrates whose oxidation is catalyzed in vivo. It has also been shown lZ25j that 8n adhesion protein invohed in ~ i n ~ ln ~g ~ p h o has ~ ~the e ss ~ ~ c t u and re amine oxidase, yet, there is at present no catalytic activity of a co~per-contai~iing u ~ d e ~ s t a n d i nofgthe relationship between these functions.

ammonia monoo~genase copper-containing arnine oxidase ~ o c ~ r e ooxidase ~ e catechol oxidase ~ o c h r o m oxidase e dopamine ~ ~ ~ o n o o ~ g e n ~ s e electron nuclear double resonance electron paramagnetic resonance extended X-ray ~bsorptionfine structure galactose oxidase he~ocyanin lysine tyrosyl quinone magnetic circular dichroism rionnal hydrogen electrode nuclear magnetic re so^^^^^ nitrous oxide reductase ~eptidylan~ido~lyeolate lyase peptid~lglycine~ - a ~ i d a t en^^^ in~ ~ p t i d y ~ ~ l y~c"i~~y~der o x y l a t~i no~~ o o ~ ~ e n a s e particulate methane monooxygenase soluble methane m o n o o x y ~ ~ n a s ~ superoxide dismutase 5-a~ino-2,4-d~hydrox~~~enyljtanine topaquinone ( 2 ~ 4 , 5 - t r i h y d r o ~ ~ h e n y l a l a n ~ n e ~ u ~ # n e ) X-ray absorption near edge structure

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231.

s

European Synchrotron Radiation Facility, BP-220, F-38043 Grenoble Cedex, France

1. I N ~ R O ~ U ~ T I O N 1.1. Spectroscopic Classification of Copper Ions 1.2. The Cupredoxin Fold 1.3. The Overall Architecture of the Multi-Copper Oxidase Family

764 765 767 767

2. ~O~ ~ T ~ ~ IN~ THE ~ ~U U ~R r I - ~C O SP ~OXTDASE ER FAMILY 2.1. Ascorbate Oxidase 2.1.1. Occurrence and Putative Biological Role 2.1.2. Structure of' Ascorbate Qxidase from Green Zucchini Squash 2.2. Laccase 2.2.1. Occurrence and Putative Biological Roles 2.2.2. Structure of the Laccase from Coprinus cinereus 2.3. Nitrite Reductase 2.3.1. Occurrence and Function 2.3.2. Structure of Nitrite Reductase from Achromobacter ycloclastes 2.4 Human C e ~ l o p l a s m i ~ 2.4.1. Occurrence and Putative Physiological Functions 2.4.2. Organization of the Human Ceruloplasmin Molecule isulfide Bridges, Cleavage Sites, and Glycans

771 771 771 771 772 772 773 774 774

3. ~ N S T ~ ~ ~C T U ~ CEQO S :A G ~ ~~ ~ IFACTORS O N V and VIII 3.1. Roles of Factors V and VIII in Blood Coagulation

779 179

763

775 776 776 776 778

LINDLEY

764

3.2. Overall Configuration of Factor V and VIII Molecules 3.3. A Model for Factor VIII Based on Human Ceruloplasmin

779 780

4. ST~UCT~~E- UNCTION RELATIONS~IPS 780 4.1. Copper Binding Sites 780 4.1.1. Type I Copper Binding Sites 780 4.1.2. Type I1 Copper Binding Sites 783 4.1.3. Type I11 Copper Binding Sites 784 4.2. Organic Substrate Binding Sites in Ascorbate Oxidase and Laccase 787 4.3. Mechanism of Oxygen Reduction by the Trinuclear Copper Center 788 4.3.1. Fully Reduced Trinuclear Center in Ascorbate Oxidase 789 4.3.2. Addition of Hydrogen Peroxide to Ascorbate Oxidase 789 4.3.3. Overall Mechanism of Oxygen Reduction 79 1 4.4. Azide Inhibition in Ascorbate Oxidase and Human Ceruloplasmin 791 4.4.1. Addition of Azide to Ascorbate Oxidase 793 4.4.2. Addition of Azide t o Human Ceruloplasmin 794 4.5. Ceruloplasmin and Ferroxidase Activity 794 4.6. Binding of Organic Substrates to Human Ceruloplasmin 797 4.7. Ceruloplasmin and Hemosiderosis 800 5. P ~ R S ~ E C T I ~ S 5.1. Evolutionary Aspects of the Multi-Copper Oxidase Family 5.1.1. Sequence Alignments and Phylogenetic Trees 5.1.2. Structural Comparisons 5.1.3. An Alternative Evolutionary Pathway 5.2. Open Questions O~~EDG~ENTS B ~ E ~ T I AND ~ N DEFINITIONS S

801 801 801 803 803 805 806 806

It is now recognized that copper is a vital requirement for the growth, development, and function of most living organisms and that the metabolisms of iron and copper are intricately linked. The multi-copper oxidases constitute a family of enzymes of which the prime members are laccase (benzendiol oxygen oxidoreductase, EC 1.10.3.21, ascorbate oxidase (L-ascorbate oxygen oxidoreductase, EC 1.10.3.31, and ceruloplas~n(Fe(l1) oxygen oxidoreductase, EC 1.16.3.1). All three enzymes normally contain at least four copper atoms compri:ing a mononuclear blue copper center and a trinuclear copper center some 12-13 A distant.

PPER OXIDASES

765

The mononuclear copper accepts an electron from a substrate molecule, which is therefore oxidized, and transmits it to the trinuclear center, Oxygen binds to the trinuclear center and, following the transfer of four electrons, is reduced to two molecules of water. In the context of living systems this reaction is very important because it is a source of free energy and avoids the release of partially oxidized forms of the dioxygen molecule, such as hydrogen peroxide and the hydroxyl radical, which are very destructive to cells. These prime members have multiple subunits, and each subunit has a folding pattern based on the cupredoxin fold (see Sec. 1.2). Table 1lists the representative members of the multi-copper oxidase family and shows the diversity in size, copper content, and biological function. The smaller members of the family, which can be considered as building blocks for the larger members, comprise one cupredoxin subunit, normally containing a single blue copper center, and are involved in electron transport. The larger members have several subunits, a large variation in the number and type of copper atoms, and functions varying from oxidation to blood coagwlation. Some of the larger members lack a trinuclear center, and, indeed, may lack copper completely, but their Ihreedimensional structures are all based on subunits having the cupredoxin fold. Such members include nitrite reductase (EC 1.7.99.3) and the blood coagulation factors V and VIII. This chapter will describe the main structural features of multi-copper oxidases as determined by X-ray crystallographic methods and discuss possible mechanisms by which these enzymes function. No attempt will be made to correlate the great wealth of spectroscopic, physicochemical, and biochemical data that have been published in the literature. Useful reference texts include “Biochemistry of Copper” by Linder [I] and “Multi-Copper Oxidases” by Messerschmidt [21.

1 .I.

Spectroscopic Classification of Copper Ions

Malmstrom [3] devised a method of classifying the different types of copper ions in terms of their visible, UV, and EPR signals as follows; High absorption in the visible region, E > 3000 M-l cm-’ at 600 nm. EPR spectrum with All < 95 x lo-* cm-l. Type IT or normal Cu2+:Undetectable absorption. EPR line shape of usual low molecular weight Cu complexes, All > 140 x 10-4cm-1, Type TIT CuZf: Strong absorption in the near-UV with A, = 330 nm. No EPR signal; pair of copper ions, which are antiferromagnetically coupled. Type I Cu2+:

The optical and EPR signals disappear upon reduction of copper by suitable electron donors. The type I coppers are normally coordinated by three strong ligands, a cysteine, and two histidines, and may also have one or two weaker ligands such as methionine sulfur or oxygen. In the oxidized form of azurin from Akaligenes deni-

766

LINDLEY

The Blue Copper Oxidase Family and Homologues No. of residues

Principle source of protein

Name

No. of Cu atoms

Types of copper

Function

Chloroplasts, plants, and algae Bacteria Fungi and plants Higher plants Yeast Denitr6ying bacteria Plasma Plasma Plasma

Plastocyanin hurina Laccase Ascorbate oxidase

99

1

129

1

I I I, II,2 x 111 (I,11, x2III) x 2 + 1 As per laccase? (I, 11) x 3 2 x I, 11,2 x TII + CU’ = type II? E type II?

Electron transfer in photosynthesis Electron transfer Lignin degradation Oxidation of ascorbate Input of iron to cells Reduction of nitrite Probably multifunctional Blood coagulation Blood coagulation

540 552 x 2

4 (4 x 2) + 1

Fet3 Nitrite reductase Ceruloplasmin Factor V Factor VIII

564 340 x 3 1,046 2,196 2,332

9

2x3

6

1 1

“Other members of the azurin family include pseudoazurin (bacteria), amieyanin (bacteria), and phytocyanin (plants).

767

MULTI~COPPE~ OXIDASES

trificans 1451, a main chain carbonyl oxygen atom coordinates weakly to the copper. Type I1 copper is normally three or four coordinate with histidine and water (hydroxyl) ligands. Type 111coppers are usually coordinated by three histidines and a bridging ligand such as oxygen or hydroxyl. The trinuclear site in the multi-copper oxidases comprises a pair of type I11 coppers and one type I1 copper atom. The classification of Malrnstrom will be used throughout this chapter. 1.2. The ~ u p ~ e ~ o x i ~ The multi-copper oxidase family is comprised of structural domains, all of which are based on the cupredoxin fold r61, This folding pattern is typified by the structures of the small blue copper proteins, plastocyanin and azurin, and comprises two p sheets arranged in a sandwich configuration. Fig. 1shows the p sheet topology and hydrogen bonding pattern observed in plastocyanin “71 (PDB code lplc); each sheet comprises essentially four strands. In azurin [41 (PDB code 2aza) there is an extra strand, 5, in the sheet involving strands 2b, 8, 7, and 4. Ribbon diagrams of plastocyanin and azurin are shown in Fig. 2. Whereas other common sheet structures, such as superoxide dismutase, the immunoglobulins, and the fibronectin domains contain only anti-parallel strands, the cupredoxin fold has two parallel strands-1 and 3 in the first sheet and 2b and 8 in the second. In addition, strand 2 participates in both sheets, crossing over from one sheet to the other. The cupredoxin fold is also adopted as a subunit structure by two respiratory proteins. Thus subunit 2 in cytochrome c oxidase has a cupredoxin fold with a binuclear copper site that transfers electrons from cytochrome c to the oxidase active site [8,9] (PDB code 2occ). The copper’atoms are each bound to a histidine residue and are bridged by two cysteines. A main chain carbonyl oxygen atom and a rnethionine residue are also involved with one of the copper atoms in the pair. Quinol oxidase also possesses a cupredoxin domain which is lacking copper, but this domain in Escherichia coli fl0l (PDB code lcyw) has been engmeered so that it has six ligmds binding a binuclear copper moiety 1111.

verall Architecture of the

ulti-Copper Oxidase Family

A simple schematic representation of the overall structural relationships between the cupredoxins, ascorbate oxidase (A01 [12,131 (PDB code laoz), ceruloplasmin (CP) [14] (PDB code lkcw), nitrite reductase (NR) [ E l (PDB code lnic), and a putative model for blood coagulation factor VIII [161 is given in Fig. 3. The X-ray structure of the laccase from Coprinus cinereus (Lac) 1171 (PDB code la651 indicates that the laccases can be classified with AO. It is also probable that the Fet3 protein from yeast [I81will have such a configuration. This protein has been shown to be a membrane-bound cell surface ferroxidase 1191 providing a link for copper-regulated iron uptake. In the figure, the triangles indicate a single cupredoxin unit and the black circles copper

LlNDbEY

768

ing

7

2a

6 FIG. 1. The cupredoxin fold illustrated by the p sheet o r g ~ i z a ~ i oand n h y ~ o g e n - b o n pattern ~g in p l ~ t ~ y a n[71. i n J3 Strands 6 , 3 , 1 and 2a form the first sheet whereas the second comprises strands Zb, 8, 7, arid 4.In azurin the second sheet has an extra strand, 5. The two sheets pack together in the manner of a sandwich.

MULTI -COPPER OX I DAS ES

769

FIG. 2. The cupredoxin structure illustrated by ribbon diagrams of (a) plastocyanin 171and (b) azurin [4].The ribbons trace the 01 carbon backbones o f the polypeptide chains. The additional strand 5 in the azurin structure is clearly seen.

770

LINDLEY

Factor Vlll

FIG. 3. Schematic diaaarn indicating the structural relationships between niembers of the multi-copper oxidase family [141. The black circles represent copper atoms. Domains 1, 2, and 3 of ascorbate oxidase (AO) are topologically equivalent to domains 1, 2 , and 6 in ceruloplasmin (hCP), although domain 2 lacks copper. Domains 1and 2 in nitrite reductase (NR) are equivalent to domains 1 and 2 in A 0 and hCP, but whereas it is the even domains in hCP that contain copper, in NIZ the copper is in the odd domains. The mononuclear copper site in NR between domains 1and 2" is equivalent to the trinuclear centers in A 0 and hCP. In coagulation Factor VIII the three type A domains correspond to pairs of domains in hCP, 1 and 2, 3 and 4, and 5 and 6 reBpectively. The factor VIII B domain is partially or completely excised during activation, but may otherwise serve to modulate the interaction of factor VIfI with other components involved in the coagulation pathway. (Adapted with the pernlission of the Society of Biological Inorganic Chemistq from Fig. 8 in reference 1141.)

atoms. Thus, the AO monomer comprises three cupredoxin domains with a type I mononuclear center in domain 3 and a trinuclear copper center (a type TI and two type I11 coppers) at the interface of the first and third domains. Human ceruloplasmin (hCP) has six domains with mononuclear coppers in domains 2, 4, and 6 and a trinuclear copper center between the first and last domains. Domains 1,2,and 6 in hCP are topologically equivalent to domains 1, 2, and 3 in AO, but note that domain 2 in AO does not contain a type I copper center. NR is a trimer of two cupredoxin domains,

wi ULYI-COPPER

0x1DASES

777

but unlike hCP the type I copper centers are located in the odd domains and not the even. In addition, there are type II copper centers at each dimer interface, 1 and 2”, 2 and l‘,and 2’ and 1”.

2.

~ ~ O UCTURES ~ ~ IN S THE T MULTI-COPPER OXI FAMILY

2.1 . Ascorbate 2.1.l. Occurrence and Putative Biological Role

Ascorbate oxidase (AO) is found in higher plants, but cucumber (Cz~cumtssativus) and gTeen zucchini squash (Cucurbitapcpo rnedullosa) are the most common sources. At the cellular level the enzyme is most abundant in the cell wall and the cytoplasm [20], but its role in vivo is still under debate. The enzyme reduces ascorbate and other compounds that contain a lactone ring with an enediol group adjacent to a carbonyl group, as shown in Fig. 4. Since in vitro catechols and polyphenols are also substrates L211, AO may be involved in biological processes such as fruit ripening, A role for plant respiration has also been suggested. 2.1.2. The Structure of Ascorbate Oxidase from Green Zucchini Squash

The molecule of A 0 comprises 552 residues organised into three cupredoxin domains with a trinuclear copper center between domains 1and 3 and a mononuclear copper in domain 3, as indicated schematically in Fig. 3. The X-ray crystal structure of the fully oxidized form of A 0 from green zucchini squash has been obtained at a resolution of 1.9 [E3].The asymmetric unit comprises a dimer, which i s also thought to be present in solution, and in the crystal the subunits (molecules) are arranged as homotetramers with 222 symmetry. In addition to the eight copper atoms, four from each subunit, the dimer also has an additional metal atom between the subunits ligated by a histidine residue from each subunit. This metal may help to stabilize the tetramcr in the crystal and has also been assigned as a copper atom. Fig. 5 shows the organization of a single subunit in AO,

o

w

FIG. 4. Reduction of ascorbate to dehyciru-ascorbic acid (L-ascorbate) by ascorbate oxidase.

772

LINDLEY

FIG. 5. Molecular structure of ascorbate oxidase from green zucchini squash. Domain 1is at the lower right hand side of the figure, domain 2 at the upper right, and domain 3 on the left (a rotation o f 180" with respect to Fig. 3).

Domain 1 (residues 1-128) comprises two 4-stranded B sheets, domain 2 (residues 124-3351 has a six-stranded and a 5-stranded P-sheet composition, and domain 3 (residues 336-552) is built up from two 5-stranded sheets that form the sandwich. Compared with the simple cupredoxin fold (Fig. 2) there i s a large insertion between strands 2a and 2b, and strand 5 has two components 5a and 5b with an insertion in between. There are three disulfide bridges, two are interdomain, C19-C201, C81C538, and the third is within domain 2, C180-Cl93. There are also three putative sites €or N-glycosidic linked carbohydrate chains in the zucchini enzyme, at N92, N325, and N440, but in the crystal structure only N92 shows any density for an N acetyl glucosamine group.

m e and Putative Biological Roles c) are produced by various fungi and certain types of plants, notably er tree. Although they consist of a single polypeptide chain with

773

MULTI -COPPER OX IDAS ES

some 500 amino acid residues, their molecular weights are often enlarged because of an extensive carbohydrate coating (the molecular weight of the tree enzyme approaches 125 kDa). The fungal laccases are extracellular enzymes that may play a role in lignin degradation [22,231. They are also believed to play significant roles in pathogenesis, sporulation, and the development of fruiting bodies by mediating adhesion between cells [241. A variety of industrial oxidative processes, such as delignification, dye bleaching, and fiber modification, currently use polluting technology, and laccases are being increasingly used as nonpolluting replacements [25,26]. On the other hand, the tree laccases are implicated in wound response by catalyzing the hardening of sap, and by lignin synthesis [27,28]. The hardening reaction involves the oxidative polymerization o f urushiols, which are catechols found in latex [291. Fig. 6 shows a typical polymerisation reaction catalyzed by a laccase. 2.2.2. Structure of the Laccase /+om Coprinus cinereus

The carbohydrate moieties, presumably because they cover the surface in a partially disordered manner, make the production o f crystals suitable for X-ray analysis very problematic. Currently, only one laccase structure, from the fungus Coprinus cinereus, hau been reported [171. The enzyme was expressed in, and secreted from, an Aspergillus oryzae expression system [301. Suitable crystals for an X-ray analysis could only be grown after deglycosylation of the enzyme using a mixture of endoglycosidase F and N-glycosidase I", and this was undertaken in the presence of 20 mM EDTA. The resulting structure, Fig. 7, was found to be type II copper-depleted, presumably as a result of the presence of the EDTA at the deglycosylation stage. The overall structure is essentially the same as AO, comprising t h e e cupredoxin type domains, a mononuclear copper center in domain 3, and a multi-copper center between domains 1 and 3. Domain 3 has four short helical regions in addition to the 8-sheet sandwich, and the last of these, near the C terminus of the molecule, is anchored by a disulfide bridge to domain 1, C85-C487. A second interdomain disulfide bridge links domains 1and 2, C117-C204. An extensive loop involving residues 284-327 links domains 2 and 3. An N-linked N-acetylglucosaminegroup i s present on N343.

(with 1Junsohmted bands)

Urushiol I J ~ c , ~ , ,+, ~0 2

+ 4Ht

Polynie~sa~on -+

hC,,d,,

+ 2H20

FIG. 6. Typical polymerization reaction catalyxed by laccase, the oxidation o f urushiols. The substrate is oxidized to a free radical by the transfer of one electron to the laccase; the free radical then undergoes polymerization.

774

LINDLEY

FIG. 7. Overall sti-ucture o f the laccase from Coprinus cinereus. The type I1 copper-depleted cluster can be seen at the center of the figure, below is the type I copper which is ligated by two histidine residues and a cysteine.

2.3. Nitrite Reduclase 2.3.1. Occurrence and Function Nitrite reductases (NR) participate in the denitrification pathway whereby nitrate is converted to nitrite, nitric oxide, nitrous oxide, and finally nitrogen [311:

The process of denitrification occurs anoxically and uses energy; it has become increasingly important in eutrophic ecosystems 1321. It is essentially the opposite of nitrogen fixation and in soil results in the loss of plant nutrients. Understanding this process is therefore as important as understanding other key components of the nitrogen cycle, including nitrogen fixation and photosynthesis. There are two classes of nitrite reductase depending on whether a heme group or copper i s used at the active sit,e, and copper-containing enzymes have been isolated from a number of organisms, including Aclzromobacter cycloclasles strain IAM 1013. The NR from A. cycycloclastes

775

participates in the reduction of nitrite to NO and the reaction consumes two protons and one electron.

NR 2H'and le-

NO+W,O

The electron is initially provided by ascorbate, although the ascorbate itself does not appear to cause the reduction of the NR but transfers the electron to pseudoazurin. This type I copper-containing protein then donates the electron to NR in a specific copper-to-copper electron transfer pathway. 2.3.2. Structure of Nitrite Reductase fi.ont Achromobacter cycloclastes The first X-ray structure of a copper containing nitrite reductase, from Achromobacter cycloclastes, was reported in 1991 I151 at a resolution of 2.3 8.The crystallographic asymmetric unit is a monomer comprising two eupredoxin type domains. However, the monomers are tightly associated around a crystallographic three-fold axis to form a trimer, as shown in Fig. 8. Domain 1contains a type I copper

FIG. 8. Trimeric structure of nitrite reductase from Achrornobactercycloclastes. Domains 1,l' and 1" (see Fig. 3) are striped. A sulfate ion resulting from the crystallization procedure is located at the center of the molecule on the threefold axis.

776

LINDLEY

center and there is a type I1 center between each domain 1 and the adjacent domain 2 in the trimer as shown schematically in Fig. 3. In the monomer residues 8-160 and 161-340 form domains 1 and 2 respectively and the two domains are stacked next to one another so that the /3 sheets form a four-layered sandwich with the domain axes inclined at an angle of approximately 40". This means that the type I copper centers are at the external surface of the trimer (cf. the type I centers in ceruloplasmin) and accessible to a large molecule, such as pseudoazurin to facilitate copper-to-copper electron transfer. Each domain contains a short helical region, from residue 141 to 149 in domain 1 and 203 to 213 in domain 2, and these two helices are sufficiently close that their interaction may stabilize contacts between domains. Each domain also contains an extensive loop region between strands 1 and 2a. In domain 1, the loop comprising residues 47 to 62 participates in interactions between domains 1 and 2 in the monomer, whereas the loop in domain 2, comprising residues 183 to 202, takes part in interactions with an adjacent monomer in the trimer.

urnan Ceruloplasmin

2.4.1. Occurrence and Putative Physiological Functions uman ceruloplasmin (hCP),a copper-containingglycoprotein with a molecular weight of approximately 132 kDa, is found mainly in the plasma and accounts for 60% or more of the total plasma copper. The enzyme is an acute phase reactant that can exhibit a two- to three-fold increase over the normal plasma concentration of 300 pgl mL. It is composed of a single chain of 1046 amino acids with a carbohydrate content of 7%-8%. Since the discovery of ceruloplasmin in 1944 by Holmberg [33], an overwhelming number of articles have been published dealing with the different facets of ceruloplasmin in terms of its possible functions and multifunctionality. In spite of these extensive studies, the precise functions of hCP have not yet been defined, but it has been associated with ferroxidase activity, amine oxidase activity, pro- and antioxidant activities, copper transport [34-371, inhibition of rnyeloperoxidase activity [38], and others. The physico-chemical properties, the biological roles, and the oxidase activity o f hCP have been the subjects of' several comprehensive reviews (see, for example, [39-421).

2.4.2. Organisation of the Human Cer-uloplasrninMolecule The X-ray structure of hCP r141 shows that the molecule is comprised of six domains organized in a triangular array. This organization is schematicallyrepresented in Fig. 3, whereas Figs. 9a and b show the traces of the polypeptide chain viewed almost parallel and perpendicular to the pseudo-three fold axis, respectively. There are six integral copper atoms. Three of these copper atoms occupy mononuclear sites in domains 2, 4, and 6, whereas the remainder form a trinuclear cluster sited at the interface of domains 1 and 6. The trinuclear center and the mononuclear copper in

777

FIG. 9. Molecular structure of human ceruloplasmin as shown by ribbon diagrams of the polypeptide chain, viewed almost parallel (a) and perpendicular (b) to the pseudo three-fold axis. In both cases the even domains are striped,

LINDLEY

778

domain 6 form a cluster essentially the same as that found in the structure of ascorbate oxidase, strongly suggesting an oxidase role for hCP in the plasma. The oddnumbered domains-1, 3 and 5-comprise approximately 190 residues whereas the even domains are smaller with approximately 150 residues. The folding pattern for both the odd and even domains is based on the cupredoxin fold, the main difference being the insertion of large loops between the first and second strands in both odd and even domains and between strands 2a and 2b in the odd domains. In the odd domains there is also a stretch of irregular structure in place of strand 5. The domains are oriented so that they are all the same way up, but the even-numbered domains point inward toward the pseudo-threefold axis reminiscent of the feet of a tripod, whereas the odd domains point outward; this configuration has a significant implication on the location of the mononuclear copper atoms (vide infra). The similarity of the two sets of domains in hCP can be demonstrated by superposing the backbone a carbon atoms and evaluating the root mean square displacement between topologically equivalent atoms. Thus, for the odd domains of hCP 168 and 149 M carbon atoms of domains 3 and 5, respectively, match the equivalent atoms in domain 1with rms fits of only 0.98 and 1.03 For the even domains 146 and 143, %-carbonatoms of domains 4 and 6 match with those of domain 2 with even better fits of 0.87 rf and 0.88 A, respectively. The fit between the odd and the even domains is less pronounced. For example, only 92 o! carbon atoms of domain 2 superimpose with those of domain 1with an rms fit of 1.77 The large loops at the top of the molccule probably play an important role in the function of cemloplasn6-n in that they sterically inhibit the approach of large molecules to the oxidation sites, ie., the mononuclear copper atoms which in contrast to NR are located in the domains oriented inward.

A

A.

A.

2.4.3. Disuljide Bridges, CLeauage Sites, and G l p m s

There are five disulfide bridges in hCP, one each in domains 1-&and one free cysteine residue, C221, located in domain 2 on the extended loop between the first and second strands. The disulfide bridges anchor the last strand of the respective domains. In the odd domains the disulfide bridges link strands 7 and 8 with the cysteines being separated by 25 residues. For domains 2 and 4 the disulfide bridges link strands 2b and 8, with some 80 residues separating the cysteines. All of the S-S bridges are located close to the relatively flat bottom of the hCP molecule. Ceruloplasmin is readily cleaved into three fragments with molecular weights of 67,50, and 19 m a , respectively 1431. The 19-Wa fragment corresponds almost exactly t o domain 6 . However, the cleavage site for the larger fragments occurs at residue 479, a residue in domain 3 on an exposed loop prior to strand 6. Ceruloplasmin exhibits heterogeneity with respect to its carbohydrate moieties but possesses at least three glycan chains ending in a sialic acid 144,451. In the predominant heteromer two biantennary and one triantennary chains are attached through asparagine residues sited within the 67-kDa fragment at N119, N339, and N378. The beginnings of these chains can be modeled from the X-ray structural data

MULTI-COPPER OXIDASES

779

and in the case of N119 the first sugar residue is clearly visible. A fourth chain, which is also triantennary, can be attached at N743 in domain 5, but there is no evidence for this chain, in the current crystal structure.

3.1

Roles of Factors V and \/I11 in Blood Coagulation

Factor VlII (FVIII) plays an essential role as a cofactor in blood coagulation, and its absence is responsible for the most common bleeding disorder, hemophilia A 1461. FVIII is synthesized in hepatocytes as a mature single-chain polypeptide of 2332 amino acids, but circulates in the plasma as a population of non-covalently linked heterodimers in a profactor form bound to von Willebrand factor. During coagulation FVIII is proteolytically activated to FViIIa by trace amounts of thrombin or factor Xa (FXa) and greatly accelerates the proteolysis of factor X to FXa by factor IXa (FIXa) on the activated platelet surface. In complex with another phospholipid bound cofactor, factor Qa (FVa), FXh cleaves prothrombin to thrombin; FVa is homologous to FVIII in both structure and function. Finally, thrombin cleaves fibrinogen to insoluble fibrin to stabilize a hemostatic clot. Feedback control is mediated by activated protein C, which inactivates FVIIIa and FVa by further proteolysis.

3.2.

Overall Configuration of Factor V and Vlll Molecules

FVIII is a large multidoinain protcin with an overall configuration, Al.al.A2.a2.B.a3.A3.Cl.C2, where A l , A2 and A3 are three homologous domains approximately defined by residues 1-336, 375-719, and 1691-2025, respectively; a l , a2, and a3 are short acidic peptidcs; is a large heavily glycosylated domain that comprises residues 741-1648; and C l and C2 are homologous domains at the C terminus each with approximately 165 residues. There are also seven disulfide bridges, two each within Al, A2,and A3 and one in the C1 domain. On or before secretion, proteolytic processing occurs within the B domain to give heterodimers consisting of heavy (Al.al.M.a2 with variable extensions of the B domain) and light chains (aXA3.Cl.C2) which are associated via a divalent cation-dependent interaction. During coagulation, thrombin causes further cleavage and, critical for W I I a activity, the Al.al, A2.a2, and A3.CLC2 fragments remain in a non-covalently associated moiety. Thus the active FVIIIa molecule may be in the form of a heterotrimcr with a structure Al.al*A2.a2*A3.Cl.C2, as shown in Fig. 3. FV shows clear homology to WID with a structure of the type Al.A2.B.A3.Cl.C2. There is 35-409'6 sequence identity between all of the corresponding domains in FV and FVIII, with the exception of the B domains, which appear unrelated. In addition, FV lacks the short acidic sequences a l , a 2 and 133.

LINDLEY

780

3.3.

ode! for Factor Vlll Based on Human C ~ r u l o p l a s ~ ~ f l

The structural information described above has been combined with the known S~IXICture of human ceruloplasmin to construct a model for FVIIIa [16]. This model replaces an earlier model L471 based on the structure of NR, which, though useiul in terms of overall shape prediction, suffers from the defect that the arrangement of the subdomailis is almost certainly incorrect. In the model of FVIIl domain A l , A2 and A3 correspond to the pairs of hCP domains 1 and 2, 3 and 4, and 5 and 6, respectively. Although models are not substitutes for high-resolution structures, in the absence of other structural information, they can often provide a means not only of understanding structure-function relationships but of suggesting further experimental approaches. The model of FWII based on hCP enables a number of observations to be made. Thus, five of the six disulfide bridges within the A-type domains are homologous to those in hCP (Al: 153-179 and 248-239; r42: 528-554 and 630-711; A3: 1832-1858) and will therefore be located near the bottom, flat surface of the FVIII molecule. The sixth is nonhomologous and occurs toward the top of the molecule in domain A3, 1899-1903. The three acidic peptide regions and the large €3 domain can also be predicted to lie near the bottom surface and will therefore be accessible to serine proteases such as thrombin and activated protein C. FVIII appears to contain one copper atom 1481 and this can be predicted to be of type IT (Sect. 4.1.2). Finally, and of prime importance, the FVIII model readily enables plausible mechanisms explaining why various mutations give rise to clinical disease [161.

4. ~ T R U ~ T U R E - F U N C ~RELATIONSHIPS IO~ 4.11

The Copper

Table 2 summarizes the ligands involved in the copper binding sites in the multicopper oxidases. 4.1.1. Type I Copper Binding Sites

In the cupredoxin copper binding domains, the type I “blue” copper site is located at the top of the domain. The ligands comprise a histidine residue situated at the beginning of strand 4,and cysteine, histidine, and methionine residues located on the loop between p strands 7 and 8 and are arranged in a distorted tetrahedral configuration as shown in Fig. 10.The histidine residue on the loop between strands 7 and 8 is exposed to the solvent region and is likely to act as the primary site of electron transfer. The Cu-Cys interaction is responsible for the typical blue color through a cysteine $ 7 ~ +Cu d(x2 -?/”I charge transfer transition [491. The distances between the copper and the nitrogen atoms of the histidines and the sulfur atom of the cysteine are normally around 2.1 but the copper-to-methionine sulfur distance is


781

TAEZLE 2. Summary of the Copper inding Sites in the Multi-Copper Oxidases a) Type I Copper Binding Sites

Enzyme (domain)

Ligand (atom)

AO(3)

Lac(3)

NR(1)

hCP(2)

hCP(4)

hCP(6)

H(NG1) C(S$ H(N81) M(Sy)1

445 507 512 517

396 452 457

95 136 145 150

276 319 324

637 680 685 690

975 1021 1026 1031

_ I

-

b) Type I1 Copper Binding Sites Ligand (atom)

H(Ns2) H(N12) H(NE~) OH- or water a

Enzyme

A0

Laca

60 448

L

-

'yes

NR

hCP

FVIII

100 135 306b Yes

101 978

99 1957

__

-

Yes

?

Laccase from Coprinus cincreus i s type I1 copper-depleted. From domain 2 related by symmetry operator (y, z, x).

e I11 Copper Binding Sites

Ligand (atom)

Enzyme

A0 106

450 506 Bridging 62 104 508

Lac 111 399 40 1 45 1 Asymmetric 66 109 453

hCP 163

980 1020 Bridging 103 161 1022

782

LINDLEY

FIG. 10. The type I copper binding site in plastocyanin [71. The ligands to the copper comprise H37 at the beginning of p strand 4 together with C84, H87, and M92 located on tbe loop between p strun+ 7 and 8. The copper lig%ndsdistance are Cu - N61 (F37) = 1.91 A, Cu Sy (684) = 2.07 A, Cu - N61 (H87) = 2.06 A, and Cu - S , (M92) = 2,8,2 A. The corresponding distances for azurin in the oxidized form [4] are Cu - NS1 (H56) = 2.08 A, Cu - Sy (Cll2) = 2.12 A, Cu - NS1 (H117) = 2.01 A and Cu - S6 (M121) = 3.13 A.

longer about 2.8-3.0 A. Indeed, in certain laccases and the second domain of ceruloplasmin the methionine residue is replaced by an aliphatic residue, leucine, with a van der Waals contact between the copper and one of the leucine methyl groups (Fig. 11). A leucine (or phenylalanine) residue at this position in the fungal laccases is thought to contribute, at least in part, to an elevated redox potential, since the removal of the potential donor function of the axial methionine destabilizes Cu2+with respect to Cuf [50,5ll. It has also been reported that in hGP this copper is in the reduced form 1521, is unlikely to play a physiological role in the function of the enzyme, and may be a relic of the evolution of the protein. There are three mononuclear copper centers in hGP and the distribution of these centers within the even domains gives interatomic separations of around 18 A, since these domains point inward toward the pseudo-threefold axis of the molecule. Had Nature organized that these coppers were in the odd domains then these interatomic separations would have been significantly larger. The distance of 18 is well within the range for electron transfer F531, and there are clear electron transfer pathways linking the mononuclear copper atoms 1541. For example the pathway between the domain 4 and 6 copper atoms is Cu4H685.. , H-bond.. . E971 1972 D973 L974 M975-Cu6.

783

FIG, 11, The environment of the domain 2 mononuclear copper Center in human CeruloPlasmin. This copper is coordinated by two histidines, I1276 and 13324, and a C Y s ~ e ~c319, e, in an almost environment. The leucine residue, L329, does not coordinate to the copper. In human cel.uloplasmin it has been proposed that this copper is in the reduced state

For H685 the N6 ring atom is bound to Cu4 and the NE hydrogen bonds to the side chain of the conserved glutamate E971. The presence of aspartic acid between the hydrophobic residues I972 and L974 suggests that electron transfer can take place either dong the main chain or through the side chains of D973 and electron reaches Cu6, it can be transferred to the trinuclear copper center though C1021 and either €3.1020 or €31022 (vide infra). A similar pathway exists between Cu2 and Cu6. Electron transfer from Cu4 to the trinuclear center would be expected to be slower than from Cu6 directly because of the longer pathway. 4.1.2.

Type I1 Copper Binding Sites

In general, the type 11 sites are coordinated by two or three histidine residues and a water molecule or hydroxyl ion. In NR the type I1 center has an almost regular tetrahedral geometry (Fig. 12), and links two subunits in the trirner. Thus, two histidine residues, 100 and H135, are located in domain 1of one subunit, whereas the third, H306, arises from domain 2 of a different monomer in the trimer. The distance between the type I and type I1 centers is 12.7 A. The fourth ligand is a water molecule and points towards a solvent access channel. It appears to be replaced by nitrite under suitable soaking conditions in the crystal, indicating that the type I1 copper site is the active center for reduction 1551.

784

LINDLEY

FIG. 12. The type I1 copper center in nitrite reductase and its relationship to the type I center, At the type I1 center two histidine residues, HlOO and H135, arise from domain 1 of one monomer, whereas H306 is located in dam+ 2 of a different monomer in the trimer. The distance between the copper centers is 12.7 A.

In A 0 and hCP (and the non~copper~depleted forms of Lac), the type I1 sites should be considered as an integral part of the trinuclear centers and are co-ordinated by only two histidine residues and a solvent molecule or OH-. In A 0 the latter hydrogen bonds to a water molecule, which in turn hydrogen bonds to the side chain of Q66. In hCP, the water or OH- hydrogen bonds directly to the hydroxyl group of Y107 and the main chain carbonyl group of S102. The copper site in a type I copper from also likely to be a type 11 copper. There is no indication spectroscopic measurements, but two of the type I1 ligands, 9 and I11957 (equivalent to W l O l and I3978 in hCP), are conserved as is Y106 (equivalent to Y107 in hCP). If this is the case the type I1 copper in EVIII will be sited at the interface of the A1 and A3 domains, and this may well contribute to the association of the heavy and light chains in the active molecule. 4.1.3. Type III Copper Binding Sites In A 0 the type I copper site in domain 3 has a closest distance of approach of 12.2 A to the trinuclear copper center (Fig. 13). The trinuclear center is ligated by four pairs of

785

~UL~I-C~P OXIDASES PE~ 517

FIG. 13. Structural relationship between the mononuclear copper center in domain 3 and the t5inuclear center between domains 1and 3 in ascorbate oxidase. The Cu-Cu and Cu-O &stances (A) in the trinuclear center are as follows: Cu2 - Cu3 = 3.68 (3.73); Cn2 - OH (or 02-)= 2.00 (2.00);Cu2 - Cii4 = 3.78 (3.90); Cu3 - OH- (or 02-) = 2.06 (2.00); Cu3 - Cu4 = 3.66 (3.69); and Cu4 - OH- (or water) = 2.02 (2.03). Values in parelltheses refer to the second molecule in the dimeric asymmetric unit. Th? closest approach of the type I 5oopper atom to the trinuclear center is Cul - Cu 2 = 12.2 A; the Cul - Cu3 distance is 12.7 A.

histidine ligands, two each from domains 1and 3. Seven of the histidines are ligated by their N Eatoms, ~ whereas the eighth, II62,is ligated by its N61 atom, 8 consequence of its location at the end of a short p strand. Within the trinuclear center Cu2 and Cu3, each ligated by three histidines, are bridged by an oxygen atom (OH- or 02-1 giving rise to a distorted tetrahedral coordination. They constitute the pair of type 111 coppers, with strong antiferromagnetic coupling, and are EPR-silent. There appears to be no oxygen atom in the middle of the copper cluster as predicted by certain spectroscopic measurements. The average values of the Cu-Cu, Cu-N, and Cu-0 distances are 3.74 A, 2.09 A and 2.02 respectively. The Cu-N and Cu-0 distances are comparable to binuclear model copper compounds, but the Cu-Cu separations appear to be too long for bonding despite the fact that magnetic interactions are possible. Several groups have reported that copper can be selectively removed from the blue copper oxidases, resulting in the loss of the type I1 EFR signal. This is normally achieved under reduced conditions in the presence of a chelating agent such as EDTA (see Sec. 2.2.2). However, Messcrschmidt et al. [56] have shown that if copper is removed from crystals o f AQ using the method of Avigliano et al. [57J, as modified

LINDLEY

786

by Merli et al. [58], then copper is lost from all three sites in the trinuclear center, both type I1 and type 111,although the type I1 site appears most depleted; the copper I site is not affected. The loss of the type I1 EPR signal may indicate that all three coppers in the trinuclear center are equivalent under certain conditions. The trinuclear center in the crystal structure of Coprinus cinereus laccase is type 11 copper-depleted, as shown in Fig. 14, and contrasts the case of A 0 where copper depletion occurs at both type 11 and typc I11 centers. This loss of copper has profound structural and spectroscopic implications. First, the two type I11 copper atoms are separated by 4.9 (cf. 3.7 and 5.1 in fully oxidized and reduced AO, respectively). Second the bridging oxygen moiety (OH-) is asymmetrically placed, being some 2.1 A and 3.1 A from Cu2 and Cu3, respectively, and forming a hydrogen bond with a nearby solvent molecule. Spin-pairing between the two type I11 coppers is therefore unlikely and the lack of a corresponding EPR signal suggests that type I1 copper depletion is accompanied by reduction of both hype I11 copper centers. Geometrically, Cu2 is five-coordinate with one of the histidines, H399, which would normally bind to the type I1 copper, rotating across to form a fourth histidine ligand through its Nc2 atom; the N61 atom hydrogen bonds to a nearby water molecule. On the other hand, Cu3 is three-coordinate with three histidines bound in a planar trigonal environment, typical of Cu+. The type I1 copper atom is not simply replaced

A

A

A

FIG. 14. The type I1 copper-depleted trinuclear center in the laccase from Coprinus cinereus.


787

by a solvent molecule as might be expected. €364 and H399, which would normally constitute type I1 copper ligands, are in van der Waals contact with one another, with a closest approach of 3.3 A leaving no space for type I1 copper binding. It is likely that both main and side chain structural rearrangements would be required to accommodate the type 11 copper atom. Clearly, further detailed structural information is required on the fungal and tree laccases in both their normal and copper-depleted states. The trinuclear cluster in hCP, and its relationship with the domain 6 type I copper, is essentially the same as that in AO. Domain 1 donates two pairs of histidines, N101-Hl03 and H161-15163, with a serine residue separating the components of each pair, while the remaining pairs are donated by domain 6, H978H980 and W1020- 1022, and are separated by phenylalanine and cysteine residues, respectively; in all cases the pairs of histidines bridge two copper atoms. The cysteine residue at 1021 coordinates to the domain 6 mononuclear copper, so that there is a good electron transfer pathway between this center and the trinuclear cluster. As is the case for AO, there would also appear to be an oxygen atom (OH-) bridging the two type 111 coppers, although the X-ray data at 3.0 cannot be unequivocally interpreted.

inding Sites in Ascorbate Oxidase and

4.2.

Lacease An examination of the surface o f A 0 reveals a binding pocket for the organic substrate in the vicinity of the type I copper. Within this pocket three residues in particular seem to be important, i.e., W163, W362, and H512, as shown in Fig. 15. I3512 lies at the bottom of the pocket with its N61 atom bound to the copper; the Nz2 is therefore available for substrate binding. The aromatic ring of W362 is approximately parallel to the imidazole ring of H512 and its NS1 atom is accessible for hydrogen bonding. The aromatic ring of W163 can be considered as forming another wall of the pocket. The L-ascorbate molecule can be modeled into the pocket [13] with the lactone ring oriented parallel to the aromatic ring of W163 and the side chain pointing toward the outside of the A 0 molecule. In this model, the hydroxyl oxygen atoms of the L-ascorbate could form hydrogen bonds with the 512 and the Ncl of W362. The transfer of an electron, leading t o oxidation of the substrate, to the type I copper and thence through the pathway ~ 5 0 7 - ~ 5 or 06 I1508 to the type 111 copper atoms in the trinuclear center can be readily envisioned. In Lac the type I copper atom is located at the base of a shallow groove, about 6 A from the molecular surface. The walls of the groove are defined by three loops, residues 152-165, 336-340, and 387-395, and the residues equivalent to W163 and W362 in A 0 (the residues implicated in substrate binding) are A172 and F459 in laccase. The relatively large size of the groove may reflect the broad substrate specificity shown by the fungal laccases.

788

FIG. 15. The putative organic substrate binding site in ascorbate oxidase. The N E and ~ N E ~ atoms of W362 and I1512, respectively, are available for hydrogen bonding to the hydroxyl groups of the substrate prior to oxidation. W163 forms part of the wall of the cavity.

hanism of Oxygen Reduction by the Trinuclear

When an electron reaches the trinuclear center it contributes to the reduction of a molecule of oxygen to two molecules of water, with a total of four electrons being required for the complete reduction. In A 0 there are two obvious channels by which an oxygen molecule can reach the trinuclear center. One is oriented to the type I11 pair of copper atoms and the other to the type I1 copper and its associated oxygen ligand; in the A 0 crystal structure, both channels are occupied by water molecules. How does the oxygen molecule bind to the trinuclear cluster? Unfo~tunately,it has not been proven possible to study oxygen binding directly in the crystalline state using X-ray methods, but a series of soaking experiments 1591 have elegantly indicated a putative mechanism for oxygen binding and reduction.

789


4.3.1. Fully Reduced Trinuclear Center in Ascorbate Oxidase

The structure of the reduced form of A 0 has been elucidated to 2.2 A resolution by soaking crystals in a reducing buffer (dithionite) under anaerobic conditions [591. First, there are significant increases in the Cu-Cu separations inothe trinylear cluster. Thus, the Cu2-Cu3 distance increases dramatically from 3.7 A to 5.1 A, whereas, the Cu2-Gu4 and Cu3-Cu4 distances change from 3.8 A to 4.4 A and from 3.7 A to 4.1 A, respectively. Second, the oxygen (OH-) atom between the Cu2 and Cu3 atoms is released (Fig. 161, and these two atoms move toward their respective histidine ligands and become three-coordinated-a preferred geometry for the Cu cation. The geometry of Cu4 is virtually unchanged in the oxidized and reduced forms of A 0 as is that for the mononuclear copper center. +

4.3.2. Addition of Hydrogen Peroxide to Ascorbate Oxidase

The structure of the peroxide form of A 0 has been elucidated at 2.6 A resolution by soaking the native crystals in buffered solutions or hydrogen peroxide [591; concen-

FIG. 16. The trinuclear copper center in>he fully reduced f y m of ascorbaie oxidasp The inter-copper distances are Cu2 - Cu3 = 5.1 A, CuZ - Cu4 = 4.4A and Cu3 - Cu4 = 4.1 A. This Fig. is adapted from Fig. 11in reference [Z] with the permission o f World Scientific Press.

trations greater than 20 mM caused the A 0 crystals to deteriorate rapidly. As indicated in Fig. 17, the peroxide binds to Cu2 and the bridging oxygen (0 In a similar manner to the reduced form, there are increases in the Cu2-Cu3 and Cu2Cu4 distances, from 3.7 to 4.8 and from 3.8 to 4.5 A, respectively. The Cu3-Cu4 distance is almost unchanged at 3.7 A. The Cu2 atom adopts a distorted tetrahedral geometry and Cu3 is at the apex of a flat trigonal pyramid. The bound peroxide molecule is accessible through one of the solvent channels indicated above, and this presumably delineates the route for oxygen binding. The peroxide molecule binds in close vicinity to I3506, consistent with the concept that the electron transfer pathway from the inononuclear copper to the trinuclear center involves C507 and either HSO6 or H508. There is also evidence of copper depletion in the trinuclear center when peroxide binds; the Gu3 and Cu4 sites are only slightly depleted, but the Cu2 site occupancy in the crystal is reduced by about 50%. In conjunctioii with this depletion, H506 appears t o move further away from the copper binding site, thus exposing it further. This may explain the instability of' A 0 toward hydrogen peroxide.

A

A

A

Cul

FIG. 17. The trinuclear copper center jn the peroxide comglex of ascorbate oxidase; The intercopper distances a1-e,~Cu2 - Cu3 = 4.8 A, Cu2 - Cu4 = 4.5 A and Cu3 - Cu4 = 3.7 A ; the Cu OP1 distance is 2.0 A. (Adapted from figure 12 in ref. 121 with permission of World Scientific Press.)

PPER OXIDASES

791

4.3.3. Overall Mechanism of Oxygen Reduction

The structures of the fully reduced and peroxide forms o f A 0 suggest the mechanism of oxygen reduction shown in Fig. 18 [Z]. Stage (i) is the resting state of the enzyme with all coppers fully oxidized. In the first step (ii) the type I copper center is reduced by the transfer of an electron from a substrate molecule. This electron is then transferred to either Cu2 or Cu3 in the trinuclear center via C507 andlor H 5 0 ~ / ~ 5 0After 8. the transfer of four electrons, all four coppers are reduced and the hydroxyl bridge between Cu2 and Cu3 is released with concomitant increase in the Cu2-Cu3 separation (iii). At this stage, dioxygen binds to Cu2 in a similar manner as observed for peroxide and the transfer of two electrons from the copper pair leads to a hydroperoxide intermediate (iv). Two additional electrons can then be transferred, one from Cu4 and one from the mononuclear copper center. At this stage (v) the 0-0 bond breaks, the first molecule of water is released, and CuZ is left in the reduced state bound t o an oxygen radical; such a radical has been detected in laccase by EPR measurements. The Cu3 and Cu4 are both oxidized and may spin-couple so that they are EPR silent. The catalytic cycle continues through fbrther reduction of the mononuclear copper and transfer of the electron to Cu3 via the 6507 and H508 pathway. The fourth electron is then donated by Cu2 to the oxygen radical, resulting in the release of a second water molecule (vi). The enzyme then reverts to step (iii) and recycles around until all substrate molecules are oxidized. Alternatively, if only four electrons are available the cycle can go from step (v) back to the resting state (i) whereby the second water remains bound as a bridging ligand between Cu2 and Cu3. Although there will be a substantial conformational change leaving and entering the resting state, the conformational changes during the cycle will be small, with the intermediate structures being close to that of the reduced enzyme. The four protons required in the reduction of the dioxygen molecule can be supplied from bulk solvent close to Cu2 and Cu3, which can access the trinuclear site through the two channels described previously. Thus, the seminal work of Messerschmidt and his colleagues has provided a plausible working model for the mechanism of oxygen reduction by the multi-copper oxidases. 4.4.

Azide Inhibition in Ascorbate Oxidase and Human Cer~lopla~min

Curzon and Cummings [60 I have identified seven categories of inhibitors of the multicopper oxidases: inorganic anions, carboxylate anions, -SH compounds, chelating agents, hydrazines, 5-hydroxyindoles, and a miscellaneous group including divalent and trivalent metal cations. The inorganic anions include two of the strongest inhibitors of oxidase activity, cyanide and azide, both with an inhibitory constant, K,M 2 x lo6 M for hCP. Indeed, azide has been frequently used in attempts to distinguish between ceruloplasmin catalysis of Fe(I1) oxidation and other ferroxidase activity in the plasma.

LINDLEY

792

I I

f

(i) Resting state of enzyme

I

H506

(vi)

Reduction of Cul, transfer of electron to Cu3, and release of second water

+le

0'. OH'

HO-Cu4"

(ii) Reduction of Cu I

(v) Release of first water and formation of O * radical intermediate

-

HO Cu4'

O-0-B

cu2;; CO3"

(iii) Fully reduced enzyme with di-oxygen adduct

I

I

I

(iv) Hydroperoxide intermediate

FIG. 18. The mechanism of oxygen reduction by ascorhate oxidase proposed by Messerschmidt. (Adapted from figure 14 in ref. T2l with permission of World Scientific Press.)

793


4.4.1. Addition of h i d e to Ascorbate Oxidase

X-ray studies of the azide complex with A 0 [59] clearly indicate that there is little structural change at the mononuclear binding site but significant changes at the trinuclear center. Two azide anions bind to the same type 111 copper atom as utilized by peroxide, Cu2, as shown in Fig. 19.0Thereare corresponding increases in the Cu2Cu3 and 43x12-Cu4 distances from 3.7 A to 5.1 and from 3.8 to 4.6 respectively; Cu2 becomes fivefold cothe Cu3-Cu4 distance is relatively unchanged at 3.6 ordinated with the two azide moieties at the apices of a trigonal bipyramid. Access to the Cu2 site for azide is the same as that for peroxide and presumably also for oxygen. The geometry at Cu4 i s little changed, but as is the case for peroxide, Cu3 is three-coordinated in the form of a Rat trigonal pyramid. Contrary to predictions from extcnsive spectroscopic studies (see, for example, 16111, there is no azide anion bound in the form of a IJ. bridge between the type 11 and type 111 copper atoms.

A

A.

A

A,

Q

FIG. 19. The trinuclear centeroh the wide comple? of ascorbate oxidase. The intercopper distances are Cu2 - Cu3 = 5.1 6, Gu2 - Cu4 = 4.6 A and Gu3 - Cu4 = 3.6 A;the Cu azide distances average around 2.0ti A. (Adapted from fig. 13 in ref. [Z] with permission of World Scientific Press.)

LI NDLEY

794

4.4.2. Addition of Azide to Human Ceruloplasmin The binding of azide (and fluoride) to the resting enzyme appears to change the EPR signal from the type 11 copper [62,63], These results, together with binding and kinetic studies 164,651, indicate that one anion binds at low concentration but two at high concentration. X-ray studies on crystals of hCP soaked for 5 days with 10 mM sodium azide 1661 have shown that at least one azide anion binds to hCP; attempts to increase the azide concentration or soaking time led to a significant deterioration of the X-ray diffraction patterns, The binding of the azide is accompanied by a color change in the crystals from blue to pale green. The azide anion binds to one of the type I11 copper atoms in the trinuclear center in a very similar manner to that found for one of the two azides (A21 in Fig. 19, see below) that bind in A 0 [591. In addition, the crystallographic studies can also be interpreted in terms of the loss of the oxygen atom bridging the type I11 copper atoms and an increase in their interatomic separation. Such observations reinforce the similarity between A 0 and hCP with respect to their oxidative mechanisms.

eruloplasmin and Ferroxidase Activity uman ceruloplasmin is unique in being able to oxidize both organic and inorganic substrates. Furthermore, the value of the apparent Michaelis constant for all substrates varies within a range of 10’ (ie,, 0.2 pM to 280 mM), whereas the value for V, varies by only an order of magnitude 1421. Such findings have prompted the suggestion that the limiting step in catalysis is a function of the rate of reduction of the blue and not the affinity of the substrate for the binding site of the protein. sieh [39] have proposed three major groups o f substrates to describe the oxidase action of hCP:

1, Fe(II1, the substrate with the highest V, and the lowest Km 2. An extensive group of bifunctional aromatic amines and phenols that do not depend on traces of iron for their activity. This group includes two classes of biogenic amines, the epinephrine and 5-hydroxyindole series, and the phenothiazine series. 3. A third group of pseudosubstrates comprising numerous reducing agents that can rapidly reduce Fe(II1) or partially oxidized intermediates of class (2) substrates. Following refinement of the native hCP structure, it was discovered that the enzyme possessed at least two additional metal binding sites 1541. These sites were found in domains 4 and 6 about 9-10 from the respective mononuclear copper sites. It was shown that for the enzyme as isolated, purified, and crystallized for the X-ray studies, the metal was also copper, but with an occupancy of only around 50% (i.e., in all the molecules that make up the crystal, copper was present in roughly half the total number of sites); these additional sites are termed the “labile binding sites” (vide

MULTI-COPPER

ASES

795

infra). For the domain 6 labile site, the ligating residues are E935, H940, and D1025 from domain 6 and E272 from domain 2, as shown in Fig. 20. The corresponding ligands for the domain 4 site are H602, E597, and D684 from domain 4 and E971 from domain 6. No corresponding site is found for domain 2 where the equivalent histidine residue has been replaced by Y241 and the remaining domain 2 residues are E236 and N323 with E633 from domain 4. X-ray studies were then undertaken with crystals of hCP soaked with the cations Cu(II), Co(II), Fe(II), and Fe(I1I) r541. Crystals were soaked under a variety of conditions, although not all were useful due to significant deterioration of the diffraction patterns. The following observations can be made:

FIG. 20. The domain 6 “labile” binding site in human ceruloplasmin. This site is surrounded by three negatively charged residues and a hisiidine and could possible accommodate more than one cation. The histidine, H940, i s some 3-5 A away from one o f the histidines, H1026, that is bound to the mononuclear integral domain 6 copper, thus facilitating electroii transfer. In the native enzyme, as crystallized for the X-ray studies the labile sites are partially occupied by copper.

796

LINDLEY

(1) Cu(1I). Soaking the native hCP crystals with CuClz (1mM for 60 h) leads to an increase in occupancy of the labile copper sites. At least two other minor copper sites were also found on the outside of the protein, but whether these sites, or those of the labile copper, are physiolo~callyimportant in terms of copper transport i s not clear. (2) Co(11). Soaking native hCP crystals with CoClz (1rdM for 72 h) appears to cause the replacement of the labile copper by Co. However, X-ray data alone cannot determine the oxidation state of the metal. The site is rich in oxygen donors consistent with the presence of Co(II), but alternatively the Co(11) could be oxidized to the kinetically inert Co(lII), which then remains in the labile sites. (3) Fe(I1) and Fe(lII). Prolonged soaking of native hCP crystals with both Fe(I1) and Fe(I1I) cations produces striking color changes (from sky blue to bright orange in the case of Fe(II)SQJ, but the crystallinity and hence resolution of the corresponding diffraction patterns deteriorates dramatically, making structural changes impossible to interpret. However, short soaking times (1mM FeS04 for 3 h and 1mil4 FeC1, for 21 h) lead to the removal of the labile copper and the appearance of iron at sites in domains 4 and 6 near the outside of the molecule; these are termed the “holding” sites and their environment is defined by the four negatively charged residues, depicted for domain 6 only in Fig. 21. The side chains E935 and E597 in domains 6 and 4, respectively, appear to move away Erom the labile positions toward the holding sites. The presence of iron in the holding sites has been confirmed by additional experiments exploiting the X-ray anomalous scattering of iron.

ne obvious interpretation of these observations is that the labile sites act as the sites of substrate oxidation. In this respect it is worth noting that “940 in domain 6 (and N602 in domain 4) is approximately topologically equivalent to one of the two tryptophan residues, W362, thought to delineate the substrate binding site in AO. For Fe(I1) the oxidation mechanism would first involve the cation occupying a labile site (first displacing any Cu(1I) present by reducing it to the labile Cu(I)) and then releasing an electron. The oxidized Fe(II1) would then translocate to the holding site. E935 in domain 6 (E597 in domain 4) may play an important role in the translocation process. The released electron would be transferred to the trinuclear copper center causing partial reduction of a molecule of dioxygen. Iteration of this procedure would lead to further electrons being transferred to the trinuclear copper center and the possibility of more than one ferric iron being stored at the holding site. Domain 4 provides a second, albeit slower, electron transfer route and a second holding site near the outside ofthe molecule. Whether the holding sites are accessible by small chelating molecules for Fe(IP1) prior to the uptake of the iron by apotransferrin, or by apotransferrin directly, remains an important question. For Go, if oxidation takes place, the relatively kinetically inert Co(1II) would remain in the labile sites, but for Fe(III), the cation would displace any copper present in the labile site and then translocate to the holding site, but without electron transfer. Under the soaking conditions used there are no significant signs of Fe(II1) cations binding at the domain 4 holding (or labile) sites. These experiments cIearly show a


797

FIG. 21. The domain 6 “holding” site in human ceruloplasmin. The X-ray studies indicate that E935 rotates away from the labile site toward the holding site (not shown in the figure) and may play a central role in the movement of the metal cations. The electron density for E931 i s poorly defined and this residue has been modeled as an alanine.

feasible mechanism for iron oxidation, at least in vitro, and are consistent with a ferroxidase role for hCP in plasma.

inding of Organic Substrates to Human Ceruloplasmin p-Phenylenediamine (pPD) is known as a classic essay for the determination of the oxidase activity by hCP. It has been shown that the principal product of pPD androwski’s base 1391; the reaction proceeds via formation of the

LINDLEY

798

quinonediimine intermediate C671, as shown in Fig. 22. For biogenic amines, such as epinephrine (also known as adrenalin), norepinephrine (noradrenalin), and seroto-

2

om

I OH

S e ~ o ~ o ~ i ~

ydroxy i ~ d o l e - ~ - ~ c eacid tic

FIG. 22. Oxidation of p-phenylenediamine and biogenic amines.


799

800

LINDLEY

FIG. 23. The domain 4 binding site for aromatic diamines in human ceruloplasmin. The loop at the top of domain 4 linking strands 1 and 2a has been omitted.

4.7, ~~r ul opl as mand in Hemosiderosis As discussed in Sec. 4.5, a clear mechanism exists for hCP to be involved in the oxidation of Fe(I1) in vitro, but it is the role of hCP as a ferroxidase in the blood that has been a subject of intense debate and interest. In 1968 it was reported [691 that pigs made copper-deficient by dietary restrictions were unable to release iron to the p l a m a and had low levels of ceruloplasmin. In this respect, hCP is believed to mediate the release of iron fram cells and to ensure that it is safely incorporated into, for example, apotransferrin. The presence of free Fe(1I) in the environment is dangerous because it can participate in Fenton and Haber-Weiss reactions to yield the biologically deleterious OH' radical. On the other hand, Fe(II1) at physiological pH is almost insoluble, so that a chaperone-type role for hCP, particularly in acute phase circumstances, is not unreasonable. More recently, mutations in the ceruloplasdn gene, linked with aceruloplasminemia and neurovisceral hemosiderosis [70-731, have given further credence to the proposition that hCP may be involved in the release o f intracellular iron and its oxidation prior to uptake and transport by transferrin. In certain cases, systemic hemosiderosis appears to be caused by incomplete expression of the hCP molecule, with the polypeptide chain being prematurely terminated. An

MULTI-CO~~ OXIDASES E~

801

examination of the crystal structure clearly shows why this should be the case if the incomplete enzyme is secreted into the bloodstream. Thus, if the chain is terminated at residue 991 [701, three residues that bind to the domain 6 copper (C1021, H1026, and M1031) will be absent. It is therefore unlikely that domain 6 would bind a mononuclear copper, and this would have a profound effect on the ability of the truncated enzyme to act as an oxidase if secreted into the bloodstream. Furthermore, two ofthe histidines that bind to the trinuclear copper center would also be missing, i.e., H1020 and H1022, as would more than 50% of the residues that form hydrogen bonds with domain 1. The truncated enzyme is therefore also likely to adopt a more open conformation than the intact hCP and be more susceptible to proteolytic cleavage. Loss of ferroxidase ability and increase in susceptibility to proteolytic cleavage would be respectively consistent with a gradual accumulation of intracellular iron leading to systemic hemosiderosis and aceruloplasminemia such as the failure to find hCP in liver extracts by the Western blot and ELISA methods.

Aspects of the

u l t i - c ~ ~ pOxidase er Family

6.1 .l. Sequence Alignments and Phylogenetic Trees

The evolution of the multi-copper oxidase family has been investigated in a number of ways, but only recently has sufficient X-ray structural information become available to use a comprehensive structural alignment approach [741, The structure and evolution ofthe small blue copper proteins were studied by R y d h r751. This study was then extended to include the blue multi-copper oxidases (but not NR) and the blood coagulation factors “s6 l. The approach was based mainly on sequence alignments and the construction of phylogenetic trees, but some use was also made of the structural information then available, At this time the X-ray structure of hCP was not known, although excellent structural predictions had been made based on the structure of A0 [771 and NR [781. Ryd6n and Hunt [761 found that, despite the fact that the multicopper oxidase family contains proteins varying radically in size, with different numbers of copper atoms and with functions ranging from electron transport to blood coagulation, it was probable that of all the enzymes in the family may have evolved from a common ancestral protein. Fig. 24 is a simplified summary of the evolution of the multi-copper oxidase family. Ryden and Hunt [761 identified six mechanisms that could be involved in the evolutionary process:

- domain enlargement in which a single domain increases in size from around 100 residues up to 210 domain duplication that allows for a size increase from 170 to around 1000 residues - segment elongation whereby a small se,grnent undergoes successive multiple duplications that can increase the chain size 50-fold -

802

LINDLEY

FIG. 24. Evolution of the blue copper oxidase and related copper proteins based on sequence alignments, structural comparisons, and the construction of phylogenetic trees [7ti].

- domain recruitment in which a domain coded elsewhere in the genome is added to the peptide chain

- subunit formation leading to multi-subunit proteins - glycosylation, which can greatly increase the size of the protein molecule They suggested that a small blue ancestral protein first undergoes a domain enlargement. This increase in size is followed by domain duplication with the development of an active oxidase site between the domains to give an ancestral oxidase protein. This development would require the addition of ligands t o bind a trinuclear copper center. Tn the Ryd4n and Hunt model, the two-domain ancestral oxidase would correspond to domains 1 and 3 and 1 and 6 in A 0 and hCP, respectively (see Fig. 3). Further duplication of one of the domains in the ancestral oxidase together with loss of some intradomain copper centers would lead to the plant and fungal oxidases (A0 and the laccases). Alternatively, two double domain duplications and loss of some intra- and interdomain copper centers would give hCP. Ryd4n and Hunt also proposed that the size increase to the present molecule of hCP (and other larger members of the family) could be to prevent its clearance from the circulation by the kidneys and/or to bestow a multifunctionality of the enzyme.

~ULTI-COPPEROXIDASES

803

5.1.2. Structural Comparisons The approach of Murphy et al.[741 is based on a detailed structural comparison using a simultaneous structural superposition technique. The superposition involved various single-domain members of the family, including the cupredoxin domains in quino1 oxidase and cytochrome c oxidase, with the multi-domain members AO, hCP, and NR. The structure of laccase was not known at this time. Murphy et al. identified a conserved core of 65 residues among a total of 23 domains (15 crystal structures) with an overall sequence identity of 18.5% and a rms difl'erence between topologically equivalent main chain a carbon atoms of' 2.05 A. These conserved cores include residues forming eight strands of the p sandwich (1, 2a, 2b, 3, 4, 6, 7, and 8 ) and three of the four residues that can potentially provide ligands to a type I copper atom. A structural similarity tree (Fig. 251, derived from the rms difference between the pairs of a-carbon atoms, partitions the cupredoxin domains into five classes: - single-domain electron transfer

proteins pseudoazurin, plastocyanin, and amicyanin - the CuA domains of bovine and bacterial cytochrome c oxidase and the related copper-deficient domain of quinol oxidase - the structures of azurin from Alcaligenes denitrificans and Pseudomonas aeruginosa - first domains of AO and NR and the odd domains of hCP - even domains of hCP, domain 2 of NR, domains 2 and 3 o f A 0 and rusticyanin

A sixth class can be defined by reducing the size of the common core to include the phytocyanins [751, stellacyanin, and the cucumber blue protein. 5.1.3. An Alternative Evolutionary Pathway There is general agreement between the phylogenetic and structural similarity trees. Both indicate that of all the cupredoxins, azurin is closest to the blue oxidase domains; pseudoazurin, plastocyanin, and amicyanin form a distinct more remote cluster. However, Murphy et al. I741 argue thal it is unlikely that the ancestral two-domain structure proposed by Bn and Hunt (761 could have existed as a monomer. The relative orientation necessary for function is maintained either by a complete sixdomain structure, as i s the case for hCP, or the presence of the second domain and a short c rig linker (which effectively replaces the other three domains), as is the cast? As an alternative, they suggest that after the initial duplication to give a two-domain structure (consisting of one each of domain classes IV and V and corresponding to domains 1and 2 in both A 0 and hCP in Fig. 31, a further development took place to give an NR-like trimer. The larger members of the fam evolved from this six-domain trimer. The formation of the oxidase function in hCP would arise from four mutations to give a total of eight histidine residues at the NR trimer interfaces required to bind the trinuclear copper center. After two of the three oxidase sites were lost and vestigial histidine residues

804

LINDLEY afn2 aoz3

hcp2 hcp4 hcp6 NC

aoz2

afnl aozl hcpl hcp3 hcp5 aza azu Cyw

arw

occ

aati

§i~~erpos~tion of c u p r e ~ o domains x~ after ~ u r p h et y al. [74] offl~ins 1. Single domain electron transfer proteins.

2.

Code Source

Poplar piaslocyanin Chlamydomonas reinhardtii plastocyanin Enteromorpha prolifera plastocyanin Alculigenes faecalis pscudoazurin Methylobacterium extorquens pscudoazurin Paracoccus denitrificans amicyanin Alcaligenes denitrqicuns azurin Pseudomonas aeruginosa azurin Thiobacillusferroxidans rusticyanin

pic plt pcy pza pmy aan aza azu ruc

PDB IpIc PDB 2plt PDB7pcy PDB lpza PDB lpmy PDB laan PDB 2aza PDB4azu PDB lrcy

Multi-domain enzymes *. Alciiligenes faecalis nitrite ieductase ( 2 ) Zucchini ascorbate oxidase (3) Humdn cenrloplasmin (6) Escherichia coEi quinol oxidase (1) Bovine cytochrome c oxidase ( I ) Puracoccus denitrificans cytochrome oxidase (1)

afn aoz hcp cyw occ arw

PDB 2afn PDB laoz PDB lkcw PDB lcyw PDB 2occ PDB larw

Ref.

[90]

112, 131 [14]

[lo]

[S] [911

* Number of domains in parentheses. FIG. 25. Evolution of the multi-copper oxidases based on a structural similarity tree (after Murphy el, al. [741 and reprinted with the permission of Cambridge University Press).

interface between domains 2 and 3 and H816 and H818 at the interface of domains 4 and 5 are remnants of these oxidase sites. The hydrogen bond interactions between hCP domains are more conserved between domains 1and 2 , 3 and 4, and 5 and 6 than between 2 and 3 etc. This is also consistent with a two-domain precursor consisting of domains 1and 2 in Fig. 3. In the case of the three-domainA 0 structure, this evolution


805

involved the replacement of three domains by a short linker peptide, contrasting the scheme of Ryd6n and Hunt [76j whereby a single domain is added to the ancestral two-domain structure. The fact that both the second and third domains in A 0 belong to the same structural similarity class, class V, is consistent with the Murphy et al. “741 hypothesis. Both schemes require the loss of type I copper sites. However, it should be noted that in the NR dimer, the domain containing a type I copper is domain 1and falls into the same class (class IV)as the odd domains in hCP, which do not contain copper. On the other hand, the second domain of NR, which lacks copper, can be classified (class V) with the even domains of hCP, all of which contain a mononuclear copper center. This can be explained in terms of the need in NR to have the type I copper towards the outside of the niolecule so that it can interact with pseudoazurin for electron transfer. In hCP the type I copper centers must be on the inside so that they can participate in specific oxidase function. The evolution of the conserved structural cores of the domains appears to be independent of the presence or absence of copper.

X-ray structural studies have enabled a significant number of questions to be answered on the multi-copper oxidase family of enzymes, particularly the number, location, and functions of the various copper sites. However, many key questions remain, particularly with regard to mechanisms of electron transfer, the precise mechanism of oxygen reduction, the details of the substrate binding sites, and the interpretation of spectroscopic and kinetic measurements. Indeed, the space required to pose all of the questions could exceed the current chapter! For ceruloplasmin, many outstanding questions center on the in vivo functions of this complex molecule. The X-ray studies strengthen the case for a multiple function capability-exaniples include ferroxidase activity and oxidation of aromatic diamines and biogenic amines-but throw little light on its capacity to act as a copper transporter. It is possible that the labile sites and other sites on the surface can act in this regard, but the trinuclear copper center and the mononuclear coppers in domains 2,4, and 6 appear to be an integral part of the structure. Of course, when the enzyme undergoes catabolism copper will be released into the environment and can possibly be adventitiously acquired by other molecules, but the structure contrasts sharply with that of serum transferrin [791, which i s specifically designed to transport iron. Another major question concerns the role of ceruloplasrnin with respect to ferroxidase activity. Does it in reality mediate the release of iron from cells as indicated in Sec. 2.4.6? Or i s it, as has been suggested by Mukhopadhyay et al. [Sol, the human equivalent of the yeast Fet3 enzyme 181-4331 which appears to participate in the acquisition o f iron by cells in yeast? In terms of the copper centers, is there a need for three mononuclear coppers or does one or more of them result from the fact the enzyme is in a process of evolution? This case has been made for the domain 2 copper

806

LINDLEY

[521, but the binding studies undertaken in See. 2.4.5 suggest roles for both the domain 4 and 6 mononuclear coppers. There are many other aspects of ceidoplasmin and the other members of the family that require clarification.

There are many colleagues who deserve acknowledgment for contributing to this chapter, but I would particularly like to thank Albrecht Messerschmidt, Elinor Adman, Michael Murphy, Gideon Davies, Slava and Irina Zaitsev, and Graeme Card. I also thank my colleagues at the GLRC Daresbury Laboratory and the UK Research Councils (MRC, BBSRC, and EPSRC) who sponsored the Joint Structural Biology Programme. My colleagues at the ~ S have ~ also F been very tolerant and given me the opportunity to spend time in the preparation of this work. Many of the figures have been drawn with the SETOR suite of programs 1921 using atomic coordinates extracted from the Brookhaven Protein Data base [991 at the web site “http:/l www.pdb.bnl.gov/”. This database was transferred to the Research Collaboratory for ioinformatics (RCSB) at “http://wm.rcsb.org/” on July 1, 1999.

Paracoccus denitrificans amicyanin Akaligenes faecalis nitrite reductase ascorbate oxidase zucchini ascorbate oxidase Paracoccus denitrificans cytochrome oxidase azide am Alcaligenes denitrificans azuriii Pseudomoms aeruginosa azurin azu CP ceruloplasmin cyw Escherichia coli quinol oxidase dopa ~,4-dihydrox~henylalanine EDTA ~thylenediamine-~, N,N’,“-tetraacetate EPR electron paramagnetic resonance F factor hCP human ceruloplasmin kcw human c e ~ ~ o p l a s i ncode ~~-~~~ Lac laccase d-lysergic acid diethylamide nitrite reduckase occ bovine cytochrome c oxidase E n ~ e ~ o ~prolifera o r ~ ~ plastocyanin ~ a pcy

aan afn A0 aoz arw


807

Protein Database poplar plastocyanin Chlamydomonas reinhardtii plastocyanin Meth,ylobacterium extoripens pseudoazurin p-phenylenediamine Alcaligen,es faecalis pseudoazurin root mean square Thiobucillus ferroxidans rusticyanin ultraviolet

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80

LINDLEY

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~ULTI-C~P OXIDASES ~E~

43.

44. 45.

46. 47. 48. 49. 50. 51. 52.

53. 54. 55. 56. 57.

58. 59. 60.

809

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810

LINDLEY

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MULTI-COPPER OX1DASES

81 1

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'Biophysics Section, University of Rosario, Suipacha 531, S2002LRK Rosario, Argentina 2LANAIS RMN-F (CONICET - University of Buenos fires), Junin 956, C1113 AAD Buenos Aires, Argentina

1. INTRODUCTION

814

2. PROTEINS WITH KNOWN STRUCTURE 2.1. The Cupredoxin Fold 2.2. Blue Copper Proteins 2.2.1. Plastocyanin 2.2.2. Amicyanin 2.2.3. Pseudoazurin 2.2.4. Azurin 2.2.5. Rusticyanin 2.2.6. Phytocyanins 2.2.6.1.Plantacyanins 2.2.6.2. Stellacyanins 2.3. Blue Oxidases 2.3.1. Laccase 2.3.2. Ascorbate Oxidase 2.3.3. Ceruloplasrnin 2.4. Nitrite Reductase 2.5. The Binuclear CuASite

816 816 818 820 824 825 825 826 827 828 828 830 830 830 831 831 832

3. PROTEINS WITH UNKNOWN STRUCTURE 3.1. Blue Copper Proteins 3.1.1. Auracyanins

833 833 833

813

814

VILA AND FERNAND~Z

3.1.2. Halocyanin 3.I.3. Uclacyanins 3.2. Blue Oxidases 3.3. NzO Reductase

834 834 834 835

4. STRUCTURE-FUNCTIONRELATIONSHIPS 4.1. Description of the Coordination Sphere of the Metal Ions 4.2. Spectroscopic Studies 4.3. Mutagenesis Studies 4.3.1. The Cysteine Ligand 4.3.2. The Histidine Ligands 4.3.3. The Axial Ligand 4.3.4. The Hydrogen Bond Network 4.4. Redox Potentials 4.5. Electron Transfer Mechanisms

835 835 836 838 838 838 839 839 839 840

5. P E R S ~ E C T ~ E S

84 1

1. I

NOTES ADDED IN PROOF

842

AC~O~EDGMENTS

842

~BRE~ATI~ AND N SDEFINITIONS

842

~ E ~ ~ R ~ N C ~ S

843

UCTION

Copper is the second most abundant transition metal ion in nature, second to iron [l], Copper, like iron, is a redox-active metal, and although essential, it is potentially toxic i2l. The free copper concentration is controlled because Cu(I1) ions may enhance cellular damage induced by reactive oxygen intermediates. In this way, its accumulation in living cells requires active transport mechanisms through copper compiexation, which takes place through metallothioneins in animals [31 and by phytochelatins in plants (41, The action of selective copper metallochaperones may regulate the free Cu(lI) concentration in yeast to approximately one copper atom per cell 151. Copper in living systems exists in two stable redox forms: Cu(1) and Cu(ITf.This redox couple is adaptable to the biological redox potential range. In terms of the hard and soft acid-base theory proposed by Pearson, Cu(1) is a soft cation and it better binds so€t sulfur ligands, whereas Cu(1I) is an intermediate cation, and coordination to nitrogen imidazoles i s preferred [6,7]. The coordination sphere of electron transfer copper sites are thus largely constituted by ligands with nitrogen (His) and sulfur (Cys, Met) donor atoms. The oxidized form is a strong Lewis acid, thus allowing the metal ion to catalyze hydrolytic reactions, as Mn(1I) and Zn(I1) ions do (see Chapters 8 and 19). However, it is rarely found in this role. Rather, it is used as a one-electron carrier, often in reactions involving oxygen activation.

815

Cl~sificationof Copper

Coordination geometry

EPR spectrum

Biological function Examples

eta1 Sites in Proteins

~ononuclear ~ononuclear Distorted trigonal Square planar Distorted t e t r ~ e d r ~ istorted tetragonal N,S,O N,O Strong band (- 600 nm) eak band (- 450nm) Axial or rhomb~c A, 5 90 x lO-*em-l (4lines) Electron transfer Blue copper proteins Cu-nitrite reductases Blue oxidases

Axial

A,/ L 140 x IO-*crn-' (4lines) Catalysis Non-blue oxidases Nitrite reductase Cu,Zn-superoxide dismutase

"The coordination geometry refers to each of the two copper ions.

inuclear inuclear Trigonal ~ l a n ~ a istorted tetrahedrala Disto~tedtrigonalu N Strong band (-- 330 nm) 530 am) ~e~ band (800am) A,, 5 30 1 0 - ~ Nondetectable (7 lines) Catalysis Oxygen transport Hernocyanin Tyrosinase Blue oxidases

Electron transfer Cytochrome c oxidase N 2 0 reductase

816

VILA AND ~ ~ R N A N D E Z

any iron-containing proteins have equivalent copper-dependentanalogues as a consequence of the differential use of metal ions by nature according to the atmospheric oxidizing level E81. Redox potentials of hexaaquo Cu(II)/Cu(I) are higher than As a consequence, iron was used as an electron those of the ~ e ( I I I ) ~ e ( Icouple. I) transfer metal ion in early stages at low oxygen concentrations, and copper use came later. In neutral aqueous solution and in seawater, Cu(I1) salts are more soluble than Cu(I) ones. In contrast, the oxidized form is less soluble for the ~ e ( I I I ) ~ e ( I I ) couple. Higher O2 levels thus favored iron precipitation and copper mobilization. Due to its later appearance and bioavailability in evolution, copper is often found in the extracellular space whereas iron occurs mainly within cells. Copper sites in proteins may be classified as follows (Table 1): Type 1- blue (mononuclear electron transfer centers, with characteristic spectroscopic features and high redox potentials), Type 2 (mononuclear catalytic centers) lly Type 3 (binuclear centers, with two Cu(1I) ions a n t i f e ~ o ~ a ~ e t i c acoupled); CUA (binuclear electron transfer centers, with characteristic spectroscopic features) The first three categories were defined by Malkin and ~ a l m s t r o maccording to their spectroscopic features 191. Other existent copper sites may be considered as combinations of the four already mentioned, such as the trinuclear copper sites, which consist of type 2 and type 3 centers [lo1 (see Chapter 16). This chapter deals with copper centers in proteins which exclusively behave as electron transfer functional units, i.e., type 1 (blue) and CUA(purple) sites. Type 1centers are present in the so-called blue copper proteins ( bacteria and plants, as well as in multi-copper enzymes, present in different organisms 211-141. The latter also posscss a catalytic center, in which one or more non-blue copper ions are involved. The binuclear purple CUAcenter is the electron entry port of terminal oxidases, and it has not been found in single-domain soluble proteins. The copper ligands in blue and purple copper sites are exclusively amino acid residues. This fact argues in favor of the adaptability of the Cu(II)/Cu(I)redox couple, which seemingly does not need a prosthetic group (as iron does).

WITH KNOWN S T ~ U ~ T ~ ~

BCPs have also been termed cupredoxins, by analogy to the iron-containing, electron transfer proteins called ferredoxins (described in Chapter 10) 13.11. They are single domain proteins exhibiting a p barrel fold defined by two f! sheets that may contain from 6 to 13 strands following a key Greek motif (Fig. 1) 111,151. The cupredoxin fold differs from the typical Greek key p barrel in that the first and third p strands are arranged in a parallel fashion to their respective p sheets. In plastocyanin, one sheet is

CTRON-TRANSFE~PROTEINS

817

defined by strands 2, 1,3 and 6; and the second one by strands 2’, 8, 7 and 4 (Fig. 1). As a result, there is a kink in the second strand such that its first half (2) i s part of sheet A while the second half (2’) is part of sheet €3. A variable helical content is present in proteins from different sources (see below for details). The copper binding site is located in the “northern” region of the molecule, following a description coined by Guss and Freeman [161. This fold has been recycled by evolution to yield more complex enzymes. Electron transfer copper centers present in enzymes are located in cupredoxin-like domains [15,171. This is the case in multi-copper blue oxidases, which consist of three (ascorbate oxidase and laccase) or six (cemloplasmin) cupredoxin-like domains (see Chapter 16). Copper nitrite reductase (NiR) is a trimer with two cupredoxin domains each [18]. The CuA-containing subunit of respiratory terminal oxidases (where the electron entry port is located) displays a cupredoxin fold [19,2Ol.

lil

et

i

FIG. 1. Ribbon diagram of poplar plastocyanin, from I361 (PDB: 1PLC). The copper ion and the copper ligands are indicated in dark. Tyr83, surrounded by the acidic patch is also indicated in dark. Numbering corresponds to the strands. The pictwre was prepared with R Sayle, Glaxo).

818

VILA AN

NAND

BCPs consist of a single polypeptide chain of 90-150 residues that bind a single type 1 copper center (Fig. 2). Some BCPs transfer electrons between c o n t i ~ o u donor s and acceptor molecules, whereas others ferry electrons over long distances. present in the three kingdoms of life. TKis suggests the existence of an ancestor prior to the divergence of archea, bacteria and eukarya, i,e., at least 1.5 billion years previous to the beginning of aerobic life [Zl]. Instead, blue oxidases are found higher e u k ~ o t e s . CPs may be grouped into different families, accordingto phylogenetic analyses, initi~lyproposed by d6n 122,231 (the evolutionary relationship between blue oxidases is discussed in Chapter 16): 1. The plastocyanin family (plastocyanin,amicyanin, pseudoazurin, and halocyanin); . Auracyanin

and uclacyanin) 5. The phytocyanin family (plantacyanin, stella~yanin9 The elucidation of the structures of the metal sites of blue copper proteins was pursued in the 1970s through spectroscopic studies by Gray, S 0 1 o ~ and o ~ co-workers ~ , who predicted the presence of a Cys ligand and described the metal site as a d tetrahedron. These predictions were confirmed by the crystal structure of oxidized plastocyanin, solved by Freeman and co-workers in 1978 [26]. The copper ion sits in a depression close to the protein surface, without being solvent~exposed,thus preventing the adventitious binding of external ligands and unwanted side reactions (Fig. 1).The metal coordination geometry in most type 1 sites is a flattened tetrahedron (Fig. 3A) [10,11]. Three copper ligands are strictly conserved in all redoxins: one Cys and two His residues, identified as the equatorial ligands. The residues bind the copper ions through their imidazole N6 atoms. The most common axial ligand is a Met res , but it may be replaced by a Gln in ) bond exhibits variable lengths in stellacyanins (Fig. 3C) [27]. The copper-Sj;s dif€erent proteins (Fig. 3A-B), and is generally weaker than any of the bonds with the equatorial ligands. Three out of the four copper ligands (Gys, His, axial) are present in a loop linking two p strands, that exhibi ariable length and composition. This loop has the general sequence C y s - ~ ~ - H i s - ~(or ~ Gln) (Table 2, Fig. 2), with the Cys and Met residues located in the transition from the p sandwich to the loop ructure and length of this loop influences the metal site structure from this loop is solvent-exposed and surrounded by a hydrophobic patch. The other His residue is located inside the protein, in an adjacent p strand, upstream in the sequence (Fig. 2). ype 1 copper ions display a trigonal geometry in some proteins. A fifth, weakly interacting axial Gly ligand, located opposite the Met, is present in azurins, giving rise

COPPER IN ELECTRON-TRANSFER PROTEINS

819

Pcl Pc2 WC AuBl AZ

Rc CBP St T J m e Ucel

Pel Po2

h i PsAz Hc AuBl Az

RC CBP St Urn63

Uccl

FIG. 2. Sequence alignment of representative blue copper proteins: Pel (poplar plastocyanin), Pc2 (Anubuenu uariubilis plastocyanin), h i (Paracoccus denitrificuns amicyaninj, psAz (AZculigenesfuecalis pseudoazurin), Hc ( ~ u t r o n o b u c t e r ipharaonis u~ halocyanin), AuBl (aurallus rusticyanin), CBP (cucumber cyanin B1 from Ghloroflexus auruntiacus),Az (Pseudomonus ueruginosa azurin), Izc ( ~ h ~ o b ~ i ferrooxiduns basic protein), St ( c ~ c u r n ~stellacyanin), er Urne ( ~ ~ e c y aTJcel ~ n ~(Arubidopsis , thuliana u ~ l a 1). c Residue ~ ~ n~ ~ b e r corresponds ~ g to poplar plastocyanin. p strands are indicated by underlining for those proteins whose three-dimensional structures are known. Cys residues marked in bold are involved in disulfide brigdes. The position of the copper ligands is indicated with an asterisk.

820

VILA AND F ~ R ~ A N D ~

Met8

is87

His84

G

is117

is94 ly45 c

His457

FIG. 3. Schematic pictures of the metal sites of (A) poplar plastocyanin, from [36] (PDB: 1PLC); (B) cucumber plantacyanin (CBP), from [ 1191 (PDB: BCBP); (C) cucumber stellacyanin, from [27] (PDB: ISER); (D) Pseudomonm aeruginosa azurin, from [lo61 (PDB: 4MU); (El Coprinus cinereus lacease, from [31] (PDB: 1A65).

to a trigonal bipyramidal polyhedron (Fig. 3D) [30]. Blue siLes in multi-copper enzymes may lack any axial ligand [31]. This situation gives rise to a trigonal planar geometry, with only three copper ligands (Fig. 3E). The hydrogen bond network in the metal site is largely conserved. In particular, the peptide NH of an Asn residue next to the upstream His ligand is hydrogen-bonded to the bound Cys sulfur atom in most BCPs (Table 2). This hydrogen bond is relevant for maintaining the geometry of the metal site. In some BCPs, a second hydrogen bond is formed between the S,(Cys) and a backbone NH located two residues ahead of the Cys in the sequence (Table 2). This residue is substituted by a Pro in other BCPs, thus lacking this second hydrogen bond. 2.2.1. Plustocyanin

Plastocyhns (Pc’s) are present in photosynthetic bacteria, algae, and plant chloroplasts. The name was introduced by Katoh and Takamiya in 1961when isolating this

821

COPPER IN ~ L ~ ~ T R O ~ - T R A N SPROTEINS FER

TABLE 2

Ligand Loop Topology in Blue Copper Proteins and Blue Oxidases Schematic sequence of the C-terminal ligand loop C+P-H---+++M hicyanin C + P - H w M Plastocyanin Pseudoazurin Halocyanin C+ Sy . . . . . . H N Azurin

_. -

(NH. . .Sy) Cys bonds

Extraloopa

Within loopb

Asn54

-d

Asn38 Asn41

___

-

-d

Asn47

Phell4

C

c

Sy . . . . . .HN Plantacyanin Stellacyanin Uclacyanin Rusticyanin

d

d _ I

c

Asn40 Asn47

PheEIl Val91

-c

-c

Ser86

Ille136

Laccase Ascorbate oxidase

-e __

Ile454 He509

Nitrite reductase

Asn96

-d

M-H-C e

“N-H . * .S hydrogen bond from a backbone amide group of a residue next to the upstream His ligand. bN-R. . . S hydrogen bond from a backbone a i d e group of a residue located in the (n + 2) position with respect to the Cys ligand in the binding loop. This residue is highlighted as in the schematic sequence. ‘No structure available. “Hydrogen bonding interaction not possible due to the presence of a proline residue in (n + 2) position with respect to the Cys ligand. eHydrogen bonding interaction not possible due to the presence of a proline residue in (n + 1) position with respect to the upstream His ligand.

VILA AND F ~ R N A N D ~ Z

822

blue protein from spinach chloroplasts [321. Pc is responsible for ferrying electrons from cytochrome f (in the cytochrome b s / f complex) to the clilorophyll-containing pigment P700’ (belonging to photosystem I) in higher plants, cyanobacteria and algae [33l. More primitive organisms used cytochrome c6 instead of Pc, and when copper became available, Pc increasingly replaced cytochrome c6 [33,341. The cyanobacterium Anabaena is capable of synthesizing either cytochrome c6 or plastocyanin depending on the copper concentration in the powth medium [351. The structure o f Cu(I1) poplar (Populus nigra var. italica) plastocyanin, reported by Freeman arid co-workers in 1978 l261, was the first structure of a blue copper protein, subsequently refined to 1.6 and 2.33 A (Fig. 1) [16,361. X-ray and solution NMR structures of several Pc’s from higher plants, algae, and cyanobacteria structures are only available for Cu(1) plastocyanins [42are available 137-421. “I!& 441, because the paramagnetism of the oxidized form has hitherto impeded the assignment of the NMR signals near the metal center [45,2841. Plastocyanins from higher plants are acidic proteins due to the presence of a characteristic acidic patch not conserved in Pc’s from cyanobacteria [43,44,461. This recognition site could have developed later in evolution 1471. The copper site geometry is a flattened tetrahedron in which the three equator(Fig. 3A, ial ligands Cys84, His37, and His87 are strongly bound (rcu-x < 2.18 Table 3). The Ss atom of Met92 is found 2.82 from the Cu(I1) ion in poplar Pc [361. The conserved Phel4 residue makes contact with Met92 and may help in fixing the position of the axial ligand [161. The Cu(II)-S8(Met)distance is variable in Pc’s from different sources, ranging from 2.69 h in the U. pertusa Pc 1481 to 2.94 A in the protein from D. crasszrhzzorna 1381. Asn38 (Table 2) is hydrogen-bonded to the Cys sulfur thiolate [161. Pc’s display redox potentials from 340 to 370 mV, which are altered at low pH. As a consequence, the protein is almost redox-inactive at pH 4.5 [49,501. The metal site structures of Cu(I1) Pc are identical at pH 6.0 and 4.2. Upon reduction at low pH, His87 is protonated and detaches from the copper ion, which then becomes tricoordinated (Fig. 4) [51]. Two protonated forms are in equilibrium under these conditions [50,511.13.crassirhizorna Pc is an exception, since a x-7~stacking interaction between the “northern” His and a Phe residue inhibits the rotation of the imidazole ring, and thus the structure of the reduced form does not change r381 and the redox potential is not pH-dependent L5.21. Two possible binding sites were suggested from the structure of poplar Pc [16]: a flat hydrophobic (adjacent) patch located at the “ n ~ r t h e r n end ’ ~ of the molecule surrounding Wis87, and a (remote) acidic patch (D42E43D44 and E59E60D61) than encircles the conserved Tyr83, which protrudes on the “east side” of the protein surface, and is adjacent to the bound Cys in the sequence (Fig. 1).The reactivity of both sites has been probed with small metal complexes (121, and through mutagenesis 153,541, and 8 still remains controversial. The remote acidic patch was identified as the binding and electron transfer electron entry site when Pc interacts with cyt bG/f 153,55,561. However, a recent -based model of the complex between Cd(II)-subst~tutedPc and cytochrome f

A

A)

823

COPPER IN E L ~ C T R O N - T ~ A N S FPROTEINS ~R

TABLE 3 Bond Lengths of Type 1 Copper Sites Protein (source) Plastocymin (poplar)

Cu(II), pH 6.0 CulI), pH 7.0 Cu(I), pH 3.8 Amicyanin ( A delenttr 1fr~csn.7)

Cu(IIl, pK 7.0 &(I), pH 4.4 Pseudoazmin ( A . fuecolrs) Cu(II), pH 6.8 Cu(I), pH 7.8 CulI), p l l 4.4 Axurin ( A dcnrtrEficum) CU(II), pN 5.0

Rusticyanin (T ferro .cidms/

Cu(IIj, pH 4.6 &(I), pH 4.6 Plantacyanin (curumber)

C u m) Stellacyanin (cucumber) Cu(II), pM 7.0 Laccase (C cinrt e1r.s) CU(I1) Axorbate oxidase (zucchini) Cu(IT), pT-1 6.0 Cu(I,, pH 6.0 Nitrite reduetaqe ( A . fuecali.;} Cu(IIj, pH 4.0 Cu(I), pH 4.0

(A)

NS His

SyCys

NSHis

A i d ligand

His37

Cys84

Hi987

Met92

1.91 2.12

2.11

2.07 2.17 2.13

2.06 2.39 3.15

0.36 0.47 0.70

1PLC 5PCY 6PCY

36 51 61

His53

cys92

His95

1.95 1.91

2.11 2.09

2.03 5.45

0.30

IAAC lBXh

69 73

His40 2.16 2.16

Cys78

His81

2.12 2.29 3.09 His117 2.00

0.43 0.37 0.77

lPAZ ipzA

2.19 His46 2.08

2.16 2.17 2.16 CysllZ 2.14

2.82 2.87 2.52 Met98 2.90 2.90 Met86 2.76 2.91 2.42 Met121-Cly4§ 3.11 (SS Met) 3.131 (0 Gly)

lPZB

82 a7 87

0.13

2AZA

30

2.13

2.26

2.05

0.14

-

105

Hi985 2.04 2.22

Cys138

His143

2.26 2.26 cys79 2.16 Cys89 2.18 His452 2.27

1.89 1.95 His84 1.95 His94 2.04 His457 1.87 His512 2.08 2.08 His145 1.98 2.08

3.23 (S6 Met) 3.23 (0Gly) Met148 2.88 2.74

His39 1.93 His44 1.96 His396 1.91 His445 2.11 2.12 His95 2.06 2.07

cys507

2.08 2.14 Cys136 2.08 2.19

CU"S

-

PDB 1D Ref.

0.33 -

lRCY 112 1A3Z 115

0.39

2CBP 119

0.33

lJER

27

1A65

31

Met89

2.61 GlnW

2.21

Met517

2.87 2.95 Met150 2.64 2.58

-

-

lAOZ 146 lhS0 147

0.48 0.64

lAS7 168 lAQ8 168

1571 has suggested that electron transfer between Pc and cytochrome f may proceed through His87 [285]. In addition, His87 is the electron exit port through which Pc gives an electron to photosystem I [58,591, even if the acidic patch is also essential in controlling binding to photosystem I [601. Binding to photosystem I increases the copper redox potential by 45 mV, presumably by inducing a conformational change

VILA AND F ~ ~ N ~ N D

824

Cu(l) ~ l ~ s ~ o c y a npi n , FIG. 4. Schematic picture of the active site conformatianal change in plastocymin at low pH. The structures were taken from refs.[36] (PDB: 1PLC) and [5l] (PDB: GPCY),

in the metal site 1581. In cyanobacteria lacking the acidic patch (Fig. 21, there i s no complex formation 1411.

2.2.2. Amicyanin In 1981, Tobari and Harada isolated a BCP from ~ e t ~ . y ~ o ~ ~ extorquens c t e r i u ~M I , that they called amicyanin (Ami) 1611. Related proteins were later characterized from Paraccocus denitrificans [621 and Paraccocus uersutus 1631. Their biological task is to accept one electron from methylamine dehydrogenase (MADH, which catalyzes the oxidative deamination of primary arnines) [62,641 and to transfer it to cytochrorne ~ 5 5 This ~ . chain is activated in methylotrophic bacteria when methylamine is the only source of carbon and energy, thus inducing the synthesis of W H and amicyanin 1621, Specific inactivation of the h i gene results in complete loss of the ability of P. denitrificans to grow on methylamine 1651. h iis not synthesized by cells grown on methanol or succinate (see Sec. 2.2.3) 1661. Amicyanins are 20 residues longer than Pc's due to the presence of an extension of the p barrel at the NH2 terminus which folds into an additional j3 strand connected by hydrogen bonds to strand 6 L67-701. This strand is interrupted by a p bulge. The protein fold presents a striking similarity with Pc, with minor d3erences confined t o the PIT-terminal extension, and a shorter Cys-Met loop (Table 2). h i lacks the acidic patch present in higher plant plastocyanins and possesses a larger hydrophobic patch surrounding the exposed His ligand. The metal site is a distorted tetrahedron, similar to that of Pc, with a longer (2.90 1691. An intramolecular hydrogen bond from the NCu(~I)-S~(~ distance et) terminal His ligand to a Ghi side chain further stabilizes the copper site. The structure of apoamicyanin from P. denitrificans shows few changes compared with the holoprotein 1671. The loop comprising the three ligands in amicyanin is the shortest among BCPs (Table 2).

A)

IN ~ L ~ ~ ~ R O N - T R A NPROTEINS SF~R

825

The redox potential of amicyanin is pH-dependent, with a pKa of 7.5. This is consistent with the fact that in reduced amicyanin at pH 4.4, His95 detaches from the Cu(1) ion, is protonated, and rotates 180” around the Cp-C, bond [71-733. h i c y a n i n forms a binary complex with MADH, decreasing its redox potential by 73 mV [741. h i c y a n i n docks to MADH through the hydrophobic patch surrounds supposed to mediate electron transfer ing the northern His ligand complex does not change in the 6.5-8.5 r751. The redox potential of pH range, apparently due to the absence of the His flip occurring in free Ami 1731. The structure of the ternary complex formed by W H , apoamicyanin, and cytochrome e551 reveals that the association between H and Ami is very similar to that observed in the binary complex [76]. h i is in contact with both M A ~ Hand the cytochrome, but there is no contact between the cytochrome and MADE 2.2.3. Pseudoazurin

Pseudoazurin (psAz) is a soluble periplasmic redox protein containing approximately 120 residues found in denitrifying bacteria and methylotrophs. It was first isolated from Alcaligenes faecalis by Beppu and co-workers in 1981 171,781, and later named pseudoazurin by Ambler and Tobari [66]. PsAz is synthesized in methylotrophs instead of amicyanin at high copper concentration or when cells are grown in methanol [66].In denitrifying bacteria, pseudoazurins donate an electron to nitrite reductase under anaerobic conditions C79,801. Pseudoazurins have an extra C-terminal fragment with respect to plastocyanin (Fig. 2) that forms two helices packed to an eight-stranded p barrel similar to the plastocyanin fold I811. Three-dimensional structures are available for pseudoazurins in their native oxidized, reduced and metal free f o m s [81-881, as well as for mutant proteins [89,901. The copper ion i s coordinated to His40, Cys78, His81, and Met86 in a distorted [82] than the tetrahedral arrangement. The CU-S~ (Met861 bond is shorter (2.76 corresponding bond in plastocyanin from poplar (2.82A), resulting in the copper atom lying further out of the plane (0.43 A) of the three equatorial ligand (Table 3 ) . An active site protonation involving His81 occurs at low pH, in the reduced protein, as in Pc and Ami 129,871. A large number of conserved lysine residues surround the hydrop~obicpatch where the exposed His ligand protrudes from the protein surface. Mutation of these Lys residues impairs pseudoazurin binding to nitrite reductase (911. This region is thought to work as docking area with a conserved acidic patch on nitrite reductase [921, and a model for an electron transfer complex between pseudoazurin and nitrite reductase has been proposed [86,931.

A)

2.2.4. Azurin

Azurin (Az)was first isolated from P. aeruginosa in 1958 by Horio E941. Az is present in the periplasmic space of several Gram-negative bacteria, such as pseudomonads,

826

VILA AND FERNANDEZ

methylotrophs, and Alcaligmes strains. In vivo studies have implicated Az in electron transfer processes related to the bacterial response to redox stress in Pseudomonas E951. Other roles, such as electron donor to NADH in Methylomonas [961; or to alcohol dehydrogenase in P. putzda, have been also suggested [97]. Yamanaka et al. noticed early on that the characteristic blue color of azurin was lost by copper depletion and the apoprotein could be reconstituted upon addition of Cu(I1) 1981. At the same time, Malmstroni, Vanngsrd et al. showed that the distinctive EPR spectrum was lost upon protein denaturation and concluded that the protein fold determined the metal site structure [991. Since then, azurin has been the most exploited BCP for the generation of point mutants, metal substitution, spectroscopic and crystallographic studies [loo\. The structure of Az was first solved by Adman et a1. at 2.7 A resolution [loll and Baker achieved atomic resolution in 1988 f301. Azurin possesses a 30-residue insertion between strands 5 and 6 of the barrel, forming a helix and a “back flap”, the most distinctive topological element of azurins. Azurins contain a disulfide bridge in the southern part of the molecule between Cys3 and Cys26 that connects the ends of an antiparallel loop. Disruption of this bridge by mutation of the Cys to Ser destabilizes the protein structure and impairs the metal binding ability rl021. The copper ion in azurin adopts a trigonal planar bipyramidal geometry. Two axial ligands are arranged in a trans fashion: Metl21, with a long Cu(II)-S8distance (3.13 A)>and 61~4.5,with its main chain carbonyl oxygen at 3.11 A from the copper ion) (Fig. 3D) [30].The Cu(I1)-Glydistance i s too long for defining a covalent bond, and this interaction may he considered as weakly ionic in bonding terms 11031. The distances to the Met and Gly ligands vary in proteins from different sources 196,1043. The crystal stixctures of azurin at different pM values in the oxidized and reduced forms reveal minor changes in the metal site structure (Table 3) 1105,1061.Apoazurin instead exhibits a higher flexibility of the His ligands upon metal removal [ 1071. The thiolate sulfur of Cys112 is hydrogen-bonded to the main chain N Phell4 (Table 2) [30J. The latter hydrogen bond is conserved in all Az’s except in Azis02 from Methylomonas strain J [961. 2.2.5. Ruslicyanin

Cobley and Haddock suggested in 1975 that a BCP, rusticyanin (Rc), was part of the respiratory chain of Thiobacillus ferrooxidan,s [1081, a Gram-negative eubacterium that uses air and Fe(l1) as the sole energy source, and it grows optimally at pH 2.0. Rc may constitute up to 5% of the total soluble protein in the cell. A high molecular weight cytochrome transfers electrons from Fe(1I) to rusticyanin, which passes them to a cytochrome c4, the primary donor of a terminal oxidase fl091. Rustieyanin has a redox potential of 680 mV at pH 2, the highest value observed in single-domain type 1 copper proteins LllO]. The protein owes its name to its oxidizing power, which is sufficient to oxidize Fe(1I) 11111. The stability at low pN values is another outstanding feature of Re.

COPPER IN E L E ~ T R ~ ~ - T R A N S PROTEINS FE~

827

Rusticyanin is the largest BCP known, with 150 residues (Figs. 1and 5). The sandwich i s composed of a six- and a seven-stranded p sheet [112,113]. A long helix (residues 11-20), unique to the Rc structure, is packed against the six-membered sheet by hydrophobic interactions (Fig. 5). The structure reveals a higher degree of intersheet connectivity than in other cupredoxins, providing a rationale for the protein stability at low p pts a distorted tetrahedral geometry, with an axial The copper ion at a Cu(II)-Sa(Met)distance of 2.88 A, Small differences in the metal site are observed upon reduction, consistent with a constrained copper center (Table 3 ) [114,1151. The Sy(Cys) atom is hydrogen-bonded to a backbone NH from Ser86, which replaces the most common Asn (see Fig. 2 and Table 2). Its side chain OH forms a tight hydrogen bond with Gln139 (next to the bound Cys), contributing to the rigidity of the metal site 11161. Rc forms a tight complex with the soluble diheme cytochrome c4 [1171. Complex formation decreases the copper redox potential by 100 mV and alters the metal coordination, probably by deprotonation of the exposed His143. This potential drop would be needed to favor the electron flow from Rc to cytochrome c4 [117]. 2.2.6. Pktytocyunins

Phytocyanins are plant proteins from nonphotosynthetic tissues. This family was defined upon tho phylogenetic studies by Ryden r221, albeit their biological function

FIG. 5. Ribbon diagram of T. ferruxidans rusticyanin, from lllZ1 (PDB: IRCY). The copper ion and the copper ligands are indicated in dark. Numbering corresponds to the 13 strands. The picture was prepared with RavMol 2.5 (R. Sayle, Glaxo).

828

VILA AND F ~ R ~ A N D E Z

is unknown. In contrast to plastocyanins, which are located in chloroplasts, phytocyanins are targeted to the endoplasmic reticulum through classical signal peptides [1181. Their crystal structures have revealed common features, such as the open structure or the fi barrel, arising from a twist in the p sheets, a higher solvent accessibility of both His ligands, and the presence of an invariant disulfide bridge [27,1191. Notwithstan~ng,they may be classified into three distinct types, according to Nersissian et al.:plantacyanins, stellacyanins, and uclacyanins [1181, based on the domain organization of the precursor and mature proteins, the glycosylation state, the identity of the axial copper ligand, and the spectroscopic properties of the proteins.

- Planlacyanins ~ l a n t a ~ a n i n(formerly s termed cusacyanins) are basic, nonglycosylated, singledomain BCPs. The most studied one is cucumber plantacyanin, best known as cucumber basic protein (CBP) [120,1211. Plantacyanins have also been isolated from spinach, beet, and asparagus 11221. CBP exhibits an eight fi stranded cupredoxin fold, in which the second sheet is substantially twisted (Fig. 6) r1191. The two sheets form a sandwich, but they lack the continuity to form a barrel. A small number of hydrogen bonds provides interstrand connectivities, and a short helix (residues 45-52) is packed against the second p sheet. The end of the helix is connected by a disulfide bridge (Cys52-S-S-Cys85) to the adjacent large loop between strands 7 and 8, where three of the copper ligands are located. The disulfide bond is feasible due to the twist in p sheet 2 and may be fundamental in stabilizing the protein tertiary structure (Fig. 6). The metal site in oxidized CBP adopts a distorted tetrahedral geometry. The C u ( I I ) - S ~ ( ~ edistance t) is the shortest observed in a blue copper site (2.61 A), whereas the Cu-S,(Cys) bond is 0.1 A longer (Fig. 3B) llP9,2861. The imidazole rings of both His ligands are solvent-exposed, this being a distinctive feature of phytocyanins.

2.2.6.1

2.2.6.2. Stellacyanins Stellacyanins (St) are unique in that they are the only group of BCPs with a Gln axial ligand instead of the ubiquitous Met [27]. They are glycoproteins with variable carbohydrate content. This family includes the original protein from the lacquer tree Rhus uernicifera named stellacyanin (Ru St) by Peisach et al. [123], St from cucumber /124], mavicyanin from green zucchini 1125,1261, and umecyanin from horseradish roots [127,12$], Ru St, mavicyanin, and umecyanin have been isolated as singledomain proteins. Instead, cucumber St in its mature form is a chimeric protein that contains a plantacyanin-like copper-binding domain and a C-terminal extension rich in hydroxyproline and serine residues, typical of sequences found in cell wall structural glyeoproteins 11241. The presence of such exkensions in the other three stellacyanins cannot be discarded because their genes have not been isolated. These evidences, together with the finding of stress-inducible genes in Arabidopsis thaliana 1129I and loblolly pine 11301, led Nersissian and co-workers to propose that stellacyanins may be cell w d l proteins involved in plmt defense systems r1181.

829

COPPER IN E L E ~ T R ~ N - T ~ A N SPROTEINS FE~

FIG. 6. Xibbon diagram of cucumber plantacyanin, from 11191 (PDB: ZCBP). The copper ion and the copper ligmds are indicated in dark. Numbering corresponds to the p strands. The picture was prepared with RasMol 2.5 (R. Sayle, Glaxo).

Stellacyanins display lower redox potentials (184-280 mV) than other 1118,1311,bearing a stronger reducing power as the Cu(1) forms. Ru St displays the lowest redox potential among BCPs in a wide pH range. RZIStellacyanin was characterized early [123,1321, but its structure could not be solved until 1996 due to its high carbohydrate content f- 40%), which thwarted all crystallization attempts. Modeling 11331, mutagenesis L1341, and NMR studies [1351 supported the idea of a Gln axid ligand. The crystal structure of the non-glycosylated copper binding domain of cucumber stellacyanin was finally solved and refined to 1.6 resolution 1271. The structure resembles the fold exhibited by cucumber plantacyanin. The metal site is a distorted tetrahedron, with an axial Gln ligand at 2.2 from the Cu(T1) [271 ion coordinated through its E-Q atom (Fig. 3C) [ 1361. As a result of the stronger axial interaction, the copper ion i s displaced 0.33 from the equatorial plane. The two His residues are solvent accessible [1371 and as a result the Cu(1I) ion sits only 3 A from the protein surface [27]. The metal site structure of Cu(U stellacyanin experiences nonnegligible changes upon reduction 1118,1381. This may be atlributabfe to the unusual flexibility of its metal site [1391. No structures are available for any phytocyanin in the reduced form yet,.

A

A

830

VILA AND FERNANDEZ

Multi-copper blue oxidases are enzymes widely spread in bacteria, ftingi, plants, and animals that catalyze the four-electron reduction of dioxygen to water through four sequential single-electron oxidation events of a substrate [14,1401. They contain at least one blue copper center and a trinuclear center composed by a type 2 and a type 3 copper site, and all the hitherto characterized enzymes are glycosylated. The three currently well-defined members of this family are laccase (Lc), ascorbate oxidase (AQ), and ceruloplasmin (Cp). The occurrence, biological roles, structural and mechanistic features of blue oxidases are described in detail in Chapter 16. In the following section we will describe the type 1 sites present in these enzymes. Their functional role is the sequential uptake and transfer of electrons from substrate molecules to the catalytic trimclear unit. Blue oxidases share the same residue spacing in the C-terminal ligand loop (Table 2), even when there is no axial ligand 1171. 2.3.1. Laccase

Laccase (pdipheno1:dioxygen oxidoreductase, EC 1.10.3.2), involved in lignin degradation 11411, is the simplest blue oxidase known. The first report of this enzyme isolated from the exudates of the Japanese lacquer tree Rhus vernificei-a dates back to 1883 [1421, and fungal laccases were also characterized by the end of the 19th century F1431. Laccase consists of a single polypeptide chain (of 500-600 residues) with three cupredoxin-like domains. The type 1 site i s located in domain 3. The redox potentials of blue sites in laccases range from 390 (Rh. uern,icifera) [1311 to 790 mV (P~lyporus pinsztus) 11443. This site accepts electrons from the substrate, t,ypically diphcnols, a~ldiainines,or aminophenols, and then transfers them over 13 to the trinuclear catalytic center where 0 2 is reduced to water. Sequence alignment indicates that fungal laccases possess a Phe or a Leu residue instead of a Met in the axial position. The crystal structure of type %depleted Coprinus cinereus laccase shows a type 1Cu(1I)ion in a trigonal planar geometry with two His and a Cys ligand, without any axial ligand 1311. Lcu462 is located at 3.5 from the metal ion, and it i s unable to form a bond. As a consequence. the copper ion lies in the plane defined by the equatorial triad ligand (Table 3, Fig. 3E).

A

2.3.2. Ascorbale Oxidme

ate oxldase (AO) (L-asc0rbate:dioxygen oxidoreductase, EC 1.10.3.3) is wide among higher plants, being also present in bacteria and probably fungi 114,1401. This enzyme can reduce ascorbate and other substrates, but its physiological role is uncei-tain. AO from green zucchini squash is a homodimer, and each subunit contains 552 residues forming three cupredoxin domains. Domain 3, formed by two 5-

COPPER IN ~L~CTRON-TRANSFER PROTEINS

831

stranded sheets and 5 helices, includes the blue center [145,1461. The metal ion is bound to His445, Gys507, His512 and Met517 with a plastocyanin-like geometry [1461, which is slightly altered upon reduction [1471 (Table 3). 2.3.3. Ceruloplasmin

Ceruloplasmin (Cpj (Fe(I1j:dioxygenoxidoreductase, EC 1.16.3.11 is a particular oxidase in that it contains six copper sites per molecule and is the only blue oxidase found in vertebrates [1481. Three of them are type 1 copper sites, whereas the other three define a trinuclear copper center. Several biological roles have been suggested for ceruloplasmin (copper transport, amine oxidase, antioxidant), and recent evidence supports a ferroxidase activity in plasma [149J. The human enzyme is a single polypeptide chain of 1046 residues. The structure solved by Lindley and co-workers reveals that the three type 1 sites are bound to domains 2, 4, and 6 IlSO]. The spatial relation between the catalytic center and the nearest blue site (the one located in domain 6) resembles that found in AO. Two of the blue copper sites (from domains 4 and 6) possess a classical CysHisJKet ligand set, whereas the one located in domain 2 lacks the axial ligand, resembling the trigonal type 1 site of laccase. Spectroscopic evidence on human Cp supports the idea that one of the blue sites, likely the one that lacks the axial ligand, is always in the Cu(I) form, being functionally inactive due to its high redox potential of at least 1.0 V [l5l I. This situation is met in Cps from mammals, whereas all three blue copper sites display physiologically accessible redox potentials in nonmammalian Cps 11521. This view is coiisistent with pulse radiolysis experiments that indicate that only one of the blue copper centers in human Cp participates in the intramolecular electron transfer to the trinuclear center 11531.

Copper nitrite-reductase (Cu-NiR, EC 1.7.99.3) is an enzyme containing type 1 and type 2 copper sites that catalyzes the reduction of NO; to NO in the denitrification step of the bacterial nitrogen cycle 11541. The type 1 center is the electron entry port t o the enzyme, and the type 2 copper defines the catalytic center where nitrite is reduced 1154-1571. The enzyme i s found as a homotrimer, with two bound copper atoms per monomer [18,157-1591. Each subunit (- 345 residues) folds into two cupredoxin domains (see Chapter 16). The type 1 Cu center is located at the top of the molecule. Its features differ between enzymes from different sources, and Cu-NiRs have then been classified into blue (Alcaligenes xylosoxidans [160,1611, Pseudomonns aureofaciens 11621, Hyplzomicmbium sp. I1631 and Fusarium oxysporurn [1641), and green Cu-NiRs (Achrornobacter cycloelastes I1651, Rhodobacter sphaeroides [1661, and Alealigeizes faecalis strain S-6 1771). The type 1 copper geometry is a distorted tetrahedron

VILA

832

AND F ~ ~ N A N D ~

with a CysHis2Metligand set (Table 31, with a shortened Cu-S(Met)bond and altered ligand-copper~lig~d bond angles compared to other BCPs, which give rise to the different colors (see See. 4.2). The catalytic site is a tetrahedral copper bound to three His and, in the absence of nitrite, to a water molecule. The two copper centers are 12.5 A apart and are bound by adjacent residues in the sequence, because His135 binds type 2 Cu and Cys136 is a type 1 ligand. The type 1 copper accepts an electron from an external donor and then donates it via a Cys136-His135 intramolecular pathway to the catalytic center located in the same subunit. Ligand replacement in the type 1site reduces the eMiciency of electron transfer with pseudoazurin 1157,167,1681. Green NiR’s (fromA. cycloclustes and A. faecalis) recognize the electron transfer donor psAz through specific electrostatic interactions between an acidic patch and a lysine-rich ring in psAz’s, as already discussed in See. 2.2.3 186,91-931. Instead, blue Cu-NiR’s (from P. uureofuciens and A. xylosoxidans) lack the negative charge region, and azurins may be their electron transfer partners [104,1623.

inuclear CuA Site Cytochrome c oxidases (ferrocytochrome-e:dioxygen oxidoreductase, EC 1.9.3.11 are terminal oxidases present in most aerobic organisms that reduce dioxygen to water in a redox process coupled with membrane proton translocation (see Chapter 15) “169,1701. The bovine enzyme possesses two heme groups (heme-a and h e m e q ) , a ~ o ~ o n ~ ccoppctr l e ~ site r (CUB)coupled to the iron of heme us, and a binuclear electron transfer copper center called Cun. The latter is located in subunit 11, which protrudes to the cytosolic side 8 above the membrane surface, whereas the other metal sites are sunk about 13 A below the membrane [20,171]. The location of CuAis consistent with its role as primary electron acceptor from a soluble cytochrome [172,173]. Subunit I1 in the homologous bacterial quinol oxidases lacks the Cu, center but retains a high degree of sequence similarity to those in cytochrome oxidases, and displays a cupredoxin fold [169,174], CUAsites have been engineered into this subunit 1174,175], as well as in blue copper proteins 1176-1781, thus confirming the evolutionary link. The structures of cytochrome oxidases from bovine heart (a 200-kDa complex formed by 13 subunits) r20,171] and from Paruccocus denitrificans (a four-subunit enzyme) 1191 are available [287]. The soluble CuA subunit of the cytochrome ba3 from Thermus tiiermophilus [1791 has been solved to 1.6 resolution IlSOl, as have the structures of two engineered CU, sites [175,177]. All exhibit a characteristic cupredoxin fold. he structure of the CuA center has been the subject of intense debate [1811. CuA i s a binuclear copper center with six ligands: two Gys, two His, one Met, and a us peptide carbonyl group (Fig. 71, In the CuA-subunitfrom T . t h ~ ~ o p h i l cytochrome bas, the copper ions are bridged by the two sulfur atoms of CysP49 and Cys153, forming an almost planar Cu2S2rhombic diamond structure with a metal-to-metal

A

833

COPPER IN ~ L E ~ T R O N - T ~ A N SPROTEINS ~ER

4

FIG. 7. Schematic picture of the binuclear Cu, site from the soluble subunit I1 of T. thennophilus cytochrome bcz3, from [1801 (PDB: 2qUA). The metal-ligandedistes are as follow!: CUl-sy(C149)z.= 2.35 Ai C~l-$,(C153)= 2.42 A 9~&3,(C149)=z 2.29 A Guz-s,(C153) z= 2.27 A; f&1~-N~(H114) = 2.11 A Cuz-N9(?4?[157)= 1.88A; Cul-Sb(Ml60) = 2.46 A Cu,-O(QlSl) 15: 2.62 A. The Cu1-Cu2 distance is 2.51 A.

distance of 2.5 [180]. The structure is not symmetrical, since one of the copper ions binds also to the N61 atom of His114 and Met160 (at 2.48 A), with an almost tetrahedral geometry, whereas the other copper coordinates to His157 and the backbone carbonyl of 6 1 ~ 1 5 1(at 2.62 A) adopting a trigonal coordination. The possibility of a copper-copper bond has been raised 1182,1831.All of the metal ligands are Iocated in loops between p strands. Sequence alignment indicates that HisllC, Cys149, His157, and Met160 correspond to the classical ligands of a blue site. These features are conserved in all known Cu, centers, with some subtle differences that may tune the electronic structure of the copper ions [1771. In this respect, the weak Met and Glu ligands may contribute to maintain the site architecture and to regulate its properties [1841. CUA exists in two redox states: [Cu(II)Cu(I)l and [Cu(I)Cu(I)l. The oxidized species is a fully delocalized class I11 mixed-valence pair [1851, i.e., with two Cu"" ions, as early revealed by EPR spectroscopy ll86l. However, ENDOR 11871 and NMR studies El88-1911 indicate difTerent electron spin densities for ligands of the different copper ions. The binuclear site experiences minor changes upon reduction [192], thus energy that makes it a more efftcient electron resulting in a low reo~gan~zation transfer unit than blue copper sites [193,194],

3.1.1. A u r ~ a n i n s

Three blue copper proteins collectively named auracyanins have been isolated from ChEorofZexus aurantiacus, a pho~o~rophic green gliding bacterium, by ~ l a n ~ e n s h i p

VILA AND FERNANDE~

834

and co-workers [1951. Auracyanins A, B1 and B2 exhibit different molecular masses due to the carbohydrate content of the B forms 11961. Auracyanin A may be the electron donor of the photosynthetic reaction center in C. aurantzacus, due t o the absence of soluble c-type cytochromes in this organism El961 Auracyanins exhibit common sequence features both with plastocyanins and azurins E1971. Sequence alignment suggests that His58, His128, Cys132, and Met132 are the copper ligands (Fig. 2) [2881.

3.1.2. Halocyanin

A 15.5-kDaprotein named halocyanin (Hc) was isolated from the membrane fraction of the haloalkaliphilic bacterium Natronobacterium pharaonis, a strain obtained from the brines of North African lakes [211. This strain requires high salt conditions and normally grows between pH 8.5 and 3 1. The gene coding for the halocyanin precursor revealed a sequence typical of signal peptides of bacterial lipoproteins [1981. The finding of a blocked NH2 terminus of the mature protein supports the hypothesis of a posttranslational modification. Re is the only blue copper protein hitherto purified from an archaebacteria. Notwithstanding the sequence similarity with plastocyanin, which suggests that the copper site possesses a His2CysMet ligand set (Fig. 21,Hc displays the lowest redox potential among BCPs (183 mV at pH 7.3). However, this potential exhibits a pronounced pH dependence [199I. 3.1.3. Uclacyanins

Uclacyanins (Ucc) are glycosylated phytocyanins that share a four-domain organization with stellacyanins that is not found in plantacyanins [1181. They have been isolated from A. thaliana by Nersissian, Valentine, and co-workers. Sequence alignment suggests that a Met is the axial ligand, as in plantacyanins, even if they are evolutionarily closer to stellacyanins.

3.2.

Blue Oxidases

There are numerous multicopper oxidases hitherto nonextensively characterized. Bilirubin oxidase (bi1irubin:dioxygen oxidoreductase, E.C. 1.3.3.5) is a monomeric 66-kDa fungal glycoenzyme with high sequence homology to other blue oxidases 1200,2011 capable of oxidizing bilirubin to biliverdin in vitro, as well as other tetrapyrroles, diphenols, and aryldiamines. A CysHis2Metligand set binds the blue copper ion [200,202,2031. Fet3 (also called Fet3p) is an extracellular membrane-bound, glycosylated protein from yeast, presumably with a ferroxidase role, being essential for iron uptake 1149,2041. Fet3 possesses a blue site, apparently lacking the axial ligand, and a trinuclear catalytic copper site, as identified by absorption and EPR spectroscopy [205,2061. A three-dimensional model was recently proposed r2071.

COPPER IN ~LECTRON-TRANSFERPROTEINS

835

Phenoxazinone synthase catalyzes the oxidative condensation of two molecules of 4-methyl-3-hydroxyanthranilicacid to actinocin, the phenoxazinone chromophore of actinomycin [208,209]. Sequence and spectroscopic data indicate the presence of the classical conserved copper binding domains of oxidases [2101. Sulochrin oxidase [211] and dihydrogeodin oxidase [212,2131are Eungal enzymes involved in the biosynthetic pathway of grisans.

Nitrous oxide reductase (N,OR, EC 1.7.99.6) catalyzes the two-electron reduction of NzO to Nzas a component of the denitrification pathway in soil and marine bacteria [2141. The physiological electron donor is presumably a soluble type c cytochrome or a pseudoazurin. NzOR’s are dimeric, soluble periplasmic enzymes that may contain a variable copper content. A CuA-likedinuclear center has been identified by EPR, and NMR spectroscopies in the NzOR from Pseudomonas stutzeri, which contains four copper atoms [215-2171. The first suggestion of the mixed-valence character of the CuA center stemmed from an EPR study of this enzyme [186,2181. A second binuclear copper center, called Cuz, is present that exists in two redox states: [Cu(II)Cu(IT)Iand [Cu(II)Cu(I)] [219]. Cuz was proposed to be the catalytic center of the enzyme, but Farrar et al. have suggested that Cu, and Cuz may be variants of a single dinuclear electron transfer center that could undergo a two-electron redox change from [ ~ u ( ~ I ~ C u (to I I )LCu(I)Cu(I>l I 1289-2921.

Four-co~}rdinatedCu(I) complexes are mostly tetrahedral, whereas Cu(I1) tends t o adopt tetragonally distorted coordination geometries due to the Jahn-Teller effect [221]. In blue copper centers, both Cu(1I) and Cu(1) forms exhibit distorted tetrahedral geometries, with minor changes upon reduction. One view suggests that the protein folding around the metal site induces an “entatic” 1222,2231 or “rack” state 113,2241 that restricts the possible coordination geometries, imposing the Cu(I) coordination geometry on Cu(I1). Solomon and co-workers have proposed that the strong Cu-Cys bond entails the metal site a geometry in such a way that a long CuMet bond is imposed in both oxidation states, opposing to the distorting J a h n ” r ~ ~ ~ e force [225,226]. The resulting small conformational change upon the redox reaction decreases the reorganization energy, thus enhancing the electron transfer rates 12931. heoretical calculations have challenged this paradigm, suggesting that these sites are not conformationally strained [227,2281.

836

pec~roscopicStudies Cu(II) is an open shell transition metal ion with a d9 configuration, with one unpaired electron located in the d$+ orbital. Instead, Cu(I) possesses a d10 closed shell, being diamagnetic. As a consequence, the reduced ion is devoid of most spectroscopic properties, and its study has been limited to X-ray absorption [138] and photoelectron spectroscopies (2251. On the other hand, the unusual coordination features of blue copper sites endow them with unique spectroscopic properties relative to normal Cu(I1) complexes. Oxidized BCPs display a strong absorption at 600 nm, assigned to a xCys -+Cu(1I) LMCT transition [229-2311. Its intensity is attributed to the orientation adopted by the d,z+,2 orbital, which favors overlap with a 7~ orbital of the S,(Cys) and gives rise to a strongly covdent Cu(I1)-Cysbond 1231,2321.A second band, at about 450 nm, has been assigned as a pseudo-oCys --3 Cu(I1) LMCT transition based on resonance Raman experiments 1233,2341.The relative intensity of these bands is a distinctive feature of each BCP (Table 4) L2351. The frequency of lower energy absorption bands in the near IR region corresponding to ligand field transitions can be correlated to the metal site geometry r236). No electronic transitions can be detected in the near-IR region below 12,000 em-’ at room temperature in Pc, but three bands are observed in the CD spectrum at 11,200, 9,150 and 5,000 cm-’ [251. These low-energy absorptions provided the first evidence of the distorted tetrahedral geometry of the blue Cu(I1) sites. Eight d-d transitions were later resolved in the low temperature optical spectra r2321. EPR spectra of oxidized blue sites exhibit characteristic small A;, values (63 x lo-* cm-l in Pc, compared with 164 x 10’4cm-’ in CuCl:-), as early recognized by M a l ~ s t r o mand VanngArd 12371, This is attributable to the strong covalency of the ~u(II)-thiolatebond, which reduces the electron spin density on the copper ion [232,2381, as supported by theoretical calculations I2331 that indicate a 42% Cu d+;s character for the HOMO in Pc vs. 61%for CuClZ-. XAS data 1238,2391indicate a high degree of covalency, and magnetic spectroscopies such as ENDOR and NMR, have confirmed the existence o f a large electron delocalization onto the coordinated CYS [240-2421. Blue copper proteins can display either axial or rhombic EPR spectra (Table 4). Lu et al. have correlated the occurrence of a rhombic EPR signal with an increase in the intensity of the 450-nm LMCT band [235]. Blue centers with rhombic EPR spectra and a higher E ~ ~ ratio ~ / have E been ~ ~ referred ~ to as “perturbed” blue sites 1233,2351. This perturbation may arise from a tetragonal (as in CBP, p&, or CuNiR) [225,2431 or a tetrahedral (as in St) 12441 distortion (Fig. 3B,C). The tetragonal distortion is evidenced by a coupled angular movement of the Cys and Met ligands (Fig. 3B), resulting in a stronger interaction with the axial ligand and a concomitantly increased Jahn-Teller effect [2431. Green NiRs, in which the high intensity of the 450-nm band alters the traditional blue color, bear the largest tetragonal perturbation in the series [2431. The different colors exhibited by blue and green NiR’s result from a different orientation of the axial Met 11611. Instead, in St, the tetrahedral

COPPER IN ~ L E ~ T R ~ N - T ~ A NPROTEINS SF~R

837

TABLE 4 Sp~ctroscopicProperties and Redox Potentials of p up red ox ins

CT bands

Axial 460 Plastmyanin (spinach) ~ c y ~ (P.i ~ n ~ ~ ~ t r ~ c ~al ~ s )464 Rhombic 450 Pseudoazurin (A.Jueculis) 460 Axial Halocyanin ( N . pharuonis) 454 Rhombic A ~ r a ~A~(C. i uurantiacusl n 460 Azurin (A. denitv$cans) Axial 445 Rhombic Rusticyanin (T.ferroxiduns) 448 Rhombic CBP (C. sativus) Rhombic 450 Stellacyanin (R.vernifificem) Axial 451 Umecyanin (horseradish) 448 Rhombic Mavicyanin (zucchini) 450 Axial Uclacyanin ( A . thaliunu) 458 Rhombic Cu-NiR (A. cycluciastes) "% =1&450/%00)

590 520 1180

420 900 580

1000 1240

1100 460 810 1000 2530

597 595 593 600 596 619 600 593 608

606 599 600 585

4900

4610 2100 4190

3000 5100 2100 2900 4080 3400 4000 3150 1890

0.12 0.11

0.41 0.10 0.30 0.11 0.47 0.43 0.27 0.13

0.19 0.32 1.34

366 294 270 133 240

27 680 306 187 290 285 320 247

'All redox potential values were measured at 25°C and pH 7.0, except those of rusticyanin (pN 3.2)and auracyanin (pN 8.0).

273 62 77

199 196 273

110 273 131 127 125 118 156

VILA

838

A N D FERN AND^^

distortion is limited to a displacement of the copper ion towards the axial Gln ligand out of the NNS equatorial plane [244]. In both tetrahedral and tetragonally distorted sites, the stronger interaction with the axial ligand effectively reduces the strength of the Cu(1I)-Cysbond 1242,2451, indicating that an interplay between the axial ligand and the bound Cys determines the electronic structure of the metal site 112421. The absence of axial ligand in laccase, gives rise to a stronger Cu(I1)-Cysbond [2461.

4.3. ~ ~ t a g e n e sStudies i$ Site-directed mutagenesis studies in BCPs during the last decade have been fundamental to our understanding the way in which the coordination chemistry of these metal sites affects their electronic structure, redox potentials, and electron transfer properties [loo]. Most of the mutagenesis studies have been performed in azurin due to the success in obtaining high-yield expression levels of this protein in E. enli. Although mutations in the copper site alter its spectroscopic features, the protein tolerates such mutations and retains its overall structure.

4.3.1.

Th.e Cysteine Ligand

Mutation of the Cys ligand into an Asp in azurin gave rise to a nonblue protein, confirming that coordination to the Cys ligand is essential to the electronic structure of type 1sites [2471. The engineered Asp binds the metal ion in a bidentated fashion, and the copper ion adopts a distorted square pyramidal coordination, with a minor shift from its position in the wild-type protein [248,2491.

4.3.2. The Histidine Ligands The role of the conserved His ligands has been thoroughly explored by Canters and coworkers. The Hisll'lGly azurin mutant leaves a pocket in the protein surface around the metal ion, giving rise to a type 2 center [250,251]. This pocket can be filled with small molecules that bind the copper. Addition of imidazole gives rise to a compound spe~tros~op~cally indisting~isha~le from the wild-type protein. This mutation allows direct redox reactions of the copper center with organic substrates, suggesting that the exposed His ligand in BCPs is essential for electron transfer and protects the metal site from unwanted redox reactions [252]. Mutation of the Gys-Met loop in amicyanin introducing the His-X4-Metsequences present in other BC that the exposed His residue is more subject to conformational changes, and is able to adapt to different conformations to fit into the metal coordination sphere [281. The His46Gly mutation in azurin instead gives rise to a type 2 site without significantly altering the protein structure [253,2541. Reconstitution of a type 1 site is not as straightforward as in the His11761y mutant, probably due do the reduced accessibility of the vacant position and the need for a more rigid ligand conformation. sp generates a distorted blue site, weakening the @u(II)-@ysbond The ~ i s ~ 6 A mutant

ANSFER PROTEINS

~~~

through displacement of the metal ion from the equatorial plane toward [255,256]. 4.3.3. The Axial Ligand ~ u m e r o u s~utagenesisstudies o Ps have been oriented to elucidate the role of the axial ligand, which is the nonconserved copper ligand in type 1 sites [134,257-2641. The absence of axial ligands (when a residue without metal binding capabilities is introduced) gives rise to a trigonal type 1 site, as naturally found in laccase [257,261]. The introduction of a Met ligand in laccase generates a plastocyanin-like site E2461. The engineering of metal-binding residues in the axial position gives rise to different situations. The Met121(3ln mutant in azurin mimics the s metal site [134,244,2651.Met 121His and Met 1 2 1 6 1 azurins ~ experience a e His and Glu residues are protonated at on [258,259,262-264)266]bec deprotonated, these residues bind to the and do not bind the copper io copper, giving rise t a perturbed type 1 site or to a type 2, respectively, as increases [258,25~]. ther ~ u t a n t sleave a vacant site that can be occupied et ligand may also water molecules or small external ligands, suggesting that th ations of the axial rve to protect the copper against unwanted reactions [267]. et have also been explored in msticyanin [268]. 4.3.4.

The Hydrogen

The Cys ligand is involved in hydrogen bonding through its sulfur atom. CPs, a conserved Asn residue is part of this hydrogen bond net is not present in rusticyanin, umecyanin, and uclacyanin (Table 2 Asn in azurin and plastocyanin alters the redox potential and the stability of' the metal site [269,270]. Ser86 replaces the Asn in rusticyanin, and this residue is hydrogen-bonded to the coordinated Cys138, as well to (3111139, thus further stabilizi~gthe met ro located two residues upstream from the bound Cys lack a second hydrogen bond. The Pro80Ala mutation in p s h leaves a hole filled by a water molecule that hydrogen-bonds to the cysteine sulfur [89,~0].

edox potentials exhibited by blue sites are higher than that of the aqueous Cu(II)/ (I) redox couple, spanning a broad range of positive potential values from 180 to 790 mV. Several stmctural factors influence the redox potential: the identity of the ~], axial ligand [225,~~6,257,271]) the solvent accessibility of the metal site [ 1 ~ 7 , ~ 7the charge dipole distribution around the copper E1131, and the hydrogen bonds Cys sulfur atom [~9,90)116)269,270]. Stellacyanin (with a Gln axial li

VILA AND FERNAN

840

solvent-exposed metal site) exhibits the lowest redox potential over a broad pH range, whereas the highest redox potentials are found for laccases, which lack the axial ligand, or rusticyanin, in which the site is embedded in a hydrophobic environment E1121. The introduction of large hydrophobic residues in the axial position raises the redox potential, whereas insertion of negatively charged residues lowers it by stabilizing the Cu(I1) state [268,2721. The high redox potentials exhibited by laccases have been attributed to the absence of an axial ligand, even if fungal laccases display disparate potential values C2461. The influence of the axial ligand is far from being negligible, but other factors may clearly surpass the effect of this residue. lectrochemical studies suggest that the enthalpic contribution to the redox potential prevails on the entropic term [2731. The enthalpic term seems to be directly controlled by the metal coordination environment, whereas the entropic change may be more linked to the solvation and dynamics properties of the metal site. Blue sites possessing weaker or null axial interactions (Az,Pc, and iaccase) show more negative reduction enthalpies than plantacyanins and stellacyanins, with a stronger axial ligand field E2731.

lectron Transfer Mechanisms Electron transfer rates between two redox centers at fixed separation and orientation in proteins is described by the following expression from Marcus theory [274]:

where (-AGO> is the electron transfer driving force, h is the nuclear reorganization parameter, and H m the electronic coupling between reactants and products at the transition state. The unique coordination features of' blue copper and CuAsites confer them low reorganization energies, as required for eilicient electron transfer [193,194,224,2751. @Psuse the exposed His ligand located in the hydrophobic patch as a module molecular electron transfer. Other electron transfer mechanisms to more remote sites can be operative, as in plastocyanin. Two possible electron trmsfe ways have been defined for Pc (see Sec. 2.2.1): the adjacent one, through whereas the remote one involves a longer route (13 provides a short pathway (6 A) via the Tyr83 side chain, in the acidic patch. Notwithstanding, both pathways exhibit comparable electron transfer rates in vitro [121. It was suggested that the covalency anisotropy in blue copper centers resulting from the strong copper-Cys bond may play a major role in defining a hole mperexchange pathway through this ligand [55,2761. lectron transfer in ruthenium-labeled BCPs has allowed Gray, Winkler, and coworkers to explore different electron. transfer pathways in azurin 12771. Directional electron transfer through the Cys ligand and across a p sheet provided efficient path-

A),

C T R ~ N - T R A N S F ~PROTEINS R

841

ways 12781. Weakening of the CufII)-Cysbond in the His46hp mutant I2561 impaired electron transfer [279], and mutation of the Cys almost abolished long range electron transfer [249]. Instead, Farver and Pecht exploited the disulfide bridge in azurin to study electron transfer from the S-S moiety to the metal site [2801. lntramolecular electron transfer is fundamental to the mechanism of blue oxidases. The catalytic trinuclear center in A 0 is situated about 13 from the blue site [1461. The bound Cys is placed between the two sequential His residues coordinated to the type 3 copper pair, offering an optimal arrangement for electron transfer through the metal centers [226,2811. Intramolecular electron transfer in Cu-NiR is supposed to take place via an electron transfer pathway essentially identical to that operating in AO, but with a faster rate due to a favorable entropic contribution 1282I. Kinetic studies on cytochrome c oxidase have revealed the following linear sequence of electron transfer events, as follows [1721: cytochrome c ---$ CUA--+heme-a -+ heme-ag --+CuB-r O2--+ HzO The electron transfer rate from cytochrome c t o the CUAcenter is about LO5 s-l. The electron is then transferred to heme-a (located 19.5 apart) with a rate of 2 x lo4 s-l, and later shuttled to the catalytic heme-a3/CuBcenter (at 13.2 A) rapidly (2 x lo5 s-'), where O2 is reduced 12831. Two electron transfer pathways have been ~ a short one through a coordinated His C1931, and a proposed from C L to~ heine-a: longer one involving one of the Cys ligands, based on the higher covalency of the ChACys bond 21831. In all cases, pathway analysis does not favor the hypothesis of direct electron transfer from GUA to the binuclear heme-a&uB site.

5. P

ECTIVES

The ubiquity of BCPs in nature highlights their suitability for electron transfer tasks. Thc precise biological function of most o f them is still unknown, and this defines a challenge for the near future. The availability of sequenced genomes will allow the discovery of novel cupredoxins, and an integrated view of their function may be obtained. Some unanswered issues covered in this chapter should be pursued in the next years. These are the few structures of BCPs yet not available, and the structure of the intriguing N2O reductase. The next step should be the structural analysis of interprotein electron transfer complexes and the way in which these binding processes tune the potentials of the redox sites. Finally, the recently uncovered CUAsite will undoubtedly be explored through mutagenesis and spectroscopic techniques t o assess the structural factors determining its redox potential, electronic structure, and rigidity.

842

VILA AND FERNANDEZ

The solution structure of the oxidized plastoqanin from SynecchocystisPCC6803 has been solved through NMR by using paramagnetic constraints 12841. The crystal structures of spinach plantacyanin [2861, the full T. thermophilus cytochrorne baa oxidase 12871, C. aurantiacus auracyanin 12881 and NaO reductase [289,2901 have been solved. The fold of auracyanin B is similar t o that of azurin, whereas the metal site coordination resembles that from plastocyanin I2881, confirming the predictions outlined in Section 3.1.1. The structure of P. nautica N 2 0 reductase 1-evealed that CuAand Cuz sites were different metal centers. The Cu, site is a tetranuclear copper cluster, in which the copper ions bridged by sulfide ligands [289,2901, evidence supported by Resonance Harnan specti-oscopy[291,2921. A thorough analysis of the coordination geometry features of blue copper sites compared to inorganic copper complexes has been recently reported by Gray, Malrnstrijm and ~ i l l i a m s[2931. A CuA center has been found in the menaquinol NO reductase from BuciZlus uzotoforman,s ~2941.

Mejandro J. Vila thanks the Fundacicin Antorchas, the University of Fogarty International Center (NIH) for supporting studies on elect orchas and ANPCyT for per proteins. Claudio 0. FernGndez thanks F'undacion s Freeman is gratefully s u ~ p o ~ i nhis g research on blue copper proteins. Prof. acknowledged for stimulating discussions. COF thanks Prof. Elinor Adman for helpful comments.

amicyanin ascorbate oxidase azurin blue copper protein cucumber basic protein circular dichroism c ceruloplasmin CP Cu-NiR copper-dependent nitrite reductase nuclear double resonance ~ N D Oelectron ~ electron paramagnetic resonance EPR halocyanin Hc

h i A0 AZ BCP CBP

COPPER IN EL~CTRON-TRANSF~R PROTEINS

HOMO IR Lc

mcrr

H MCD NiR N20R NMR Pc PSh RC St Ucc Ume XAS

843

highest occupied molecular orbital infrared laccase ligand-to-metal charge transfer methylamine dehydrogenase mawetic circular dichroism nitrite reductase nitrous oxide reductase nuclear magnetic resonance plast ocyanin pseudoazurin rusticyanin stellacyanin uclacyanin urnecyanin X-ray absorption spectroscopy

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ns

tai

European Synchrotron Radiation Facility, BP - 220, F-38043 Grenoble Cedex, France

1. INTRODUCTION

2.

3.

858

OWN X-RAY STRUCTURES: ALBUMIN AND COPPER-ZINC S U P ~ R O ~ D~ EI S M U T ~ E 2.1. Serum Albumin 2.2. CuBn Superoxide Dismutase

858 858 860

STRUCTURES: M E T ~ L O T ~ I O N E I NAND S P~~-TRANSPORTING ATPases 3.1. Metallothioneins 3.2. Menkes Cly-Transporting ATPases

860 860 862

4. ~TRUCTU~E-FUNCTION R~~TIONS~IPS 4.1. Copper Binding Sites in S e w Albumin 4.2. St~c~re- unction Relationships in Superoxide Dismutase 4.2.1. Nature of the M e t ~ - B i n ~ nSites g 4.2.2. Mechanism for Dismutation of the Superoxide Radical 4.2.3. Access to the Active Site and the Dirneric Nature of Cu/Zn Superoxide Dismu tases 4.3. Copper Binding Sites in Metallothioneins 4.4. Copper Binding Site in the Menkes' Cu-Transporting A'I'Pase

862 862 863 863 865

5.

874

857

871 a72 873

a58

LINDLEY

AC~NOWLEDG~ENTS

874

AND ~BRE~TION S DEFINITIONS

875

RE~ERE~CES

877

The purpose of this chapter is to describe a number of proteins that bind copper but that cannot readily be classified with the family of proteins that are involved in the transport and activation of dioxygen (Chapter 15) or the multicopper oxidases (Chapter 16). Such proteins include albumin, copper-zinc superoxide dismutase (SOD), the metallothioneins, and Menkes’ copper-transporting ATPases. In the case of albumin and SOD, single-crystal X-ray structures have been determined. In addition, for SOD extensive NMR studies have been undertaken (e.g., El]). For Menkes’ Cu-transporting ATPases and the Cu-binding metallothioneins the structures have been determined only by NMR methods. NMR techniques normally produce a batch of similar structures distributed in space around a common framework, so that the structural information is not as precise as that determined by high-resolution X-ray methods. This lack of precision is adequately compensated by the application of such methods to solutions of macromolecules, obviating the need to grow single crystals of a quality suitable for the X-ray technique. In addition, NMR techniques can be readily adapted to studying molecular flexibility and the dynamics of reaction mechanisms. Another area of research that has become of intense interest over the past few years concerns copper chaperones. These chaperones transport copper within cells to those proteins that require copper such as SOD and cytochrome c oxidase, and thereby prevent cytoplasmic exposure to copper ions during transit. In this chapter we do not attempt to give a comprehensive account of the very active research that is being undertaken in this field, but we hope that the references cited will enable readers to pursue more in-depth studies as required. A useful overall text for the proteins described in this chapter is given in El.

AY STRUCTURES: ALBUM1 E DISMUTASE 2.1. Serum Albumin Serum albumin (HSA) is the most abundant of the plasma proteins with a concentration in humans between 3.0 and 4.5 g per 100 mL out of a total serum protein concentration of about 5.7 - 8.0 g per 100 mL. One of the functions of serum albumin is to impart a sufficiently high osmotic pressure to plasma to match that of the cell

COPPER PROTEINS OF VARIOUS FUNCTIONS

859

cytoplasm, but it i s also involved in the transport of fatty acids, bilirubin, drug molecules, and metal ions. HSA i s not glycosylated,as are most of the plasma proteins, and plays no role in immunosuppression. The protein consists of a single polypeptide chain of 585 amino acids with a molecular weight of around 65 m a . Some 61% of the amino acid residues are conserved among the bovine, rat, and human serum albumins. Garter et al. [31 first reported the X-ray structure of human HSA at low resolution, 6.0 A, in 1989.This was followed in 1992 [4]by higher resolution structure determinations at 3.1 A for the wild-type HSA and 2.8 A for a recombinant form expressed in yeast; a more comprehensive review of the structural studies can be found in L5I. The overall structure of HSA is shown in Fig. 1[PDB code: lAO6I. It is composed of three structurally homologous domains (labeled I, TI, and 111in Fig. 11, consistent with the amino acid sequence, which repeat to form a triangular molecule with sides of roughly 80 A and an average depth of about 30 A. Each domain contains two mainly helical subdomains and approximately 67% of the total HSA molecule is helical with the remainder comprising turns and extended polypeptide chain. The structure contains 17 disulfide bridges that primarily occur between a-helices and often cause distortion in the local helix structure. There is also a free thiol residue at Cys34.

FIG. 1. Molecular structure ofohurnan serum albumin [4] as determined by X-ray crystallography at a resolution of 2.8 A. The three structurally homologous domains are labelcd I, 11, and 111,

860

LINDLEY

uperoxide Dismutase CuiZn SOD catalyzes the very rapid two-step dismutation of the toxic superoxide radical (0, ’ 1 to molecular oxygen and hydrogen peroxide. These processes involve the alternate reduction and oxidation of the active site copper [61: SOD-Cu(I1) -t- 0,

’

SOD’-Cu(I) + 0,’

+ H+ F-t SOD’-Cu(1) -t- O2 + H+ .Ft S~D-Cu(I1)-t HzOz

It is now apparent that most cells in eukaryotic organisms probably contain CuiZn SOD and that the enzyme may play an important role in shielding intracellular components from oxidative damage. For reviews, see 111 and L7-101. Eukaryotic s normally exist as homodimers with two subunits each containing some 150 amino acids and an overall molecular weight of around 32 kDa, but prokaryotic SODs can be monomeric or dimeric. There is considerable sequence homology among the SODs from the various species so far examined and within the mammalian specieshuman, rat, pig and horse-the homology is around 80%. The residues directly or indirectly involved in metal binding appear to be completely conserved. The X-ray structure of bovine erythrocyte CuiZn SOD was reported in the early 1980s at a [11,121 (PDB code: 2SOD). In this structure, the two identical resolution of 2.0 subunits, each containing 151 residues, are related by a crystallographic twofold axis, so that the active sites are facing away from each other giving a distance o f 33.8 between the copper atoms. Within each subunit the polypeptide chain is folded into two 4-stranded antiparallel p sheets (cf. the topology of the cupredoxin fold in the chapter describing the multi-copper oxidases) which then pack together to form a Battened cylinder. Figure 2 shows the main chain topology for one subunit in the dimer as well as the positions of the two metal atoms. The first sheet is composed of strands 1,2,3, and 6 and the second of strands 5,4,7, and 8. The overall motif formed by the seven strands, 2 - 8 inclusive, is almost identical to that found in the immunoglobulins 1131. The active site Cu and Zn atoms are located at the bottom o f a deep cleft on the outside of the subunit. The cleft is formed by two large loops, one between strands 4 and 5 and the second between strands 7 and 8.

A

3.

OWN NMR STRUCTURES: METAL~OTHION ~ ~ N K ECOPPER-T~ANSPORTING S’ ATPases

3.1. ~~tallothioneins Metallothioncins (MTs) are small, cysteine-rich, metal-binding proteins, first isolated in 1957 by Margoshes and Vallee [14] and subsequently found in a wide variety of human and animal organs, in most eukaryotic species, and in plants. The mammalian MTs have a single polypeptide chain of some 61 amino acids including 20 cysteines, but no aromatic amino acids or histidines. Most of the cysteine residues are found

861


c

FIG. 2. The polypeptide fold found in a subunit of SOD [11,121. There are two p sheets that pack together to form a flattened cylinder. Each sheet contains four anti-parallel strands; the first sheet is composed of strands 1, 2, 3, and 6, and the second of strands 5, 4,7, and 8. The metal atoms are located in a deep channel on the outside of the subunit between two large loops.

either adjacent to one another or within one or two residues of another cysteine and are directly involved in metal binding. There are at least two distinct functional genetic cistrons for most mammalian species, expressing two forms of the protein (MTI and MTII) that difyer by only a few amino acids. Both f o m s appear to have an asymmetric two-domain structure. At least three functions have been ascribed to the NITS: metal sequestration, temporary binding and long-term metal storage, and intracellular (and extracellular) metal transport. However, it would appear that the capacity to sequester metals that might bind elsewhere and thereby interfere with normal cell processes is likely to be the most important function. The presence of an excess of free ionic Gu in cells would lead to oxidative damage and the affinity of NITS for copper is second only t o that for mercury. For the structure, see Fig. 7 in See. 4.3.below.

862

LINDLEY

u-Transporting ATPases Menkes’ disease, first characterized by Menkes et al. in 1962 [151, is caused by a disorder in copper metabolism r16,17 I. Specifically it involves an X-chromosome linked recessive disorder in cellular copper export and normally results in death in early childhood. There occurs a severe and long-term deficiency of copper that results in progressive neurological deterioration, probably due to the impairment of crosslinking of elastin and collagen by the copper protein lysyl oxidase. The gene responsible for Menkes’ disease encodes a CPx-type ATPase 1181 that is localized in the trans-Golgi network of cells and is thought to move copper across intracellular membranes into the secretory pathway [191. The complete ATPase is a complex multidomain, transmembrane protein, but close to the N terminus and within the cytosol there are six metal binding domains, mbdl-6, each containing a GMTGxxC sequence motif 120-223. It has been established 1231 that each of the N-terminal domains binds one Cu(l) ion selectively relative to other metals such as Cd, Co and Zn, and since patients suffering from Menkes’ disease are deficient in copper these N-terminal domains may somehow be specific for binding copper. A %-residue fragment of Menkes’ copper-transporting ATPase, corresponding to mbd4, has been expressed and purified and its solution structure established by NMR techniques (P B codes: lAW0 and 2AWO) [241. The structure (Fig. 3 ) comprises a four-stranded antiparallel sheet packed against two helices and this u-P ferredoxin-like fold [25] is also found in a number of functionally diverse catalytic, RNAbinding, and ~NA-bindingproteins.

~ L A ~ I SH O NI PS 4.1.

Copper Binding Sites in Serum Albumin

Albumin possesses two major binding sites for metals: the free cysteine at C34 and a site close to the N terminus. The free cysteine residue, 634, is highly reactive with an unusually low pKsp1 of 5.0 compared with 8.5 and 8.9 for cysteine and glutathione, respectively 1261. In most preparations of albumin some 30-35% of C34 is blocked by cysteine or glutathione. This may help to stabilize the protein against dimer formation whereby C34 participates in a disulfide bridge. 634 also binds metals such as Au, g, Cd, and, to a lesser extent, Cu. ether the binding of Cu at 634 is physiologically significant is not clear. The N-terminal site has a high affinity for both Gu(l1) and Ni(I1) and in this regard His3 plays a central role, L271; albumins that lack a histidine at this position, such as canine albumin, have a much lower affinity for these metals, p~esumably accounting for the greater susceptibility o f dogs to copper poisoning. In the crystal structures of albumins so far determined, the first three residues at the N-terminal region are disordered, preventing a detailed definition of the metal binding site and implying a flexibility o f the polypeptide chain that may be necessary to accommodate


863

FIG. 3. Overall view of one conformer of the mbd4 domain in the Menkes' Cu-transporting AT'Pase [24].

metal cations. However, the site is thought to involve the N-terminal nitrogen atom, the next two peptide nitrogen atoms, and a nitrogen atom of the side chain of His3. Such a site has been simulated with the tripeptide glycylglycyl-L-histidine-N-methylamide I281, as shown in Fig. 4. In the albumin structure it is also possible that the side chain of Asp1 could fold around to give a further carboxylate ligand to the metal atom. In contrast to ceruloplasmin (see Chapter 161, copper on albumin is readily exchangeable, particularly with amino acids that may be present in serum and other body fluids 129,301.

4.2. 4.2.1.

Structure-Function Relationships in Superoxide Dismutase Nature of the Metal-Binding Sites

In the oxidized form of bovine SOD the Cu(I1) has four histidine ligands-H44, H46, H61 and H118 (Cu-N distance = 2.17 A averaged over the two dimers in the asymmetrical unit)-arranged in an irregular tetrahedral distortion from square planar geometry. The Zn is tetrahedrally surrounded by histidines 61, 69, 78, and aspartic

864

FIG. 4. Structure of the copper-bindingpeptide, glycylgly~y~-L-hist~din~-~-methy~amide simulating the coo5dination state and peptide conformation in albumin f28.l. The Cu-N distances average 1.99 A. (This figure was produced using the drawing program provided by Cerius, Molecular Simulations, Cambridge, UK, with data extracted from the Cambridge Stmctural Database [ 711.)

acid 81 and is completely buried in the structure. His61 bridges the two met& in an , (average for the two almost planar manner, giving a Cu-Zn separation of about 6.2 & dimers) and with the implication that this residue is completely deprotonated in the oxidized form of the enzyme. Figure 5 shows the residues in the bovine SOD structure [12], which surround the active site channel. One rim of the channel is formed by residues 131, 134-139, and 141, whereas residues 56,58-60, and 63 form the second rim; the metals and their ligands form the channel floor. A network of hydrogen bonds fixes the orientations of both the main and side chains of the metal-liganding residues to form a specific geometry for the active site. A molecular surface representation shows that the active channel contains at least one pit in its narrowest part. This pit is formed by the exposed surface of the Cu(11) atom (5.2 with respect to a 1.4-A probe corresponding roughly to the diameter of a water molecule) and parts of H61, H118, T135,and R141. In the bovine SOD structure, solvent molecules occupy this “Cu pit” in diflerent ways in different bunit its. 1x1subunit Y (nomenclature of PDB entry 2SOD [ll]), a water molecule is 2.49 A from the Cu and acts as a fifth ligaad, giving the copper an almost square In the remaining three subunits in the asymmetrical unit the p ~ ~ i den~ronment. a l

A2

865

COPPER PROTEINS OF VARIOUS FUNCTION§ N137

F1G. 5. Active site channel of bovine (Bos duurus) superoxide dismutase [11,12]. Residues 131, 134-139, and 141 form one rim o f the channel whilst residues 56,58-60, and 63 form the other rim. The metals, and their ligcnds form the floor of the channel. H61 bridges the Cu, and Zn atoms (Cu-Zn distance is 6.2 A), and is completely deprotcpated in the oxidized form of the enzyme. A water molecule in the Y subunit is situated 2.49 A away from the copper, and can be considered to be a fifth ligand giving rise to a square pyramidal environment for the copper atom.

water is further away from the copper at an average distance of 3.64 A and can form hydrogen bonds with the side chain of R141. In combination these two waters simulate the position that could be occupied by a superoxide radical and the nature of the pit seems specific to a moiety of this size and shape. 4.2.2. Mechanism for Dismutution of the Superoxide Radical

The X-ray structure, in conjunction with biophysical arid biochemical studies, enabled Taker and colleagues [12]to propose a mechanism for dismutation that first involves the reduction of Cu(I1) by superoxide. Molecular oxygen is then released and the &(I)-bridging histidine bond broken with concomitant protonation of the N E ~ atom of H61. The reduced &(I) protein then reacts with a second superoxide radical to form HzOz and the reoxidized enzyme.

866

LINDLEY

Since the original work by Tainer and colleagues [ l l , l Z I , numerous crystallographic and solution structural studies have been undertaken with CdZn SODS in order to further investigate and refine thc reaction mechanism and other characteristics. Table 1summarizes the Cu/Zn SOD structures, including both prokaryotic and eukaryotic species that have been deposited in the Protein Database (PDB) I311. With respect to Table 1,it should be noted that the precision of the geometrical data will in general improve as the resolution of the structure increases. For example, the wide variation Of 2.34 to 3.17 observed in the Cu-Nz2 (bridging histidine) bond distances in the five dimers in the asymmetrical unit of the human SOD K136E mutant (PDB code: IFUN) may be attributed, at least in part, to the relatively low value of the resolution, i.e., 2.85 A. However, the possibility that some of the subunits contain reduced copper or that there is a mixture of Cu(lI) and Cu(I) should also be taken into account. Most recently, Hart et al., L32] have reported studies involving a wild-type yeast SOD with reduced Cu(1) in three different forms as follows; 1. Under 15 atm of oxygen, so that the copper is partially oxidized 2. In the presence of azide which binds to the Cu(I), and 3. A mutant, in which H48, which is bound axially to the reduced Cu(1) in the azide complex, is replaced by cysteine

These authors 1321 have elegantly correlated the various studies to yield the reaction cycle shown in Fig. 6. In the resting state of the SOD enzyme (A), the Cu(I1) is in a characteristic distorted square planar conformation with respect to the histidine ligands. A superoxide radical enters the active site cavity ( the bound water molecule, binds directly to the copper ion and donates its electron to yield Cu(1).The histidine bridge breaks, oxygen diffuses out of the active site channel, The copper moves into a trigonal planar conformation with and water reenters (6). the remaining three histidine ligands, characteristic of Cu(I), and with this new environment the Cu-Zn separation increases by roughly 0.5 A. A proton, probably from the bulk solvent, facilitates protonation of'the Nz2 atom of H63. A second superoxide radical enters the active site cavity (D), displaces the water molecule nearest the copper, and forms hydrogen bonds with the next nearest water molecule and the ~ of H63. Electron transfer from the copper to the superoxide protonated N E atom coupled with proton transfer from H63 and this water molecule lead to the formation of hydrogen peroxide, E, which then diffuses from the active site and is replaced again by water. The imidazole bridge reforms and the copper moves back into a distorted square pyramidal configuration (including the bound water molecule) to complete the catalytic cycle. In this mechanism the four waters in the active site channel form a hydrogen-bonded chain that can act as a proton shuttle. Many of the crystal structures listed in Table 1 are fully compatible with this mechanism, but those of reduced bovine SOD at pH 7.0 and 5.0 150,511 appear at iirst sight to be in contradiction. However, it appears that in these crystal structures the existence of the histidine bridge between the copper and zinc cations may be dominated by steric rather than electronic factors. Ferraroni et al. [5U point out that the


867

TABLE 1 Configuration of the Copper Atom in CuEn Superoxide Dismutase Structures Determined by X-ray Crystallography, and NMR as Reported in the Protein Data Base [311a.b Code

Species'

2ms

Actinobacillus pleuropneumoniae Human

lAzv

Resolution (A) and AUd 1.90

D

Geometry, and histidine ligands'

2.50

M M

trig. pl. trig. pl.

3H 3H 4H 3H 3H 3H

2.30

D

A squ. pyr. B squ. pyr. A trig. pl. B squ. pyr. Asqu. pyr. B squ. pyr. squ. PI. squ. pl. trig. pl.

4H 4H 3H 4H 4H 4H 4H 4H 3H

1.90

D

A trig. pl. B trig. pl. A squ. pl.

B trig. pl. 1BA9 lBZO 1CB4 lCBJ 1COB 1ESO lFUN 1JCV

Humanf Pon,yfish symbiont (space group R32) Bovine (space group C222,) Bovine (space group P212121j Bovine, Zn(I1) is replaced by Co(I1) Escherichia coli Human Yeast At 93 K (space group R32)

N~~

1.65

D

2.00

D

2.00 2.85

1.55

M 5D M

(A)

&-NE~ bridging His 2.93 (H85) 3.00 (H85) 2.65 (H63) 3.12 (H63) 3.44 (H63) 3.16 H(61)

2.37 (H61) 2.15 (H61) 3.20 (H61) 2.21 (H61) 2.16 (H61) 1.99 (H61) 2.65 (H61) 2.34-3.17 (H63) 3.16 (H63)

CU-H~O

Cu-Zn

(A)

(A)

-

6.47 6.41 6.24 6.44 7.07 6.65

2.19 2.39 (3.50) 2.55 2.89 2.38

-

6.32 6.18 6.59 6.07 6.12 5.96 6.51 6.16-6.50 6.62

Ref. 33 34 35 36

37 37 38 39 40

41

LINDLEY

868

Table 1 (continue Code

Species'

wcw

Yeast at 298 K (space group H32) HUmm ICu site is partially occulpi-ed by ~d(11)lSDA

1MFM

Bovine 1SDY 2SOD 3SOD

1sos 1SPD

lSRD lSXC lsxN

1sxs 1SXZ

Yeast Bovine Bovine Human HUnlan

1.70

M

trig. pl.

3H

3.16

aeteristics of the ~ r i ~ ~ n g X

L1

L2

Y

L3

Z

Hp' His

56 2

L4

L5

Ref.

€kiss I: Oxidoreductases Superoxide dismutase family Bovine

6.3

fi, His

Human

5.5

HiSg W S

Frog (Xenopush i s )

6.0

His, BiS

Spinach (Spinaciaoleraceaj

Yeast

6.1

6.5

1 7

His, His

14

1 7

His, His

14 8

His

His

56 2

1 7

His@ His

14 8

His, His

56 2

His, His

14

Bisfi

8

E S

56 2

Hise His

14

Hisp His

56

His ((2

-

His p

1

Ria

7

&is,

1

His

7

8

8

His

1

His

22

His

8

His

7

2

54

Glass 111: Hydrolases Escherichia coli dkalirie phosphatase

3.9

His Aspp

His,

0

103

ABP

?"hr, HiSp

256 166

ser

50

Glu, (C)

-

His (6) ?

13341

ZINC SITES IN ~ ~ T A L L O E N Z Y ~ E S

Phosphotriesterasefamily Pseudomonas diminutu

Escherichiu coli Bucillm cereus phosphdipase C

897

Zn*

3.3

Zn zn* Zn

3.35

Zn*

3.3

Zn Penicilkum citrinum P1 nuelease

Zn*

3.2

Zn Esche&hia coli endonuelease IV

Zn*

3.4

zn p-Lactamase farnily Bmteroides fi-agilis Bacillus cereus

Stenotrophomonas maltophilia Pseudomonas sp. carboxypeptidase Gz 111 Aminopeptidasefamily Bovine lens

Zn Znh Zn Znh Zn Zn” Znl Zn2 Znl Zn2

3.5

3.7-4.4

His Hisg

1

IliS

32 3 12 3

107

4

His, His, His, His, His,

33

hPP

36

35

His

39

3.3

2.9-3.0

27 48 55 113

1

His

60

77

cys

41

1

His q s

60

His

ASP

73 135 208 58

ASP Asp

17

77 3.4

111 28 110

1 0 34 28 1 4

His Glu

41

76

HzO?

A

Table 3 (continued)

Escherichia coli methionine-1 Pyrococcus furiosus methionine-2

Human methionine-2

Col c02 co1 co2 Col

2.9

2.8 3.2

COZ

Aerornonas pro feolytica Streptomyces griseus

Znl Zn2 Znl

3.5 3.6

ZnZ Escherichia coli UDP-sugar hydrolase

Znl

3.3

Zn2 Purple acid phosphatase family Kidney bean

Rat Porcine (uterfenin) Human protein phosphatase I

Fe(II1) Zn Fe(1II) Fe(I1) Fe(IlI) Fe(l1) Fe(II1) Mn

3.3 3.1

3.3 3.5-4.0

Glu (C) Glu 30 Glu, (C) Glu 92 Club ( C ) Glu, 94 His tC)

10 62 10 59 10 68 34 19 34 11 1

126 32 186 33 196 32 103 61 114 62 40

ASP,

169

31

100

His,

34

28 36 37 38 37 38 1

2

Tyr

84 2 94 2 94 25

His Tyr His

157 36

31

48

Asp (6) His (C) ASP ( C )

TYr His Asp His

167 34 167 34 179 74

ZINC SITES IN METALLOENZYMES

Human brain calcineurin

899

Fe(II1) Zn

3.1

Asp Asp

1 31

His Asn

25 48

ASP(GJ Hisg

53 74

Hi.

HpO

13421 1671

81

Class V: Isomerases Human glyoxalase II

Zn Zn"

3.3-3.5

His ASP

1

0

His His

23 38

His (6) HzQ

'Amino acid residues that bridge the two metal sites are shown in italic bol~face.R is the distance between the metal atoms. See footnote in Table 1 €or other definitions.

900

AULD

have been found to bridge such sites. The class I11 hydrolases have by far the most representatives of this type of zinc site. While His and Cys predominate as ligands of catalytic and structural zinc atoms, respectively,Asp and His predominate in cocatalytic zinc sites where the frequency is Asp > His > Glu> Gys. Ser, Thr, Asn, Lys, Tyr, and backbone carbonyls (e.g., Trp) have been found to be ligands to the bridging zinc sites. The ligands to these sites often come from nearly the entire length of the protein. The metals in these sites may therefore be important to the overall Fold of the protein as well as catalytic function. The ligands are often at the ends of fi sheets or come Irom short “l00p’~stretches of amino acids connecting f3 sheets and v. helices. 2.3.1. HeteronucZear Cocatal.ytic Sites

Weteronuclear bridging sites are found in several cases. Zn/Mg are seen in alkaline phosphatase and lens aminopeptidase; Fe(III)/Zn in the purple acid phosphatase family and Cu(II)/%nin the superoxide dismutase (SOD) family (Table 3). The role of zinc in the SOD family is generally considered supportive to that of copper, which undergoes oxidation-reduction during catalysis. However, zinc may be important to substrate specificity.Thus, the zinc-deficient SOD has been proposed to participate in both sporadic and familial amyotrophic lateral sclerosis by an oxidative mechanism involving nitric oxide E621. The purple acid phosphatases are a group of nonspecific phosphomonoesterases that have been found in animal, plant, and fungal sources 1631. The characteristic purple color of this subclass of acid phosphatases comes from a phenolate-Fe(II1) charge transfer transition at 560 nm. The presence of Fe(II1) is universally found in these enzymes. The 35-kDa mammalian purple acid phosphatases (PAP) or tartrate-resistant acid phosphatases ( T W ) contain an Fe(III)-Fe(II)iron center 1641 in contrast to the Fe(II1)-Zn(11)center found in the 110-kDa kidney bean enzyme [651. The Sey/Thr human protein phosphatase 1and calcineurin also contain a very similar cocatalyiic Fe(III)-M(~I) (where M is Zn or Mn) site to that of the PAPs [66,671.In this case, there is no Ty-r-Fe(1II) interaction but the general ligand nature, the distance between metals, and the presence of a bridging Asp residue is common to all of these enzymes (Table 3). A mechanism has been proposed for the PAPs in which the phosphate ester binds to the Zn(I1) site and the phosphate bond undergoes nucleophilic attack by an Fe (1II)”coordinatedhydroxide ion [631. The E. coli methionine aminopeptidase-l (MetAP-1) [681, the hyperthermophile P~YI-OCOC~US fiiriosus methionine aminopeptidase-2 (MetAP-2) f691, and human methionine aminopeptidase-2 E701 have been isolated as dicobalt enzymes. The physiological metal for thcse enzymes is still not certain. Zinc works as well as cobalt in the yeast aminopeptidase-1 [71] and recent studies of the E. coZi MetAP-1 indicate that it functions as an Fe(I1) enzyme [721.

ZINC SITES IN M ~ T A L ~ O E N Z Y ~ ~ S

901

2.3.2. P-Lwtamases: Cocatalytic or Catalytic Zinc Sites ?

The majority of' p-lactamases utilize an active site serine in the hydrolysis of the plactam ring. However, there are now new pathogenic bacteria that have a metallo-plactamase that contains zinc. Several X-ray structures have recently appeared on this class of zinc enzymes from Bacillus cereus [73-751, Bacteroides fiwgilis [76,771, and Stenotmphomonas maltophilia r781. These structures are similar in the need of at least one zinc site that has the characteristics of a catalytic zinc site. Thus, the first structure of the B. cereus enzyme had one zinc coordinated by three histidines 86,138, and 149 and a water molecule in a tetrahedral arrangement 1731. These histidine residues are conserved in all members of this class of enzymes. These crystals were grown at pH 5.6 in 0.1 M ZnS04 in a citrate, cacodylate buffer. At this pH the binding constant for the second zinc is 29 mM [791. NMR studies indicated that when one zinc binds it coordinates to three His ligands and when a second zinc binds it coordinates to a fourth His ligand [791. Growing the crystals at pH 7.0 in the presence of 0.5 mM ZnSOd in Tris buffer yields an enzyme with two zincs bound [75]. The second zinc, Zn", binds to Asp90, Cys168, and His210 and two water molecules in a trigonal bipyramidal coordination geometry. The Cys ligand is not conserved in all the 6lactamase structures. One of the metal-bound waters is bridged in sequence to Asp90 and the catalytic Zn site. However, this water lies closer to the catalytic Zn than to Zn". In two independent molecules in the crystal the Zn to Zn* distances are 3.9 and 4.4 while the distance between the catalytic zinc and the shared water remains constant at 1.9 A. This distance is reasonable for zinc hydroxide. In addition, Asp90 remains in hydrogen bonding distance of the waterhydroxide in both molecules, indicating that it may play a role of a general acid-base catalyst in the reaction. The first reported fl-lac%amase structure that contained two zinc sites was for the B, fmgilis enzyme 1761. This enzyme was crystallized at pH 7.0 in a 10 pM ZnClz, Hepes buffer. This enzyme has the same type of'structure for the two zinc sites as is found in the two zinc €3. cereus enzyme except the distance between the Zn and Zn" is much shorter, 3.5 A, and the shared water is equidistant between the two sites. The structure of the S. maltophilia enzyme WM obtained on crystals grown in 5 mh!i ZnSOI at pH 7.0 "2731. It also contains two zinc ions separated by a water molecule. Once again the structure of the catalytic zinc site is essentially identical to that found in the B. frugtlis and B. cereus enzymes. Zn is bound to three His residues, 84,86,160, and the bridging water molecule in a tetrahedral geometry. Zn" binds to the wnserved Asp88 and His225 but the third protein Ligand is a residue unique to S. maltophilia. The change in coordination from the Cys168 to the His89 ligand causes the Zn" site to have a distorted trigonal bipyramidal geometry. Thus, while the Asp88 and Wat2 remain apical ligands, the positions of the three planar ligands, Watl, and His225, have rotated 76" about the Watl-Zn* bond [781. The importance of the second zinc site to catalytic activity is still not clear. This is the first cocatalytic zinc site in which there is no bridging amino acid. The second zinc site is not highly conserved in the few enzymes that have been examined. The mono zinc B. cereus enzyme is active and the Aeromonus hydrophila AE06 enzyme is

A

A

902

AULD

inhibited by Zn with a K, of 46 pM 1801. The first zinc binds to this enzyme with a dissociation constant lower than 20 nM. The mutation of Cys168 to Ser yields an enzyme crystal structure in which only the catalytic zinc site is found 1811. The kcat values for this enzyme is reduced 140- to 1500-fold while the k,,JK, values are reduced 970- to 3700-fold depending on the substrate used 1821. This reduction in activity could be due to the loss of a residue that played a role in the transition state in catalysis. Thus, in summation, all of the B-lactamases are dependent on the presence of one zinc that has the characteristics of a catalytic zinc site. The second zinc is not universally important to the activity of the p-lactamases and its importance to the B. fragilis enzyme is still questionable. The variable picture observed in the metallo-plactamases could reflect an evolving zinc binding site.

rotein Interface Zinc has recently been found to bind at the interface of protein subunits and between different proteins (Table 4). 2.41. Zim Binding Sites in Enzymes

A remarkable catalytic zinc site is found in glyoxalase I where the C-terminal domains of one monomer interact with the N-terminal domains of a second monomer to form two zinc binding sites at the interface of the subunits [83]. The active site zinc is coordinated by four protein residues and one water molecule in a square pyramidal coordination geometry. The base of the pyramid is formed from 61n33 and 61u99 of one subunit, 6 1 ~ 1 7 2from the second subunit, and a water molecule. The apex of the is126 from the second subunit. Such a geometry could be viewed as octahedral with one axial ligand missing. 0th EPR and XAFS studies suggest a distorted octahedral coordination in so n for the cobalt and zinc enzymes 184,851. The sixth site may be a water molecule. Another remarkable feature of this zinc enzyme is that while zinc binds more tightly than Mg (3 x vs. 1x low6), the Mg enzyme is fully active 1861. Thus, both the metal coordination geometry and the activity of the Mg enzyme is quite unusual for catalytic zinc sites in zinc enzymes. Recent mutagenic studies of the zinc ligands suggested that the metal li may be directly involved in catalysis 1871. Thus, both the E172Q and double mutant still bound zinc but the catalytic activity was decreased by lo5- and 1Os-fo1d, respectively. Crystallographic results of the enzyme coniplexed with a transition state analogue S-(~~hydro~y-~-~-iodophenylcarbamoyl)glutathione that mimics the enediolate intermediate that should form along the reaction pathway are consistent with this hypothesis [881. In this structure the two oxygen atoms of the ~y~roxycarbamoyl moiety displace two zinc-bound water molecules that are observed in a nontransition state complex. In addition, the 6 1 ~ 1 7 2carboxylate oxygen-Zn distance has increased from 2.0 t o 3.3 in the complex. The zinc ion is envisioned to play a Lewis acid role in catalysis by directly coordinating the enediol

A


903

LE 4 Protein Interface Zinc Sites"

Enzyme/protein

Class

Nitric oxide synthase I Bovine endothelial Human endothelial Human inducible Human glyoxalase I V E. coli signal transducing protein, PTS ILAG'" 11 I1 E. coli PTS IL4G'";glyeerol kinase Superantigen family Staphylococcus enterotoxin C2 (SEG2) Staphylococcus enterotoxin type A (SEA) Staphylococcus enterotoxin type A (SEA) Staphylococcus enterotoxin type D (SED) Staphybcoccus enterotoxin type D (SED) Streptococcus enterotoxin type C (SPEC) Streptococcus pyrogenic exotoxin A (Sped11 III Tonin crystals Human Psoriasin (SlOOA7) Shaw T1 tetramer Human interferon-a2B dimer Human interferon+ dimer S. aureus Toxic shock syndrome toxin-1 Human prolactin receptor/growth hormone

*Gand L;

L1

X

L2

4

QSP

4

*P

4

Qsp

65

Glup

14

Hi,

14

Hisg

3

His, His, His, His,

1 1 3 1 3 1 3 0 0 3 60 0

Y

L.?

L'I

z

34 27 37 37

GlU, iC) His, 33 His, 28 His 39 His CYS 26 Glu, His, Hisp His

are the second subunit or protein zinc ligands and 2 is the spacer for these ligands. See footnote in Table 1for other definitions.

Ref.

904

AULD

intermediate, and the freed zinc ligand Glu172 is proposed to facilitate proton transfer between the adjacent carbon atoms of the substrate "31. A novel Cys site is found in the nitric oxide synthase enzymes (Table 4). In endothelial nitric oxide synthase, eNOS or NOS-3, a zinc ion is found tetrahedrally coordinated to pairs of symmetry-related Cys residues near the bottom of the dimer interface [891 (Table 4). The Cys ligands, Cys96 and CyslOl, are part of a small threestranded antiparallel sheet that orientates the Cys ligands in the same direction directly across the antiparallel strands. The zinc site is further stabilized by H bonds between the Gys ligands and the backbone amide NH of Leu102 and Gly103 as well as bond of the ainide NH of CyslOl to the carbonyl of Asn468. Thc zinc is positioned equidistant from each heme (21.6 A) and each tetrahydrobiopterin, H4R (12 A). Serl04, two amino acids removed from one of the Gys ligands, H-bonds directly to the pterin side chain hydroxyl group. The metal center is believed to act in a structural capacity to maintain the integrity of the pterin binding site. It is also centered in the most electropositive region of eNOS. It could therefore provide a binding site for the electronegative reductase domain. In addition, if one of the Cys ligands has nucleophilic capacity it could undergo S-nitrosylation [go]. A precedent for this is the DNA repair protein E. coli Ada in which a zinc-bound Cys can be methylated irreversibly in the DNA complex [911. The nitrosylated Cys might then release zinc which may be controlled by the redox status in situ [921. The crystal structure of the E. coli expressed human endothethial, eNOS, and the inducible form iNOS or NOS4 also have the same zinc binding site [93]. An independent study of human iNOS expressed in E. coEi found that the zinc site was not present after refolding [941, similar to an earlier study on the murine inducible, iNOS or NOS-2, where a disulfide was found [951. However, in the presence of zinc and reducing agents the Zn(Cys)* site formed readily [94]. These Cys residues are conserved in 20 mammalian e, i, and n NOS enzymes, suggesting that the site may occur in all forms of NOS 1891. A tetrahedral zinc binding site is observed in the crystal form of the E. coli signal-transducing protein IIAG1', PTS II.AGlc, [961 and in its complex with glycerol kinase [971. In both cases, the signal-transducing protein supplies two His residues with a spacer arm of 14 and the third Glu ligand comes from either a neighboring molecule in the crystal or the glycerol kinase. The fourth ligand in both cases is a water molecule (Table 4). The site is said to be geometrically equivalent to the zinc binding site in thermolysin (Table 1).If this site contained a suitable acid-base catalyst it might display hydrolytic activity. Although the biochemical effect of 0.01 mNI zinc on the inhibition of glycerol kinase by PTS IIAG1' has been demonstrated, the physiological role for zinc ions in PTS and protein interactions remains to be established [961. 2.4.2. Zinc Binding Sites in Superantigens

Staphylococcal enterotoxins belong to a family of proteins termed superantigens because they form complexes with class I1 MHC molecules, enabling them to activate a number of T-cell lymphocytes f.981.Zinc has been shown to be an important com-


905

ponent to their function. Thus binding of enterotoxin A and enterotoxin E to HLA-DR is completely abolished by low concentrations of EDTA [981. This binding is coinpletely restored by the addition of a 2 pM excess of Zn2+but not by Ca", M g + , or other metal ions. The Cd substituted Staphylococcus aureus enterotoxin type A (SEA) has a metal binding site composed of Serl, Ifis187, His225 and Asp227 1991. The amino acids involved in binding the Cd ion are arranged in octahedral coordination geometry. An approximate square plane is formed from the ol-amino group and yO of Serl, and N of His187 and His225. Asp227 ligates from beneath the plane and a water from above. Crystals o f the zinc containing protein revealed Zn binding between two molecules in an asymmetrical unit. His187, His225, and Asp227 bind to zinc in a tetrahedral geometry [loo]. The fourth ligand is His61 from a neighboring molecule in one SEA molecule and water in the other site. Mutation o f either His225 or Asp227 to Ala results in more than 10,000-foldreduced binding of MHC class I1 molecules while the His187Ala mutation only leads to a 10-fold reduction [loll. Modification of ligands in the short spacer would be expected to greatly reduce or eliminate zinc binding. Zinc binds in solution to SEA with a Kr, of 0.3 pM [lOOl. The zinc binding site seen in the crystals of SEA could indicate this protein can form dimers in the presence of zinc. However, gel permeation chromatography in the presence of Zn" concentrations as high as 100 pM showed no evidence of dimer formation [loo]. In contrast, the same experiment performed with Staphylococcus aureus enterotoxin type D (SED) yielded dimers at a 1 pM zinc concentration. SED crystallizes as a Znz'-dependent dimer with two high-affinity zinc sites located between the C-terminal p sheets of the two monomers C1023. Each zinc is tetrahedrally coordinated by His118 from one SED molecule and Asp182, His220, and Asp222 from the other. A similar zinc binding site is observed in the superantigen SEC DO31 (Table 4). Another zinc site is formed in dimers of SEA when the crystals are soaked with 10 mM ZnClz [1041, which corresponds to the zinc binding site in the solvent-exposed region between the interface of two domains of SECZ [1051. The zinc ion is coordinated to Asp83, HisllS, and His122 of one molecule and Asp9 of an adjacent SEC2 molecule (Table 4).Atomic absorption spectroscopy indicates that zinc also binds in solution. In SEA two of the ligands are identical to SECB, Asp86, and Hisll4, while a water molecule and Glu 39 replaces the other ligands [1041. A second zinc site is also found in SED Cl021. This time the zinc is bound at the interface through His8 and Glu12 of one monomer and His109 and Lysll3 of another. The zinc site in SEC2 lies within the binding region of the MHC class I1 molecules and thus has been suggested to be important to complex formation [105]. A similar type of zinc binding site is found in the superantigen streptococcal pyrogenic exotoxin A, SpeAl 11061 (Table 4). In this case, it was demonstrated that the zinc site was present when the crystals were grown at pH 7.9 but not at pH 5.7 where the zinc is expected not to bind as well. Possible modes of binding MHC class II and T-cell receptors to SpeAl were proposed 11061. It should be noted that a zinc binding site, similar to the superantigen sites, was observed in crystals of rat submaxillary gland tonin that had been grown in zinc containing mother liquor [1071. The zinc binds to the catalytic His57, His97, and

906

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Wis99 of one molecule and Glu148 of an adjacent one. The presence of zinc was reported to inhibit the enzyme at pH 6.5. The structure of the zinc binding sites in the dimeric form of psoriasin (SlOOA7) also contains three His and one Asp ligand [lOSl. This 22.7-kDa homodimeric protein belongs to the SlOO class of calcium-binding EF-hand proteins. Two zinc binding sites occur per dimer through the formation of a tetrahedral site consisting of the N-terminal His17 and Asp24 of one monomer is86 and His90 of the other. This binding site is apparently weak since it is reported to have a dissociation constant of 100 pM.

2.4.3. Other Protein Interface Zinc Binding Sites

A highly conserved zinc protein interface site occurs in the N-terminal, cytoplasmic tetramerization domain (Tl) of voltage-gated Kc channels [t09]. The crystal structure of the Shaw T1 tetramers reveals a four-layered protein scaffolding,Within layer 4 on the proposed rnombrane side of the tetrarner there are four zinc ions coordinated is and two Cys from one monomer and one Cys from another. The zinc is tetrahedrally coordinated by CyslO2 and CyslO3 from layer 4 and His75 from layer 3 of one monomer and Cys81 from layer 3 of another monomer. This site (CCx2,Cx& of one moner) is conserved for 62 members of the Shaw, Shab, and Shal T1 domain sequences but not for the Shaker T1 domain [log]. The physiological function of this zinc site is unknown. Some of the protein interface zinc sites have been obtained from solutions containing concentrations of zinc of about 50 mM, which may induce a zinc binding site. Thus, ciystals of the human interferon-ann (huIFN-a2B)are obtained by the hanging drop method containing 40 mM zinc acetate [ l l O I. The most extensive interactions in the dimers found in the crystal occur in the vicinity of the zinc binding site. This site is formed from adjacent Glu residues 41 and 42 located on a 310 helix. A distorted tetrahedral zinc coordination sphere is completed by the identical glutamates from a twofold symmetry-related molecule. The biological role of such a dimer is unknown. Gel filtration experiments indicate that huIFN-a2Bis a monomer up to 50 pM and the presence of 1mM Zn does not shift the equilibrium toward the dimer Ill01. The asymmetrical dimer observed for human interferon f3 (huIFN-f3)is obtained under conditions where no zinc is added to the crystallization buffer [lll].His ligands 93 and 97 reside on an CI helix in molecule B. Molecule A provides Hisl21, and a water completes this four-coordinate zinc site. However, gel permeation experiments in the presence of zinc did not show any evidence of a dimer in solution. The dimers form in crystal structhe crystal on contact surfaces opposite to those found in the IFN-a2~ ture. The residue Glu43, which H-bonds to zinc ligand Hisl21, has been identified as part of the binding site of monoclonal antibodies [ll21. The presence of zinc might thereEore modulate this interaction. Although zinc is not required for biological activity of toxic shock syndrome toxin-1 (TSST-l), it does potentiate its mitogenicity at submicromolar concentrations 11131. Crystals grown in the presence of 0.2 M zinc acetate reveal a Zn site at the interface formed from His74 and His135 of neighboring molecules (Table 4).

907

ZINC SITES IN ~ ~ T A L L ~ ~ N Z Y M E S

3. T ~ A ~ K I NZINC G

The original definition of a metalloprotein required the metals to be bound so firmly that they are not removed from theprotein by the isolationprocedure [1141. Inherent to these studies was the determination of the metal type and stoichiometry by some analytical means. The metal was then removed and the apoenzyme examined functionally to see if the absence of the metal impairs the function of the protein and addition of the metal to the apoenzyme restores the function. m i l e this definition still is reasonable, the modern advances in sequencing and structural analyses have led to identification o f potential zinc binding sites without ever determining if the enzyme needs zinc for function. The availability of a putative zinc-binding motif from a structural reference, combined with a family of sequence-related proteins, can lead to prediction of the need €or zinc in the function of a protein where this was never suspected (see below).

termination of the Number of Zinc Enzymes Information on zinc binding sites in metdoenzymes and related proteins has become increasingly available as the interest in how zinc affects biological function has accelerated. This is particularly apparent over the last decade (Fig. 2 ) . In 1989 there were only 12 reported structures of zinc binding sites in enzymes. At the time of this

r FIG. 2. Time course o f the determination of zinc binding sites in metalloenzymes.

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perspective there are about 12 dozen structures. In addition, the discovery of RNAand ~ ~ A - b i n d i nproteins g that contained zinc 11151 led to the designation of such binding sites as “zinc fingers” [1161. This picturesque term alone led to the identification of many zinc binding sites in proteins whose function was assumed to be DNA binding but may be involved in protein-protein interactions 11171 or have yet undiscovered functions. In 1992 the number of zinc enzymes, based on the original criteria of what constituted a zinc enzyme, was estimated to be about 300 [ZI.If one now broadens the definition of zinc enzymes to include those proteins that likely will bind zinc due to the presence of zinc-binding motifs found in zinc reference sites, the number of zinc enzymes will be in the thousands. This is due to the explosion of information gained from modern DNA sequencing techniques. Thus, the number of zinc proteases based on this criteria has increased from about 100 in 1989 to 1518 [1181 in November 1999 (Fig. 3). Since the number of known zinc binding sites in zinc proteases is about one-half of all the known class I11 hydrolases (Table 1) and the number of hydrolase sites represents about one-half of the total number of zinc reference sites, the total number of zinc enzymes is likely of the order of 6000 encompassing different species of all phyla. As the number of sequenced genomes increases this number also increases.

FIG. 3. Time course of the discovery or zinc peptidases.


3.2.

909

Zinc Binding Site Motifs as a Means for the Discovery of New Zinc Families

Zinc binding site motifs can be highly conserved, not only in the identity of the ligands and their spacing but in the neighboring amino acids in the linear sequence 171. This leads to the formation of a family of zinc enzymes that may have a similarity in their overall primary structure and some function in common. 3.2.1. AminopeptidasesiLTAd Hydrolase

Even in the first few structures that identified some of the common characteristics of zinc binding sites in enzymes such families could be formed 171. Thus, the neutral protease Bacillus thermoproteolyticus or thermolysin was the head of a f m g y of bacterial neutral proteases that included the B. cereus, B. slearothermophilius, B. subtilis, and B. amyloliquefuciens which had a high degree of overall sequence identity. The crystallographic structure for the B. cereus enzyme [1193 is very similar to that for thermolysin 11201. The ligand binding site for both of them is composed of His142 and His146 and Glu 166 (Fig. 4). The amino acid residues Asp170 and Asn165 H-bond to the His residues of the short spacer and Glu143 is believed to be a general acid-base catalyst. This is therefore a very compact catalytic zinc site. When one compares this group of enzymes to the Staphylococcus aureus and Pseudomanas aeruginosa neutral proteases the overall sequence similarity drops to 49% and 28%, respectively, but the same catalytic zinc site is confirmed by crystallographic studies [121,122]. The same metal binding site appears t o be conserved in the mammalian neutral endoproteases and the mono zinc minopeptidases (Fig. 4 L71), but this has yet to be confirmed by structure determinations.In the case of the aminopeptidases it would appear that new secondary interactions must stabilize the zinc binding site since the Asp residue is no longer always conserved. The presence of an amino acid segment in leukotriene & hydrolase that is homologous to the putative zinc binding domain of intestinal aminopeptidase predicted the presence of zinc and a hitherto unrecognized aminopeptidase activity in LTA, hydrolase [71, even when the primary structures of these enzymes are only about 20% conserved. On this basis, the leukotriene & hydrolase was shown to contain 1g-atom zinc/mol protein, to exhibit aminopeptidase activity and t o be inhibited by bestatin and captopril, specific peptidase inhibitors [123,1241. In addition, mutagenic replacement of the proposed LT& hydrolase zinc ligands causes complete loss of zinc and abolishes its activity toward both types of substrates, suggesting that these residues are the likely zinc ligands 11251. The fact that aminopeptidase inhibitors also inhibit the LTA, hydrolase activity leads to new avenues for drug development based on inhibition of these enzymes. It also highlights the difficulty in designing specific inhibitors for metalloenzymes since the primary target is often the metal itself.

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910

rabbit ne~tralend5pr5tease rat n@utralend5pro~eas~

G F D

N T L G

G F D

N T L G

~a~ a~inopeptidaseN

Q W F

L W L N

Q W F

L S L K

Q W F

L W L N

S W T

F W L N

FIG. 4. Catalytic zinc binding site of the thermolysin family as a reference for the aminopeptidase family and LT& hydsolase. Only a few representatives are given for the endoprotease and aminopeptidase families.

3.2.2. Astacin FamilyJMatrixMetalloproteinases

These zinc classifications can also predict new zinc binding sites. In 1988 Astacus protease, or astacin as it is now called, was identified as a zinc protease [126]. The , in one amino acid segment suggested that this presence of two histidines, zinc site could be similar to that of thermolysin (Fig. 4).However, it was not identical since no Glu was found 19 amino acids removed from the proposed second His ligand. Two other zinc proteases, Serratia protease and fibroblast collagenase, in addition to Astacus protease, did have a very similar clustering of potential zinc ligands, (1261, By 1990 the search o€ protein and translated gene banks with this potential zinc binding site signature retrieved a small number of proteins, all of which were believed to be zinc proteases [1271. These included Serratia protease, protease €3, the snake venom protease Ht-d, and all of the collagenases, stromelysins, and gelatinases sequenced at that time. The recognition of the homology of both mouse kidney and human intestine (then known as PPH hydrolase) meprin to Astacus protease led in fact to the naming of the immediate astacin family of zinc proteases [128]. By 1992 the use of this putative zinc signature led to the identification of 33 proteases that defined four major groups of homologous proteins [1291. This discovery was rapidly followed by the X-ray crystallographic structure of astacin that confirmed the prediction of this new catalytic zinc site [130]. Within 2 years the

ZINC SITES IN ~ E T A L L O ~ N Z Y ~ E S

91 1

structures of a member of each of the astacin subfamilies was determined, i.e., matrix metalloproteinase-1 [131], the snake venom protease, adamalysin I1 11321, and the alkaline protease of P. aeruginosa I281. Today these groupings are too large to list in one figure. As of November 1999, the MEROPS database file listed 80 members of the immediate astacin family, 115 members of the matrix metalloproteinases, 148 members of the snake venom and ADAM families, and 21 members of‘ the serralysin subfamily [llSl. Remarkably, these proteases not only have the same catalytic zinc binding site but also are topologically “homologous” even though they would not have been considered homologous based on their primary sequences. The zinc binding site in this superfamily is the smallest site known since all zinc ligands (His x3 H i s y5 His) (Table 1) and the presumed catalytic glutamate residue are supplied from an 11amino-acid segment. 3.2-3. Cytidine Deaminases

Cytidine deaminase from E. coli i s a dimer of identical subunits (31.4 kDa) each containing a single zinc atom coordinated to three ligands: His102, Cys129, Cys132 11331. Other members of this family that indicate the s m e binding site may exist as judged from their primary structure are the deaminases from H. in,flueazae, Arabidopsis thaliana (II), and the mRNA editing enzyme AIPOBEC-1 (1341. However, the cytidine deaminase from B. subtiEis is a tetrameric enzyme of identical subunits (14.8 kDa) that contains 1mol of zinc per mol of enzyme subunit. at present 12 members of the tetrameric class of cytidine deaminases. In all of these protein sequences there is a Cys in place of the His found in the dimeric cytidine deaminase crystal structure 2134,1351 (Fig. 5). The conservation of amino acid residues around the ligands is quite good for both the dimeric and tetrameric proteins. In particular, the Ala and Glu residues C-terminal to the third His ligand of the dimeric cytidine deaminase is conserved in the postulated third Cys ligand sequence for the tetrameric enzymes. Proof of this postulate awaits a three-dimensional structure determination. 3.2.4. Common Motifs in P-Lactamases, Glyoxalase 11, and Arylsulfatases

A recent protein database search using the zinc-binding motif T/S retrieved three families of enzymes; 7 p-lactamases, 14 glyoxalase 11, and 6 prokaryotic arylsulfatases [1361. These enzymes perform diverse tasks. The metallo-p-lactamasesare zinc-dependent hydrolases. Glyoxalase 11 is the second of two enzymes in the glyoxalase pathway. It is a thiolesterase that catalyzes the hydrolysis of S-u-lactoylglutathione to form glutathione and D-lactic acid 11371. The sulfatases hydrolyze sulfate-ester bonds in a wide variety of structurally different compounds. The eukaryotic sulfatase, human arylsulfatase A, contains a magnesium in its active site and is structurally related to the zinc-containing alkaline phosphatase E1381, and both have been suggested to be part of a superfamily of metalloenzymes [1391. Much less is known about the structural nature of the prokaroytic arylsulfatases [1361. Based

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imeric c ~ i d i n edeaminase

Arabidopis thaliana I1 Tetrarneric cytidine dearninase S P

S P

P A P S P

S P M P S P T P T P S P

FIG. 5. Comparison of catalytic zinc sites in dimeric and tetrameric cytidine deaminases 11341.

on the possibility of a common cocatalytic zinc site, the authors concluded that the catalytic mechanisms of these three classes of enzymes arose through convergent evolution. Since only the Bacillus cereus [731 and Bacteroides fragilis p-laetamases 1761 had published three-dimensional structures at the time the sequence comparison was made, the authors examined these sequences looking for a common cocatalytic zinc site like that ofthe p-lactamases. The first two His residues in the sequence used for the search bind to the catalytic zinc in the p-lactamase family (Table 1) while the Asp binds to a second zinc, Zn", in a proposed cocatalytic site (Table 3). Fib,rure 6 shows a short-hand version of their results listing the major amino acids that appear in the sequence neighboring the putative zinc-binding ligands. In terms of the original zinc-bindingmtifused to search, T/S)lw[rrD, there is a reasonable degree of equivalence in the three classes of enzymes for this region of the primary stnxdure. Thus, the two hydrophobic amino acids and a Thr that are Nterminal to the first His ligand in the catalytic site of the p-lactamases also occur in the glyoxalase I1 and arylsulfatase family. The C-terminal side of these peptides shows a somewhat less identical type of set of amino acids. There is a preponderance of Gly residues in the p-lactamase and glyoxalase I1 families while more hydrophobic amino acids occur in the arylsulfatase fmily. A major difference occurs C-terminal to the Asp ligand where the 6-lactamase family mainly has Arg while the glyoxalase I1 and arylsulfatase families have His conserved in all of their sequences, This is particularly interesting in view of the recent human glyoxalase I1 structure [137] where this His

913


zn 99

Zn 101

Zn* 103

56 Zn

58 59 Zn* Zn*

PLactamase

Human glyoxalase n

54 Zn Arylsulfatase

GS

L5

D6

H6

L, F3

Zn 162

Zn* 181

p-Lactamme

T6 E3

Human glyoxalase I1

6'

4

D6

G6

R3

' 5

' 6

L4 3'

c,l

G13R11

110 Zn

Arylsulfatase

L4 L4G6

H, E, As TS

R2

y, H,

ss T,

P-Lactamase Human glyoxalase fI

Arylsdfatase

P-Lactamase

Human glyoxaiaseI1

No amino acid bridging ligand bridging ligand Asp134

FIG, 6. Comparison of the zinc ligands in the p-lactamase family with those proposed for the glyoxdase I1 and prokaryotic arylsulfatases. Data are taken from [136]. Only the major amino acids found in the sequence are given.

along with the Asp residue is a ligand to the Zn" atom in its cocatalytic site. The other two His residues bind to the first zinc as in the P-lactamase enzymes. Three other peptide segments were compared for the three families again searching for correspondence to the p-lactamase family. His223 and Cysl81 bind to the Zn" site in the p-lactamase family and His162 to the catalytic zinc. It is now known that two of these amino acids bind to the same zinc sites of the glyoxalase I1 enzymes (Fig. 6). HisllO of human glyoxalase I1 binds to the first zinc and His173 to Zn" [781. The conservation o f Gly and Thr residues djacent to the HisllO and His162 ligands of the glyoxalase and P-lactamase families are also consistent with these zinc sites having similar structures (Fig. 6). However, the two ligand sites do differ beyond this region. The arylsulfatase family also has a high conservation of the Gly residue and an inversion in the positions of the small amino acid and the carboxylate containing residues on the C-terminal side. There is not quite as good a corre-

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914

spondence of the His223 @-lactamaseland His173 (glyoxalase 11)ligand sites. Outside of the fact that there is a Gly and a hydrophobic position conserved N-terminal to the His ligand there is not a lot of similarity between these peptides for the p-lactamase and glyoxalase 11families, making the assignment of this residue as a ligand solely on the basis of primary structure difficult. The arylsulfatase family differs even more. The Cys ligand seen in the j3-lactamase family is only conserved in 11of the 14 glyoxalase 11sequences, in agreement with this residue not being a zinc ligand in the glyoxalase I1 enzyme family. The arylsulfatase f ' d y has a conserved His ligand at this position but the adjacent residues differ in chemical properties from the j3-lactamase family. In addition, it should be noted that Stenotrophomonas (Xanthomonas) maltophilia P-lactamase has a His at this position but does not use this residue to bind to Zn" [781. Instead it uses a His residue adjacent to the Asp ligand to the Zn" site, His104. Since this residue is highly conserved in the arylsulfatase family (Fig. 6), this His may be a ligand to the Zn" atom in a cocatalytic site for the arylsulfatase family in a manner similar to that of the S. maZtophiLia j3-lactamase [781. Asp134 serves as the bridging ligand between the two zinc sites of the glyoxalase I1 family. This residue comes from a highly conserved region of this family of zinc enzymes 11371, The postulated cocatalytic site of the f3-lactamases does not use an amino acid to bridge the two sites; thus, no attempt was made to look for this residue in the glyoxalase I1 and arylsulfatase families [1361. The authors use of a particular zinc-binding motif has been highly successful in both predicting and properly placing five of the seven protein ligands for the cocatalytic site of the glyoxalase I1 family. There is a reasonable expectation that the arylsulfatase family may also use many of these same ligands, although based on new information gained from the structure of human glyoxalase the conserved His in the arylsulfatases that is equivalent to the is59 of glyoxalase I1 should be considered as a ligand to the Zn" site. In addition?a search for the presence of the equivalent to the bridging Asp134 would be useful. An earlier study based on a comparison of fewer glyoxalase I1 and p-lactamase sequences successfully predicted six of the seven ligands correctly for the glyoxalase I1 family, including the second Asp based on the analogy to the B. fragilis P-lactamase crystal structure 11401. The authors also demonstrated that Arabidopsis thaliana glyoxalase I1 contained two bound zinc atoms. However, they did not envision Asp134 as a bridging ligand nor did they suggest His59 as a ligand to either site. Nevertheless both of the above studies demonstrate the power of comparative sequence studies.

mbers for the Metallopeptidase Family ~etalloproteaseshave been arranged into 14 clans that break down into 54 families or subfamilies [141,1421. One of the clans is listed as metallopeptidases not yet assigned. With the number of sequences contir;uing to increase rapidly this gives incentive to find new families of zinc proteases or confirm assignments already proposed.

ZINC SITES IN M E T ~ L L O ~ ~ Z Y M E S

915

A novel putative zinc protease, S2P, was recently cloned from human and Chinese hamster ovary cells that is able to cleave sterol-regulatory element binding proteins (SREBPs) [1431. The SREBPs are membrane-bound transcription factors attached to the endoplasmic reticulum membranes in a hairpin fashion [1441. In order to activate transcription, the N-terminal domain of the SREBP must be released from the membrane so that it can enter the nucleus. This release is accomplished in a two-step proteolytic event that is regulated by sterols. The protein contains the zinc-binding motif HEIGH from position 170 to 174, and mutagenesis studies suggest that Asp467 is the third ligand 11451. All of these ligands are in hydrophobic sequences, suggesting that the active site may be located in the membrane where it would cleave its target, a Leu-Cys bond in the first transmembrane helix of SREBP. Computer database search and alignment tools using the S2P protein as a reference have retrieved 28 proteins that have been arranged in 6 subfamilies [146]. Two of the regions most highly conserved is the thermolysin zinc-binding zsG/Al:3and the region of the postulated third Asp ligand 28G15.Arnino acid residues at the positions where total conservation was not achieved were generally very similar in chemical nature and the sequences were generally hydrophobic. At present the only metalloprotease catalytic zinc site that has an Asp as the third ligand is Streptimyces caespitosus endopeptidase, which has a short spacer of 5 amino acids (Table 1E1471). If confirmed, the S2P family would have one of the longest spacer arms for the third ligand, i.e., nearly 300 amino acids. Another smaller family of metalloendopeptidases has also been suggested to have a third Asp ligand 11481. Sequence alignment of 6 proteins and mutagenesis studies of deuterolysin suggest His128, His132, and Asp164 as the catalytic zinc ligands.

utagenesis as a Means of Detecting New Zinc Binding Sites With the knowledge gained on the types of ligands and spacing characteristics of zinc sites [7,611 it is not unreasonable to mutate suspected zinc-binding ligands in a protein known to depend on zinc for function and use functional activity, metal binding, or binding parameters of inhibitors thought to bind to the metal site as a means to assess the effect of the mutation. This approach has been undertaken several times using a variety of the criteria given above, and in some cases structures are now available to evaluate the results of the mutagenesis experiments. The binding affinity for human growth hormone (hGH), for the extracellular domain of the human prolactin receptor (PRL), is increased 8000-fold in the presence of 50 pM Zn2+ [149]. In the course of this study four residues from the growth hormone were mutated (HislSAla, His2lAla and Glu174Ala, Asp171Ala) and their effect on the dissociation constant, K, for the binding of hGH to PRL determined. The Aspl7lAla mutation had no effect on K,, while the other three mutations weakened this constant by 135-,91-, and 356-fold, respectively. These three residues were therefore chosen to be ligands to the zinc site. His18 and His21 were believed to be on an Q!

916

AULD

helix based on modeling of the hGH sequence to the structure for the porcine GH. The spacer of two would be unusual for such a support structure (Table 1).The fourth ligand to the zinc was believed to be His188 from the prolactin receptor due to a >2000 fold increase in K,. upon its mutating to Ala 11491. A crystallographic study of this complex indicates that the growth hormone supplies two ligands, His18 and 611.1174, while the prolactin receptor supplies the other two, Asp187 and His188 11501. His21 of the growth hormone implicated by mutagenic studies 11493 is not a ligand but serves to orientate the Glu174 ligand Il501. The physiological significance of this zinc binding site is questionable in view of the need of free zinc concentrations in the range 10-100 @!I. On the basis of site-directed mutagenesis studies, two models of zinc binding were proposed for Pseudomonas diminuta phosphotriestorase. One predicted His55 is 57 as ligands to one zinc site and His254 and His257 as ligands to the socond zinc, with His230 as a bridging ligand between the sites 11511. The second model proposed His55 and His57 and His201 as ligands to a catalytic metal binding site and His254 and His257 as ligands to a structural metal binding site [152]. Cys residues were excluded from being ligands. These studies did not have the benefit of multiple sequence alignments to aid in the identification of these residues. The Xray structure of the cadmium-substituted enzyme revealed a number of surprising features 11531. The carbamylated Lys169 bridging ligand between the two cadmium sites is a unique zinc-bridging ligand although functionally it resembles the carboxylate-containing Glu and Asp residues normally found in cocatalytic sites I81 (Table 3). This type of ligation is found in the cocatatalytic site of nickel urease from Klebsiella aerogenes C1541. His55 and His57, along with Lys169, Asp301, and a water molecule, make up the first cadmium binding site, whereas the more solventexposed site uses His201, Wis230, Lys169 and two water molecules as ligands. His254 and His257 are 5.0 A and 7 7 A from the metal cluster and not ligands to the zinc as was anticipated by both site-directed mutagenesis studies. However, the imidazole ring of His230 forms a stacking interaction with the imidazole of His254 and this in turn participates in a similar interaction with His257 11531. These interactions could be important to the formation and stabilization of the metal cluster. Mutagenesis studies of the p-lactamase from B. frugilis led the authors to conclude that a second zinc binding site in this enzyme was composed of four Asp ligands: Aspf31, Asp90, Asp152, and Asp183 11551. Only one of these proposed ligands, Asp90, is conserved in the five sequences available [731, indicating that the postulated zinc binding site would not be part o f the general P-lactamase structure-function relationships. The X-ray structure of this enzyme shows that this second zinc is bound to the side chains of Asp90, HisZl0,and Cys168 residues [76]. Only Asp90, the one conserved Asp, of the four postulated Asp residues directly ligates the zinc. The structure reveals that the carboxylate of one of the other predicted Asp hgands, Asp183, makes a hydrogen bond to a His ligand of the first zinc site while another buried Asp, Asp152, has no direct contact but likely aids in positioning a Trp residue near the three His ligands to the first zinc site. The mutagenesis studies therefore revealed the importance of these residues to structure


917

and/or function of the B. fragilis enzyme but led to the wrong conclusion in identifying them as direct ligands to the zinc. A combination of sited-directed mutagenesis and homology searches using a suspected zinc-binding motif was used in the search for the metal binding site for Wad,the u-Ala-D-Ala dipeptidase of vancomycin-resistant pathogenic Enterococcus [156]. This led to the proposal of Hisl16, Asp123, and His184 as the ligands to the catalytic zinc site. Sequence searches using the suspected zinc binding motif found that the S. albus G dipeptidase, the Sonic hedgehog protein, r proteins also contained this potential zinc-binding motif. The first structural studies of the S. albus G dipeptidase suggested Hisl54, His194, and as ligands to the zinc [1571. However the Brookhaven Protein Data Base entry by these authors as of 1996 indicates that the ligands are His154, Aspl61, and His197 in agreement with the assignment of the S. albus G and VanX dipeptidases to the same zinc peplidase family [1561. On the basis ofthis X-ray structure the authors further suggested that Glu181 could be the catalytic base for VanX. While an X-ray structure has been reported for Sonic hedgehog revealing an unsuspected zinc binding site I 1581 no hydrolytic function is known for this protein. X-ray structural analysis of Enterococcus faeciurn D-Ala-u-Ala dipeptidase 11591 reveals a catalytic zinc binding site composed of His116, Asp123, His184, and a water molecule (Table 1)that is Hbonded to Glu181, the proposed base catalyst L156j. In summary, whenever possible, the combined use of sequence alignments based on suspected zinc binding ligands in combination with site-directed mutagenesis should give the best chance of correctly predicting zinc binding sites in proteins.

4.

STRUCTURE-FU~CTIONRELATIONSHIPS

4.1. Inactivation of Metalloenzyme Catalysis Since the zinc-bound water is one of the most critical components of the catalytic zinc site, there are three ways that one can envision inhibiting the metalloenzyme. Thus, one can bind a second zinc ion to the catalytic metal-bound water, displace the water by an inhibitor designed to bind to the zinc, or remove the zinc (Fig. 7). 4.1.1. Inhibition of Metalloenzyme Catalysis by Zinc

Normally when an investigator suspects that an enzyme is a metalloenzyme attempts are made to inhibit the enzyme with a chelator and then try to reverse the inhibition by adding excess zinc or another metal ion. If the excess zinc ion doesn’t reverse the inhibition the wrong conclusion is sometimes reached that the enzyme didn’t depend on zinc for activity. Over the years there has accumulated much anecdotal information that zinc inhibits zinc enzymes, but how it does this has not been examined often. The most thorough study of its mechanism of inhibition has been done on CPD A.

918

AULD

Remove Zinc

FIG. 7. Inhibition of zinc enzymes.

Within a few years of its discovery CPD A was demonstrated to bind a second zinc in range 7-10 by equilibrium dialysis [1601. Kinetic studies show that zinc and lead both competitively inhibit peptidase catalysis in the micrornolar range [161]. The apparent inhibition constant for zinc is 5.2 pM at pH 8. This inhibition is strongly pH-dependent (Fig. 8). Over the pH range 510 zinc is distributed among five protonation states, Zn2+ cation, Zn-mono-, di-, tri-, and tetrahydroxide. The inhibition pattern follows closely the formation curve for the zinc monohydroxide species for the pH range 6.5-8. In this pH region the active form of the enzyme, EH, predominates. Above 9 inhibition decreases as the zinc monohydroxide species is converted to its higher di-, tri-, and tetrahydroxide forms. In the pH region below 6.5, the strength of the zinc inhibition is suppressed below the level

6

7

1

PH FIG. 8. pH dependence of zinc inhibition of carboxypeptidase A. (From [161].)

919


expected solely for the formation of the Zn monohydroxide species. However, this is the pH region in which the active enzyme form EH i s converted to EH2 [162]. The experimental data fits very nicely to a mechanistic scheme where inhibition occurs through the binding of the zinc monohydroxide species to the active EH species of the enzyme -independent constant for this zinc inhibition is 0.71 JAM.The derived pKa of 6 for the inhibition studies agrees with the corresponding value obtained in peptide hydrolysis experiments for the group, EH2, whose ionization leads to formation of the catalytically active form of the enzyme 11621. The ionizable ligand, EH2,is assigned to the 61~270,since chemical modification decreases of this residue with CMC (l-cyclohe~l-3-(2-mo~holinoethyl)carbodiimide) the binding affinity of CPD A for zinc and lead by more than 60- and 200-fold, respectively [1631. A bridging interaction between the Glu270-coordinated zinc hydroxide is implicated by the ability of Zn and Pb to induce a marked increase in the 560nm absorbance of the Co enzyme within 20 ms of mixing lo4 M Zn or Pb with 10* M cobalt enzyme E1631. Such an increase in absorbance is observed when azide displaces the catalytic metal-bound water 11641. In the case of zinc this species is transient since the stability constant for the zinc for the catalytic metal binding site is more than 300-fold greater than that of the cobalt enzyme. However, in contrast to the Zn ion, Pb does not displace the catalytic cobalt ion, and the mixed dimetallic CoCPD PbOH' complex is stable for days. Based on spectrokinetic and chemical modification studies, a binding model was proposed to account for. the metal ion inhibition (Fig. 10) E1611. The zinc monohydroxide ion binds to the ionized carboxylate of Glu270 and displaces the zinc-bound water. This bridging interaction explains both the loss of metal binding upon chemical modification of 6 1 ~ 2 7 0or lowering pH and the increase in absorbance in the Go enzyme upon binding of Pb or a Zn ion. Removal of the Glu270 carboxylate through protonation or chemical modification should decrease inhibitory metal binding.

ydroxide Complex EH*ZnOH+ Ki,,im Zn

*+==

9.05

11

7x?07M 9.75

ZnOH' ;;t Zn(OH),

-

-l.0l

- 5 .m Zn(0H)j -Zn(OH):-

FIG. 9. Kinetic scheme for zinc inhibition of carboxypeptidase A.

920

AUL

~ydroxide Bridge

X"

Elu 270 -

co

His 69

Zn2+

O,C

- Glu 72

His 196

Inhibitory Metal

Catalytic Metal

FIG. 10. Schematic of inhibitory zinc binding site in carboxypeptidase A. (Adapted from [1631.)

~ e p l a c e ~ eof n tthe catalytic cobalt-bound water molecule with the hydroxide anion should lead to an increase in visible absorbance (as is observed when azide binds to the metd). A water molecule and a chloride anion complete the tetrahedral zincinhibitory site since chloride enhances the inhibition by zinc and lead. The for the charge-neutral Zn(0W)Cl and Pb(0H)Cl are both about 5 x M [161]. If the inhibitory metal monohydroxide complex is charge-neutralized by a multidentatr? ligand that can bind to other active site residues, an even lower inhibition constant can be anticipated. Such inhibitory complexes could be very important to regulatory processes andlor toxicological processes involving Pb or other heavy metals with zinc enzymes. This model of zinc inhibition proposed for carboxypeptidase [161,163] was recently con$rmed in its entirety by two different crystallography groups [31,1651. Thus, a 1.7-A structure of the crystalline carboxypeptidase A that had been dialyzed vs. 0.33 mM ZnClz revealed a second zinc bound in a distorted tetrahedron composed of the carboxylate of Glu270, a water molecule, a chloride ion, and a hydroxide ion his hydroxide forms a 114" angular bridge between the inhibitory and the zinc ions. The inhibitory zinc holds the hydroxide at the same location as the catalytic zinc-bound water molecule in the uninhibited enzyme, Superposition of the benzylsuccinate inhibitory complex on the ZnOH+-inhibited complex demonstrates that the carbonyl group of the inhibitor and the i n h i b i t o ~zinc ion are almost the same, indicating that the second zinc ion should interfere with proper positioning of the substrate [165]. This is consistent with the competitive inhibition observed with cond zinc binding site was also observed in crystals of themolysin s have been compared with those for c a r b o ~ e p t i d a s A e E1651. The atdytic zinc distance is 3.2 A for thermolysin and 3.3 A for carboxypeptidase A. A waterhydroxide bridges the two sites with a similar geometry: 116" in i o ~ is thermolysin and 114"in carboxypeptidaseA. In thermolysin the ~ o o ~ d i i l a tsphere completed by Tyr157, is231, and one of the carboxylate oxygens ofGlu166. The latter is ~ a ~ i c u l a r interesting ly because it is the third ligand to the catalytic zinc as well. This inhibitor site therefore resembles the cocatalytic sites seen in many hydrolytic

92f


enzymes (Table 3). It therefore means that finding multiple metal binding sites in structural sites should always be accompanied by functional studies to discern whether the metal site is active or inhibitory. 4.1.2. Inhibition of Nonmetalloenzyme Catalysis by Zinc

Zinc inhibition of zinc proteases i s now very common. However, zinc also inhibits a variety of enzymes that do not have catalytic zinc sites (Table 5). The inhibition can be in the low nanomolar range in some cases. In these situations it is possible that the zinc could be involved in regulating the activity of the enzyme. Thionein has been demonstrated to reverse the inhibition of glyceraldehyde-3-phosphatedehydrogenase by zinc ions, suggesting the possibility that apometallothionein can be involved in a regulatory capacity with zinc as its partner [1671. The fact that zinc inhibits enzymes has also been used to design new serine protease inhibitors [168,1691 (Table 5). The type of zinc ligand is important to the inhibition. Thus, the zinc-bound bis(5-amidino-2-benzimid~olyl)methane( and its derivatives inhibit several human serine proteases with an inhibition constant The presence of zinc increases inhibition by the agent in the absence of of 1-25 d. zinc up to 10,000 times. For example, the K; for BABIM inhibition of trypsin decreases

TABLE 5 Zinc Inhibition of Enzyme Catalysis

Carboxypeptidase A Aeromonas hydrophila p-lactamase Renin HIV protease type Phosphomannose isomerase Nitric oxide synthetase 6-Phosphogluconate Caspase 3 Caspase 3 Tyrosine phosphatase Aldehyde dehydrogenase Human factor Xa Bovine trypsin "BAUIM as ligand; 'keto-BABIM as ligand.

500 46,000 24,000 12,000 6,400 30,000 21 100 4 10

200 150 1"

< lb

922

AULD

from 19 pM to 5 nM in the presence of 100 nNI zinc ions at pH 8.2 [168]. The crystal structure of trypsin cocrystallized with BABINI and zinc shows zinc tetrahedrally coordinated to two nitrogens of BABIM and two of the catalytic triad residues, His57 and Ser195. The type of zinc binding site found is reminiscent of that observed for the inhibition of zinc proteases by zinc ions [161,163,165,1661.Thus, the zinc is bridging two catalytic components His57 and Ser195 for trypsin and Glu270 and a zinc-hound hydroxide for carboxypeptidase A. In these enzymes the zinc hydroxide and serine oxide are the nucleophiles and the glutamate carboxyl group and His imidazole are the likely general acid-base catalysts. 4.1.3. Inhibition of Metalloenzyme Catalysis by Removing the Metal-Bound Water or Metul

The importance of zinc enzymes in both normal and pathological processes makes them targets for drug design. The active site zinc in these enzymes is a particularly attractive target because the strength of a chelator-metal interaction increases the potency and specificity o f an inhibitor. Zinc metalloprotease inhibitors are usually designed to inactivate the enzyme by displacing the zinc-bound water and forming a stable ternary complex with the enzyme and active site zinc. The success of such chelating inhibitors is demonstrated by the clinical efficacy of the ACE inhibitor captopril, ([2S]-N-~-3-mercapto-2-methylpropionyl~~~-proline), which has had a major impact on hypertension treatment 11701. The success of this approach led to the design of many types of metal-binding agents that displace the metal-bound water and are designed to mimic the transition state of the enzyme. Popular groups,are hydroxamates and thiolates for the C-terminal side, phosphoryl goups for the Nterminal side and carboxyalkyl and phosphinic acids as transition state or tetrahedral intermediate mimics 11711. Specificitycan be built into the inhibitor on either side of the metal-binding ligand. Many of the crystal structures of zinc enzymes reported in the literature are in fact ternary complexes where the zinc-bound water has been displaced. Compared with the extensive research on inhibitors that bind tightly to metalloproteases, relatively little is known about the molecular mechanisms of how a metal binding agent or chelator removes zinc 1172,1731. n-Penicillamine (D-PEN),the drug used for many years in the treatment of rheumatoid arthritis and Wilson’s disease, was recently shown to be capable of removing the metal from zinc proteases [174,1751. It is a member of the Cys family of amino acids, It differs from D-CYSby the presence of two methyl groups on the f3 carbon. Initial rate assays on the inhibition o f metalloproteases, carboxypeptidase A, thermolysin, and matrilysin by the PEN and Cys inhibitors reveal that the penicillamine andogs are slightly stronger inhibitors for the matrix metalloprotease, matrilysin, but relatively weak for all enzymes. DCys is the most potent inhibitor of carboxypeptidase A and thermolysin. The disulfides of D-PEN essentially do not inhibit. In the course of these studies n-PEN was observed to inhibit carboxypeptidase A in a relatively fast time-dependent manner. Thus, repeated mixing o f a preincubated mixture of D-PENand CPD-A in a stopped-

S IN M ~ ~ A L L O E N Z Y M E S

923

flow instrument leads to a iirst-order decrease in activity with a half-life of 40 s t o a new constant level. The inhibition by u-PEN is relatively weak (Kl about 1mM at zero time and 0.2 rnM after equilibration). Two mechanisms might account for the action of D-PEN.Usually time-dependent inhibition is thought of as the forming of a tighter enzyme-inhibitor complex. However, D-PENmight also be catalyzing the release of the zinc. In this case, the zinc would be free and converted to a Zn(D-PEN)2complex by the time the second step is complete. The chromophoric chelator, 4-(2-pyridylazo)resorcinol (Pf4.R) was used to investigate this possibility (Fig. 11).In the absence of D-PEN very little metal is removed by 1.8 mM PAR. However, in the presence OC D-PEN, zinc is transferred to PAR in less than 500 s. This is a 400-fold increase €or the zinc off rate constant for the free enzyme. D-CYS, on the other hand, decreases the rate of zinc transfer as might be expected if it formed a stable ternary enzyme-zinc-D-Cys complex. The interaction of D-PENand D-CYSwith the active site metal has been examined by replacing the active site zinc by a chromophoric cobalt atom [1751. Both inhibitors perturb the d-d transitions of CoCPD in the ,500- to 600-nm region within milliseconds of mixing but only the COCPD-D-C~S complex initially displays a strong S --+Co(IX) charge transfer band at 340 nm indicative of a metal-sulfur bond. ~ i l e the D-CYScomplex i s stable, the CoCPD-D-PENcomplex breaks down to apoenzyme and CO(D-PEN)~ with a half-life of 0.5 s. Thus, the addition of two methyl groups on the @-carbonof n-Cys to yield n-PEN changes the mechanism of inhibition from formation of a tight ternary complex to catalytic removal of the active site metal. Complete removal of zinc and thus inactivation of the enzyme can be accomplished in these systems at low D-PENconcentrations if a secondary scavenger chelator is added to the system [1741. Such chelators bind metal that has been released from the enzyme but do not participate in the release [172,1731. This is the basis of

100

s

0 mM D-PEN

20

0

0

FIG. 11. n-PEN catalyzes the removal of zinc from carboxypeptidase A as indicated hy the chromophoric chelator, 4-(2-p~rr.idylazo)resorcinol(PAR).

AULD

924

chelation therapy. In the case of carboxypeptidase A, 2.5 pM thionein (apometallothionein) inhibits catalysis by only about 10% over a 15-min period consistent with its action as a secondary cheiator (Fig. 12). However, in the presence of 250 ph4 D-PEN and 2.5 pM thionein total inhibition is achieved in less than 15 min. D-PEN accelerates zinc equilibration between carboxypeptidase A and thionein. Consequenti~ly, the in vivo potency of D-PEN as a zinc enzyme inhibitor can be augmented by coadministration of secondary chelators or through the presence of natural chelators such as thionein or other metal-binding species. From a physiological perspective, such a mechanism of inhibition would be essentially irreversible, given the minuscule levels of free zinc believed to be present in the body 11761. U-PEN is the first drug found to inhibit a metalloprotease by increasing the dissociation rate constant of the active site metal. The ability of D-PEN to catalyze metal removal from carboxypeptidase A and other zinc proteases suggests a possible mechanism of action in arthritis and Wilson’s disease, and may also underlie complications associated with its clinical use.

ffect of ~caffoldingon Catalytic Activity The binding constants for imidazole and acetate binding to zinc are not particularly strong, e.g., pK1 for acetate is about 1.5 [3]. Binding of four carboxyl groups to a zinc would then be expected to have an overall binding constant of lo6. the binding constant for EDTA is lox7due in large part to the proximity effect of

100 80

0

FIG. 12. R-PEN inhibits by mediating the equilibration of zinc between carboxypeptidase A and thionein.


925

the four carboxylates. The fact that so many zinc enzymes bind zinc with picomolar binding constants yet have imidazole and acetate as their ligands likely reflects their proximity and restricted mobility in the protein. The zinc binding sites frequently have an cl-helical or P-sheet structural region of the protein that supplies the zinc ligands, particularly in catalytic zinc sites 1611. There is a strong correlation between the short spacer length and the type of secondary support structure that supplies the ligand (Table 1). When an a-helix support structure is used, as in matrix metalloproteinases, the spacer consists of three amino acids juxtaposing the ligands in an orientation suitable to establish a tetrahedral-like coordination sphere. In contrast, if the secondary support structure is a @-sheetas in carbonic anhydrase, the spacer is one. This allows the ligands to come from the same side of the sheet. The stability and the function of the metal site is also likely influenced by the second shell of residues in the vicinity of the metal binding site. The secondary interactions of the ligands with hydrogen-bonding groups of the side chain groups of the amino acid residues or the carbonyl oxygen of the backbone peptide chain may be critical to the formation and stabilization of the zinc sites containing oxygen and nitrogen ligands. Comparative structural studies of four OC the first known zinc enzymes-carbonic anhydrase, mrboxypeptidase A, alcohol dehydrogenase and thermolysin-led to the identification of carbonyl and carboxyl “orienters” [1771. The particular interaction between a y or 6 carboxyl group of Asp or Glu, respectively, and a histidine ligand to the zinc was referred to as a new catalytic triad [178]. Such interactions occur, for instance, between the 6-carboxylate of Glu117 and the His119 ligand of carbonic anhydrase and the y-carboxylate of Asp170 and the His142 ligand of thermolysin. 4.2.1. Carbonic Anhydruse

There are currently X-ray crystal structures for catalytic zinc binding sites of five of the mammalian carbonic anhydrases (Table 1).The zinc ion is coordinated by the imidazole side chain of His94, His96, and His119 in all of these mammalian carbonic anhydrases (Fig. 13). The three His ligands of these enzymes readily allow formation of zinc hydroxide, which can then add OH- to COZ to form HCO, [1791. The amino acids adjacent to these zinc ligands are also highly conserved in comparison with the rest of the protein. Thus, while the similarity scores for this set of proteins is 60-99%, the metal binding site ligand sequence is 80-100% with the majority of the sequences being above 90%. In terms of identity of amino acids the overall structures are 3595% identical while the metal binding site sequences are 60-100% conserved. One of the reasons for this is that H bonds are formed between Gln92 amide nitrogen and His94 and between the Glu117 carboxyl group and His119 [1771. These interactions likely stabilize zinc binding and influence the function of the zinc in catalysis. In addition, the backbone carbonyl of Asn244 accepts an H bond from His96 and the hydroxyl group of Thr199 accepts an H bond from the zinc-bound water [180]. These residues are also conserved in all the carbonic anhydrase I-V enzymes.

926

AULD 94

96

119

Consensus CAHI-human

CAHl-macne CAHl-macum

CAHl-horse CAHI-mouse CAHl-sheep CAHLrabit CAHZmouse CAH2- human CAH2-rabit CAHlbovin

CAH2-sheep CAH2-rat

CAH2-chick CAH3-mouse CAH3-rat

CAH3-horse CAH3-human CAH4-rat CAH4-rabit CAH4-mouse CAH4-human CAH4-bovin CAH5-human CAHS- rat

CAH5-rnouse FIG. 13. Zinc ligands of the mammalian carbonic anhydrases I-V. Data obtained from a Smith-Walerman search using the sequence of human CA I on the Bioccelerator computer at the European Molecular Biology Laboratory (EMBL) at Heidelberg. Names are from the searches and denote both type and species of CA.

Secondary ligand intcractions are postulated t o be involved in strengthening the metal complexation or modulating the nucleophilicity of the zinc-bound water 11781. Both have been shown to occur in the case of carbonic anhydrase through the examination of functional and structural consequences of mutating the orientating residues 61n92, Glu117, Asn244, and Thr199 [180-1821. The results indicate that the zinc affinity is reduced by about a factor of five- to tenfold when a native H bond is eliminated. The effects also appear to be additive. Thus, the combined mutations of Gln92Ma and Glu117Ala leads to a 40-fold increase in the dissociation constant for zinc while the individual mutations lead to four- and t,enfold increases, respectively [180]. The weakened binding of zinc is largely accounted for by an increased off-rate constant for zinc dissociation.


927

These secondary protein interactions would also be expected to affect the charge on the zinc. The binding of hydrogen donating or accepting groups to the zinc ligands might therefore modulate the pK, of the catalytic zinc-bound water, resulting in changes in the nucleophilicity of the water and the ease with which it can expand its coordination shell or allow the zinc-bound water or hydroxide to be displaced. The pH dependence of the carbonic anhydrase II-catalyzed hydrolysis of p-nitrophenylacetate was used to examine this postulate. In the wild-type enzyme it is characterized by a pK, of 6.8, which is believed to reflect the ionization of the zinc-bound water molecule [ 1801. In the case of the Gln92Glu mutation in carbonic anhydrase I1 a neutral hydrogen bond acceptor is replaced by a negatively charged acceptor. The positive charge on the zinc should therefore be reduced, which in turn should make the ionization of water more difficult. The results of the functional studies confirm this relationship since the kinetically determined pK, for water increases by 0.7 unit for this mutation [l80]. The reverse effect would be expected for a mutation that removes this negative charge since this should increase the charge on the zinc leading to a lower pK, for ester hydrolysis. %placing 6 1 ~ 1 1 7 with Asp led to a slight decrease of 0.2 unit consistent with a less favorable interaction of the Asp117 carboxylate with the His119 imidazole group. The results with Glu117Ala did not agree with the expectations. The pK, values for the native and 61u117Ala mutant are 6.8 and 6.9, respectively. The value for the Ala mutant should have dropped by as much as 1 unit due to the loss of the anionic interaction with the His ligand. However, the X-ray studies demonstratc that the 6 1 ~ 1 1 7carboxylate-His119 interaction is replaced by a chloride ion in the case of the Glul17Ala mutant 11821. The kinetic assays didn't contain any chloride ions but did use 0.1 M sulfate for controlling ionic strength [ISOl. If sulfate can bind in the same region as chloride, His119 may still have an interaction with an anion species. A n even greater surprise occurred in the mutant 61u117Gln [1831. This mutation would be expected to remove the negative charge imposed on the His residue by the natural Glu carboxylate and have only a subtle effect on the structure. However, the resultant CA 11 displayed greatly diminished activity and ability to bind inhibitors, and the value of the kinetic pK, for esterase hydrolysis is believed to be greater than 9. Since the X-ray structure of the inhibited enzyme looked the same, His119 was proposed to be an anion that is stabilized by the Gln amide bond. If this were to happen a negative charge would be added to the zinc and the pK, of the zinc-bound water would be expected to increase. However, it is not clear why a neighboring amide bond should induce a His imidazole group to lose its second proton. In addition, the spectrum of the mutant Go enzyme doesn't have the high-extinction (275 cm-lM-'), multibanded (500, 550, 630, 660 nm) form that is seen when water ionization reduces the charge on the metal [1831. Instead it displays a broad absovtion peak near 560 nm, with a maximum absorption coefficient of' about 90 cm-I M-I, that could be indicative of a five to six coordinate species in solution. The importance of the Thrl99-zinc hydroxide interaction was demonstrated by examining the pH dependence of the carbonic anhydrase II-catalyzed hydrolysis of p nitrophenylacetate [1811. Mutation of this residue to a Ser results in a 0.4-unit

928

AULD

upward shift in the kinetic pKa characterizing this profile and a decrease in the pH independent value of kcadKmof threefold. However, removal of the hydroxyl group and the methyl group by the Thrl99ALa mutation raises the pK, 1.4 units and decreases activity 60-fold, suggesting that the hydroxyl group is important to stabilization of the transition state. The hydrophobic residues at positions 93, 95, and 97 are also highly conserved for this family of enzymes (Fig. 13). These residues are generally Phe for positions 93 and 95 and Trp for 97. The aromatic residues would be expected to restrict the motion of the His ligands [184,1851 and/or provide an environment of low dielectric constant that would enhance electrostatic interactions 11861. A cassette mutagenesis technique was used to select CA I1 enzymes mutated at positions 93,95, and 97 that retained high zinc affinity [1841, The best variant enzymes contained Ile, Phe, Leu, and Met at position 93; Ile, Leu, and Met at position 95; and "rp or Val at position 97, in general agreement with what is found for this class of enzymes (Fig. 13). When small hydrophilic residues are introduced at these positions the zinc affinity s-Val mutant compared to the native Phedecreases. Thus the Thris-Trp enzyme has a 110-fold increased K,, for zinc essentially due to an increase in the off-rate constant of 400-fold. The kc,dKm for the COz hydrase and esterase activity is decreased 90- and 30-fold, respectively, but the pK, for esterase activity doesn't change. The better zinc binding and higher catalytic efficiency correlate with the combined volume of the residues at positions 93, 95, and 97. The results of recent studies of the cobalt- and copper-substituted mutants have indicated that these residues may be important to retaining a tetrahedral geometry for the zinc site [1851. Studies of the effect of mutating the ligands that bind zinc indicate that this enzyme is capable of catalysis with different ground-state geometries. Thus, the coordination geometry of the Hisll9Asn and His94Asn mutants both become trigonal bipyramidal while the corresponding Gln mutants remain tetrahedral [1871. The k,,J Knl values for esterase activity and COz hydrase activity for the Hisll9Asn decrease only 3- and 12-fold, while those for His94Asn decrease 7- and 30-fold, respectively. These results compare favorably with the tetrahedrally coordinated Gln mutants where the esterase activity and COz hydrase activity for the Hisll9Gln decrease 2and 16-fold and those for Wis94Gln decrease 125- and 344-fold, respectively, The zinc affinities of these mutants and those for the corresponding Asp and Glu mutants all are within a factor of 5 of each other but all bind zinc about 104-foldmore weakly than the wild-type enzyme [187,188]. The kinetic pKa values for these mutants increase 0.5-1.8 units higher in agreement with model studies suggesting that higher coordination numbers for zinc sites should have higher pX, values for metal-bound waters [1891. Thus, the change in the inner ligand type for carbonic anhydrase I1 can lead to a much more weakly bound zinc, but the resulting catalytic zinc site can adapt to several different ligands and in some cases retain reasonable activity by changing the ~ o u n d - s t a t coordination e geometry. In no case does one obtain higher activity than in the native enzyme.

929

ZINC SITES IN ~ ~ T A L L O ~ ~ Z Y M E S

Comparison of the structures of matrilysin, thermolysin (TL), and carboxypeptidase A reveals both similarities and differences in their active sites. A common feature is a catalytic zinc atom that is coordinated by three protein ligands and a nearby ionizable carboxylate group of a Glu residue that is considered to act as a nucleophile or general base (Fig. 14). The fourth ligand is water in the active enzyme. The zinc site is closely similar with regard to the orientation of the metal ligands, the proposed catalytic glutamate residue, and the position at which inhibitors bind r190-1921. However, the type of the ligand and the scaffolding of the zinc site is not the same (Fig. 14) [3]. The catalytic zinc of matrilysin is made up of three His residues 11901, whereas the zinc atom of thermolysin and CPD A contains two His and one Glu 1120,1931. Furthermore, the secondary interactions of the zinc ligands with adjacent side chain carboxylate groups observed in TL and CPD A is not observed in matrilysin. In CPD A: 6 1 ~ 2 7 0is considered to assist the ionization of the metal-bound water based on chemical modification, crystallographic, spectrokinetic, and NMMR studies [3,5,37,1941. The pH dependence of matrilysin catalysis demonstrates that ionization of a group with a pKa of 4.3 is needed for catalytic activity 11951. Glu198 was viewed as the l&dy amino acid residue that could be responsible for this pK, based on the mechanisms proposed for TL and CPD A. The active form of matrilysin, devoid of its prodomain, was expressed for mutagenesis studies 11961. This enzyme allows the determination of the effect on catalysis induced by modifying the chemical properties of active site residues. The suspected group responsible for the acid pKa, Glu198, was mutated into Asp, Cys, Gln, and Ala, and the zinc binding properties, kinetic parameters, and pH dependence of each mutant were determined to examine the role of

3

CARBOXYPEPTIDASE A, CPD-A

THERMOLYSIN, TL

MATRILYSIN

FIG. 14. Scaffolding of the zinc binding sites in carboxypeptidase A, thermolysin, and matrilysin. (From [196].)

930

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Glu198 in catalysis. The mutations chosen either modify (Asp and Cys) or eliminate (Gln and Ala) the general base properties of residue 198. All of the mutants bind 2 mol of zinc per mole of enzyme, indicating that Glu198 is not crucial t o the binding of the catalytic zinc to the enzyme 11961. The value of h,,JKm for the Glul98Asp mutant is only 4-fold lower than that of wild-type enzyme at the pII optimum of 7.5, whereas that for the Glul98Cys mutant is decreased by 160-fold. The Glu198Gln and Glu198Ala enzymes containing the mutations that eliminate the nucleophilic and acid-base properties of the residue are still active having lower hcat/Kmvalues of 590- and 19OO-fold,respectively. The decrease in activity of all the mutants is essentially due to a decrease in kcat. The high activity for the Asp mutant of the corresponding glutamate is also observed in other MMPs, such as gelatinase A and collagenase [197,1981. The high and detectable activity of Glu mutants observed in MMPs i s in contrast to mutations of the corresponding glutamate of the thermolysin-like neutral endopeptidase 24.1 1 E1991, the neutral protease of B. stearothermophilus MK232 [200], the neutral protease of B. subtilis 12011, and mouse arninopeptidase A [202]. No residual activity is detected toward synthetic peptides, casein, or gelatin even when the Glu residue is replaced by an Asp. Similar results are found in the case of the serine protease from Bacillus amiloliquefaciens, in which mutations of the Ser and His located in the catalytic triad decreases hcat by a factor of 2 x lo6 12031. In addition, the mutation of the nucleophilic residue SerlO2 to Cys and Ala in alkaline phosphatase decreases IZc,JK, by 2 x lo* and 4 x lo5, respectively [204]. The marked contrast in the present result on matrilysin and those of the thermolysin-like enzymes implies that the conserved glutamate in matrilysin and other MMPs may play a different functional role in their catalysis from that proposed for the thermolysin-like enzymes. The lower catalytic efficiency of the Glu198Ala and Glu198Gln mutants in comparison with the Glul98Asp and Glu198Cys enzymes suggests that acidbase properties of residue-198 are still important in catalysis. The kcaJKmvalues of the mutants as a function of pH display broad, bell-shaped curves that are similar to those of the wild-type enzyme [196]. The acidic pKa value is not greatly affected by the change in the chemical properties of residue 198. The similarity in the pH profiles for the mutant and wild-type enzymes indicate that the ionization of Glu198 is not responsible for the acidic pK,. Ionization of Lhe zinc-bound water may be the group responsible for this pKfi since the three His ligands and the scaffolding of the matrilysin catalytic zinc site are different from those observed in carboxypeptidase A and would predict a lower pKa for the metalhnund waler. In addition, the MMPs have a second zinc site that is removed from the catalytic zinc by 12.5 A (Table 3 ) . It is composed of three histidine ligands (His146, Hisl61, and His1741 and one aspartate (Asp148) tetrahedrally coordinated to the zinc with spacing intervals of 1,12, and 12 (Table 2). This zinc site is unique to this subclass of the extended astacin family. Sequence alignment of the MMPs and other homologous members indicates that these four ligands and spacing intervals are highly conserved Ell. Conservation of the residues adjacent to all of the ligands is much higher than

ZINC SITES IN M~TALLOENZYMES

931

that of the overall conservation observed for this large group of homologous proteins. This conservation may reflect the importance of these residues in the structure andor function of these proteins. This zinc site has the characteristics of a structural zinc site since it is fully coordinated, there are no bound water molecules, and a relatively short sequence of the protein provides all four zinc ligands [71. However, while the two zinc atoms are 12.5 A apart, several of the conserved amino acids adjacent to the third and fourth His ligands of the second zinc site (e.g., Ala160,162,173,and Phe175) form the environment surrounding the predicted catalytic residue 6 1 ~ 1 9 8[Sl, The properties of the “structural” zinc site in the MMPs and in other similar sites may therefore influence function indirectly through effects on local conformation. If the zinc-bound water is the nucleophile in the reaction, the role of 6 1 ~ 1 9 8in catalysis may be to stabilize the transition state or act as a general acid catalyst after the ratedetermining step. The pKa of this group may be higher than is normally found since its environment is made up of residues that are hydrophobic. 0

4.3.

Mechanistic Studies of Catalytic

Universally H2Q is a ligand and the critical component of the catalytic zinc sites (Table 1) (91. The water is activated by ionization, polarization, or displacement (Fig. 15). The nature of the direct ligands as well as the second shell of amino acid interactions with the zinc-binding ligands and the makeup of the active center cavity will dictate how the zinc-bound water will be involved in catalysis.

Dis~iace~ent FIG. 15. Activation of zinc-bound water in catalytic zinc sites [91.

932

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4.3.1. Carbonic Anhydmse

he class IV lyase carbonic anhydrase (CAI i s likely the best example of an ionization activated zinc-bound water mechanism. It is a ubiquitous enzyme involved in the physiology of COX transport between metabolizing tissues and the lungs 11791. Sequence analyses suggest that there are several distinct forms in vertebrates: three cytosolic forms (CA I, 11, and 111); two membrane-bound forms (CA IV and I); a mitochondria1 form (CA V); and a secreted salivary form (CA VI). Crystal structures of five different forms of this enzyme [20S-2101 (Table 1) have shown that the catalytic zinc is located in a 15-A-deep active center cavity nem the middle of the enzyme molecule. One part of the cavity is dominated by hydrophobic residues while another segment is more hydrophilic [179], The catalytic zinc is tetrahedrally coordinated to the N E of~ His94 and His96 supplied from a P-strand ~ncompassing residues 88-108 and a N61 of His119 from a second P-strand extending from 113 to 126 [2051 and a water molecule. The amino acid ligands and adjacent amino acids are highly conserved throughout this large family (see See. 4.2.1 and Fig. 13). The function o f carbonic anhydrases is to catalyze the conversion of carbon dioxide into bicarbonate. The generally proposed mechanism for these enzymes involves the nucleophilic attack of the zinc-bound hydroxide on the carbonyl oxygen of COz (Fig. 16 [179,211,212]). Spectroscopic studies early on showed that metalbound water could be easily displaced by a wide variety of inhibitors and indicated the importance of the metal-bound water to catalysis [213-2151. Studies of the effect of changing both direct and indirect ligands to the zinc suggest that the role of zinc in the mechanism is to stabilize the developing charge in the transition state of the hydration of COX (see Sec. 4.2.1. and 1212,2161). The bicarbonate formed in this

19

H

HI19 H96

FIG. 16. Mechanism of carbonic anhydrase catalysis. (Adapted from [177,2121.)


933

manner must then be displaced by water and a proton removed to regenerate the active form of the enzyme. The last step requires proton transfer from this deep well to the surface of the protein being mediated by both buffers and side chain residues 11791. His64 has been implicated for several of the isozymes and His67 for CA 111 12171. Studies of the proton shuttle in murine CA IV have suggested that several proton transfer pathways may exist between the zinc-bound water and the bulk solution f2181. Several His residues have been implicated in this proton shuttle for CA I1 (His3, His4, Hisl5, Hisl7, and I-Iis64), and the absence and/or change in the nature of these residues in other CA forms could refiect the different levels of catalytic activity observed for these enzymes [2191. The studies of the catalytic reaction for this family of enzymes is important not only for the determination of its catalytic mechanism but also for the design of metal binding sites. It indicates in particular that while catalysis is driven primarily by the nature of the zinc binding site, the nature of the larger catalytic cavity must also be considered in the design of a metal site for any particular reaction. 4.3.2. Metalloproteuses

Carboxypeptidase A and its endopeptidase relative, thermolysin, are likely examples of polarization-assisted catalysis (Fig. 14). The zinc-containing pancreatic exopeptidases carboxypeptidase A and B are important in the degradation of food proteins leading to the formation of amino acids. The carboxypeptidases complement the actions of chymotrypsin, pepsin and trypsin by allowing the production of essential amino acids such as Phe, Trp, Lys, and Arg E2201. In addition to the pancreatic forms of carboxypeptidase from human, bovine, crayfish, rodent, and porcine sources, carboxypeptidase activity has been identified in such diverse sources as orange peel, yeast, fungi, molds, barley, spleen, kidney, and connective tissue. Several new caxboxypeptidases have been identified by gene sequencing and their activity in various tissues. Thus, the human mass cell E-, M-, and N-carboxypeptidases are believed to be involved in immune/inflammatory and hormone processing U71 and references therein). The amino acid sequences of members of the carboxypeptidase A family are between about 60% and 80% conserved L2211. The His and Glu ligands to the zinc, Cys residues that should form disulfides, and other amino acids proposed to be important to specificity and catdysis in the CPD A family are all conserved. X-ray crystallographic analysis of bovine CPD A and its transition state and substrate-analogue complexes in conjunction with kinetic and chemical modification and mutagenic studies has led to the assignment of a number of active site-residues in catalysis. Thus, Arg145 is postulated to be the site of interaction for the free a-carboxyl group of the substrate and Glu270 is the principle catalytic moiety (1192,2211 and references therein). Argl27 assists as an electrophile by forming a hydrogen bond between the guanidinium group and the oxygen of a putative tetrahedral intermediate based on both crystallographic 1222,2231 and mutagenesis studies [224,225J. Two conserved Tyr residues, Tyr198 and Tyr248, have been assigned roles in substrate binding. An

934

AULD

unusual feature is the presence of a cis peptide bond between Ser197 and Tyr198 [2261. Since Tyr198 has been assigned a role in peptide binding and His196 is a ligand to the zinc, the energetics of a substrate’s interaction with Tyr198 may be directly relayed to the catalytic zinc. The catalytic zinc site is composed of His69 (N61), 6 1 ~ 7 2( 0 6 1 and O E ~ His196 ), (N61), and a water molecule. The first two ligands, separated by a short spacer of two, are at the ends of a reverse turn in an 01 helix while His196 is the last residue in a ppleated sheet extending from amino acids 191-196 12261. This site is highly conserved throughout the extended carboxypeptidase family 171. Analysis of the individual kcat and K , profiles are supportive of a three protonation state model EW,&EH@E C1621. According to this scheme, when the enzyme is in its EH, form below the acidic PKEH~ it can bind substrates but not hydrolyze them. The ionization of the group EH2 with a pKa of 6.0 transforms the enzyme to its active form EH. Further ionization of the enzyme to the E form, occurring with a pK& of 9.0, markedly reduces substrate binding and therefore catalysis. Several reasons lead to the assignment of ~ K E to Hthe ~ ionization of the carboxyl group of Glu270 and its subsequent interaction with the water ligand of the zinc, stabilizing the active site structure. This is consistent with crystallographic studies that show the interatomic distance between the zinc and the carboxylate is 4.5 A, a distance consistent with an H bond between the metal-bond water molecule and 6 1 ~ 2 7 0[2261. Chemical modification of Glu270 with CMC inactivates the enzyme [227]. ~ e m p e r a ~ u jump re studies provide evidence for a conformational change coupled to the formation of the EH2 form o f the enzyme, which might reflect movement of Glu270 away from the metal C2281. Spectrokinetic and NMR studies on CoCPD show that anions bind to the EH2 form of the enzyme. This is reasonable if the prot(~nationof Glu270 breaks its interaction with the water and thus allows its displacement by anions [164,229-2311. On the other hand, the enzyme is inhibited by zinc hydroxide by binding to the EH form of the enzyme (see Sec 4.1.1. [161,1631). This is also consistent with this model since the positive ZnOH+ can bind to the negative Glu carboxylate oxygen and displace the metal-bound water forming a Zn0-Zn bridge (Fig. 10). Calculation of the coordination geometry of the Cd-substituted enzyme from PAC spectra [232,233], and molecular calculations on a model system [2341 are also in agreement with this assignment. The alkaline pKER2 has been assigned to the metal-bound water based on p -dependent XAFS studies of the free zinc enzyme and of its inhibitor complexes [235-2371 as well as visible spectral and NMR studies of the cobalt enzyme [231,2381. These studies of the effect of pH on the zinc coo~dinationsphere in conjunction with those of inhibiior and substrate binding allow the following mechanism to be proposed for CPD-A catalysis [5,192] (Fig. 17). In step I, the zinc acts as a Lewis acid catalyst by expanding its coordination sphere to accept the peptide carbonyl. Glu270 acts as a general base removing a proton from the rnetal-bound water, allowing the hydroxide to attack the peptide carbonyl. Arg127 likely plays a role in stabilizing the negative charge in the transition state. In the step 11, Glu270 acts as a general acid catalyst by donating a proton to the leaving amine as the metal-bound tetrahedral

ZINC SITES IN ~ E T A L L O ~ N Z Y M E S

935

\

R2

FIG. 17. Mechanism of carboxypeptidase A catalysis. (From 131.)

intermediate collapses to products, 111. In step IV, the N-terminal product leaves and water returns to the metal with the C-terminal product still bound in a salt-bridged manner to 611.1270. The enzyme complex is thus poised for the reverse reaction, synthesis of a peptide bond. In this state, the zinc-bound water is “vulnerable” to the displacement of the water by anions. The zinc-bound water therefore plays a role in both the acid and alkaline pK, values of carboxypeptidase A. It needs to be in its protonated state, [ZnL3(HzO)lf,to be catalytically active. In this way it can poise the ionized carboxylate 6 1 ~ 2 7 0for catalysis. Ionization of the water decreases the charge on the zinc, making it a poorer Lewis acid for interaction with the substrate. A general acid-base role for Glu143 in thermolysin catalysis is also proposed based on X-ray crystallographic and computer graphic analyzes of thermolysin bound with inhibitors [1911 and the proximity of 6 1 ~ 1 4 3to the zinc-bound water molecule 11661. This role for the glutamate in catalysis is generally believed to pertain to the MIMP family of proteases as well. However, our recent mutagenesis studies indicate that this may not be its role in catalysis (see Sec. 4.2.2). 4.3.3. Alcohol Dehydrogenases

The dimeric alcohol dehydrogenases (ADH) (EC 1.1.1.1)contain both a catalytic and a structural zinc site (Tables 1 and 2). These zinc enzymes are NAI)+-dependent and catalyze the reversible oxidation of alcohols t o aldehydes. Seven human ADH genes have been identified and designated as ADH1 through AI)H7 12391. The M D 1 to ADH5 and ADH7 encode six subunits of the ADH enzymes that are designated by the Greek letters a,p, y,7c, x and u. The gene product of ADH6 has

936

AULD

not been observed. Polymorphism occurs for the ADH2 (p) and ADH3(y) loci resulting in nine distinct human subunits. These enzymes have also been classified, based PI,p2, p3, yl,yz on sequence identity and substrate specificity 12401, as class I (a, containing isozymes), class I1 (d, class 111 ( x ) , and class IV (0). Class I11 has also come to be known as glutathione-dependent formaldehyde dehydrogenase [141. The first crystal structure reported was that of the horse enzyme, denoted EE, and considered to be part of class I 145,2411. The three-dimensional structure of several human isozymes have been reported recently. These include class I, P1[3,, fk&, p3p3 ADH R42-2441, class 111, xx ADH 1141, and class IV, 00 ADH 12451. The crystal structure of cod liver ADH has also been reported [2461. This enzyme has a substrate specificity similar to that of class I enzymes but a sequence similarity closer to that of class 111 ADH 12471. Many structural and mechanistic studies have been performed on horse liver enzyme. Each subunit of the dimeric enzyme is divided into a coenzyme binding domain and a catalytic domain that are separated by a cleft containing a deep pocket zinc ions reside in the catalytic domain. The catalytic zinc is ligated to 7 and Cys174 and a water molecule in a tetrahedral coordination geomes the first residue of a short a helix and the last residue of a p strand, whereas His67 is supplied from the end of a p strand and Cys174 from the middle of an a-helix [2481. Since zinc does not undergo oxidation-reductionchemistry due to its filled d-shell the zinc works in conjunction with the coenzyme NADCto catalyze these reactions. The zinc-bound water must be displaced for these enzymes and the Lewis acid nature of the zinc can then polarize the carbonyl bond of the aldehyde substrate for hydride transfer from the coenzyme. When the coenzyme binds it triggers a major change in conformation of the enzyme [249]. The two coenzyme binding domains have similar orientations while the two catalytic domains are rotated relative to each other. When NAD is bound the cleft between the catalytic and coenzyme binding domains “closes” around the coenzyme [250]. In the absence of the coenzyme the cleft is considered open L241l. In the open conformation solvent is accessible to the catalytic zinc and the fourth ligand is water. The induction of the closed form by coenzyme permits the ~ s p l a c e ~ eofn the t water by the alcohol or aldehyde substrate and places the zinc-substrate complex in a more hydrophobic environment. The interconversion of these two states have been observed kinetically in the process of coenzyme and substrate binding [251,2521. importantly, the conformational changes places the zinc-bound substrate in the proper orientation to the C-4 position of the cofactor nicotinamide ring for optimal hydride transfer F2531. The major changes in conformation that occur make it difficult to determine ly to participate in catalysis. However, it was recognized early on 1 could be a possible proton shuttle system in this f a i l y of enzymes 12411, and mutagenesis studies of the yeast and human PIPl support this is of view [2541. This amino acid is not conserved in all the ADIls. ~ u t a ~ n e sstudies man rcn enzyme indicate that it does not serve this role. The authors 47 might substitute for His51 in this case [2551. His47 is conserved in

S IN ~ ~ T A L L O ~ ~ Z Y M € S

937

class I1 and I11 and the cod liver enzymes that do not have His51 [14,2461. This His is believed to be close to the 2’ hydroxyl of the nicotinamide ribose in the closed conformation and might serve as a base catalyst 1141. Sequence alibmments indicate that His47, His51, or both are conserved in all ADHs except human class V and deer mouse class VI 12461. Although this enzyme has been studied for a long time, the dynamics of the system have made it difficult to assign the pK, values of about 7 and 9 in the pH profiles of coenzyme binding to functional groups in the enzyme L2561. Candidates for these groups are the imidazolium of His51 or His 47 and the catalytic zinc-bound water.

echanistic Studies of Cocatalytic Zinc Sites Cocatalytie sites occur in enzymes with two or more transition metals in their catalytic sites. These metals are generally about 3 ik apart and bridged generally by Asp, Mu, and His amino acids and sometimes an additional water molecule (Table 3). The bridging amino acids and HzO could have critical roles in catalysis. Thus, their dissociation from either or both metal atoms during catalysis could change the charge on the metal, promoting its action as a Lewis acid or allowing interaction with an electronegative atom of the substrate. Alternatively, the bridging ligand might participate transiently in the reaction as a nucleophile or general acid-base catalyst. In this manner, the metal atoms and their associated ligands would play specific roles in each step of the reaction that works in concert to bring about catalysis.

4.4.I. Alkaline Phosphatase and Related Phosphatases

E. coEi alkaline phosphatase is a dimeric enzyme that contains 4 mol of zinc and of magnesium per 80-kDa protein (2571. It catalyzes the nonspecific hydrolysis of phosphomonoesters. It has a cocatalytic zinc site in each subunit made up of two zinc atoms and one magnesium that form a nonequilateral triangle with the metals as the apices [258]. The ligmds to these metals and the adjacent amino acids are highly conserved for a large f d y made up of representatives from bacteria, yeast, and mammalian sources [8]. One metal site has the properties of a catalytic zinc site being composed of two ligands, Asp327 and His331 supplied from a short a-helix, a third protein ligand His412 supplied by a 3! strand, and a water molecule (Table 1). The second zinc, Zn”, and Ihe Mg are bridged by Asp51 (Table 3). This is the first zinc site where it is known that a reactant amino acid in catalysis, Serl02, is a ligand to a metal, Zn”. Several other phosphate-hydrolyzing enzymes also have cocatalytic sites resembling E. coli alkaline phosphatase (Tables 1 and 3). The overall primary structures of the mammalian and E. eoEi enzymes are only 25-30% but their putative metal-binding ligands are cntirely conserved I81 and the amino acid sequence has been fit to the E. coli enzyme 12591. However, there are some

938

AULD

differences in the active center that have been investigated due to the 10-foldgreater activity of the mammalian enzymes [2601. The E. coli enzyme has a salt bridge between the Asp153 and Lys328. The human enzyme has a His at both positions [2611. The mutant E. eoli enzyme Aspl53His displays many of the features characteristic of the mammalian enzyme, including the low activity in the absence of magnesium and a time-dependent enhancement of activity in its presence. The X-ray structure indicates that the octahedral Mg binding site of the wild-type enzyme has been converted to a tetrahedral Zn binding site in the mutant enzyme [260,261]. In the wild-type enzyme, Asp153 W-bonds two water molecules bound to the Mg site. The structure of the phosphate complex is also different from the wild-type enzyme likely due to changes in the conformation of Lys328 resulting from mutation of Asp153 into His153. The increased activity in the presence of Mg is believed to be due to the displacement of the third Zn by magnesium with a concomitant change in the coordination site. Mutation of the bridging Asp51 ligand to Asn has profound effects on activity and structure of the cocatalytic site C2621. At pW 8 the activity of the mutant enzyme is about 1%of the wild type. At pH 9 and in the presence of Mg the activity returns in a time-dependent fashion. The X-ray structure indicates that the low-pH, low-ma~esiumform of the enzyme no longer has a third metal binding site. The activation by Mg at high pH in this case is postulated to be due to Mg binding to the Zn" site. Much was learned and proposed for the mechanism of this enzyme from a combination o f X-ray crystallographic, NMR, and kinetic studies on the Cd- and Co-substituted enzymes ([258,263,264] and references therein). It was known early on that the rate-determining step was pH-dependent. In the alkaline pH region the release of the non-covalently bound product phosphate (E P -+E + Pj is rate limiting while in the acidic pEs region the breakdown o f the covalent phosphoryl intermediate (E-P +E = Pj is postulated to be rate limiting. SerlO2 is the nucleophile in the first step of the reaction. In the E P complex the phosphate ion is coordinated to both Zn and Zn" and with two of its oxygens to the guanidinium group ofArgl66. The phosphate is further H-bonded t o the amide of Ser102 and a water molecule that is coordinated to the Mg [258]. Other participants that appear to stabilize this complex are Lys328 and Asp153. Recent studies have shown that mutation of Serl02 to Gly, Ala, or Cys decreases activity lo4- to 105-fold, with only the Cys mutant having an effect on the position o f the phosphate [2041. The breakdown of the Ser phosphoryl intermediate, E-P, is believed to be through a zinc-bound waterhydroxide on the catalytic zinc in the proposed mechanism. Mutation of one of the ligands to the catalytic zinc site, His33lGln, yields an enz.pe in which the covalent phosphate intermediate can be observed in the crystal structure [265]. The structure shows the zinc-bound water on the catalytic zinc to be in a position for apical attack on the SerlO2 phosphoryl bond. Recent studies have also proposed the vanadate complex of the enzyme as a transition state complex [2661. The vanadate ion is bound in a trigonal bipyramidal geometry with the active site SerlO2 and water molecule in opposite apical positions. The equatorial oxygens are stabilized by interaction with the guanidinium group of Arg166.


939

4.4.2. Aminopeptidases

Aminopeptidases, widely distributed in bacteria, yeast, plant and animal sources, catalyze the hydrolysis of a wide variety of N-terminal peptides and amino acid derivatives. The structures of several aminopeptidases have been reported (Table 3) [68-70,267-2691. These include both cocatalytic zinc and cobalt sites. In addition, a cocatalytic zinc site has also been observed in a bacterial carboxypeptidase [2701. The most extensive steady-state and pre-steady-state kinetic, spectral, and structural studies have been performed on bovine lens leucine aminopeptidme (BLAP), and Aeromonas proteolytica aminopeptidase (AAP). Both enzymes exhibit similar kinetics [271], and their C- terminal catalytic domains have similar folds. However, the cocatalytic zinc binding sites of these two enzymes diEer notably, which may underlie differences in their catalytic mechanisms [2721. The zinc binding site of the hexameric BLAP is composed of two zinc atoms separated by 2.91 A [267]. Znl is defined as the fast-exchange, weak binding site in which Mg can substitute for Zn 12731. It is coordinated to the carboxylate 0 6 1 o f h p 255, the carboxylate 062 and backbone carbonyl of Asp332 and the bridging carboxylate Or2 of 61~334.Zn2, the second zinc, is defined as the tight-binding, slowexchange site. Zn2 is coordinated to the carboxylate 0 6 1 of Asp255, the carboxylate 0 6 1 of Asp273, the side chain amino N of Lys250, and the bridging carboxylate 611.1334. No bound water molecules are observed in the free enzyme or in any of its inhibitor complexes of these crystals, and there are no nucleophilic amino acids in the active site which are in a position for direct attack on the scissile carbonyl bond of the substrate 1273,2741. A general base-catalyzed mechanism is still favored using the zinc ligand Asp255 as the general base acting on an active site water molecule and Lys262 as the general acid catalyst [267,272,273,2751.It has been proposed that Zn2 is involved in substrate binding while Znl along with Arg336 are the electrophiles that polarize the scissile carbonyl bond [275]. The cocatalytic site of E. coli alkaline phosphatase serves as a precedent for containing a ligand that is involved in catalysis (see Sec. 4.4.1 [8,2581>.In this case, SerlO2 forms a phosphate intermediate during catalysis. Alternative mechanisms have been suggested based on results of inhibitor studies using the transition state analogues, L-leucinephosphonic acid and the gem-diolate analog L-leucinal E274,2761. In the L-leucinephosphonic acid complex one of the phosphorous oxygens bridges both zinc ions while the second phosphorous oxygen is closer to Zn2. In the L-leucinal complex the amino terminal nitrogen is bound to Zn2 and one of the gem-diolate oxygens is bound to Znl while the other bridges both metals. In the course of these studies a new crystal form was found in which a water molecule does bridge the two metal sites in the unligated form 12771. These results lead to the proposal of a modified mechanism in which a metal bound hydroxide is the nucleophile and the carbonyl of the $ubstrate is polarized by both zinc ions acting as Lewis acid catalysts. These studies also observed a bicarbonate ion or carbonate molecule bound near Arg336 in the unligated form of the enzyme

9-40

AULD

[2771. This led to the discovery of bicarbonate acting as general base catalyst 12781 in the structurally similar aminopeptidase PepA 181. The bicarbonate is believed to be bound to Arg336 {BLfW numbering) since no activation is seen in the Lys, Ala, Met and Glu mutants. These studies also indicate Arg336 is not essential to catalysis. The bicarbonate is proposed to facilitate proton transfer from the zinc-brid@ng water nucleophile to the peptide leaving group. Mutation of Lys282 Lo Ala reduces activity 10,000 fold suggesting it may be important to stabilizing the transition state. The structure of Aeromonas proteolylica aminopeptidase has been determined in its free state I2681 and in complexes with a hydroxamate inhibitor [a721 and butaneboronic acid 12791. The two zinc atoms are separated by 3.5 A. In this case, Znl, is coordinated to both carboxylate oxygens of Glu152, the Ne2 of His256 and a bridging water and a bridging carboxylate 0 6 1 o f Asp117 12681.Zn2, is bound to both carboxylate oxygens of Asp179, the Nr2 o f His97, and the bridging water molecule and carboxylate 0 6 2 of Aspll7. The cocatalytic zinc sites of AAP and BLAP differ in several details. are bound to both sites in AAP whereas no His residues are involved in uses both an Asp carboxylate and a water molecule as bridging ligan uses the carboxylate oxygens of a Glu residue, one oxygen of an Asp residue, bridging water molecule. BLAP uses a Lys residue t o bind Zn at the tightly bound site, These combinations o f ligands as well as the difference in interatomic distances o f the two zinc atoms could lead to differences in the charge on the zinc, which in turn could influence catalysis. ine inhibitor complex with The results of a ~ " i o d o - ~ - p h e n y l a l ~hydroxamate have led to a proposed mechanism For the AAP-catalyzed hydrolysis of peptides [272]. Both the hydroxyl and carbonyl oxygen atoms of the hydroxamate bind to Znl while only the hydroxyl oxygen, albeit at a shorter distance, binds to Zn2. In the complex the distance between the zinc atoms increases to 3.7 A. In addition, the carboxylate oxygen of 6 1 ~ 1 5 1 hydrogen bonds the hydroxylamine nitrogen in a snanner seen in several of the matrix rnetalloproteinases [190,280] and thermolysin r281l. The Bimilarity of these structures led the authors to propose Glu151 as the general base catalyst in AAP [272]. The importance of the zinc-bound water to catalysis has been indicated by studies of fluoride ion inhibition 12821. Fluoride inhibits in an uncompetitive fashion that is weakened as a group in the ES complex ionizes with a pKa of 7.0. The zinc-bound bridging water molecule is proposed to be the g o u p with this pKa based on the belief that substrate binding should break the bridging water bond and allow fluoride to displace it. Such uncompetitive inhibition by chloride and bromide has been observed for therrnolysin catalysis [283,2841. On this basis, the preferred mechanism for AAP catalysis involves general base catalysis by Glul51 on the zinc-bound water as is proposed for carboxypeptidase and thermolysin (Fig. 17).

ZINC SITES IN M TALLO~NZY MES

941

Technological advances in gene sequencing, mutagenesis, and X-ray structure determination should continue to lead to more information on possible zinc binding sites and crystal structures of biological metal binding sites. Thus, the search for putative zinc sites can be increasingly guided by the growing number of zinc sites available for reference. Since a family of proteins that have this site will likely conserve the zincbinding ligands, this should be the first level of comparison. In addition, there may be a catalytic group or a possible secondary ligand orienter that is important to the stability of the zinc that i s conserved in the vicinity of the proposed zinc ligands. This can also narrow the search for prospective family members. After such a group of proteins has been compiled, the expression and purification of a member of this group can be carried out. Analytical measurement of zinc by an established analytical means that provides information on the metal to protein stoichiometry will give the first indication of the validity of the postulate. Methods such as zinc blotting should be avoided since they are not quantitative and are artifact-prone. Thus, the original studies indicated that several known zinc enzymes did not exchange with 66Zn and several non~etalloproteinsdid bind "Zn [285-2871. XAFS can give information on the type and number of zinc ligands. Mutagenesis of putative zinc ligands in conjunction with zinc binding and enzyme activity measurements will provide further evidence for the predicted zinc binding site. In these experiments some form of affinity chromatography or affinity labeling of the expressed protein should be made to avoid ambiguous answers due t o a mixture of active and inactive proteins, some of which bind zinc. The ultimate verification of the zinc binding site will need structural determination by X-ray crystallography or NMR. The ability to rapidly determine structures is both a blessing and a potential curse. Since zinc can be added to crystallization solutions and may in fact aid in forming crystals, weak zinc binding sites can readily be determined in the crystalline state that may not be physiologically important. Zinc should be demonstrated to bind in solution to the system under inspection and a measure of the binding strength should be obtained. Since zinc in the human body fluids will likely be bound t o some macromolecule, in order for it to bind to the protein being examined it must be able to compete with other zinc proteins in its environment. Dissociation constants weaker M are of questionable physiological significance since many metabolites than could compete with such a weak zinc binding site. Future mechanistic studies of zinc metalloenzymes will continue to benefit from a combined approach using mutagenic, structural, and transient state kinetics to examine the system. However, the next decade or two will also likely begin to bring answers to how zinc is stored, transported, and distributed and how it influences the earliest stages of development. Thus, information should become available on the role of zinc in morphogenesi~,cell division, and difrerentiation. Based on its past history we can expect zinc to turn up in many unanticipated situations.

AULD

942

A

AAP ACE ~~

CA captopril CMC G G D-PEN EDTA

IUB LT& M E ~ O ~ S MetAP-1 MetAP-2

MHC MlVP NAD NADP NMR nNOS, NOS-1 PAC

PAP PAR peptidase

PKC PMI PRL protease proteinase PTS

NS AND DEFINI~IONS Aeromonns proteolytica aminopeptidase angiotensin-converting enzyme a distintergrin and metalloprotease domain (human form of snake venom metalloprotease) alcohol dehydrogenase bis (5-amidino-2-benzimidazolyl)methane bovine lens leucine aminopeptidase carbonic anhydrase (2S)-~-1~-3-mercapto-Z-methylpropiony~-~-proline l-cyclohe~l-3-(2-morpholinoethyl)c~bo~n~ide cobalt carboxypeptidase carboxypeptidase A u-penicillamine ethylenediaminetetraacetic acid endothelial nitric oxide synthase human growth hormone human immunodeficiency virus class I1 major ist to compatibility molecule human interferon inducible nitric oxide synthase International Union of Biochemists leukotriene hydrolase system for classification of peptidase sequences methionine aminopeptidase-1 methionine aminopeptidase-2 major histocompatibility complex matrix metalloproteinase nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate nuclear magnetic resonance neuronal nitric oxide synthase perturbed angular correlation of y-rays purple acid phosphatase 4-(2-pyridylazo)resorcinol enzyme acting on peptides protein kinase C phosphornannose isomerase prolactin receptor enzyme acting on proteins; see also proteinase enzyme acting on proteins; see also protease phosphoprotein transfer system

ZINC SITES IN ~ E T A ~ ~ O E N ~ Y M ~ §

SEA SEC SED SOD SpeAl SREBP TL TRAP TSST VanX Wat W S zinc blot

943

Staphylococcus aureus enterotoxin type A Staphylococcus aureus enterotoxin type C Staphylococcus aureus enterotoxin type D superoxide dismutase superantigen streptococcal pyrogenic exotoxin A sterol-regulatory element binding protein thermolysin tartrate-resistant acid phosphatases toxic shock syndrome toxin dipeptidase of vancomycin-resistant pathogenic enterococci water X-ray absorption fine structure method to detect zinc binding by 65Zn autoradiography

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945

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286, L. Serrano, J. E. Dominguez, and J. Avila, Anal. Biochem., 17Z7 210-218 (1988). 281 L. A. Schiff, M. L. Nibert, and B. N. ~ i e l d sProc. , ~ ~ tAcad. Z . Sci. USA, 86, 41954199 (1988). 288. H. W. Park7 S.R. Boduluri, J. F. Moomaw, P. J. Casey, and L. S. Beese, ~c~eri'ce, 275, 1800-1804 (1997). 289. . F. Schmid and 3.R. Herriott, J. MoZ. Biot., 103, 175-190 (1976L 290. A. Guasch, M. Coll, F. X. Aviles, and R. Huber, J. Mol. Biol., 224, 141-157 (1992). 291. E. Schl~genhau€,R. Etges, and P, Metcalf, Structure, E;, 1035-1046 (1998). 292. . Lacy, W. Tepp, A. C. Cohen, B. R. DasGupta, and R. 6 , Stevens, re Struct. Biot., 5, 898-902 (1998). 293. D. B. Lacy and R. C. Stevens, J. Mot. BioZ,, 291, 1091-104 ~ 1 9 9 9 ~ . 294. J. M. Ghuysen, Evolution of DD-peptidases and beta-laetarnases, in A n t ~ ~ ~~ ~o ~~ ic ~ ioft Bacterial i on Cell Surfme Assembly and Fanctian (P. Actor, L. ~ a n e o ~ ~ o oM.r eL., ~ ~ i g ~ eM, n sR., J. Salton, and C.D. S h ~ e k ~ ~ n Eds.), American Society for Microbiology, Washington, C, 1988, pp. 268284. 295. F. X . 6omis-R~th,L. F. G e s s , J. ~ ~ ~ ~I, May, e r X.~ Lee, ~ n , and VV. Rode, J. Mol. Biol., 239, 513-544 (1994). 296. T. Kumasaka, M. Yamamoto, H. Moriyama, N,Tanaka, M. Sato, Y. Katsube, Y. ~ a ~ T.~ ~ ~ w i o r i -aS a ~~oS h , ,Iwanaga, and T. Ueki, el: ~ ~ o € yo), 119,49-57 (19961. 297. VV. Gong, X. Zhu, S. Liu, NI, Teng, and L. Niu, J. Mol. Biol., 283, 657-668 (1998). ~, G. P. B o ~ r e 31-1. n ~Bartu~iik, ~ ~ ~ 298. N.Mnskos, 6. F ~ r n a n d e ~ " C aR.t ~Muber, G. A. Ellestad, P. Reddy, M. F. Wolfson, C. T eh, €3. J. Castner, R. avis, €3. R. Clarke, M. Petersen, J. N.Fitzner, Cerretti, C, J, March, . J. Paxton, R. A. Black, and W. Bode, Proc. . Acad. Sci. ~ S 95,~ , 3408-3412 (1998). 299. B, Lovejoy, A. Cleasby, A. M. Hassell, K. Longley, M. A. Luther, D. Weigl, 6. ~ e G A. eB. ~~ ~ ~ ~I) ~.r Drewry, o y , M, H. Lambed, and S. B. Jordan, Science, 263, 375-377 (1994). 300, I?. J. Moy, P. K. Chanda, S. Cosrni, M. R. Pisano, 6. Urbano, d. . Powers, ~ ~ o c ~ e m37,i s1495-1504 ~~, (19981. uxley, S. J. Davies, Y. Mushtaq, M. Pieper, H. ' ~ s e h e ~ c hW, e~ 301. ode, and F. X. Gomis-Riitb, Eur. J. Biochem., 247, 356-363 (19971, Marcy, G, C. Guca, S.P. Salowe, B. L. 302. P. R. Go K, ~ a ~J. P.~ S~ringer, n , and €3. A. Bush, 3. Johnson, 303, 3. W. Becker, A. I. Marcy, L. L. Rokosz, M. 6. Axel, J. J. ~ i ~ ~P.~M. eCameron, r ~ d6. ~K. Esser, W. M. Hagmann, J. D. EEermes, axid J. P. Springer, Protein Sci., 4# 1966-1976 (1995).

. Z. Ye, L. L. Johnson, D. J. Hupe, . F. ~ ~ J. E, ~ R. Rubin, A. Pavlovsky, C. Humblet, and T. L. Bfundell, S t ~ ~ 4,C375-3863 ~ ~ ~ (1996). ~ , 305. F. X. 6omis-Ru~h,U. Gohlke, M. Betz, V. Knauper, 6. Murphy, C. LopezOtin, and W. Bode, J , Hal. B i d , 264, 556-566 (1996). 306. B. Lovejoy, I&, S. Cam, C. Luong, C. Broka, R. T. Wendricks, J. A. C~pbell, lker, R. ~ a r ~ E-f.n ,Van Wwt, and M, F, ~ ~ o ~ ~ Nature Struct, B i d , 6, 217-221 (1999). 307, . Wilson, F. B. ~udolph,and F. A. ~ ~ o Science, c h 252, ~ ~12~$-12$4 304.

(1991). 308. T. me inn el^ S. Elanquet, and F. Dardel, J. Mol, B i d , 262, 376-386 ( 1 ~ 9 6 ~ . 309. M. K, Chan, W. Gong, I?. T. Rajagopalan, B. Hao, 6. M. Tsai, and D. Pei, ~ ~ ~ c ~ 3G5 e ~1 3z 9§0 ~t ~~3 9, 0(1997). 9 310. A. Bechr, I. Schlichting, W. Kabsch, D. Groche, S. Schultz, m d A. F. Wagner, N ~ t ~ Struct. r e Bid., 5, 1053-1~5$(1998). 311, T. F. KO, C. C. Liao, W. Y. Ku, K. F. Chak, and H. S. Yuan, Structurej 7, 91102 (1999). 312. D. J. Hosfield, Y. Guan, B. J. aas, R. P. Cunningham, and J. A. Tainer, Cell, 98, 3 9 7 ~ (1999). 0 ~ B. Birknes, K. Jynge, S. Wmsen, A. ~ o r d v i k ,6. 313. wewenda, ~ ~338, 357-360 ~ ~(1989).e , 314. A. Volbeda, A. Lahm, F. Sakiymna, and D. Suck, EMBO J., IQ, 1 6 0 ~ - 1 6 ~ 8 (1991). 315. D. M. Burrgissw, B. Thony, U. Redweik, D. Hess, C. W. Heizmann, R, Huber9 and W. Nm, J. M'l. B i d , 253, 358-369 (1995). 316. T. Ploorn, B. Thony, J. Yim, S. Lee, H. Nar, Mr. Leimbacher, J. Richardson, R. Huber, w d G. Auerbach, J. ~~1~ B i d 9 286, 851-860 (1999). 317. P. T. Erskino, N. Senior, S. Awan, R. Lambert, 6. Lewis, I. 3 . TicMe, M. S ~ ~P. aSpencer, ~ , P. Thomas, M, J. W a ~ e P. ~ M. , S h o o l ~ n ~ n - J o r ~S.a n ~ P. Wood, and J. B. Cooper, Nat. Struc. B i d , 4, 1026-1031 (1997). Goker, G. Lewis, 318. P. T. Erskine, E. Nor~on,J. 11. Cooper, R. Lambert, Spencer, M. Sarwar, S. P. Wood, M. J. Warren, and P . S € i o o l i n ~ n ~ J o ~ d a n ~ l ~ c ~ 38, e ~4266-4276 ~ § t ~(1999). , t ~ ,17726-17734 319. S, Kmlin, Z. Y. Zhu, and K. D. &din, B i o c ~ e ~ i §37, (19981. 320. L. Chantidat, D. Leroy, 0.Filhol, A. Nueda, M. J. Benitez, E. M. Chmbaz, G. Cocket, and 0. ~ i d e b e rE~ ,~ B O J., 38, 2 ~ ~ 0 - 2 9 4(1999). 0 J., 16, 3396-3404 (1997). 321. M. Hyvonen and M. Saraste, . N. Jones, B. P. Kaine, and M. A. Weiss, S t ~ u ~ t;,t 5~6-569 ~ r ~ ~ 322. (1998). ~ o 38, 1c ~ ~ ~9 ~ 1 e(314~~~96~ ~~. ~ 323' S , ~~~n~~ and P. A. Frey, ~ ~~~~

AULD

958 324.

. Raaijmakers, 0. Vix, I. Toro, S. Golz, B. Kemper, and D. Suck, ~ M B O J., 18, 1447-1458 (1999).

325. 326. 327. 328. 329. 330. 331. 332.

. Fourmy, F. Dardel, and S. Blanquet, J. Mol. Biol., 231, lQ78-1089 (1993). . Fourmy, T. Meinnel, Y. Mechulam, and S. Blanquet, J. Mol. Biol., 231, 1068-1077 (1993). . Cai, R. Zheng, M. CaErey, R. Craigie, G. M. Clore, and A. M. Gronenborn, Nature Struct. Biol., 4, 567-577 (1997). J. A. Tainer, E. D. Getzoff, K. M. Beem, J. S. Richardson, and D. 6 . Richardson, J. Mol. Biol., 160, 181-217 (1982). . E. Parge, R. A. Hallewell, and J. A. Tainer, Proe. Nature Acad. Sci, USA, 89, 6109-6113 (1992). K. D. Carugo, A. Battistoni, M. T. Carri, F. Polticelli, k Desideri, 6. Rotilio, A. Coda, and M. Bolognesi, FEBS Lett., 349, 93-98 (1994). Y. Kitagawa, N. Tanaka, Y. Hata, M. Kusunoki, 6. P. Lee, Y. Katsube, K. ada, S. Aibara, and Y. Morita, J. Biochern. (Tokyo), 109, 477-485 (1991). Djinovic, 6. Gatti, A. Coda, L. Antolini, G. Pelosi, A. Desideri, M. Falconi, rmocchi, 6. Rotilio, and M. Bolognesi, J. Mol. Biol., 225, 791-809

333. N. L. Ogihara, H. E. Parge, P. J. Hart, M. S. Weiss, J. J. Goto, B. R. Crane, J. Tsang, K. Slater, J. A. Roe, J. S. Valentine, D. Eisenberg, and J. A. Tainer, B i o c ~ e m i s t35, ~ , 2316-2321 (1996). 334. Y. L. Chen, S. Park, R. W. Thornburg, L. €3. Tabatabai, and A. Kintanar, iochernistry, 34, 12265-12275 (1995). 335. M. M. Benning, J. M. Kuo, F. M. Raushel, and H. M. 33, 15OQl-15007 (1994). 336. J. L. Vanhooke, M. M. Benning, F. M. iochemistry, 35, 6020-6025 (1996). 337, J. L. ~uchbinder,R. 6. Stephenson, M. J. D Scanlan, and R. J. Fletterick, Bioche~istry,37, 5Q96-51Q6(1998). 338. T. H. Tahirov, H. Oki, T. Tsukihara, K. Ogasahara, K. Yutani, C. P. Libeu, Y. Izu, S. Tsunasawa, and I. Kato, J. Strruct. Biol,, 121, 68-72 (1998). 339. T, Ecnofel and N. Strater, Nature Struct. Biol., 6, 448453 (1999). ndqvist, E. Johansson, H, Kaija, P. Vihko, and 6. Schneider, J. Mol. 340. 291, 135-147 (1999). 341. , Guddat, A. S. McAlpine, D. Hume, S. Hamilton, J. de Jersey, and J. L. rtin, Structure Fold Des., 7, 757-767 (1999). 342. 2. P. Griffith, J, L. Kim, E. E. Kim, M. D. Sintchak, J. A. Thomson, M, J, Fitz~bbon,M. A. Fleming, P, R. Caron, K. Hsiao, and M. A. Navia, Cell, 82, ~ Q 7 - 5 ~(1995). 2 343. . Y. Zhang, I. M. ardon, J. 0. Hui, K. L. O’Connell, R. A. Poorman, A. 6. Tomasselli, and R. L. Neinrikson, biochemist^, 30, 8717-8721 (1991).


959

~ s ~ 344. T . N. Wells, F. Goulin, M. A. Payton, and A. E. Proudfoot, ~ i o c h e ~32, 1294-1301 (1993). 345. A. Persechini, K. McMillan, and B. S. Masters, Biochemistry, 34, 1509115095 (1995). 346. J. M. Perry and M. A. ~ ~ l eProc. t t Null. ~ ~ Acad, Sci. ~ ~ 95,A 11101, 11106 (1998). 347. W. 6. Niehaus, S. B. Richardson, and R. L. Wolz, Arch. Biochem. Biophys., ,333, 333-337 (1996). . K. Perry, M. J. S ~ ~€3.hR., E3t~~n~cke, 6. E3. Salvesen, P, Duriez, G.G. Pokier, and Y. A. Hannun, J. BioE. Chern., 272, 1 8 5 3 ~ ~ 8 (~~393 ~ ? ~ . 349. A. Teplyakov, K. Polyakov, G. Qbmolova, €3. Strokopytov, I. Kuranova, A. CPsterman, N. Grishin, S.Smulevitch, 0. Zapitko, 0. Galperina, M. Matz, and V. S ~ ~ ~ a Eur, n o v3.~B i o c h ~ ~208,281-288 ,, (1992). 350. X. Cbeng, X. Zhmg, J. W. P ~ u ~ a t and h , F. W, Studier, Proc. Natl. Acud. Sci. USA, 91, 40344038 (1994).


Department of NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

1. INTRO~UCTION 1.1. Hisb~calPerspectives 1.2. ~ e ~ ~ of t iZinc o iFinger ~ D o ~ ~ n s 1.3. Focus of this Chapter

962 962 962 963

2. ZINC FINGER PROTEINS WITH KNOWN STRUCTURE 2.1. Zinc Finger Domains C ~ n t ~ nOne in~ Zinc Atom 2.1.1. C2H2 2.1.2. C2HC 2.1.3. GATA 2.1.4. Zinc Ebbon

964 964 964 965 970 9-71 972 972 975 975 975 976 976 577 578 979 979

2.1.6. Integrase 2-1.7. BTK 2.1.8. XFA 2.1.9. Copper Fist 2.1.10. BIR 2.2. Double Zinc Finger Proteins 2.2.1, C4 Nuclear Hormone Receptors 2.2.2. GAL4 ~ i ~ ~Cluster c l e ~ 2.2.3. LIM 2.2.4. RING

961

FOLKERS, ~

962

~

~ AND ~

A

2.3. ~ ~ ~ b r a n e - ~ iDouble n d i ~~~i ngc - ~ i n gDomains er 2.3.1. C1 2.3.2. FWE and ~ a b p h i ~ 3An

A

981 981 981

3. ZING FINGERS WITH UNKNOWN S T ~ U G ~ ’ ~ ~ ~

4. ~ ~ ~ U C T ~ R ~ ~ -~ ~ U ~ ~ T~ ~~ I ~~ N ~ 4.1. Evolutionary Aspects 4.1.1. Zinc Finger Proteins in Eukaryotes 4.1.2. Absence of Zinc Fingers in Prokaryotes

~

982

S

~ 983 I 983 983 987 389

990

~~F~~~~~~~

992

As early as 1869 it was realized that ziiic is an essential trace element far e ~ ~ ~ o t e s In fact next to iron, zinc is the second most abundant trace element in humans, With the identification of zinc in enzymes such as carbonic mhydrase and carboxypeptidase, where the zinc atom is present at or near the active site of the enzyme, it was initially thought that zinc was required for catalysis. In another classical zinc enzyme, alcohol ~ e h ~ ~ o g e n one a s ezinc ~ atom is in its active site and is involved in the catalytic function, while another zinc has a rather structural function. The difference between the two types of zinc binding domains is the Iigand usage for metal coordination. In the first case, zinc is bound to two histidines, one oxygen derived from a carboxyl side chain and a water molecule or three histidines and a water molecule. In cont~ast,in the latter case zinc is coordinated by four sulfur atoms of cysteine residues resulting in a tetrahedral coordination o€ the metal. At present, structural info~mationis available for 775 proteins in the Protein Data Base (PDB [I])that contain one or more zinc atoms, many of which arc enzymes. It i s not our intention to describe all proteins that contain one or more zinc atoms. Excellent reviews on many zinc binding d ~ m ~ can i n be ~ found elsewhere (12-41 and refs therein; see also Chapters 19 and 20), Instead we will focus in this review on the so-called zinc fingers.

efinition of Zinc Finger Domains We define the zinc finger as a zinc binding motif that can autonomously fold into a separate f u n c ~ i o nunit ~ or domain fan i n d ~ ~ e n d folding e n ~ unit) where the zinc is bound tetrahedrally by a combination of cysteines and Mstidines. Common motifs are

ZINC FINGER PROTEINS

963

C2312, &‘2HC,or C4, but various other combinations exist. According to this definition, we do not consider the zinc metalloproteases, the metallothioneins, or the copper-zinc superoxide dismutases as zinc fingers due to their nontetrahcdral zinc coordinatio~.~ u ~ h e ~ oproteins r e ? such as the adenovirus e2a ~ ~ A - b i n prod~n~ tein, the tumor suppressor protein p53, and the 6’ subunit of the clamp loader complex of R coli DNA polymerase I11 are not viewed as zinc finger proteins because the structural role of zinc is merely to properly position (parts ODdomains, while folding of (parts of these) proteins most Iikely can occur independent of the zinc. Finally, several proteins contain a typical zinc finger region within a more complex structure. Initially, the term zinc finger was used only for one class of proteins found in the t r ~ s c r i p t i o nfactor T fIlA from Xerzuplcs laeuis, which contains a sequence of nine ns as repeats of the C2H2 motif 151. ~ubsequently,it was found that this repeat an independent DNA binding domain. Soon after the identification of this class of zinc finger proteins, novel types of zinc fingers were identified, all of which appeared to be involved in binding to nucleic acids. This led to the d o p a thlat zinc C6-81. However, with the identi~~cation of finger domains are ~ ~ A - b i n d i nunits g proteins trhat interacted with various zinc finger domains, it became clear that these domains might also mediate protein-protein interaction f91. The zinc finger domain is quite abundant. Previous estima~~ons led to the proposal that approximately 1%of all mammalian proteins contain one or more zinc finger domains, but our analyses indicate that this may be a conservative estimate (see Sec. 4.1).

t .3. Focus of this The presence of conserved eysteines and histidines within a sequence p r o ~ d e sa strong i ~ ~ c a t i ofor n the presence of a zinc finger, especially if in~tatioiiof these conserved residues is deleterious for zinc binding and function of the domain. However, the three-dimensional structure is the only firm evidence of whether the ~ o l ~ e ~folds t ~ as d ea zinc finger domain. i othose ~ ~ zinc finger proteins At first we will s u m m ~ i s~t er u c t ~ r eand ~ ~ n c tfor for which the three~diInensiona1structure is known by taking advantage ofthe various available databases [&lo-151.We describe species distribution, the number of proteins that contain this zinc finger domain and the ~ ~ number ~ of zinc m finger ~ repeats per protein (see ables 1-31 W--l31. Fu~hermore,we specify some evolutionary aspects for zinc finger proteins: Is this domain present only in proteins from different organisms that perform identical function (orthologs), or do more distantly related protein8 that fulfill ~ f ~ ~f ~~ c et i no (paralogs1 nt ~ contain this domain (T~bles 1-4) [lo-121. Next to this functional description we will clarifl. the s t r L ~ c t u ~ aspects a~ [ll for the various zinc finger proteins: the metal coordination pattern; how zinc is coordinated by the ligands (see Fig. 2); the topology of the domain (see Fig. I), ~ n the folding ~ t o~ ~ o according ~~ o ~ ~ to the ~A~~ n El4~1 and SCBP /151 abases (see Table 1).Finally, we illustrate where the metal ligands reside in the structure (see Ef’igs.1 and 2). We will highlight the similarities and differences among the zinc

finger protein structures that provide a framework for further analysis of putative zinc finger proteins. These zinc finger domains will be f ' u n c t i ~ ~described n ~ ~ ~ (see Table 2).

The classical C2H2 zinc finger, or ~ F I finger, I ~ is characterized by the C x 2 C x 12 H x 3H consens~satt tern. This domain, c o n ~ i s t i nof~a p p ~ o ~ n ~ a30 t eresi~y dues, functions as an independent DNA binding domain. Subsequently, such DNA binding domains were found in many ti.~nscriptionfactors. At present, approxi~nately 9OQQcopies of this domain were found in 1735 proteins ~$~~~ TlQlJ.This almost certainly classifies this domain as the most abundant eukaryotic DNA binding t e g e n o ~ using e a C2 ~ o ~ a i~nn ,d e edetailed ~, analysis of the ~ o ~ p l eyeast pattern revealed the presence of 105 classical zinc fingers in 53 proteins in the yeast genome (161. Similarly, so far 189 different proteins in C. elegans were reported to contain in total 640 zinc finger domains. In both cases, these results indicate that approximately 1%of all proteins in both organisms contain one or more T F I I I A ~ l ~ e e nestimates t made for humans ff7J.So fay. zinc fingers, This is in good a ~ ~ e ~with 523 human proteins containing in total more than 3500 C2H2 domains were found. Given the total amount of human genes present in the n o n r e ~ u ~ d adatabases nt (e.g., OT and § ~ - T r contain ~ ~ ~ 5406 L and 94-17' human p r o t e ~ nrespectively; ~, [lSI, the percentage of classical zinc fingers in the genome of higher ~ tThe C 2 ~ zinc 2 finger domain has been found in e ~ k ~ y o~t ~ sieven~be higher. many important proteins. For example, YY1, SPI,EG1Z-1, and W T are important regulators of' gene expression and are involved in (early) embryonic development. Finally, it has been reported that these proteins are involved in cancer ~ o ~ n ~ a t i o n as a consequence of mutation or aberrant expression (see [19]). A solution s t ~ c t of~ the e first zinc finger was d e t e ~ ~ i ~ as n eearly d as 1988 [203 from the Saccharomyces cerevisiae protein ADR1, which regulates the expression of genes involved in carbon source metabolism. Subsequently, structures of many C2H2 single and multiple zinc ~ ~ e r - c o n t a i n iproteins ng have been solved both free [21--261 and in the presence of DNA [27-321, no large structural differen f81, At present, 34 between the free and D ~ A - b o ~proteins d have been deposited at the PDB fll. All of thesc zinc fingers share a similar fold: two i ~ e ~ l a rshaped ly antiparallel j3 strands followed by an Q helix (Fig. 1). For several proteins, e,g., $wit5 1261 and t r a m t r a c ~1291, a third p strand is found. The additional p sheet appears to be required for folding of this zinc finger domain. The zinc atom is c o o ~ d i n aby t ~the two cysteines located between the two j3 strands, with a turn hetween the two strands, while the two histidines are located in the C terminus

of the CL helix (Figp. 1and 3). Furthermore, stabilization of the structure is obtained by contacts between the sheets and the hydrophob~ccore of the a helix. ~ e n ~ r a ~ multiple copies of the C2H2 zinc finger me found (e.g., Xfin contains 37 copies). However, each finger can fold independent~yas demonstrated by the s i m i ~ ~ among the structure of the individual zinc finger domain and the c o ~ e s p o n d ~ n g [22,25]. The s t ~ ~ u roef the s various zinc finger proteins a r ! m u l t i ~ ~ structure er quite similar, with backbone atomic rms differences of maximally 0.9 and 1.2 A between the ~ n d i ~ d fingers u a ~ of Zif268 and MBP-1, respectively, and only 1.0- to 1.5-A c2ifFerences were found between the two fingers of MBP-1 a i d the three Zif fingers 1251. Although the fingers fold independently, interactions could be identified between the two fingers 0 ~ ~ f251,~ whereas ~ for - Swi5 1 no inter~ngercontacts were I ~ contacts were described between the indidetected [33]. Also for T ~ Iinterfinger n t of the variotxs finger regions within the vidual fingers c a ~ s i n ga ~ e r ~positioning major groove o f [31]. These findings suggest that, although the overall folds of the i n ~ i v i d ~ G a ~ inc fingers are alike, small d i f f e r e ~ ~with e s respect to the folding topology and the ~ n t e r ~ n gcontacts er can have substantial impact on their biological activity. The solution structure o f the C2H2 zinc fingers present in the termin in^ domain of ATF-2 is giving a new hnctional insight into this class of zinc fingers f341. The ~ t ~ ~ tofu this r e zinc finger i s highly arable with other C2H2 zinc fingers, with a backbone rnisd o f approximately 0.7 A with two different C2H2 zinc fingers. Most remarkably, this domain is present in the minimal transcription activation domain of this transcription factor and is not required for DNA binding, in contrast to all the above-described zinc finger structures. This is also the first example of a ~ i n i n ~ trans~ript~on al a ~ t ~ ~ domain a t ~ ~that n shows s t ~ in ~isolation, ~ r ~ although the C-terminal half of the domain is, in agreement with earlier structures of other a c t ~ ~ ~ ~ u nt os tr~~~,t u r e~d i. o c h e m i cdata ~ have clearly demonstrat~dthat ~ h o s p h o ~ l a t i oofn residues within the minimal activation domain but outside the zinc finger region are required for activation of transcription E351. It was proposed o f kinases are at least partially mediated by the zinc finger region, that inte~~actions arguing that also this non-~NA-bindingC2H2 zinc finger domain can mediate prot ~ i ~ - ~ r o t~nteractions. ein This finding is not novel fm this class o f zinc fingrs: homoand h e t e r ~ d i ~ e r i ~ a twas i o n reported, being critically dependent on the zinc finger regions [9].

Shortly after discovery of the C2H2 zinc fingers, the retroviral zinc finger, or C2 zinc finger or zinc k n ~ domains c ~ ~were found in tthe nucleic acid~bindin~ protei~~ of the retroviral gag protein, This protein is cleaved by viral proteases to give rise to sevcrd small proteins, including the nucleocapsid proteins, which contain one or two C2HC fingers. All members of this C2HC zinc finger protein family mcompass 4 C, which is frequently found in viral. prothe conserved pattern C x 2 C teins (for both ~~A~ [I23 and T [lo1 over 1000 proteins were detected; see

of the ~ t ~ c t and u rF ~~ n ~ i Co hn~ ~~ c t e ~ i z aoft iZinc o ~ Finger Domains

S~~~ Domain*

PDBb

dC0P fold‘

CATH classd

C2H2 C2HC C2HC GATA Zinc ribbon Zinc ribbon 6 box Integrase

WAY

C2H2

x+O

Erg WA Copper fist BfR2, BIR3 C4NHR GAL

htrovirus Few

1GAT 1TFI 1PFT 1FRE I.AUB 1BTK IXPA

GR

1KGD ID66 Lrn LCTL RING lCHC C1 1PTQ Frn lWY ~ b ~ h i l i n - 3lZBD a

Rubredoxin p Rubredoxin fi Rubredoxin Few 3 3helix -.

-

GR

___

Zn Cu txnf -

w

6.R ZnZCys6 GB RING PKC P13BP

Pl3BP

%layer sandwich

Irregular r+P 2-layer sandwich

1AAF

1C04 IQBH

CATH architerture‘

x+P Few

6 x+p

P

-

Single sheet Single sheet Irregular Nonbundle

_ I

I _

2-layer sandwich Irregular Ribbon %layer sandwich Single sheet

_c_

I I

KO.

Zn‘

Pattern”

1 C 2,5 C12,15 II 3,5 H 1 C2C4H4C

Patterns” 1-37 1-9 1-2 1-2 1 1 2 i,l+ring) 1

162C4H4C 1 C 2 C 16,20 C 2 C 1 C2C24C2,5C 1 C2C15C2C 7. C 2 cih 14,17 C 3,6 EX 1 H3H23C2C 1 H9,lO C C 9 C 1 C 2 C 17,18 C 2 C 1 CZC8C1H I C2C16H6C 2 C 2 C 13 C 2 C 15,17 C 3,7 C 9 G 2 C 2 C 2 C 6 C 6,12 C 2 C 6,8 C 2 C 2 C 16,23 R 2 C 2 C 2 C 16,23 C 2,sC 2 C 2 C 9, 39 C 1,3 W 2,3 C 2 C 4,48C 2 C 2 H 10,13 C 2 C 9,14 C 2 C 4 H 2 C 5,9 C 2 C 2,4 C 12,23 C 2 C 4 C 2 C 1636 C 2 C 2 C 2 C 13,15 C 2,4 C 4 C 2 C14,19 C 2 C

1 1

1

1-3 1 1 1-5

1 1-2 1

1

Example’

Function’

TFIEA: SPl,Zif268 Transcription nanos, BkR3 Nucleic acid binding gag, NCP7 Nucleic acid binding GATA-1 Transcription TFIXS, RBP9 Transcription TFXTB Transcription Xnf?, PML Transcription HIV integrase Integration Signaling WrK xP.4 Excision repair Amtt Transcription XI,@, MIHB Signalling GB, ER, RAR, RXR TraosLTiption gd4,PPR1, PUT3 Transcription CRP, Enigma, Lhx 1 Signaling rag 1,ring1 Signaling Signding Raf, PKC Fabl Signaling rabphilin-3A Bigrialing

DNAk Prot’

+

+ 4-

+ +

+ + +

+

Refm

+

6,19

+ +

36 45 49,50 55 61 64 67 68 72 73,774 77,78 102 110,111 61,119 127,129 126,134 137

+

+ +

+ + + + + +

+

+

“Domain: most common name of this zinc finger domain. bPDB: PDB accession number for a representative example of this class of zinc fingers, with a number referring to the paper desciibing the structure I11. ‘SCOP fold the fold according to the SCOP database [151;-: no info available. dCATH class: classification according to the CATH database [141;-: no info available, tl: mainly 0. helix; p: mainly p sheet; c1-k p: tl helix and p sheet. ‘CATH architecture: description of the architectme according to the CATH database. fZn: number of zinc atoms per domain. gpattern: consensus pattern of ligands: C and H are cysteine and histidine residues while the numbers in between indicate the minimal and maximal spacing between the ligands, c!’h represent either C or H. Consensus patterns are obtained from multiple sequence alignmentsderived from the PFAM 1121or SMART [lo] databases. Occasionally the consenms pattern is determined by omitting few putative zinc finger proteins with irregular spacing between the ligands. hNo.patterns: minimal and maximal number of domains per prohein ’Example: few proteins that contain one or mare of these domains JFunction: process in which the protein that contains this domain is most frequently involved in. ‘DNA interaction of this domain with RNA or DNA ‘Prot: interaction o f this domain with protein. mRef;reference to review(s! or to paper($ describing the structure of the domain.

968

FOLKERS, H A N ~ A W AAND , BOELENS

969

FIG. 1. Struc~ureof zinc finger domains. Ribbon diagram of r e p ~ e s e n t a examples ~~e of the three dimensional fold of the various classes of zinc h g e r domains using the PDEj ill coordinates, Side chains of the zinc coordination residues are in black, the zinc atoms are repmsented as black balls, at the N-termind end a N is depicted. The figure was produced with M ~ [159]: C2R2 (IAAY): crystal structure of the mouse Zif268 zinc ~ n ~ r complex - ~ ~11601. A C2HC (1M): NMR structure o f the HIV-1 nucleocapsid protein [381. GATA (IGAT): NMR structure of the avian erythroid t r ~ ~ c ~ ~factor t i oGATA-1 n DNA hinding domain in complex with DNA W1. Zinc ribbon (1TFI): NMR structure of the human transcriptional elongation factor 11s DNAbinding domain EsU, B-box (1FBE): NMR structure of the Xenopus laeuis nuclear factor xnff B-box domain 1631. htegrase (IAUB): NMR structure of the N-terminal domain of HXV-2 integrase f651. UTK (IEVK): crystal structure of the zinc binding domain of the human Bruton's tyrosine kinase 1671. XPA (1XFA): MMIK, strwcttire of the human repair factor XPA DNA and RPA binding domain

[7Ol. BIR ( 1 ~ B ~NMR ) : st~uctureof the liuman ~ ~ b ~~ € t ao ~r ~ t o ~ ~s ~i h s b / c - i a BER p - l )domain 3 E741.

NHR (1RGD): NMR structure of the rat glucocorticoid receptor DNA binding domain T991. ~ n ~ 1 ~~6crystal 6 ~ ~: structure s of ~the ~ a & c h a ~ ~ cereuisiae ~ , y ~ e s Gal4 protein in complex with DNA [lO6l. LIM (1CTL): NMR fitructure of the avian cysteine-rich protein [1131. RING (1CHCk NMR structure of the equine herpes virus-1 c3hc4, or RING domain [123]. C1 ( 3 PTQ: crystal. structure of the second C 1 domain of mouse protein kinase C delta [133]. mTvE ~ 1 ~ crystal ) structure : of the ~ a e ~ ~ a ~ oeereuiszae ~ y c e s phosphatidylinosital-~~~hos" phate binding FYVE domain of protein vps27 [1351. ~ b ~3A (1ZBD): h ~ crystal l ~ structure ofa complex nf the rat Eab-3a protein and rat r a b p ~ ~ n 3a ofrector domain f1377.

Table 3 in See. 4.1). Inspection of these finger proteins revealed that most of them are sequence variants of the HIV gag protein. More interestingly over 350 non-viral proteins were found in vaiious organisms that contain 62HC zinc fingers as well, such as nanos, ByrS and CNBP. Although not studied extensively, many of these p r o t e ~ ~like s , the viral nueleocapsid proteins, share the ability to bind nucleic acids 136-381. In addition, these viral zinc fingers can also mediate protein-protein interactions [39,401. While the viral counterparts generally contain either one or two copies of this zinc finger motif, the e ~ k a ~ o tC2HC jc zinc finger proteins can contain up to nine repeats. Thus far, structural i~formationis only available for the nucleocapsid proteins 141-433.The backbone of the peptide folded around the zinc involves a number of turns that are stabilized by the tetrahedral coordination of the zinc by the three cysteines and the h~stidineresidues and by hydrogen bonding (see Figs. 1 and 3). The ~ ~ t e r m i six n a residues ~ neighboring the first two liganth of this finger are highly similar to the corresponding region of the iron binding site of rubredoxin, The two form ~ndependen~ domains that interact only fingers from the ~ u c l e ~ a p sproteins id transiently with each other and do not possess a fixed orientation relative to each other 1441. 2.1.3. GATA

The e ~ t h r ~ i d - s ~ e ctransc~ption ~fic factor GATA-1 can bind to the consensus bindG ] which this transcription factor is named. However, ing site I‘ ~ / A ] G A T A ~ ~after o ~ and is required for DNAthis domain is also found in other t r ~ n s c r i p t ~factors b i n d ~ ~ gThe . proteins of the GATA family contain either one or two zinc finger domains of the C4 type with a pattern C x 2 C x17 6 x 2 C. Omichinski. et al, de~er~~ then ~ solution d structure of the C-terminal GATA-1 zinc finger domain bound to a GATA binding site l4Sl and revealed both similarities and diff‘erences with the other zinc finger proteins. The zinc binding domain is composed of two short a ~ ~ ~ ~ a r pa lsheets, l e l an c5 helix, and a long loop. The Erst two cysteines are located at the end of strand 1 and in the hairpin loop between strands 1 and 2 of sheet 1,respectively, while the other two ligands are located in the 2 helix, (see Figs. 1 m d 3). ~ y ~ r o ~ hintera~tions o~ic further stabilize the relative o~entationof the structure elements with respect t o each otber. The zinc finger domain of the GATA family is sti-ucturally related to the Nterminat finger of the nuclear hormone receptor ~ ~ A ~ ~ domain i n d (see ~ ~See. g 2.2.1.)with respect to topology and conformation of the side chains of the Zn-binding for the 6%atoms between the first finger of the DNA Iigands. ~ o t ~ bthe ~ yrmsd , biiiding domain of the glucocorticoid receptor (see Sec. 2.2.1) and the GATA zinc finger was only 1.5 .&, The remainder of the structure of GATA is completely distinct from the other zinc finger proteins, and is €armed by a long loop aid an extended strand, the latter being required for specific DNA binding. Recently, the structure of the N-terminal finger of GATA-1 has been determined L451. This domain adopts similar confor~ationas the C-terminal finger (rmsd for Ca, C, and N is 1.8A for

the zinc binding domain). However, the N-terminal finger of GKFA-1 is not required for specific DNA binding and is by itself unable to bind to DNA. It does increase DNA binding affinity and specificity and is most likely involved in protein-protein interaction. Interactions has been recorded between the zinc finger regions of GATA-1 (selfassociation), but more interestingly also with SP1, FOG, and E Although no structural information is available concerning these interactions, biochemical data indicate that residues involved in protein-protein interaction are distinct from those involved in DNA recognition and that the rcsidues invol~edin protein-protein interaction form a single continuous surface 146,471. These residues are not present in the @-terminalfinger but are conserved in all of the N-terminal fingers of GATA proteins, strongly arguing that DNA and protein binding can occur simultaneously.

2.2.4. Zinc Ribbon The transcription e~ongationfactor TFfIS is involved in r e ~ ~ a t of ~ oread~through n at specific pause or termination sites during elongation of?transcription [49,501. The nucleic acid binding domain of this elongation factor contains a C4 zinc binding site with a pattern C x2 C x24 C x2-5 C. The structure for this domain has been determined and designated as a zinc ribbon Kill it has a characteristic threestranded antiparallel @ sheet (Fig. 1). Three @ turns are pwsent, two of which form the zinc binding domain created by the f.3 turn present in ~ - t e ~ m iregion n~l that is extended in nature and the f.3 turn between strands 2 and 3 of the j3 sheet. Although this protein can bind to DNA [SZI, the structure clearly differs fi*omother zinc finger proteins, because it lacks an M- helix as Eound in many other zinc finger domains. Sequence analysis led to the proposal that this domain might be present in other proteins invofved in nucleic acid transactions such as viral prirnases, UvrA, TFIIE, subunits of II. Determination of the structures for the archaeat RNA polymerase I1 subunit 9 [RBPSI [531 and the Bacillus stearothernzophilus primase [54] confirms the presencc of a zinc ribbon in these proteins. ~ u r t h e ~ o rite ,has been shown that a similar fold was also found in the N-terminal domain of TFIIEP ~551. Rece~tly,two novel zinc ribbon s t ~ c t u r e were s added to this class of zinc finger domains: the ribosomal protein 236 1561 and in the protein kinase CK2p [571. The overall folding topology is similar with an rnisd smaller than 1.1 for the main chains of TFIIS, T ~ ~andI EBP9, ~ , in~ludingthe zinc binding domain 1531. However, differences are found that are mainly attributable to the length of the loop between strands 1 and 2, the length of the @ strands, and the N-terminal region, in which in ~ B an ~ additional @ strand is present. In the case of the L36 protein 1561that has a C x2 C 12 C 4 H zinc-coordinating pattern, it is found that this four-residue spacer and the histidine are different from that of other zinc ribbons. ~ r t h e r m o r ethis ~ protein has an additional short 31Qhelix, located between p strands 2 and 3, possibly required to compensate €or the increased spacer between the last two ligands. Although many zinc ribbons bind nucleic acids, functiona~p~otein-pi-oteininteractions have been

A

9

972

FOLKERS, HAN

identified for a number of these domains, inc~udingdimerization (protein kinase CEu( E571f. ~ t h e r m o r e TFIIB , can interact with the activation domain of ftz 1581, the u ~ i g a n d e dvitamin D receptor [591, or with RNA polymerase IE [60].

-box zinc finger is a domain that is generally present in prote~nsthat also contain a R I N finger ~ (see See. 2.2.4).B-box proteins fulfill a hnction in transcripn or as a cofactor. Some of these proteins tion either directly as a t r ~ s c ~ i p t i ofactor een shown to be oncogenic after translocation of part of the genet including the region with another transcription factor. A classical example for this phenomenon is the formation of acute promyeloc~icleukemia, where the PML gene is fused with the retinoic acid receptor a. The B-box zinc finger is most likely required for p~otein"proteininteraction rather than for NA-binding [61]. Most biochemical evidence indicates that this domain alone is incapable of binding other proteins, but finger and a coiled-coil domain a functional protein-protein together with a R I N ~ interaction unit is formed [621. The zinc coordination pattern for this -box family remained unresolved since residues were conserved in the B-box family. By analysis of the struc-box zinc finger of XNF7, a Xenopus nuclear factor, combined with studies on mutant peptides and structure calculations, Borden et al. [631 determined that one Znz+ is bound to a C x2-4 C/H x 14-17 C 3-6 R sequence, The other Cys and is residues are not involved in metal ligation; the authors suggested that these residues are involved in metal-mediated dimerization L631. The s t ~ c t u r e(Fig. 1)is composed of two j3 strands that run perpendicular to each other. The first cysteine is found at the end of an extended region, the first histidine in p strand 1,and the other two ligands reside respectively in a helical turn and in the extended region that eventually runs into the second j3 strand.

i n t e ~ a s eis involved in the site-specific cleavage of the viral NA and the inteof this protein a on of the recessed DNA to the host DNA [64]. At the N termi x23-32 C x 2 C is found that is zinc finger motif c o n t ~ n i n gthe sequence conserved in all retroviral integrases. T nction of this zinc finger is not known. Since both biochemical experi~entsand structure information revealed that this domain is a dimer in solution, it has been suggested that this domain is most likely involved in dimerization, tetramerization, and/or oligomerization, The structure of the HIV2 monomer of this domain consists of three a helices [SSI elices 1-3 form a three-helix bundle that is stabilized by the zinc molecule. The coordination is distinct from that of other zinc fingers: the first in helix 1coordinates zinc via the Ne2 atom, while the second histi the other two ligands are formed by the sulfur atoms of the two in the loop between helix 3 and a helical turn, respectively (Figs.

laay

I03 132 ~ERPYACFVEsCDRRPSRSDELTRWIRIW(I\C;aE(

301

za-ribban 2 5 2

XEEE----EEE---

I ~ ~ ~ 1$ r u ~ ~ laub

55

FLEK1EPAQEE~EK~~SNVKELSHK~GIPNLVARQTVNSCAQCQQ;tLxGEAIHGQW _ _ _ _ _IIIIIIHIIHHHHH--- HHHHHHH--- - -HHHHIMHHH-HIIH -- -------I-

t i n ~ and secondary structure of the zinc finger domuins. FIG. 2. ~ i n c - c o o ~ ~ i R aresidues Primary sequences of a representative example (denoted with its PDB code) of each zinc finger domain with the first and last residue of the domains depicted above. The zinc coordination residues m e in bold and underlined, if two zinc atoms are bound the second is also marked with a line above the residue. The secondary structure elements for the depicted zinc finger domains are derived from the ~ ~ c t u~re es c r i ~ t of ~ othe n authors; for references, see Fig, 1.

973

974

FOLKERS, ~ A N ~ A W AND A, ~

O

~

FIG. 3 Tetrahedral coordination of zinc in various zinc finger domains. Representation of tht.? metal binding site for some distinct classes o f zinc finger proteins, showing the metal coordiination by C4,62B2, CPHC, integsase, LIM, and GAL zinc fingers, e ~ p h a s ~ z i on n gzinc binding by t,lie different nitrogen atoms (in black) of the histidine ring used for zinc (large black hall) coordination. Figure is prepared using M ~ L M O LLl591.

the solution structure of the HWl dimer two distinct forms were found: one has a geometry similar to that of the above described HW2 m o ~ o ~whereas er the other monomer has R T ~alternative zinc coordination perfomcd by the N61 o€the conserved histidines [GG]. This difference is most likely caused by a helix Lo coil transition of the C-terminal part of the first helix that contains the first histidine in one of the two r n o ~ ~ o mc e~~n f ~ ~ a t i oThe n $ .second ~sLidineis in this c o n f o r ~ a t i op~~ e s e nin~ a helical turn, while in the “BW2” form it is in the loop that connects helices 1and 2.

~

975

The Bruton's tyrosine kinase domain (BTK) i s found in a limited number of protein tyrosine kinases and is always found together with a PH domain. The zinc b i ~ d i n g motif comprises only 26 residues, with a metal coordination sequence that is very unusual: H x10 CC x 9 6. In line with that, the stmcture of this domain does not resemble any other zinc finger fold and essentially consists of a long loop held together by the zinc ion [67]. The two cysteine residues in the center of the domain make a tight turn (Fig. 1). ~ r t h e r m o r contacts ~, between the BTH domain and the PI3 domain may stabilize this zinc finger region. Since the domain is always found adjacent to the PH domain, it remains to be seen whether this zinc binding motif can fold as an ~ n d e ~ n d e domain. nt

The human Xerctderinap ~ g ~ e n group ~ o A ~ protein z ~ ~ @PA) is involved in nucleotide excision repair and is thought to be involved in the recognition of damaged DNA. The central region of this protein is required for DNA-binding and can interact with RPA 70, an s s ~ ~ A - b i n d i nprotein. g This domain consists of two subdom~ns:a 64-type zinc iinger with the pattern C x 2 C x17 C x 2 C and a C-terminal subdomain 168J. The latter is composed of a sheet helix Ioop region and a helix-turn-helix region and is probably involved in ~A- ind ding as determined in NMR experiments fG9I. The zinc finger subdomain is composed of an antiparallel p sheet and a helical turn [701 or a short helix [71]. T h ~ u g hthe overall fold is distinct from that of other zinc fingers, the zinc coordination is similar. The first two cysteines are located in a p hairpin s t ~ c t u r eThe . other two cysteine residues are located In a loop region in the structure of Ikegami et al. 1 701, while in the structure of Buchko et al. 1711 the two cysteines are in a helical conformation (Fig. 1).Recently, Buchko et al. [69l refined their structure, and now it appears more comparable with the s t r ~ c t ~of r eIkegami et al f701. More interestingly, in these NMR studies using chemical shift changes in XPA upon interaction with RPA it was de~onstratedthatt the interaction between XI)A and RPA was at least partially dependent on the zinc finger domain 1691. This zinc finger motif though present in a DNA-binding domain is thus primarily required for protein-protein interact~on~= 2.1.9. Copper Fist

In fungi the expression of several genes, such as rnetallothioneins and supcroxide ~ s m u t a s e ,is regulated by cop~er-dependentt r ~ s c ~ p t i ofactors n such as Amt 1 and Acel, which can bind both copper and zinc. h t l binds the Zn(11) ion through a C x 2 cf x8 C x H motif in a distinct manner that can already be predicted from thc unusual spacing between the last two ligands 1721. In this situation the zinc is coordinated with the N61 nitrogen, whereas normally the Nt. is involved as is the case g l yrecently ~ described structure with the CBHC and C2H2 zinc fingers. ~ n t e r ~ s t ~the

FQLKERS, ~ A N Z A W AAND ~

976

of the zinc ribbon-like fold of the ribosomal protein L36 1561, and one of the histidines of K N integrase zinc finger domain [65,66J also uses the N61 as a ligand. ~ossiblyin these cases the W6f nitrogen is used to compensate for the unusual spacer length between the two ligands. The zinc binding domain, denoted copper fist, consists of a ~hree-strand~d a n t ~ p a r a ~@esheet, l with one M. helix, an extended region with a h e l i d turn arid a short 3rahelix. The four ligands are present in p strand 2, in the 01 helix, and two in an extended region following the 310 helix, respectively.

motif found excluThe ~ ~ c urepeat ~ domain o ~~~1~~ ~ is a 7~~residues-long sively in a family of proteins that act as inhibitors of apoptosis, These proteins regulate programmed cell death through inhibition of the caspases that are involved in ~ s to three of these d o m ~ n s the ~ ~ ~ pcascade. t o ~This ~ sfmily of proteins c o n t ~ one with a C x2 C x 16 K x 6 C pattern and i s mostly found together with a RING finger domain. cently, the solution s t ~ c t u ~ *ofe sthe BIR domain 2 [731 of XIAP and BIR domain 3 of ~ I 2741~ haveBbeen delermined. The BIR zinc finger domain (as shorn in Fig. 1)consists o€ two- or three-stranded antiparallel @ sheets and four a helices. Despite ~ t r ~ cdi~erences, t u ~ ~ the coordinatio~of zinc is e~sentia11~~ the same as for the classical 62I-I2 zinc fingers. The first two cysteines are located in the loop region connecting the two antiparallel strands; the side chain of histidine that contacts the zinc residue is, similar to the classical finger, found in an a helix (helix 3 ) of the BIR domain. In contrast to the classical C2K2 zinc finger, the Last zinc ligand is located in the turn region following helix 3. The residues s u r r o u n ~ n gligand side chains are all ~ ~ ~ r ~ ~ ~and h othis b i hc~, d ~ o ~ h ocore b i c is stabilized by zinc ~ i n d ~ nand g th~ou~h i n t e ~ a c ~between ~ ~ n ~ a large number of hydrophobic and aromatic residues, ~ t h o u the ~ h overall fold for the BIR domains of the two proteins is quite similar, as well. In BIR2 three ~ t i p ~ a l8l strands e~ were fomd, d ~ ~ e r e ~were ~ c eobserved s whereas the corresponding region in BIB3 from MZHB is composed o f two extended regions that run a ~ p r o x i ~ ~ tantiparallel. ely This difference mirrors the var~at~ons observed for the classical zinc fingers @-sheetregion, which is not always a perfect f3 sheet [61, for which structures with two and three strands are found (see Sec. 2.1.1 t26,291).

ouble Zinc Finger Proteins MI zinc finger motifs described so far consist of four conserved metal ligands and bind only one zinc atom. ow ever, a distinct class of zinc finger proteins binds two zinc atoms that collectively form one intrinsic zinc binding domaiii f19,751. The first examples for such double zinc fingers are the DNA binding domains of the ~ yof yeast s ~transcr~pt~~n nuclear ~ o ~ m o receptors ne and the b i n u c l e ~~ n ~ finger factors such as GAL4.Other examples of this class of zinc fingers are the Zn finger

moti€s of LIM, RING, C1, and most likely also the PHD fingers (see Table 2 in &see.3). 2.2.1, 6 4 Nuclear Hormone Receptors

Nuclear hormone receptors are ligand-dependent transcription factors that bind to estr~gens,a n ~ o ~ e thyroid ns~ mrious ~ i ~ p h ~hormones, lic such as glucoco~~coids, hormone, vitamin D, and fatty acids “761. So far more than 800 family members have been identified in various e u k y o t i c species but none in fungi or plants, In C. elegans, which is t h o u ~ hto t contain approximately three to five times fewer genes than mammals, 255 nuclear hormone receptor zinc finger domains have been identified, whereas so far a p p r o ~ a t ~ 100 l y nuclear hormone receptors have been cloned in humans. Thus, most likely more members will be identified in this family of proteins that are key regulators of many important cellular events 1761. Although the ligand binding domains for these receptors may di€fer ~ u b s t a n t i ~the y ~ DNA binding domain is conserved in almost all members of this family (77,781. The DNA binding domain contains a double ziiic finger motif, which was originally thought to form two independe~tzinc fingers. Solution structures of the GR [79], ER [SO],EAR l81l and [821 DBD’s revealed the f o r ~ a t of ~ oone ~ compact domain e ~ c o m ~ a s stwo i n ~a helices o r i ~ n t ~ almost perpendicular to each other that are connected by an extended stretch (Fig. 1). The zinc i s coordiiiated by the cysteines located in the region preceding the ct helix, i n of~ the x helices. The overall fold where the last ligand resides at the ~ * t e ~ i iend for all members is highly comparable. However, there are some functionally important differences. For the first zinc finger region the most notable differences between the various members of this family i s a short antiparallel p sheet, which is fre~uently found within the zinc binding region at various positions. Differences in the second zinc finger include the D box (region involved in ~ m e ~ i ~ ~ and t i o the n ) presence of an additional helix at the C terminus [82]. The nuclear hormone receptor zinc finger domain is required for DNA binding and is involved in homo- and het~rodime~ization 176,781. om par is on of the solution structure of NHR DBDs by NMR 179-831 and the crystal structures of Dl3Ds bound to @s their c ~ r r e s p o n DNA ~ n ~ binding sites revealed important structural d i ~ e ~ e n cf8488 I. The NMR structures showed a poorly defined second zinc finger, especially within the D box [79-$23. In contrast, in DNA-bound structures of the DBDs of the steroid hormone receptors GR [841 and ER C851 as well as of 3%-EXR ~eterodimerf86l this region i s well defined, resulting in formation of a distorted helix. This difference could be the result of the methods used fNMR vs. X-rayl, or genuine confor~~ational changes. Recently, we CS91 showed that large microsecond time scale dynamics occur in regions of KXR that are ~ ~ s o r d e r eind the solution s t ~ c t u r e182,901. ~ b s e ~ v ~ t i o n s by van Tilborg et al. C9ll showed that similar dynamics are also found in a distinct region of RAR that is involved in dimerization with RXR [911. This suggests that is funct~onal~y importan$ for the adaptation of the protein-~~otein this ~exibili~y

978

FOLKERS, HANZAWA, AND ~

~

~ t e r a c t i o nsurface. Most impo~antly,conformational changes are observed in the second zinc binding domain, including the D box, upon binding of half-site response element in NMR ex~eriments192,931. Furthe namic analysis of GR in the free and DNA-bound state provides ~ndependent evidence for conform~tio~al changes upon DNA binding i941. Together these data clearly demonstrate that part of the DNA binding domain of nuclear hormone ~ in re~eptorsis flexible and u n s t ~ c t u r e din solution, while DNA b i n d i ~results functionally important conformational ges involving the regions involved in p r o t e i n - p r o ~ i n~ ~ r a c t i oand ~ protein inte~~action. In both cases a half-site nd response element was used for comparison between free and ~ ~ A ~ b o uRXR. Given the high on cent rations used in these N M ~e x p e r i ~ e ~ t spart , of the observed conformational changes might also be caused by homodimer formation between two hatitsite p r o t e i n - ~ ~complexes. A ~ ~ l ~ o we u gdid h not observe indications for such dimerization using biochemical experiments [931, we cannot completeiy exclude il. In line with this, molecular dynamic simulations also provide evidence for conf o ~ ~ a t i o ndifferences al between ~ o n o m e r i cand dimeric DNA-boun~ER that reside mainly in regions that are most flexible in the solution structure, suggesting that the three-d~ensionalfold i s also influenced by dime~zation[95]. ~trongestproof for the functional role for flexible regions and locally induced structures upon DNA binding was obtained by two recent papers showing the structure o€ RXR bound as a homodimer "31 and the RAR-RXR lzeterodimer 1971 to a direct repeat with a 1base pair spacer. In both cases, distinct structural changes were observed in comparison with the free protein. By comparing the two complexes it appeared that different regjons are involved in DNA binding and protein~~rotein i~iteractio~s, and a distinct DNA curvature is observed 196,971. The flexibility in solution and conformational changes as a result of complex formation thus provide a structural explanation for the crucial in nuclear hormone receptor gene regulation 1761. Finally, the zinc finger region also plays a more direct role in the transcription process than previously appreciated. This is demonstrated by the phenotype and solution structure of two GR mutants that appear to be sequestering a limiting these mutants have a D-box cofactor in the absence of ligand E981. ~nterest~ngly, Conformation that is similar to the DNA-hound GR [99,1001. Together these observag influence tions suggest that allosteric changes caused by DNA b i n ~ ~ nsi~ificantly thc activity of the receptor [loll. 2.2.2. GAIA Binuckear Cluster

The DNA binding domain of the yeast transcription factor GAL4 contains a distinct and a t ~ zinc ~ binding c ~ domain, a so-called ~n2Cys6binuclear cluster that is cornposed of six cysteine residues flQ2l.These six ligands contact two zinc atoms with a ~ o ~ s epaitern ~ ~ u Cs x2 C x 6 C! x6,12 C x 2 C x l i 6,where two ~ s t e i n e bind s the two metals (Fig. 3). This ZnBCys6 binuclear cluster is exclusively found in fungal transcription factors. T h ~ e e - ~ i m e n s i ostructures n~ have been determined for m ~ ~ i p l e

ZINC FINGER P R O T ~ I ~ ~

979

GAL4 binuclear clusters including GAL4 and PUT3 both free in solution [103-1051, and bound to DNA ~ 1 0 6 - ~ 0 SAll ] ~proteins have a similar fold; for example, the rmsd for the Ca atoms between PUT3 on the one hand and GAL4, PPRI, LACS, and FIAP1 on the other hand is less than 1 A Cl091. The structure is composed of two repeats y consisting of an a helix, followed by a sharp with a twofold internal s ~ e t r each turn, and an extended region that is held together by the two tetrahedrally coordinated zinc ions (Figs. 1and 3 ) . The first two Cys are present in the o( helix while the last 6ys is located in the extended region. 2.2.3. LIM

The LIM domain is named after three transcription factors in which this domain was initially identified: Lin-1, Isl-1, and Mec3. At present more than 200 proteins have u ~~ ohm a i nis been fount?that may contain up to 5 LIM domains [110,1111. ~ t ~ o this frequently found in transcription factors, it is most likely not required for direct DNA binding. Instead homo-dimerization or complex formation with other proteins has domains. The function of some of the LIM domains, been reported for many L notably the LIM homeodomain family, has been associated with embryonic development Il101. LIM domains are also found in combination with a kinase domain in which case the I,IM domains function to interact with various other proteins which may influence the biological activity. This family of adapter proteins has a consensus sequence C x2 C x 16-23 €3 x2 C x 2 C x2 C x 16-23 C x2-3 61. Variations are frequently observed; in particular, the last cysteine i s often replaced by a histidine or even an aspartic acid without the loss of zinc binding capacity. The two zinc-binding regions foim two independent sequential zinc binding units. In fact, it has been demonstrated that the N-terminal binding module can fold independently [llZ].Both fingers consist of two antiparallel p sheets that are positioned almost p e r p e n ~ i cto ~ leach ~ other [113-1151. The metal binding site for the N - t e r ~ n a finger l is formed by the two cysteines that are present in a rubredoxin knuckle formed by strands of fi sheet 1 and a third histidine ligand at the end of p sheet 2; the fourth ligand resides in the helical turn that connect the two zinc binding modules (see Figs. 1and 3). The first two ligands of the 6-terminal finger also form a rubredoxin knuckle, while the last ligand pair is found in an a helix. The structure of the C - t e r ~ i n a l domains includes a fold that is similar to GATA-1 and to the N-terminal finger of the nuclear hormone receptor DBDs. As already pointed out, multiple copies of the LIM domain can exist in one protein. experi~ents demonst~atedthat when two such LIM domains are present, they are spatially separated and fold independently of one another 1116-1181. ~~~

NG finger motif was initidly ~ d e n t i fas i ~a c y ~ t e i n e - ~ cdomain h present in a few proteins among which the protein RING-1 (really interesting new

g e n ~ 3 after - ~ which this domain was called t1191. ~ubseqL~ent d a ~ b a s eanalysis revealed the presence of more than 800 members in ~ e t ~ o~iridiplantae? a , fungi, and viruses. In contrast to LIM, generally only a single copy o f the R~NGfinger is found in a protein. The domain, however, is frequently found tagether with one or more copies of the B-box zinc finger. The function of most ~ ~ finger N G cont~ning proteins is unknown, but they might fullill a function in the transcri~tionprocesses though direct DNA binding has not been confirmed. Based on b i o c h e ~ i cexperi~ ments a role for this domain in protein-protein interaction was proposed [611. Recent reports suggest that RING fingers might be involved in the regulation of protein de~adationmediated by the RING finger-dependent interaction with ubiquitin-conjugating enzymes, resulting in L~biquitinationI120-1221. The latter finding may shed new light on khe oncogeenic characteristics ofthe RING finger proteins when fused to other proteins. The structure determination OF the RING finger protein of the immediate early showed a novel and remarkable Iigation pattern, equine herpes vims protein (Em?, referred to as cross-brace. The metal binding is mediated by the first and third pair, respectively~and the second and fourth pair of conserved residues in the consensus RI~G finger sequence C x 2 C x9-39 C x 1-3 H x 2-3 C x 2 C x 16-36 6, x2 C. The structure is therefore not composed of two sequential units but forms a single continuous structural domain folded around the two metal binding sites t1231. The structure can be described as an a-P sandwich, consisting o f three antiparallel p strands with a helix between strands 2 and 3 (Fig. 1).The first and last ligand pair are present in the ordered loops, whereas the second and third ligand pairs are at the N-and C-terminal end of the second p strand. Despite the distinct r~spe~tively? cross-brace metal coordination, topological similarities axe found with other zinc finger proteins, including the TFIIU E1231. Structural information is also available for three other RING fingers: Z /1251, and NOT4 (W. Hanzawa, and R. Boelens, u ~ ~ u b ~ s results?. hed e zinc ligation pattern is retained, large structural difyerences among gers were repo~ed.The R I N ~ finger fold of RAG1 is quite similar (Ca rmsd, 2 A). However, this finger is part of a larger domain that a ~ m ~ r i ~ a tdomain. ion Interestingly, one zinc binding motif of this t ~ g ~ t hforms er RING finger is part of a binuclear zinc clustor composed of the four cysteines of the RING finger and one additional ~ ~ t i present ~ n ein the NG finger and in addit~on one cysteine and one histidine. The second cysteine of the first zinc motif farms a e the bridging ligand for the two zinc atoms 13-23]. cent$ we solved the s t ~ c t u r of zinc finger domain of the human NQT4, a protein involved in the negative regulation zinc ligation pattern (68 vs. ~ ~ ~ was C found, 4 ) of tr~nscription.~ t ~ o u agdistinct h it appears that this protein has a similar global folding as the classical RING fingers. ~ r t ~ ~ e r m due o r eto~ its high content of proline residues, the j3 sheet region i s not present in this RING finger. Finally, considering the conservation in the foolding topology between the other RING fingers, it is r e ~ a ~ k a bthat l e the folding of the PML RING finger was so different C1241, despite the 3% sequence similarity with other R I N finger ~ proteins. ~~~

981

oubls Zinc Finger D ~ ~ a i n s Another class of zinc fingers involved in signal t r ~ s d u c t i o ncan be classi~edas m e ~ ~ b r a ~ e ~ b iproteins n d i n ~ 11261. So far three distinct domains have been described in this group: the cysteke-rich domain Cf of' PKC, the ph~sphatidylinosi~ol~3-phosphate recognition domain FYm, and the effector domain of rabphilin-3 that regulates i ~ ~ ~ a r i were t i e s observed the activity of the small GTP hydrolase JzAB3A. ~ t r u c t u r as ~ between these distinct classes of zinc finger domains and also between these domains and L and both the ~~~~

2.3.1. 6 1

Proteins of the protein lrinase C and -mas family contain several conserved domains, two o f which are involved in cofactor binding. The first domain (Cl) is c ~ ) ~ p o s of e dtwo cysteine-rich motifs that can bind the second-messenger diacylglycerol, a cofactor involved in sti~ulationof the kinase activity of PKC [127,1281, The solution structure of the C1 domain revealed a somewhat unusual metal coordination ~ 1 2 9 , 1 ~Ligands 0 ~ . within the sequence €1 x10-13 C x 2 C x9-14 C x2 C x 4 IJ x 2 C x 5-9 C bind two zinc atoms in a discontinuous fashion. The first zinc ion is bound by the conserved residues 1,4,5, and 8 while the second zinc is bound by the i.e~aining residues (Fig. 2). The domain contains two antiparallel @ sheets formed by @ strands 1 , 4 , 6 and 2, 3, respectively, an w helix packs against p strand 1. The beginning of p strand 1and the C - t e r m i region ~ ~ together with the loop region between p4 and BS form the first zinc motif. The second zinc is bound by the ligands present in the loop between g2 and p3 and in the loop between p5 and helix 1 (Fig. 1). Interactions with agonists such as phorbol esters have been analyzed and ~~terestingly, ~1, the rewaked the i n ~ o ~ v e ~oef nthe t second sheet @2,3) r 1 3 ~ , ~ ~ solution structure of a C1 domain of Raf-1 (that is unable to bind diacylglycerol) revealed a c o n s e ~ a t ~of~the n overall fold, while clear differences a m found specifically in the phorbol ester binding site [1331. The region that interacts with lipid overlaps with the phorbol ester binding site; a larger region ~ n c ~ m ~ a sapproxisi~g mately one-half of the protein including one of the zinc binding sites (zinc 2) is involved [ 1321.

The consensus patterns for rabphilin 3A (C x 2 C x13-15 C x 2 4 C x 4 C x2 C x 1419 C x2 C) and mfvE: (C x2-4 C x13-23 C x2 C x 4 C x2 C x16-36 C x 2 C) including the short distance between fingers 1 and 2, are very sirrdar. Combined with the no~consecutivemetal coordination pattern (see Fig. 2) and the sidarities in function (membrane targeting and signal transduction), this strongly suggests that these zinc fingers belong to the s m e class [126,1341. Indeed, the secondary structure

982

FOLKERS, ~ A N ~ W AND A , BOELENS

elements and overall folds are conserved f135-1371. The structure of the FWE domain t135,136] is formed by two antiparallel p sheets and an ot helix (Fig. 1). The first zinc is coordinated by ligand pairs 1and 3 that are located in the unstructured N terminus and the first f3 hairpin, respectively. This region is most likely directly involved in membrane interactions, as detected by NMR titration studies using FtdIns[3~~-embed~ed lipid micelles. The N-terminal region is thought to be involved in dimer formation [1361, which was suggested to be crucial for PtdlnsC3IP binding [1381. The second zinc is bound by the second and fourth cysteine pair, the former found in the loop between strands 1 and 2 of the first p hairpin, the latter pair in the loop between hairpin 2 and CI helix, and within the ot helix, respectively. Apart; from membrane binding, the FntrE domain can associate with PtdIns[3]P and with lower affinity to PtdInsl51P but not with a Ptdfns 11361. Ligand binding results in conformat~onalchanges en~ompass~ng large part of the FYVE domain structure E1361. Exact details for membrane binding and phosphoinos~tidebinding remain elusive due to technical limitations encountered with membrane proteins (see f126,135,136,1381);however, the tetrahedral zinc coordination appears essential for both structure and function [136]. The structure of the rabphi~n3A eEector domain C137f is essentially the same as that of except for the presence of an additional long 01 helix at the N-terminus, a longer CI helix at the C terminus, and a di€ferent loop structure (Fig. 1). Furthermore, small differences were found with respect to positioning of the ligands within the structure elements. For biological activity of the small G protein 3A, the effector domain of rabphilin is essential; the interaction is dependent on the two helices that reside N- and C-terminally from the zinc finger, respectively. ~ e v e r t h e ~ ethe s s zinc binding domain is required for interact~onbecause pres~niably it can stabilize the positioning of the remaining rabphilin 3A structure.

Current genome projects have already resulted in a huge m o u n t of new gene products. Classification of such sequences on the basis of primary sequence is certainly helpful in the elucidation of t.heir function. However detailed insight into the function structure. of a protein can only be obtained from the complete thr~-dime~isional Zinc-finger proteins all share a sequence conservation of cysteines and histidines at a more or less conserved distance, patterns that are easily recognized by sequence homology comparison programs. Consequently, many novel putatrive zinc finger domains have been found on the basis of sequence c o ~ s e ~ a t i oA n .search for zinc T [lo], P F m E121, finger proteins within various domain databases, such as S and P ~ ~ S l T[Ill, E reveals that zinc finger domains are abundant. A summary of this analysis is given ill Table 2, including a functional description of these putative zinc finger proteins. In most cases, zinc binding and or mutational analysis strongly suggests that these proteins are indeed zinc finger proteins.

Occasionally, however, the zinc finger is primarily designated on the basis of the conservation of metal ligands (for references, see Table 2). It is beyond the scope of this chapter to describe all of the putative zinc finger proteins; the presumed or proven novel zinc fingers that are not detected by these domain classification databases are generally not included. The presence of' many different classes of proteins that c o ~ t asi~~c ah c ~ n s e n s metal u ~ ligation sequence pattern ~ ~ d e r s c o rthe e s importance of the zinc finger domain.

T and , Using the protein domain databases from Bork et al. ( ~ ~ [lo]) S o n n h a m ~ e et r al. ~~F~ [lZ]),the number of proteins that contain one or more zinc finger domains was determined in different organisms, as summarized in Tables 3 and 4. Using the total amount of proteins present in these databases (February 2000) an estimate can be given for the percentage of proteins within the i~vestigated organisms that contain a zinc finger domain. These values indicate that zinc fingers make up a high percentage of the total amount of proteins in the investigated genowever, some care should be taken while interpreting these data. Not all putative zinc finger domains need necessarily be a real zinc finger and can also be counted double, due to the presence of' multiple distinct zinc finger domains in one protein. The databases are not dways entirely no~redundantas already reported for the CBHC zinc fingers. Finally, we estimated the total number of proteins present in the databases which can introduce relatively large errors for the organisms for which the geno has not yet been fully sequenced. More reliable estimations can be made for E. cdi, jannaschii, S. cereuisiae, and, to a lesser extent C. elegans, Although most of the above described factors lead to an overestimation of the percentage OF zinc finger rotei ins, in general we can c ~ n ~ l u that d e a p ~ r o ~ m a t e5% l y of all proteins of eukaryotes might contain one or more zinc finger domains, which i s in agreement with estimations made earlier E1391. Some questions remain unanswered: why is this domain so frequently present in eukaryotes and why is it almost completely absent in prokaryotes?

Zinc is chemically inert and not involved in redox reactions, and is therefore an ideal b ~ i l d ~ nblock. g The special properties of transition metals, and especially of zinc in comparison with other ions as summarized by Berg and Shi E191, might in part explain the frequent use witshina stmetzxral unit. Zinc-fingers are small and compact modules folded around a zinc atom. ~onsequently,apart from a minimal spacer between the ligands no specific restrictions are encountered for these spacer residues,

FOLKERS, ~ A N Z A ~AND A ~

984

Hmea

Rep

'bee'

Hitsd

Clafiy"

MFP MFPV MFP

252

Double Lbuble Double Double Double Sm& Slngk Single Single Single slngk? Slngle Single

Ubr 1

161 162 163 164 165 16s 167 165 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 12 188

114 hfFP MFW B U E

MFP

W F P MFP F v P f BA AMk'

Y ~~~~

BV B

M

v is1 MFP MFP P MFP 114 FaFP

v XIIFP

25 48 18 35 132 30 17 41 98 49

18 6 10 9 36 19 26 15 114 13 109 64 45

13 19 % e

28 21

Single Single SlXlgk

sl?l&? S1ngle

Sm&e SingIe S&e Urhom Unknown Urhown unknown

Ullhat?2 Unknom L'nknOWn Unkriawn

Patternf C 2 C 8,19 C 2,4 C 4 &I 2 C 12,21 C 2 C C 2,4 C 9,12 C 1,2 C 4 C 2 H 2 H 7 C c2 c 7,12 c 2 c 5 C 3 c 7,9 H 3 C CZC2H8H3C4C1C2C C 2,4 C8, 12 C 2 C 4 C 4,16 N 3 H 5,G H* C 7.10 C 5,6 C 3 H C 2 C 13,20 C 5, 10 C c 2 c 8,lO C 2 c c2,4 c 10 c 2 c c 2 c 10,M c 2 C C 2 C 11,14 H 5 €3 c 2*4C 11c 2 c c 2 C 12,lti c 2 c c 2 c 12,18 C 2 c c2c 1 3 c 2 c C 2 C 14,15C 2 C C 2 C 17 C Z C C2H17C2 c 2 C 2.830 N 2 C C 2 C 29,361 C 2 C C 2 C 12 H 6 C c 2 c 7,11 c 2 c 5 c 2 c C 1,2 C 16,17 C 2 C 3 I-f 4,7 H C 2 C 7.5 G 7 C 2 C 4 H8 H C1,6 H 1 C 3.7 Cci'h 3,8 C1,13 C C 2 C 2 C4,5 C 2 C 2 C 15 C 4

1-3 1 1

x 1 1-2 1-3 f 1-8 2 f,2 1,7

Transcription L'biquitination Trsnscripticn Transcription

t

+

+ +

7 1-3

Unknown Chaperone Il"ranslation Signaling Hydrogenase ~ansc~p~ioR Transcription Translatien Unborn Psimase IZibosylation iOncoprotein Tmnscription Signaling Signaling Transcription 4t Transcription Methylatian

1

1 1 1 I 1 2 1 1-6 1 1

1-3

-k

~ e u b i ~ ~ ~ i n a ~ n nmseription Topopisomerase DNA repair

+ + + +

+

+

1

UDknOwn

i-

N 3 C 4 c 2 c 3 cc 2 C

1

4.

CCCCCHflCCC

1

RepliCation LJbiquitination

t

985

uMame: inoat frequent name of this zinc finger domain. *%fi reference for the zinc fuager donlain. 'Tree: species lhat contain this domain; M:inetama, P: fungi, P: plants, A: archaea bacteria, B: subaderia, V: viruses 'Hiti: number of individual proteins that contain one or more copies of this domain. Data extr:j.~&ed from the S W R S ) [lo]. PFAM (PIT121,PRQSITE (1%) L131 databases (in this order). 'C.ks: single and double: one and two zinc atom@per domain; unknown: pattern with 6,7,>8 confierved eysteines or histidines. fPattern; Cimiensufi pattern o f ligands: C and H are cysteine and hstidine while the numbers in between indicate the minimal and maxims1 spacing between the ligands,c/h represent rriular G or H. "No. p a t t m : minimal and maximum number of domains per prokin. hFunction:procesfi irrt which the protein that contain this &main is most frcquently involved. 'IQW'DNA reported interaction of this domain with RXA or DNA or possible inkackions based on sequence homology with known DNA binding domains. JProt: reported intaraction of this domsin with protein or possible interactions based on &c?quencehomology with known protein interaction domains. h&;uurw:databases used. for hnctiond characterization of the protein domains; S: SMART [lOJ, P: Pfam (121, PS: PROSITE 1131, PD: pro-DQM [ll].

FOLKERS, ~ A ~ ~ AAND W ~A O~ ~ L ~ N

986

TABLE 3 Number of Proteins That Contain Zinc Finger Domains for Which a ThreeDimensional Structure is Icnown" Name

Tree

C2H2

MFPAV WP V MFP WPAV

cam C2HC GATA Zinc ribbon Zinc ribbon

B box In tepdse BTK XPA Copper fist BU12, BIR3 C4 NHii GAL

LIM RING CI FYVE rabphiiin-3a

MA MP V

M MF F MFP M F MFP MFPV MFP MFP M

No. pmteins 1735 358 1029 123 5% 18 112 597 29 10 8

52 815 131 249 838 231 64

4

Human 523 35 0 11 5 1 43 0 11 1 0 7 101 0 66 159 61.

14 0

C.elegms A m ~ z ~ o p s z sS cer. 189 47 0 14 2 1 16 0 0 1 0 2

255 0 36 127

37 18 1

80 121

0

14 2 1 15 0 0 0 0 0 0 0 5 2 78 5

5 0

52 15 0 I1 4

1 0 0

~ . j u ~ nE. m ~Ei ~ ~ h 2 0

0 0 1 1 0

0 0 0 0 0

0 0

0 0 0

0

0 1

3

0

1

0 0 0 0 0 0 0 0

0 0

0 61 3

39 1 5 0

a 0

0 0 0 0 0 0

0

'Number of zinc finser proteins (no. protetns) for the vanous ctiasses of zinc finger protetns for which a three-dimensional structure 1s known {nume) are obtained from the SMART [lo] and PFAM fl2.I databases. The domain is fortnd In the varions organisms where tree explains the evolutinnary distribution of the indicated zinc finger domain; M : nietrzzoa,F: fungi, P: plants, A: archaea bacteria, B: eubacteria, V: muses. Distribution of these zmc: fingers over varioiis orgmisrrts is given: hiiman: Homo sapzens, C. elrgans: C ~ 7 i u r h u b ~ elegans, i ~ ~ s Arubrdopszs: A ~ b i ~ o ~ s~z s ~ ~ S. ~cer..~ Saccharomyces cereuzssae, M.jannashii: Methu.nococcusjunnuschii, E. coli: Escherichia coli.

~

a~~uwing hrge variations t ~ i - o ~ g evolution h o ~ ~ without loss of the t h r e ~ d i m e n s i o n ~ fold. Such limited restrictions on residues are more diflicult to imagine if the strmcture is coniposed of ~ u l t i p l esecondary stiuctui-e elements needed for m a i ~ ~ t ~ofn ~ c the fold. The altered specificity mutants of Zif268 found in a random selection a ~ ~ r o confi~m a c ~ that changes in the residues not involved in ligmd b~ndingcan functionally change the zinc finger [140,1411 while three-dimensional structure is m a i n t ~ n [e1421. ~ ably even a secondary structure element can be ~ ~ t r o d u c e d within the loop region without significantly altering the overall fold [1431. Further evidence for e v o ~ u t ~ variation o n ~ ~ resulting in € u n c t ~ o nchanges ~ is exemplified by the wide variety of DNA sequences that can be used as binding site for ~ T~~n, c h ( ~ aU ~ k ) the C2H2 zinc finger proteins e.g. %if268 ~ ~ C C T C C ~ C G and SPl ~ C G C ~ (see ~ C[29,1441). ) In fact, variation of only a few crucial residues can already lead to a completely altered binding specificity, e.g., ER (AGGTCA) vs. CR ( A ~ ~ C "781. A The ~ ability to change not only the sequence but also the leng%h between the ligands without significantly influencing the remainder of the structure is another putative advantage. Because of this, long and relatively u n s t ~ ~ ~ uloop red

987

TABLE 4 Number o f Zinc Finger Proteins in Various Speciesa M. junnuschii E. coli

Data

No. proteins

Humai

C. elegans

Arubtdopsis

S. cer.

PDB

6454 1339 7793 307186

1036 257 1293 18619 100000 7

746 152 898 19105

426 134 560 7857 25000 7

197 65 262 5932

4 5 9 1715

14 4289

4

05

0.3

Putative Total Current status Predrcted Estimated %

3

5

0 14

UDatsconcemng the zmc finger proteins are ohtamed from the S U T [lo] and PFAM databases 1121. The e s k w t e d percentagc (e&~nuied%) is obtmied using the total nmnount o f m e Angers (total)calculated fromt the s u m of all FDB (those fmger protans for which a three-dintensional structure has been detezmmed, see TEMP 3) ondp7Autzu~(see Table 2) zinc fingers proteins (no proteins) and the tom1 number o f piwtmis (rctrrent &tUs) present I I ~a nonredundant protem database (SWTSS-PROT+TrEMBL, 1181) for all proteins, and the genome databases of ENTREZ (http iiwww3.ncbi.nlm nlh gav,’Entrez) f ~ e ~ n a i . h uelegans, b ~ ~ ~ ~ ~ ~ rivrrvzsslne, i ~ ~ r~~ h u7 ? ~ ~ ojnnnuschiz, e~c z ~~s ~and Escherzelztu s COOL) and MIPS (http ilwww m~ p abiochem mpg de) (Homo supzens, Arubzdupsas t!~aLauna)for the c m respondtrig organisms for which the genome sequence IS not complete (based on the duta The estimated number of protcins [or the or~ani31ns obtained from the first completed chramosomcs of these orgiinisms) IS given ( p j ~dictedj

regions can be introduced that are highly mobile without infiuencing the fold o f the d dimerremainder of the protein. For example, the length of the D box ~ r e ~ u i r efor ization) of nuclear hormone receptors, formed by the flexible loop between the first two cysteines of the second zinc finger, is not constant. The presence of flexible loop structures within zinc fingers can have important functional consequences foi- dimerization and transcription regulation as evidenced by conformational changes within box of the nuclear hormone receptor DNA binding domains upon Given the limited sequence requirements for zinc finger formation, this domain is ideally suited to combine formation of a compact structure with large c o n f o ~ a tional freedom. From an evolutionary point of view it can be advantageous to combine flexible regions with R few rigid parts heId together by a simple metal coordination. This enables these domains by sub~t~tution of only a limited number of amino acids to adopt €uncti~nall~ distinct conformations while maintaining a similar fold, lhereby ~n giving rise to novel functions. A good example of this phenomenon is the ~ n c t i of 2 zinc finger of ATF2 [MI.

None of the above described classes o f zinc fingers were found in E. co2i. Even the c ? ~only ~ i few zinc finger domains, one of which is the archaecz b a c t e ~ M. u ~j f f n ~ ~ s has zinc ribbon domain of TFIIB, which is not surprising given the conservation of many basal transcription factors between archaea and eukaryotes. In fact, only 11 C2 zinc ~ ~were ~foundein eubacteri~, r ~ most 01which are found in A ~ ~ or

o

Rhizobium, two bacteria that are known to live in symbiosis with plan^, m&g it likely that these zinc fingers were the result of horizontal gene transfer. This raises the question whether the zinc finger was present in the common ancestor of at1 three domains of life (eubacte~a,archaea bacteria and the eukaxyotes) and was almost ~ ~ ~ l e tlost e l in y the archaeal and bacterial stem or whether the zinc finger has evolved after the s e p ~ a t i o nof the eubacteria from the eukaryotic and archaeal lineage. On the other hand, zinc fingers appear to collectively need the presence of two ligand pairs at a rather broad range of distances between the pairs and also within the pairs; it seems rather unlikely that this simple sequence combination would not be ~ s . other reasons might also account for the absence of present wit^^ ~ r o k ~ o tThus, a zinc finger domain in bacteria. ~ i s u bond ~ ~format~on e could in p r i ~ ~ cprevent i ~ ~ e zinc binding; however, it is t ~ i o u g that, ~ t in bacteria and in eukaryotes the c ~ o ~ l a shas m a redox e ~ ~ r o n m e n t that does not permil, this. Special c o m p a ~ m e (periplasm ~~s and endoplmmic reticulum) of the cell Elre required for Formation of disulfide bonds [145]. Given the canservation of mechanisms it therefore seems unlikely that disulfide bond formation could prevent zinc finger f ~ r m a ~ i oinnp r o k a ~ ~ t e s , Zinc is an essential trace element for both the bacteria and eukaryotes, but i s to be regx.xlated. though similar excess of zinc is toxic. Thus, zinc h o ~ e o s t ~needs mechanisms are employed, substantial differences exist with respeck to zinc homeIn the first domain of life ostatsis between the eukaryotes and bachria [4,14~,14~1. f147-1501 or stored metals are either complexed to proteins such as ~ieta~lot~iioneins a, or in organelles such as endoplasmic reticulum, nucleus, ~ i t o c h o n ~ r ichloro~~asts vacuoles 131511, while increased eMm is the main prote~tio~i m ~ h a n i $ st ~ excess of heavy metals in bacteria [246,1521. Possibly as a c ~ n s e q ~ e of n cthat ~ the o n sconcentra~ionsare ~nfavorablefor zinc actual zinc levels or tlre ~ ~ c ~ ~ a tini zinc finger formation in prokaryotes. Alternatively, it i s known that iron and copper can also bind to cysteines, albeit not as efficiently as zinc t191. Possibly the presence and concentra~ionof iron, or other transition mnetals that are also thought to be regulated di~erentlybetween bacteria and eukaryotes 11471 is not controlled accurately enough ofnzinc finger proteins. in the first g o u p to permit the f o r ~ ~ t i o Another explanation might reside from some fundamental differences between n ~ e ~ e proa s p r o ~ ~and t ee ~ k ~ o t with @ s respect to t r a n ~ c ~ p t i oregulation. karyotic transcriptioir is controlled by repressors and changes in NA architecture [1531, e u k a ~ a t i ct r a ~ c r i p t ~ oisnrestricted by c h r o ~ a t i na~higher order mtdtiprotein-DNA complex. Remodeling of chronzatin is required for t ~ ~ s c r i p tactivation, io~ bydlarge ~ ~ l t ~ p ~ complexes o t e i ~that are r e c ~ iby t c~h~r o ~ a t i n which is p e r f o r ~ ~ bound transcription factors C1541. Various zinc finger proteins (TR, GR) have been shown to bind with high affinity to chromatin 11551, Possibly thc ancestorM of the zinc finger proteins were better adapted for binding to chromatin, while the prokaryotic t r ~ ~ ~ s c r ifactors p ~ i o evolved ~ from an ancestor that appeared more successful in DNA bending.

The fact &at several putative zinc finger proteins have also been found in bacteria leaves open the p o s s ~ b ~that ~ t y their almost complete absence in p r o k ~ o ~ s is purely coincid~ntal.~ e t e r m i n a t i oof~ the t h r e ~ - ~ m e ~ss~i ~o c~t ~u of r ethese prokaryotic zinc fingers is required to better understand the evolutionary history of zinc finger domains.

5. With at least 15 different classes of zinc finger proteins considerable knowledge i s gained concerning structure and ~ n c t i o nof these domains. fingers are compact domains folded ~ o u n the d metal ion whereby the retaining of the positioning of the various structure elements with respect to one another is maintained by the tetrahedral zinc c o o r ~ ~ ~ Zinc t i ~ finger n . domains all share a conserved t e ~ r a h e ~coorr~l ~ t l in y a similar fashion as the iron in the di~iationwith a zinc ion that is ~ e ~ ~ u ebound rubredoxin fold. Most of the emphasis has been given to the role of zinc finger proteins 8s DNA binding domains, but recently it became clear that other aspects of this domain might be even more im~ortant:the interaction of zinc finger proteins with other proteins or with membranes and second messengers. An important task for the future will be to determine structures of such zinc finger domains both in isolation and in complex with their partner. The structural information, especially the conformational changes induced by ~ i o m o l e c uinteracti~ns, l~ will provide valuable insight in the mechanism underlying portan ant signal t ~ a n ~ d u c tevents i ~ n in which these domains are par~icipat~ng. It is clear that c ~ s t ~ l o g r a p h studies ic on biomolecular complexes present structural details on the recognition process, but NMR studies of these protein domains in solution s ~ ~ p l e m e the n t n e c e ~ dynamic s ~ ~nformation.Recent progress in N methodology at present allows structure determi~ationof larger complexes [mf, but more i m p o ~ a n t ~recent y developments such as d e t ~ r m i n a t i oof~a protein stmcture in reverse micclles [157,1581, make it possible to study zinc finger domains that associate with cell membranes in more physiological environments, The genome s~quencingprojects that are currently under way already resulted putative ~~ ainc in the i d e n t i ~ c ~ t i o nf a huge number of new gene products, i n c l u d ~ finger domains, without clear function. Despite our increased knowledge of zinc finger domains, secondary and tertiary structure predictions €or the putative zinc finger ~ e are not very reliable. d o ~ a i n son the basis of the primary s e q ~ ~ e nalone Therefore, further investigations are required to determine the structure for these novel domains. This will most likely lead to new excitement and further insight into the function of zinc fingers in the already large array of processes that zinc finger proteins are involved.

FOLKERS, ~ ~ ~ ~ AND A W A ,

990

ADRl ATF BIR BTK C CATH

Z

GAL

yeast transcription factor transcriptional activator of the glucoserepressible alcohol dehydrogenase (ADW2) gene activating transcription factor baculovirus inhibitor o f appoptosis Bruton’s tyrosine kinase cysteine classification of protein domains using homologous superfamilics, topology or fold groups, architecture and class: http:/iwww.biochem.uci.ac.ukibsmnicath DNA binding domain search and retrieval system http://~~.ncbi.nlm.nih.gov/~ntu.ez/ equine herpes virus RING domain estrogen receptor domain found in the proteins Fabl, YOTB, Vacl, and EE241 S. cereuzsiae transcription factor involved in galactose-induced gene expression glucocorticoid receptor guanosine 5’-triphosphate histidine S. cereuisr’ae transcription factor that contains a Z x i ~ ~ binuclear ys~ cluster human ~ ~ ~ L ~ ~ ovirus d e ~ ~ i e n ~ inhibitors of apoptosis ~ 1 ~ y L ~ ~ rEactis p o ~transcriptio~al y~~s activator abreviation Dom the first proteins that contain this domain: Lin-1, Isl-1, and Mec3 major histocompatibi~tycomplex binding protein 1 mammalian L4p homologue €3 Munich. Information Centre for Protein Sequences. http://www.mips.biochem.mpg.de/ nuclear hormone receptor global negative-regulators of transcription Protein Data Bank http:/ / ~ . ~ c s b . o r ~ / p d b / S: protein families database of alignments and http://w~~~.sanger.ac.u~’Pf~/ Pleckstrin h o ~ o l domain o ~ a C4HC3 zinc finger-like motif found in nuclear proteins thoilght to be involved in c ~ r o m ~ t i n - ~ ~ ti ra ~t es d c r i p t i or~e~~l l ~ t ~ o n protein kinme C

99 1

putative transcription factor involved in acute promyelocytic leukemia due to a chromosomal translocation, which involves PML and 31 gene PPR 1 pyrimidine pathway regulator 1, a yeast transcription factor P r o - ~ Q ~ protein domain database consisting of an automatic compilation of homologous domains http://prote~~.to~louse.inra.fr/prod~m~htm~ database of protein familics and domains: h~t~:liwww.expasy,ch/c~-binlprositei pho~phatidylinositol PtdIns ~ t d I n s r 3 ~ phosphatidylinositol-3-phosphate S. cerevisiae praline utilization transactivator PUT3 Ras-related protein RAE3 3A ~ ~ r i n e / t h r e o ~kinase i n e that associates with RAS RAFl V(d)j recombination activating protein 1 RAG1 retionic acid receptor small ~ ~ ~protein b ~ ~ n ~ MS RNA polymerase I1 subunit 9 regulates start-site selection and elonREP9 gational arrest ~ domain first idcnti~edin the protein named “redly i ~ t e r e s t i nnew gene” root mean square MnS root mean square difference rmsd RNA p o l ~ e r ~ ~ e RNAP replication protein A RPA retinoid X receptor RX€t o f Proteins: ~ t r ~ c t Classi~cation ur~ SCOP http://scop.~rc-1mb.cam.ac.uk/scop/index.~itml Simple Modular Architecture Research Tool: Smart http://sm~~.embl-~e~delber~.de/ protein sequence database: h t t ~ : l / w ~ . e x p ~ y . c ~ transcription factor thyroid hormone receptor t r ~ n s l ~ t i of ~ nEMBL s nuckotide sequence entries not yet; i n t e g r a t ~ ~ T: in S ~ ~ S S - P R Ohttp://www.expasy.ch/ Xenopus inhibitor of apoptosis XUP Xenopus nuclear factor XNF X e ~ ~~ ei ~r ~~ e ~ ngroup t o As protein ~ ~ XPA

PML

992

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f

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'Food Science and Technology Laboratory, University of Bologna, Via Ravennate 1020,47023 Cesena, Italy ' ~ a ~ e t ~i c ~ s o Center, n ~ University c ~ of Florence, Via L. Sacconi 6, 1-50019 Sesto Fiorentino, Italy

1. I ~ T R O ~ U C T ~ O ~

2. P 2.1. 2.2. 2.3. 2.4. 2.5. 2.6. 2.7, 2.8.

~

~ T ~ ~ ~ S 1002 1002 ~ e t ~ l o t h ~ o nOcc~rrence e ~ ~ s : and Biological Role ~etallothioneins:Structural Classification 1003 Structui~eof ~ u mE a~~ b, iRat, t ~ and Mouse M e t ~ l ~ Q t h i # ~ ~ i n1003 Structure of Blue Crab Metallothionein 1006 ~ t ~ c tof~Purple r e Sea Urchin ~ e t a l l o t h ~ o n ~ i n 1007 Insulin: Q c c ~ r ~ and e n Biological ~~ Itole 1007 Insulin: ~ t ~ ~C lca ~t s iu~ ~~ t i~# n 1008 Iiisulin: Structure 1009 2.8.1. The Three ConforI~a~ions: 2Zn- (or Te), 4Zn- (or T$&), ~ h e ~ ~ o l - I n ~ (or u c eEli) d Insulin Species 1009 1011 2.8.2. ~ u t a n t s 1011 2.8.3. Sequence Variation and Insulin Structure

VirrrI-IU N ~ O W ~ T ~R U C T ~ E uman ~ ~ ~ r oGrowth n a l ~ n h i ~Factor ~ t o ~ (Met~lothionei~~3) 3.2. ~ h ~ o c ~ ~ l a t i n s 3.3. Cod Insulin n 3.4. ~ x p r e s ~ i oSysteins 3.4.1. ~ e t ~ l ~ t ~ i o n e i n s 3.4.2. Insulin

3. PRO 3.1,

1002

1012 1012 1013 1013 1013 1013 1013

BA~IN AND ~ VIEZZOLI

1002

4. ~ ~ ~ U ~ T U ~ E - F ~ NRELATIQNS~IPS CTION 4.1. M e t a ~ l o ~ h i o n e ~ s 4.1.1. Interaction with Metals 4.1.2. Metal Exchange 4.8. Insulin: ~ e l a t i o n s hBetween i~ C o ~ o r m a t ~ o n~arl a n s ~ t i o and ns Biological Function

~

~

R E

E ~

1016

~ AND T DI ~ F Q I N~ I T~I ~ ~ S E

1015

I016 1016

5.1. ~etallothioneins 5.2. Insulin

~

1014 I014 1014 1015

~

~

N

1016

~

~

~

1017

The role ol' Zn2' in enzymes and proteins has been exhaustive~ytreated in Chapters 19 and 20. However, in two other classes of proteins, metallothioneins (MTs) and insulin (IN), Zn2+ has a peculiar function that is ~ f f e ~ e from n t the w e l l ~ d ~ ~ n e d catalytic, structural, and cocataIytic classification. The role of zinc for both classes is connected to storage processes: in the case of 34% to the s t o i ~ aof~the metal ion itself, in the case o f IN to the storage of the protein in an inactive form, For MTs, the mqjor intracellular z i n c ~ b ~ nproteins, d i ~ ~ an i ~ ~ o ~role a nin tzinc uptake, distribution, storage, and release has been stated. MTs are capable of forming a stable complex with the metal ion and to release it either to maintain the intracelMar zinc co~centrationat a standard level or to function as a chaperone for the synthesis of other zinc proteins, donating the metal to apoznetalloproteins or inserting it directly during their synthesis [1-71. In xnsznmals, IN is stored in the secretory granules of the pancreas as a zinccontaining hemmer. The high concentration5 of %n2' and Ca2+ in these granules [S,SIf indicate a ~ ~ ~ trole i of o both ~ aions ~ in the later stages of b ~ o ~ ~ t h e[lo] sis and they also possibly stabilize the structure of the protein when it is deposited in the pancreatic cells. However, the active form of IN that is released into the bloodstream is a monomer that does not contain zinc [lI-lSI.

In recent years, a steadily increasing number of new amino acid se~u@nces of MTs from various species is being reported. MTs are now known to occur in a11 animal

phyla examined so far as well as in certain fungi, in higher plants, and in some prokaryotes. In mammals, the genetically polymorphous proteins are most abundant in parenchymatous tissues, i.e., liver?kidney, pancreas, and intestines. There are wide variations in concentration in different species and tissues, reflecting effects of age, stage of development, dietary regimen, and other not yet fully identified factors. though MT is a &ytoplas~ic protein, it can also accumulate in lysosomes, and during development it is observed In the nucleus. Despite a lapse o f almost $0 years since their discovery and intense investigations, the p r i m a i ~physi~logicalrole of these proteins has not been elucidated in detail. Experimental evidence exists for the following roles: (1)participation in maintaining the homeostasis of‘ essential trace metals particularly zinc and copper; (2) heavy metal deto~fication;(3) storage of essential metals that can be donated to other m e t ~ l o p r o t e i ~(4) s ; protection against intracellular oxidative stress; (5) in the case of the mammalian isoform MT-3 (growth inhibitory factor, GIF), regulation of zinc-dependent processes during cell growth and di~erentiation[l-71.

~ t a ~ l o t ~ i o n eStructural i~s: Classification

The term “metallothioneins” is a collective name for a superfamily of metal-binding proteins. On the basis of the SCQP classification, the MT superfamily belongs to the class of sniall proteins f141. The superfamily contains only one family (MT) with seven species that correspond to the seven MTs of which a structure is available: human (Homo sapiens), rabbit ( O ~ c t o l ~ g cuniculus), us rat (Rattus raftus), mouse (Mus m ~ s c u ~ ublue s ~ ,crab ( ~ a ~ l i ~sapidus, e ~ ~ ebaker’s s yeast ( ~ a c c ~ a r ~ ~&yecr e v§~ s ~ ~ e ) and purple sea urchin (Strongylocentrotuspurpuratus). In accordance with other classification systems (the one adopted in 1978 by ~ o r d b e r gand Kojima C151 and subsequently extended in 1985 by Fowler et al. [16l, and the more recent one proposed by Kojima, Binz, and Kagi [171), the MT superfamily is defined as comprising all polypeptides that resemble equine renal MTs in several of their features, Such general features are low molecular weight, high metal content, characteristic amino acid composition (high Cys content, low content of aromatic amino acid residues), a unique amino acid sequence with characteristic characte~istic ~ s t r i b u t i o nof Cys, Le., Cys-X-Cys, and s~ctroscopic~anifestatio~is of metal thiolate clusters, The structure of mammalian, crab, and sea urchin MTs will be discussed in Sec. 2.3-2.5 (sec the Protein Data ase references reported in Table 1).

2.3. Structure of Human, Rabbit, at, and Mouse ~ ~ t ~ l l o t ~ i o n ~ i f f The characteristic feature of d MTs i s the occurrence of Gys-X-Cys ~ripeptide sequences where X stands for an amino acid residue other than Cys. In the absence

of ~ s u l ~bridges, de the abundance of cysteine residues furnishing polariza~lethiolate ligands gives the protein a high affinity for d" metal ions. Mammalian NITS,so far the most extensively studied, contain 60-68 amino acids with 20 Cys and they usually bind a total of 7 equivalents of divalent metal ions, e.g., Zna+ and Cd"', or higher stoichiometries of monovalent ions, e.g., Cu+. The 3 0 structures of mammalian MTs were determined in aqueous solution by 2D NMR spectroscopy using the reconstituted Cd7-MTfrom mouse f181,human [19], rabbit fzOl, and rat (211,and in crystals by X-ray d i ~ a c t i ousing ~ i the native Zn2Cd5MT 1221 from cadmium-overloaded rat liver at 2.0 resolution (Table 1). The m o n o ~ ~species ~ r ~ c of m a ~ m ~MTs, i a as ~ determined in ~olutionby and in crystals by X-ray diffraction, appear similar:.They show a dumbbell-~i~e shape with two ~ ~ r ~ sized ~ and f almost ( ~ ~ spherical ~ i domains, ~ ~ ~ each with a diameter of 1520 A, containing at their centers the respective four- and threemetal clusters: the 6terminal a domain { r ~ s i 31-61) d ~ ~ contains ~ 11Cys and binds 4 divalent metd ions; ~~~

TABLE 1

PDB References of the Available ~ e t ~ l o t ~ ~ o n e i n ~ . Protein Source

Metal bound

~ t ~ ~available t ~ r e ~

Exp. method

-

domain (31 aa)contpplesed witJt Cd fi domain (30 aa) ~ a ~ ~ pwith l ~ Cd e d

: CI *

2mhu: p domain (30 aa) cornpkxed with Cd ln&u: a domain (31 aaf c o with Cd~

~

Zmb: p domain (31 aa) complexed with Cd Imrb: CI domain (31aa)compkzed with Cd 2m-t:/?I domain (30 aa) complexed with Cd : a domain (31 aa) cov~plexedwith Cd 4mt2: overdl protein (61 aa) complexed with Cd, Ma, Zn Blue crab Cds-MT-1 (CalEirzectes supidus)

Cuy-MT

ldmd: a domain 131 aa) complexed with Cd Idmf: /?I domain (28 aa) comnplexed with Gd e x ~Cd d W R ,minimized Zdnne: p domain (28 aa)c ~ r ~ p ~ with average structure l b c : CI domain (81 aa) complexed with Cd

NMR

PJMR

l~~ a domain (36 aa) e ~ ~ with ~ Cd~ Z q j l fi domain (28 aa) mmplexed with Cd

NMR

laqq: overdl protein (40 an) complexed with Ag laoo:overall protein (40 aa) conqplemd with Ag

NMR, minimized average structure NMR NMR, minimized average structure PJMR, minimized average structure

~

taqs: overall protein (53 aa) complexed with Cu taqf: overall protein (40 aa) eompEcrxed with Gu

Ifmy: fragment (residues 9 to 48) complrned Wlth c u

e

TALLOTHIONEINS AND INSULIN

1005

the N-terminal p domain (residues 1-30) contains 9 Cys and binds 3 divalent metal ions (Fig. 1).The two protein domains are connected by a flexible hinge region formed by a conserved Lys-Lys segment (residues 30 and 31j in the middle of the polypeptide chain [231. The metal-cysteinyl interactions in the two clusters are of two difyerent types: the cysteine thiolate residue is either bridging or terminal. In the three-metal cluster, three cysteine thiolates are bridging and six are terminal? whereas in the four-metal cluster, five form bridges and six are of the terminal type. Both clusters show a tetrahedral tetrathiolate coordination, with the (M2+)3(CysS)gcluster in a cyclohexane-like conformation, and the (M2')4(C$&)11 cluster in a bicyclo nonano-like conformation (Fig. 2). Although a number of metal complexes of mammalian MTs containing seven M2+ or M3+ ions have been isolated and prepared in vitro (Zn2+, Cd", H 8 + , Pb2+,Co2+,Ni2+, Sn2+,Pt2+,Bi3+, and Tc03+ [24,25], direct evidence for the existence of the M& and the M4Sll cores has been obtained so far for the Cd2+,Zn2+,Co2+,and Fez+ derivatives only [23,26-29]. Studies with Co2+-substitued MTs have been especially useful to establish the geometry of the metal binding, as Go2+ is an excellent spectroscopic probe for the analysis of the structure of zinc wing to its peculiar electronic properties, it is capable of inducing large hyperfine shifts on the '€3 NMR signals of the neighboring protons (p-CH2 groups of the Cys residues in the case of the CoS duster of MTsj without causing dramatic line broadening [31]. In addition, due to the close similarity between the coordination chemistry of Co2+ and Zn2', one obtains Co2+ derivatives that closely mimic the native zinc systems 126,271. It was possible, therefore, to follow the cluster formation by titration 1261.

B domain

a dornRin

FIG. 1. Shown are the fl and a domains of human Cd7-MT-2 as obtained from PDB refcreiices 2mhu and lmhu. (Cd ions are shown as gray big spheres; the Sy-Cpand Cp-Cc"connectivities as orange and red sticks, respectively.) The picture was prepared using the program MOLMOL 1941. See Figure 21.1 in the color insert.

1006

BABINI AND VIEZZOLI

? MHTN

ri I STCR

1 METAL CLUSIER

FIG. 2. Ball-and-stick diagram of the mammalian three-metal and four-metal MT clusters. (Divalent metal ions are shown as black big spheres and cysteine sulfurs as gray small spheres.)

Concerning the folding of the protein, the polypeptide backbone wraps around both clusters forming two large helical turns. In the C-terminal a domain the spiral of the peptide fold is left-handed and in the N-terminal f3 domain it is right-handed. In both the NMR and X-ray structure, the local conformation in seven Cys-X-Cys segments constitutes an unusual type of secondary structure, the half turn, so far unique to MTs [321. Additional secondary structure elements include two 310 helical segments between residues 41 and 47 and residues 57 and 61. W i l e the metal centers are completely isolated from the solvent, the solvent accessibility for the sulfur atoms varies. Each domain of MT displays a solvent-exposed cleft containing three Cys molecules: residues 5, 7, 13 in the f3 domain, and 37, 41, 57 in the a domain.

2.4. Structure of

lue Crab Metallothionein

Blue crab (Cullinectes supidus) MT contains 58 amino acids of which 18 are cysteines. The 3 0 solution structure of blue crab Cd6-MT-1has been determined by homonuclear and heteronuclear magnetic resonance spectroscopy [33,34]. These structural studies reveal the presence of two separate domains, each containing a cluster of three metal ions, tetrahedrally coordinated to the sulfur atoms of the nine cysteine residues present in each domain. In total, 6 Cys molecules participate in bridging two metal ions and 12 act as terminal ligmds, The only elements of secondary structure observed are several type I fi turns and a short helix segment in the 6-terminal domain. A structure comparison of mammalian and blue crab MTs 1331 indicates that although their sequences are significantly different, the binding scheme between metals and cysteines is highly conserved in the f3 domain with only minor differences. The tertiary structure of the crab MT-1 p domain bears a close resemblance to the same domain in mammalian MT-2 but with much nmower and shallower clefts. The crab MT-1 a domain is more compact than the corresponding mammalian domain, primarily as a result of the presence of only three metal ions instead of four. No clefts are visible extending into the metal-binding cluster, and the only exposure of the cluster occurs on a surface that will be covered by the domain in the intact protein. Consequently, in crab MT the @ domain is more exposed to the solvent than the a domain 1331.

ZINC METALLOT~I~NEINS AND INSULIN

2.5.

1007

Structure of Purple Sea Urchin Metallothionein

The structure of the purple sea urchin Strongylocentrotus purpurutus recombinant Cd,-MTA has been determined by homonuclear 'H NMR and heteronuclear ['H, lL3Cdl correlation spectroscopy 1351. This MT is a 64-residue protein containing essentially the same number of metal-chelating Cys-Cys and Cys-X-Cys motifs as the mammalian MTs and forms a two-domain structure with one domain containing a three-metal cluster and the other a four-metal cluster. However, the sequential order of the a and p domains, and hence of the three- and four-metal clusters, is inverted between the two MT types. In mammalian MTs, the three-metal-contai~ing p domain corresponds to the N-terminal half of the sequence and the CI domain i s located in the C-terminal part, whereas in sea urchin MT the N-terminal a domain contains four metal ions and the C-terminal p domain three metal ions L351. Despite this domain inversion, the Cd binding affinity and the metal binding properties of the two MT types are similar. Substantial differences exist in the structural roles of the corresponding cysteine residues, i.e., in their serving as singly bound or bridging ligands. In particular, the locations of the bridging Cys along the sequences are clearly different. As a consequence, the geometries of the a-domain metal-sulfur clusters in the two species are difl'erent 1351. Besides, in contrast to all other presently known three-metal cluster-containing MT domains, the chirality of the sea urchin MTA 0 domain is left-handed 1351. 2.6.

~ ~ s ~ Occurrence lin: and Biological Role

I n mammals, IN is synthesized in the p cells of the pancreas through a complex sequence of posttranslational events and stored in tlie pancreas secretory granules as a zinc-containing hexamer. However, in response to physiological stimuli, the hormone is released into the bloodstream where it exists in the biologically active monomeric form 111-131. The hormone interacts with specific receptors on the cell surface to stimulate glucose transport from the blood to muscle, adipose, and other tissues. The failure of the pancreas to produce sufficient amounts of IN causes diabetes mellitus, a chronic dysfunction of carbohydratc and lipid metabolism. This dysfunction can also be due to mutations in the IN receptors which prevent IN from binding. Since the 1920s the hormone has been used for tlie management of diabetes. The monomeric unit is small (MW 58081, consisting of two polypeptide chains: the A chain (21 residues) and the I3 chain (30 residues). They are connected by two interchain cystine linkages (A7-B7 and A20-Bl9). but an intrachain (AG-ALf) cystine linkage is also present L361.

BABINI AND VlEZZOLI

1008

tructural Classification According to the SCOP database, IN belongs to the class of small proteins, with an insulin-like fold, classified as disulfide rich and nearly all+ 1141. This fold classification, besides other insulin-like proteins (relaxin, insulin-like growth factor, bombyxin111, includes IN from 230s laurus, Homo sapiens, and SUSscrofa. At present, 85 IN structures are available in the Protein Data Base, but in the present chapter only those concerning the zinc protein will be described and discussed (see the PDB references in Table 2). The zinc-containing proteins have been isolated in different crystalline forms, called 2-zinc hexamer and 4-zinc hexamer (even if the zinc content i s less than 3) and phenol-induced IN species [3742L.

PDB References of the Available Zinc Insulin Protein source

Exp. method

Structures available

Bovine (Bos taurus)

X-ray diffraction

Bins: complexed with Zn

Human (Homo sapiens)

X-ray diffraction X-ray diffraction X-ray diffraction X-ray diffraction X-ray diffraction X-ray diffraction X-ray diffraction X-ray diffraction X-ray diffraction X-ray diffraction X-ray diffraction X-ray diffraction X-ray diffraction NMR NMR

Iben: complexed with Cl, hbd, Zn complexed with Cl, crs, Zn; mutant complexed with 61, iph, Zn; mutant complexed with Cl, Na, Zn Ixda: conzpkxed with cl, iph, myr, z n Izei: complexed with C1, crs, Zn; mutant complexed with Cl, fyl, Zn :complexed with Cl, i$, Zn complexed with Cl, iph, Z n comnplexed with Cl, rco, Zn; mutant complexed with Cl, iph, Zn; mutant complexed with Cl>iph>,Zn,; mutant 140:complexed with C1, Zn; mutant laiy: complexed with iph, Zn I a i ~complexed : with iph, Zn

Pig {Sus scrofa)

X-ray diffi*action Neutron diffraction X-ray diffraction X-ray diffraction X-ray diffraction X-ray diffraction X-ray diffraction X-ray diffraction X-ray diffraction X-ray diffraction X-ray diffraction

4ins: complexed with Zn Sins: complexed with Z n

lzni: complexed with C1, Zn at& complexed with scn, z n 3mth complexed with Cl, rnpb, Zn : complexed with Zn, mutant :complewd with Zn 7ins: complexed with Zn :complexed with Cl, iph, Zn lwav: complexed with iph, Zn

ZINC M~TALLOTHION~INS AND INSULIN

2.8.

Insulin: Structure

2.8.1

The Three Conformations: 2Zn- (or TG),4Zn- (or T&J, Phenol-Induced (or RG) Insulin Species

1009

The crystal structure of IN was reported for the first time as the rhombohedral2Zn form [43], named according t o its zinc content [37]. In that work it was shown that three equivalent dimers associate on an approximately threefold axis to form a hexamer. The two molecules that form the dimer have nearly identical conformations and are related to each other by a twofold axis, which is perpendicular to the crystallographic threefold axis. In this form each 21-residue A chain consists of two antiparallel cx helices connected by a short extended strand (A 9Ser-A 12Ser). Each 30residue B chain consists of two extended strands connected by an a-helical segment, B9Ser-B19Cys (Fig. 3). Each Zn2+ ion lies on a crystallographic threefold axis in an octahedral environment formed by the three symmetry-related B10-His residues and three water molecules. The addition of chloride to the crystallizing medium induces a change in conformation of residues Bl-B8 from an extended conformation to an cx helix (producing a continuous a helix from BlPhe to B19Cys) in three of the six subunits 141,441.As

FIG. 3. Structural representation of the dimeric unit of a 2 Zn IN (T,) hexamer from Sus scrofa. (PDB reference 4ins; Zn ions are shown as pale blue spheres, chains A of molecule 1and 2 are shown by green and blue ribbons, chains B of molecule 1 and 2 are shown as yellow and pink ribbons, respectively.) See Figure 21.3 in the color insert.

1010

BABlNl AND VlEZZOLl

far as the coordination of Zn2+ is concerned, at one face of the hexamer there is a single octahedral Zn2+ site as in the previous 2Zn form. At the other face of the hexamer three tetrahedral cavities, which can be occupied by zinc ions, are formed. However, even if four zinc sites are available, this species, referred to as 4Zn-IN, binds fewer than three Zn2+ ions [41]. Crystals grown in the presence of phenol (added as a preservative) contain two zinc ions per IN hexamer 1441. A full X-ray analysis of these crystals showed that in all six molecules an 01 helix is present from B1 to B9. The phenol molecule, which sits in the cavity created by the A chain residues from one dimer and the structure from the adjacent dimer, evidently stabilizes this new helical c 1451. Each Zn2+ ion is coordinated by the N E of~ three symmetry-rela molecules. The coordination sphere of zinc is completed by a single, axial chloride ion, resulting in a tetrahedral coordination. Besides the monoclinic form, a rhombohedra1 form was also crystallized in the presence of phenol. In the latter case, phenol molecules occupy positions similar to those observed in the monoclinic structure, although the hydrogen pattern between phenol and the A6Cys and AllCys residues is changed 1461. A nomenclature to describe the different crystallographic conformations with respect to the extendcd (T) or helical (R) conformation o f the B4 chain was introduced by Kaarsholm [lo]. Herein, the 2Zn form i s referred to as Tg, the 4Zn form as T3R3, the phenol-induced form as &. Also mwresol, resorcinol, 4 - h y d r o ~ b e n z a ~ i dand e, 2,7-dihydrox,~aphthaleneare capable of evoking the s m c transformation as phenol 147-491. The crystal structure of the adduct of the 4-hydroxybenzamide-T~R~ IN hexarner with two molecules of 4-hydroxybenzamide per R-state monomer was also reported [sol. In this structure the two guest molecules are involved in an extensive hydrogen bonding pattern between pairs of dimers of the hexamer and two water molecules. The solution structure of the phenol-induced 36-kDa R6 hexamer was determined by " I R spectroscopy and restrained molecular dynamics [Sll. The NMR data show that the aromatic ring of residue Phe(B25) can take two different orientations in solution, one pointing inward and one outward from the surface of the monomer. However, the overall solution structure of the RB hexamer is compact, rigid, and resembles the corresponding crystal structure. The main structural difference between the monomers in solution and in the crystal is at the N-terminal end of the B-chain, which is disordered in solution but part of the extended 01 helix in the crystal phase. A transition from the extended to the helical conformation is also induced by the nontoxic phenolic derivative 4'-hydroxyacetanilide, producing a T& hexamer. In this case, the guest molecule is bound by the R-state monomer through the formation of two H bonds with A6Cys and AllCys [52].Of the two zinc ions, the one that lies in the R-state trimer has a tetrahedral coordination, whereas that in the T-state trimer can adopt both the octahedral and tetrahedral coordination, depending on crystal growth conditions.

ZINC METALLOT~IONEINSAND INSULIN

1011

2.8.2. Mutants

The dissociation rate and capability of IN to change from the hexamers through dimers to the biologically active monomers is a property relevant for IN therapy because it controls the rate of action of injected IN, It can be modulated through different methods: by controlling pH, protein concentration, metal ion content, ionic strength, and solvent composition [38,42]. Alternatively, the self-association can be reduced by the introduction of charge repulsions into the monomer-monomer interface 1531by amino acid substitution or by the removal of some of the amino acid residues responsible for the dimerization. Thanks to their low molecular weight, many of these stable monomeric mutants have been the subject of structural studies in solution, through NMR. However, most of these analogues do not bind zinc C54-631. The role of some residues in hexamer assembling, like Ser at position €39and Glu at position B13, residues that are highly conserved in almost all known sequences of IN, has been well defined. These residues are connected by a network of H bonds and H-bonded water molecules and form a polar channel, down the central three-fold axis, containing the zinc ions [42]. The electrostatic repulsion among the six close B13 Glu residues, in order to allow the hexamer formation, is overcome by the zinc coordinas and by the formation of three H-bonded pairs on behalf of the Glu13 side chains. The mutation B13 Glu +Gln leads to the formation of a hexamer that crystallizes both as 2Zn- and as a Zn-free protein, depending on the conditions. Their X-ray structures have been solved and compared with the native 2Zn (Tci) and 4Zn (T&) proteins [64]. In the case of the 2Zn B1361n IN, the quaternary structure is similar to the native protein (T6), whereas the structure of the zinc-free mutant resembles that of the 4Zn (T3R3)protein with water molecules contacting the side chain of B13 Gln. The monomeric zinc free B28 Pro+Asp mutant, which is a rapidly acting hormone for therapeutic purposes, crystallizes as R6 zinc hexamer, in the presence of phenol and rn-cresol. In the crystals of these hexamers, phenol and rn-cresol have been found associated with the aromatic side chains at the dimer-dimer interfaces [651. Attempts to mimic the effect of phenol to promote the transition T-state 4 R state were performed by substitution of B5His with Tyr. In the absence of phenol, structural studies have shown that the molecule crystallizes as a half-helical hexamer (T~Rs),under conditions that usually induce the fully nonhelical state (T6). From molecular modeling calculations it appears that the B5 His 4 Tyr mutation destabilizes the T state through loss of relevant hydrogen bonds and through steric clashes, rather than stabilizing the R state [661. 2.8.3. Sequence Variation and Insulin Structure

The sequences in different natural INS are quite similar, with the exception of copypu and guinea-pig IN, which show several sequence variations from other organisms. However, in all cases there are some groups of invariant residues: (1)a group of essentially nonpolar residues (the three cystine groups; Leu at positions A16, B6,

LI

I01

Ile; B18Val; as well as Gly at 8 and B23), which constitute the core d ensure the correct arrangement of the chain within the monomer; i m p o ~ a nfor t contacts between the two , B16Tyr, B24Phe) th erization; (3) anoth (AlGly, A5Gln, AlSTyr, residues located on the surface of the molecule, involved in interactions that may be important in maintaining the tertiary s t ~ c t u r of e the molecule and that may also be involved in activity E671.

TU

The human neuronal growth inhibitory factor (MT-3)is a 68-amino-acidcysteine- and metal-rich protein with low molecular mass (7-8 kDa). This protein, which is specific to the central nervous system, impairs the survival and neurite formatiQnof cultured neurons. On the basis of the high metal content (7 mol m e t ~ m oprotein) l and 70% sequence identity to mammalian MTs (MT-1 and isoforms), including the preserved array of 20 cysteines, MT-3 has been included in the superfamily of metallothioneins; thus the designation as MT-3. h i n o acid sequences similar to that of T-3 have also been reported for rat, mouse, pig, horse, cow and dog ~ T - 3 references therein). ~ MT-2 l isoforms, the consensus Compared to the mammalian M ~ and sequence of MT-3 shows the following differences: (1) a conserved insert of a Thr in the N-terminal region; (2) the presence of two c o ~ s e r v eproline ~ r~sidu~s in the Cys(6)-Pro-Cys-Pro(S)motif; (3) a Glu-rich he~apeptidein the C-terminal region. Besides, in contrast to ~ T - l ~ which T ~ Zusually ~ contain. seven Zn2+,MT3 isolated from human brain possesses an unusual metal composition, ie., it con~ stoitains four Cu+ and three Zn2+ per p o l ~ e p t i ~chain e E691. A ~ i m i l ametal chiometry, 4 4 Cu" and 2-25 Zn2", is also present in native T-3 isolated from bovine brain [70], ite high similarities between the primary structures of ~ T - and 3 mamma21-21, the biological properties of these pr stantial alterations might occur in the with that determined for mammalian MT-l and MT-2 isoforms. Th. is currently unknown as the distribution of the metals in isolated studies on C u ~ Z n ~ - ~ and T - 3Zn7-NIT-3 established the presence of a t w o - ~ o m ~ n structure, resembling that reported for the other mammalian MTs, with each domain encompassing a metal thiolate cluster. The biological activity of MT-3 is confined to the the metal-cont~nin~ N-terminal f3 domain (residues from 1to 32) [71,72]. In ad~ition, i n h i b i t o ~activity of the 32-residue N-term in^ domain co~tainingthree Zn2+ was abolished by a double mutation of the conserved MTthe ~ T ~ ~consensus ~ T - sequence 2 C(6)-S-C-A(9) [711.

INC ~ ~ T A L L ~ T H I O N E IAND N S INSULIN

1013

been suggested that a unique conformation within the N-terminal f3 domain might provide an interface responsible for the growth inhibitory activity.

Phytochelatins are short glutathione-related peptides that are enzymatically synthesized (i.e., they are not direct gene products) in plants and in some fungal species exposed to heavy metals 1731, and they sequester metal ions in stable intracellular peptide complexes. Phytochelatins have the general formula (y-Glu-Cys),Gly, where n, (number of y-Glu-Cys diisopeptide repeats) is typically between 2 and 14, and are designated as y-EC peptides. Chemically, sequestration of metals is thought to occur by coordination of cysteine thiolate groups, and thus phytochelatins appear to be analogous to MTs, both in role and in binding mechanism [74]. However, despite the similarities to NITS, the limited structural data available on metal-yEC peptide complexes indicate that the two species differ in structure.

3.3. Cad Insulin The amino acid sequence of cod IN 1751 contains 16 differences from that of pig IN. The protein was crystallized and only preliminary X-ray data are available to a resolution of 3 A. In spite of the 16 sequence differences between pig and cod IN, it seems that the hexameric structure found for pig IN also exists in crystals of cod IN 176,771. Other INS and IN-like proteins have been jsolated from many organisms (like Acipenser gueldenstaedtii, Anguilla rostrata, Samia Cynthia, Lymnaea stagnalis, Rattus norrregicus, etc.). The proteins have been sequenced but their structure is not available.

3.4. Expressian Systems 3.4.2. ~ e t a l l ~ t h i o n e i n s

Most of the MT structures described in the previous sections have been obtained using the native protein. In the case of sea urchin MT and mouse MT, the PDB structure refers to the recombinant proteins. Both proteins were expressed in Escherichia coli, but using two different expression systems: plasmid p P W t for cloning the synthetic gene and host strain E. coli 1B 392 Lon A 1 for expression of sea urchin MT [781, plasmid pET3d and host strain BL21 (pLysS) for mouse MT 1181. 3.4.2. Insulin

The human protein has been expressed through three major methods: two involve E. coli and consist of the cytoplasmic expression of a large fusion protein [79,801 and the

1014

BABINI AND VIEZZOLl

use of a leader sequence that allows the secretion of IN into the periplasm ISll. A third method utilizes yeast (Saecharomyees cereuzsiae) to secrete the IN precursor into the medium [82]. The last method was also used to produce and isolate mutants I53,83] Recently, a novel mini-psoinsulin, having the central C-peptide region replaced by a sequence forming a reverse turn, has been expressed in E. coli as a fusion protein. This proinsulin showed enhanced refolding yield, and human IN was efficiently obtained from that by enzymic conversion [841.

The molecular mechanism of action of MTs is not known, but a correlation between the biological properties of these proteins and the structural features can be made. Some of these correlations are discussed below. 4.1.1, Interaction with Metals

An astonishing property of MTs is that of accommodating metals of widely difTering size, such as zinc (“covalent” atomic radius, 131 ppm) and cadmium (“covalent” atomic radius, 148 ppm), in the interior of the globular domains without causing gross conformational changes in the enfolding protein and in its dynamic features [SSl. The flexibility of the protein chain is a possible explanation for this property. The folded protein shows a considerable degree of dynamic structural disorder. In fact, in the 3D structure of MT, based on the root-mean-square-deviation (rmsd) values calculated from the ensemble of the NMR conformers and the crystallographic B factors, the polypeptide loops linking the metal-bound cysteine residues are less precisely defined than what is generally observed in proteins with a high content of regular secondary structure [321. More evidence of a nonrigid structure of the overall MT structure comes from the ‘H NMR ‘H-% m i d e exchange studies of Cd7-MT, which show that all but 1Qamide protons exchangw too rapidly to be seen by this technique [86]. The ability of the folded polypeptide chain to ad,just to different metal (cluster) volumes without compromising the mode of metal coordination has been attributed to the adaptability of the peptide loop structures interspaced between the metal-bound cysteinyl residues 1851. These loops, characterized either as turn or half-turn secondary structure elements, which make up the major portion of the protein that encloses the metal-thiolate core, dlow the polypeptide chain to follow contractions or expansions of the core with minimal changes in bond angles and side chain orientations ofthe residues in the loops [851. The metal binding sequence, and therefore the metal clusters formation, has been investigated through ‘EE NMR spectroscopy in the case of the cobalt protein from rabbit E261.

ZINC ~ ~ T ~ L l ~ T ~ l O AND N INSULIN ~ l N S

1015

4.1.2. Metal Exchange

To participate in cellular metal distribution the metal clusters in MTs have to exhibit specific chemical characteristics, wh~chenable the protein to bind the metal tightly (thermodynamic stability) while featuring mechanisms that make the mehl ava~able(kinetic lability). The c ~ c u l a t e ddi~sociation on st ants of zinc and cadmium from rabbit liver Zn-MT and Cd-MT (Kd = 1.4 x M and Kd = 5x M, respectively, at pIl 7 f873 indicate that the metal clusters in MTs are thermodynamically quite stable, yet the dynamic properties of the MT structure also show the high kinetic l a b ~ t yof the metal complexes, which means that the thiolate ligands undergo both metallation and demetallation rapidly. The rate of zinc exchange betw~entwo MT isoforms, measured using radioactive “‘Zn C881, proves for zinc MT clusters a high kinetic lability. This kinetic reactivity distinguishes zinc in biological clusters from zinc in the active site of enzymes. For example, zinc enzymes, such as carboxypeptidase and alkaline phosphatase, show exchange halffives of the order of hours and days, as expected for a well-defined and rather rigid catdybic site [23J Kinetic studies indicate that the metal ion can exchange quickly by a specific mechanism and yet be bound in a coordination environment providing a relatively high t h e r m o d ~ a m i stability c t 881. In MT, st~bi~ization of zinc binding i s achieved via a total of 28 intramolecular zinc-sulfur bonds (16 in the CI domain and 12 in the f3 domain), but high reactivity is still provided by these 28 bonds in which only 20 cysteines participate. The number of bridging ligands is a determinant of the reactivity of zinc clusters E891. Indeed, the reaction rates generally decrease when the number of connectivities of the cluster increases. Zinciligand ratios smaller than 4, due to the sulfur-thiolate bridges as observed in MTs>poise the molecule fast metal~lig~nd exchange “31.

The T3R3 IN hexamer, also known as 4 Zn IN, is used in diabetes if a slow-acting preparation is requested because it is slow to dissociate to the biolo~callyactive monomer. The major factor that stabilizes the hexamer, with respect to the monomer, is the stable b ~ d of~ zinc n ~ ions. This is possible as a consequence of the T --tR transition and the accompanying formation of a 12-,& channel at the bottom of which the zinc ions lie isolated from the e n ~ ~ o n ~ e n t , The transition of this form toward the active monomer requires, prior to the d i s s ~ ) c ~ a tof i ozinc ~ from. the hexamer, that the R state undergoes a change in conformation to the T state to make the zinc ion accessible to the solvent and easily t ~ ~ s f e r a b lIn e . the absence of zinc, the hexamer begins to dissociate to dimers and then to biologically active monomeric species 1901.

1016

Meta~lothioneins,by virtue of: their large metal content, their unusual bioinorganic structure, and their remarkable kinetic lability, constitute a unique class of metalloproteins. The ubiquitous expression of these proteins implies that they provide a selective advantage, but the reasons for this have not yet been j d ~ ~ t i ~In e dfact, . more than 40 years a&er their discovery their primary evolutionary function is still a topic of discussion. Despite the fact that MTs were first identified as detoxificatioil agents capable of protecting cells from the effects of heavy metals, fmther studies indicated two major roles of the 343's in the metabolism of Zn: (1)the regulation of MT gene expression by multiple Zn-dependent proteins and (21 the relationship between Zn release from MT and the glutathione redox couple. The biological function of a new class of MTs (GIF) isolated from brain may be unrelated to Zn metabolism and/or heavy metal d e t o ~ c a t i o nt5,Sll. The structural determination and the biochemical characterization of these proteins are prerequisites for understand~~ig the molecular mechanism of their action and its correlation with their structural features. This should also be helpful in clarifjring the evolutionary significance of MTs.

Since external a ~ p l i ~ a tof i oIN ~ is used for the t r e a t ~ e nof t diabetes, research in this field has moved toward the development of' IN analogues with different abilities for s e ~ f - ~ ~ ~ One ~ ~ example a t i o ~is. the f~t-act in^ LysPro IN, characteri~edby reduced self-~ssociationand fast absorption [9Z]. A slowacting IN with protracted glucose with strong i n t e r h ~ ~ econtacts r i ~ that are responsible lowering activity is HoeQOl~ for stabilizing the crystal in the subcutaneous space 1931. However, the goal of the o n could future research is an orally applicable, instead of injectable, IN p r e ~ r a ~ i that became a reality when the interaction between IN and its receptor is completely understoo~[93].

Thanks are expressed to Prof. Milan VaB& for valuable suggestions and critical comments.

ZINC M ~ T A L L ~ ~ H I O N € IAND N S INSULIN

crs EGpeptides GIF hbd

IN iph Kd

mPb

MT rnyr

NMR PDP rco SCQP tY1

1017

rn-cresol GluCys-peptides growth i n h i b ~ t o ~ factor ~ - ~ y d ~ o ~ b e n ~ a ~ d e insulin phenol dissociation constant rnethylparaben ~etafl~~~ion~i~ rnyristic acid nuclear magnetic resonance Protein Data Base resorcinol structural classification of proteins tylenol ~4‘-hydro~yacetanilid~~

1. M. Va%k and D, W. Hasler, Cmr. ~ ~Chem. ~ Biol., n 4, . 177-183 ~ ~ ~ 0 0 ~ 2. D. A. Suhy, K. D. Simon, D. I. Linzer, and T. V. O’Halloran, J. Bid. C~ern., 274, 9183-9192 (1999). 3. R. D. Palmiter, Proc. Natl. Acad. Sci. USA, 95, 8428-8430 (1998). 4. E. J. Kelly, 6. J. Quaife, G. J. Froelick, and R. D. Pdmiter, J. Nutr., 226, 1782-1790 (1996). 5. V. Daragan and K. H. Mayo, B ~ ~ ~ 32,~~~ 4 ~8 8 -~1 1(1993). ~ 9 ~s t ~ , 6. B. L.Vallee, ~ e ~ r * litt., c ~27, e 23-33 ~ ~ (1995). 7. B. L. Vallee and W. Maret, in ~ @ ~ a Z l o t h ~ o11n1~(K. i n T. Suzuki, N. Imura, and M. Kjmura, eds.), Birkhauser, Basel, 1993, pp. 1-27, 8. L. G. Howell, Adu. Cytopharmacol., 2, 319-327 (1974). avu, 6. L u ~ d ~and e ~S., Fafkmer, Acta ~ ~ ~ 86, ~ ~70-577 ~ ~ (1977). o l . K-C. i , KO, and M. F. Dunn, ~ ~ o c h e ~ , 28, i s t 4~ ~, 2 7 ~ 4 3 10. N. C. ~ a & r s h o ~ (1989). 11. L. 6. Howell, M. Kostianovsky, and P. E. Lacy, J. Cell Biol., 42, 695-705 (1969). 12. a. F. Stcsiner, Diabets, 26, 322-340 (1976).

1018

B A B I ~AND I VlEZZOLl

13. A. Permutt, The Islets of IAngerhans (8. J, Copperstein and D. WatEns, eds.), Academic Press, New York, 1981, pp. 75-93. 14. T. J. P. Hubbard, A. 6. Murzin, S, E. Brenner, and C. Chothia, NucZ. Acids Res., 25, 236-239 11997). 15. M. Nordbctrg and Y. Kojjrna, Experientia Suppl., 38-55 (1979). 16. B. A. Fowler, C. E. Hildebrand, Y. Kojima, and M. Webb, Experientia SuppZ., 52, 19-22 (1987). 17. Y. Kojima, P. A. Binz, and J. R. IGigi, in ~ e ~ ~ Z l o t ~ i oN n e (C. i n Klaassen, ed.1, B ~ r ~ a u s eBasel, r, 1999, pp. 7-13. 18. K. Zangger, 6. Oz, J. D. Otvos, and I. M. Armitage, Protein Sci., 8, 26302638 (1999). 19. B. A. Messerle, A. Schaffer, M. VaBEiJs, J. H. Eigi, and El. Wiithrich, J , Mol. Biol., 224, 765-779 (1990). 20. A. Arseniev, P. Schultze, E, Wtirgotter, W. Braun, G. Wagner, M. VaBak, J. H. K$@, and K. W u t ~ c hJ., Mot. Biol., 201, 637-657 (1988). 21. P, Schultze, E. ~tirgotter,W. Braun, G. Wagner, M. VaSitk, J, H. Kiigi, and K, Wiithrich, J. MoZ. Biol., 203, 251-268 11988). 22, . Robbins, D. E. MeRee, M. W i l l j ~ s o S. ~ ?A. Collett, N. H. X U Q ~W.~ F. , Furey, B. C. Wang, and C. D. Stout, J , MoL Bid., 221, 1269-1293 (1991). 23. M. V d & m d R, Bogumil, in NATO ASI Series 2 ~ (N. 2 ~ t). ~ a d j ~ed.), ~ ~ ~ er Academic Publishers, Nonvell, 1997, pp. 195-215. 24. Kagi and A. SchaRer, B ~ o c h e m i s t27,8~09-$515 ~, (1988). in Metal Ions in Biological Systems (El. Sigel, 4.1, 25. M, Vag& and J. H. Ei@, Vol. 15, Marcel Dekker, New York, 1983, pp. 213-273. 26 I, Berkini, C. Luchinat, E. Messori, and M. Va%k, J. Am. Chem. Soc., 111, 7296-7300 (1989). n ~~u,c h ~ n aL. t , Messari, and M. VaS& Eur. J. ~ ~ o c h e m21, ,1, 23527. I. B e ~ ~C, 240 (1993)" 28. X, Ding, E. Bill, M. Good, A. X. Trautwein, and M, VaBBk, Eur. J . B~och~m=, 171, 711-714 (1988). 6. I-fenkeI, H. Winkler, M. 29. X. Q. Ding, C. Butzlaff, E. Bill, D. L. Pount~~ey, Vai%k, and A. X. Trautwein, Eur. J. Biochem,., 220, 827-837 (1994). 30. I. Bertini and C. Luchinat, Adu. horg. Binchem., 6, 71-111 (1985). ertini and C. Luchinat, NMR of P a r a ~ a ~ ~n ~~~ lc e inc Biological ~ l ~ ~ 31. Systems, Benjamin/Cummings, Menlo Park, CA, 1986. 32. W, Braun, M, VaBjk, A. H. Robbins, C. 2). Stout, 6. ~ ~J. H. I ~U@, and~ K. Wiithrich, Proc. Natl. Acad. Sci. USA, 89, 10124-10128 (1992). i s ~620, 33. S. S. Namla, M. Brouwer, Y. Rua, and I. M. Armitage, ~ ~ o c h e ? n 34, 631 (1995). ~G773-67$7 i s ~ ~ , 34, S. S. Namla, D. R. Winge, and I. M. Armitage, ~ ~ o c h ~ Iigands via the side chain of h g " , which ~ o r m ~ h y d r o ~ ebonds ~ ~ to a sulfur atom hi^^ the cluster and to two pterin ~ ~ g e nAs . second c o ~ ~ e c t i oisnp ~ o by~a h~y ~ e~ o g ~ ebond n b ~ ? t w ethe e ~ cluster ~ i g Sy ~ of n ~ pterin nit~ogen.These p a t ~ w ~ may y ~allow electron t ~ ~ n s ~~ ~ e rt ~ e e ers and, as the ~ ~ ~cluster - i ssjust~6 ~below~ the usurface ~ of the ~ ~ a ~ the tungsten and the ferre ~ r o t ~iti is~ a, likely ~ n t e r m e between ~ ~ L ~ e r . ~ ~ u s the ~ c~ ~~ sy t, ~ of lAO~ from ~ ~ ~ p ~ r~preceded i~ # ~t ~ o~~ . ~o ~ g h s ~ e c t ~ o s c ~~p i cn v ~ ~of this t ~ ~ r~o ~~e iMuch tn .~ oof the ~ ~early s ~ ~ c t r ~ s c oi pnivce s t ~ ~ a ~ tions were carried out on an inactive form of the enzyme, called the red t ~ ~ ~ ~ catalytic function was ~ d ~T u ~~g s tte ~ ~ at was isolated be protein ( hwdge of the ETP form evealed three or four sulfur l i ~ ~ at~ 2.43 d s

A from the tungsten, two 0x0 groups at 1.74 A,and a possible longer 0,” contact at 2.1 f3201. Subsequent studies of the ~ t ~ o n i t e - r e d u c eactive d e n z ~ showed e the presence of four or five sulfur ligands at 2.40 A,a single 0x0 group at 1.75 A, and

possibly an additional O/N contact at 1.97 A LSj. These results clearly indicate the presence of one or two VV=O groups and imply that the dioxo form of the enzyme is inactive. However, it should be noted that the AORs from P. furzosus and Pyrococcus strain ES-4 have considerable heterogeneity at the tungsten site, as revealed by the profusion of W(V>EPR signals attainable under different conditions. Many of these correspond to inactive forms of the enzyme, with only about 30% of the as-prepared ~c~~y en~ymehaving the ability to cycle between W ( ~ ~ / ( Vat)the / ~p ~h ~y s i o ~ ~ relevant potentials associated with the active enzyme. This means that the crystallographic and W studies represent an average structure of several forms of the enzyme rather than a single species t3211. The [4Fc-4S]-t state of the iron-sulfur cluster has distinctive electronic properties in that it possesses a pure S = 312 ground state. This probably arises due to either the special protein conformation in the vicinity of the cluster and/or the hydrogen bonding interactions that link the cluster with one of the pterins [3211.

2.4.2. Formaldehyde Perredoxin ~ ~ i d o r e ~ u c ~ a ~ e s

For~aldehydeferredoxin oxidoreductases (FORs1 [~0~-3071 me closely related to AORs [295-2991, with about 40% sequence identity (and about 60% similarity). In contrast to AORS, FORs can oxidise only C1-C3 aldehydes or less than C6 dialdehydes. FORs, like AORs (Sec. 2.4.11,have been isolated from the h ~ e r t h e ~ o p h i l i c archaea P. firiosus [305,306] and Thermococcus Zitoralis 113071. These two enzymes are virtually identical with 92% sequence similarity (and 87% identity). FOR from P.firiosus has been crystallized and the structure solved to 1.75 A resolution 13061. FOR is a homotetramer with subunits of 621 residues (70 m a ) , each of which contains one 4Fe-4S cluster and one tungsten coord~natedby two MPTs. FOR is a flat, plate-like homotetrmer with a hole through the center. Each monomer is spherical with similar folds to AOR and, like the latter, is composed of three domains; however, different parts of the surface are used to form the inte~actions and the two enzymes form different oligomers. The most significant differences ~ FOR has an between AOR and FOR are the dimensions o f the ~ u b s t r achannel. insertion of five residues that seal the cavity opening and create a large chamber at the bottom (by the tungsten) connected to the protein surface by a narrow hydrophobic channel. The residues closest to the tungsten are similar and conserved, unlike the other residues composing the cavity. Glutaric dialdehyde has similar dimensions to the physiological substrate of FOR. Glutarate, the oxidation product, can be soaked into a crystdl of FOR and shows up in the cavity with one carboxylic acid group near the tungsten and the other anchored to the protein through interactions with cavity side chains. As in AOR (Fig. 27), the tungsten is coordinated by the four sulfur atoms (W -, ,S = 2.5 A) of the two MPT cofacters and there are no protein side chains

bound to the metal. One other ligand, assumed to be oxygen, is coordinated to the tungsten at about 2.1 A, although this distance is uncertain due to “ripples” in the electron density caused by the heavy tungsten atom. The phosphate groups of the two pterins are (as in AOR) linked by an Mg+ bridge and also, in this case, by a Ca2* ion. FOR has been co-crystallized with the ferredoxin that, is its physiological redox partner. P. furiosus ferredoxin is small, with just 66 residues and contains one 4Fe-4S cluster. The proteins interact such that the ferredoxin 4Fe-445 cluster is as close as possible (- 15 A) to the FOR 4Fe-4s center. A straight line through the two 4Fe-4% clusters nearly passes through the tungsten; thus, it is proposed that an electrontransfer pathway exists from the tungsten to the femedoxin 4Fe-4S cluster via the FOR 4Fe-4S cluster. The EPR spectra recorded for FOR resemble those of AOR and are similarly dominated by the signal from the [4Fe-4SJi cluster, the nmjority of which has an S = W2 ground state with a minor S = 1/22component. The EPR spectra ifso reveal a considerable heterogeneity at the W(V) site I5l.

3. ENZYMES WITH U

~

K STRUCTURE ~ ~ W ~

New molybdenum o~otransferaseenzymes w e being discovered f r e ~ u ~ n tand l y regularly, especially as a consequence of the determination of genome sequences 1118,131,1373.As the explosion of genome sequence determination continues, a significant number of new molybdenum oxotransferases will undoubtedly be added to the presently known cotlection summarized in Table 1. Refitrement of the s t ~ u c t ~ r e s reported in Sec. 2 will continue, and the structural characterization of many other ~ o l y b d e n u ~oxotransferases n will certalnly be accomplished in the near future. This information, linked to the results of site-directed mutations, activity, and spectroscopic information, will provide valuable further inforInation detailing the structure-function relationships for individual members of this important family of enzymes, The challenges that remain can be exemplified by xanthine oxidase 115,217,3221. This is one of the most intensely investigated of all enzymes, not only because of its ready availability from cow milk but also because the oxidation of xanthine to uric acid produces either peroxide or superoxide (depending on the pH and the level of enzyme reduction), resulting in a range of biochemically significant inflammatory responses. Mop (Sec. 2.3.3.1.) and xanthine oxidase are stntcturally related, as demonstrated by the conservation of the amino acid sequences, in general, and of the protein segments at the molybdenum and iron cofactors, in particular [280,2811. Also, there is a strong functional relationship between these two enzymes, since xanthine oxidme is capable of catalyzing the oxidation of a wide variety of aldehydes. Furthermore, ~pectr~scopic studies indicate that the nature of the co~respond~ng m(~1ybdenum tenter of these two enzymes is very similar in the active, desulfo, and other states t2,2822841. Mop lacks the FAXI dom&n present in xmthine oxidase. Thus, the FAD domain may replace the connecting segment in the Mop and be located in the shallow

315,216 117 118 11

1~~,121 12%

127 228 129 130 131 132,133 E 18 1 ~ 136

~

137 138 139

14

141 142

143 144 14 14

147 148 1 ~ ~ , I 5

151 152 153 154 155 156

357 158 15

160 161 162 163

170

d e h ~ oxidase ~e

GARNER ET AL.

1066

Table 1 (continued~

Enzyme

Source

Ref.

Nic~tinieacid hydroxylase (dehydrogenase)

Arthrobacter oxiduns 188 Bacillus niacini 185 ~ ~ o s ~ d burkeri ium 190 Picolinate hydroxylase Arthrobaeter p i c o l i n ~ ~ ~ ~ ~ l u s 191 Quinaldic acid ~ - ~ ~ d o r ~ d~~~ ~ s oe sp. e uc~~o m n aAK-2 s 192 Serratia marcescens 193 Q u i n ~ ~ ~ e ~ 4 - o x i d o r ease duct 194-196 A ~ h ~ b u e sp. ~ eRii61a r 193 Serratia marcescens ~ ~ m ~ r nt oe s~t aus st e ~ o ~ ~ ~ 197 198,199 Pseudomanus putida R h ~ ~ o c o esp.Bf ci~~ 200 Quino~~e-4.-carboxyXaee-2~ r a b a ~ ~ e r sp. i u m1B 201 o~doreduct~se ~ u ~ L ~ p ~uicina ~ora, Xanthine dehydrogmase 202 203,204 Chicken liver ~ ~ ~ u ~ y reinhardtii d o ~ o ~ a ~ 205 206,207 Drosophila melano%aster Drosophila puesdoobscura 208 Drosophila subobscura 209 Emericelia. n ~ d ~ ~ ~ u ~ s 210 Eubaeteriu~barfzeri 211 Human 212 ~ i c ~ - o ~ oL'actylliticus cc~s 203 2 13,214 Mouse 215 Rat Ever Cow milk 21~220 X ~ ~ ~oxidase h i ~ e

Aldehyde oxidoreductase Chlarate reductase Carbon monoxide oxygenase Dye-linked aldehyde dehydrogenase 2-Furoyl-CoA dehydrogenase $-~ydmxybeilzoyl-CoAreductase 2 * ~ y ~ r o tinate ~ i ~ o ~ ~ c dehyd~ogenase

~ ~ ~ ~u cf~ ~a~ o~c o l~~ aur i us s 221 Proteus rrtirubilis 222 Pseudomonas e a r b ~ ~ ~ d ~ ~ ~ r223 a n ~ Amycolatopsis ~ e t h ~ n ~ l ~ c a 115 Pseudomonas puiida Thauera arom~ticu ~~ y c a b a ~ t e r ~ u m

224 225

2%

PROTEINS ~ O N ~ A I N ~OLYBDENUM IN~ OR TUNGSTEN

1067

Table 1 (continued) Enzyme

Source

Ref.

Periplasmic nitrate reductase

Alculigenes eutroph us Desulfovibrio desulfuricans Escherichiu coli Paracoccus denitrificuns ~ h o d o b sphaeroides ~ ~ r A ~ ~ , ~ o ~~ ai ecot l ei n~ o ~ ~ i l ~ s D r o s o ~~ ~~ l ~a ~~ ~o g ~ t e r Mammalian liver Pelobucter acidigallici Archaeoglobus fulgidus

227 228 228-230 145 231 232 233 234 235 236

Picofinic acid dehydro~enase Pyridoxal oxidam Pyrimidine oxidase Pyrogallol transhydroxy~ase Pyruvate:ferredoxin oxidoreductase Selenate reductase Tetrathionite reductase ThiosL~lfatereductase (precursor)

*Enzymesfor which structural information is available. PDB ID code: a XDM8, IDMR, 2DMR, IDMR; 1CXS; 1AA6; 2NAP; 1ALO; 1QJ2.

237 222 238 239 1TMO; 1SOX

dish- like depression between the iron, Mo1, and Mo2 domains, and in contact with a21 three. The m e c h ~ i s mof xantbine oxidase has been the subject of considerabl~attention and discussion f2,15,191,21~-219,3231,including theoretical calculations 13241. The experimental investigations have included I3C and I7Q ENDOR of MOW)species and by kinetic studies of the exchange of oxygen isotopes. Single-turnover experiments have shown that transfer of a bound oxygen atom to the substrate takes place within the reductive halfmcycleof'the reaction, during which Mo(VI) is reduced to Mo(TV). Crystallographic studies of Mop l281l demonstrate the presence of a ~ o l y b ~ e ~ u m - b owater u n ~ molecule in. the active site and suggest that this group involves the c a t ~ ~ i labile c ~ yoxygen. %his oxygen is probably t r a n s f e ~ e das ORto the substrate carbon atom in concert with the addition of H- from the ~ ~ ~ ~ t r C-H bond to the Mo=S group to form an MoXV-SHintermediate. Therefore, the crystal structure of xantbine oxidase is eagerly anticipated, especially to see the accuracy o f the above structural predictions and to provide an improved basis for describing the function of this very i m p o ~ aenzyme. ~t Further characterization of tungstoenzymes by protein crystallography is an n important priority. The two structures reported so far, the aldehyde f e ~ r e d o ~oxi[3Ml from d o r ~ u c t a s e1261 and the for~aldehyde femedoxin oxidor~du~tase ~ ~ r o ~ o~~ c u s~ (Xec. 2.4) ~ arc~strikingly 0 similar ~ in the ~ nature s of their function and their tungsten center. It i s important to know if this environment is generally applicable to the tungstoenzymes (Table 21, or whether-as for the molybdenum

1

&.

1070

4.2. ~oo~~ination Sphere of the Metal ions The results of protein c ~ s t ~ l o g r a p h investigation^, ic linked to ~ ~ ~ t r o s cstudies o~ic of the metal centers of the molybdenum oxotransferases and tungstoenzymes (see See. 21, have greatly advanced our u n ~ ~ r s t a n d i nofg the nature of the coord~~ation sphere of molybdenum in oxotransferase enzymes and tungsten in tungstoenzymes. The structural results have led to a broad classification of the molybdenum oxotransferases (as shown in Fig. 4). For details of the coordination sphere of molybdenum in the oxotransferases, the reader is specifically referred to DMSQ reductase, See. 2.3.1.1fFig. 11;TMAO reductase, See. 2.3.1.2iFig. 14; formate dehydrogenase R, See. 2.3.1,:3/F'ig. 16; dissimilatory nitrate reductase, See. 2.3.1.4./ Fig. 18; sulfite oxidase, See. 2.3.2.fFig. 20; aldehyde oxidase, Sec. 2.3.3.1.fFig. 22; CQ hydrogenase, See. 2.3.3.2Fig. 24. The tungstoenzymes, aldehyde oxidoreductase and formaldehyde ferredoxin o~doreductase,are describ~din Sec. 2.4 and the nature of tungsten center of the former is shown in Fig. 27. Members of the DMSO reductase family have the molybdenum ligated by the dithiolene group of two MPTs. In addition to these two dithiolene groups, the molybdenum is also ligated by a terminal 0x0, or sulfido, or selenido group plus a donor atom from a side chain of a serine, qsteine, or selenocysteine amino acid residue. In the case of the DMSO reductase from R. capsulatus,arguments have been advanced for the presence of an a d ~ t i o noxygen ~ atom, leading to a seven-coo~dinatesite 1246,2481 (see See. 2.3.1.1 and Fig. 11).However, there i s no clear consensus for ~ ~ n g s t o e i i zgenerally ~es appear to involve two MPT moithis p o ~ t ~ i l a[2d9,251]. te eties ligating the metal. The additional coordination of tungsten by an amino acid side chain has not been demonstrated and, beyond the two ~ t h i o l e groups, ~e the remainder of the metal's coordination sphere appears to involve at least one 0x0 group plus possibly a hydroxo group or a water mdecule. Thc oxidized form of tung~ten-subst~tutcd DMSO reductase from R. capsulatus involves the metal bound to two MGD cofactors, the oxygen of a serine residue, and an 0x0 group [511. Members of the sulfite oxidase and xanthine oxidase fmities involve the molybdenum bound by one MPT group. In the state of chicken liver sulfite oxidase that was c ~ s t a l l i z e d ~ o n s i d e r etod be the oxidized sttlrtt+-the molybdenum is bound to the dithiolene of MPT, a cysteinyl residue, an 0x0 group, and a waterhydroxo group e, 11651, However, by Mo K-edge EXAFS of the oxidized state of this e ~ z y ~evidence was obtained for two M0=0 groups and three Mo-S bands [2731. Thus, the crystallographic and spectroscopic ~nvestigationsof mdfite oxidme still require reconciliation, Aldehyde oxidase, the only member of the xanthine oxidase family to haw been structurally characterized to date, exhibits a fac-[Mo=O,=S,-QHzf center bound to the two sulfur atoms of' an MCD cofactor. There is no amino acid residue coordinated to the molybdenum [280,2811 Ars noted in the i n t r o d u c t o ~comments to Sec. 2.3., the coord~~ation environments of these metals in the various enzymes should not be regarded as having been n i~g h - r ~ s o ~ ~crystallot~on defined by the information presently reported. A d ~ t i o h I

PROTEINS C O N ~ A I ~ I~ N~~ L Y ~ D OR E NTUNGSTEN U ~

1071

graphic studies are required. These should delineate how the nature of the coordination sphere varies as a function of pH, redox state, the presence of substrate or substrate analogue, inhibitor and site-directed mutations. This structural information is a necessary prerequisite to establish the reaction mechanisms that operate for the various m o ~ y ~ e ~o ux m o t r ~ f e ~and ~ e tsu n g s t o e n z ~ e ~ .

4.3. Description of the Reaction Mechanisms There is a wealth of spectroscopic information, sometimes linked to kinetic studies, obtained by measure men^ that were performed before the results of the protein c ~ s t a l l o ~ a p hstudies ic described in Sec. 2 became available. Although the validity o f the majority of these experimental results is not questioned, it is vital that the o f each o f these studies be reconsidered to take account of the strucinter~i-et~tion tural information now available. The s t ~ u c ~ u rknowledge al will stimulate other investigations of the reaction mechanisms of the rndybdenum oxotransferases and t u n g s t o e ~ ~ eIn s .particular, an awwmess of the molecular architecture is especially valuable in generating site-directed mutations to assess the role of specific amino acids. The oxygen atom t r m f e r reaction (1)is often taken to imply that direct oxygenatom exchange occurs between the substrate and the molybdenum (or t u n ~ ~ t e n ) center. Certainly, this is compatible with the known chemistry of these elements (see See. 1.4.2) and this appears to be the case for the DMSO reductases. Thus, catalytic and single-turnover experiments [250], and resonance Raman studies I2511 have demonstrated that DMSQ reductase from R. sphaeroides is an oxotransferase, The rwonance Raman data have been interpreted in terms of the active site cycling between m o n o ~ o ~ o -and ~ o des-oxo-Mo(~} (~~ forms via n substrate-bound intermediate [251]. Similarly, for sulfite oxidase, extended Hiickel calculations favor the direct attack o f WSO, at an Mo"=O group 12741. However, it should not be presumed that oxygen-atom transfer proceeds so directly in other enzymes. There are several other ways in which net oxygen-atom transfer can be achieved and ~echanismshave been proposed for several of the molybdenum oxotransfei~a~es~ For aldehyde oxidase, the reductive half-cycle is proposed to proceed by conversion o f an HzQ molecule to OH-, followed by transfer of OH- to the substrate in concert with H- absti-actionby the Mow=S group to ~ e n e r a t ~ M o ~ - ~and H RGQzH [281]. The catalytic mechanism for CQ dehydrogenase i s envisaged to involve the formation of carbon oxide selenide (O=C-=Se),with nucleophilic attack by the M o v i - ~ ~ group yielding COz, Wf and Mo(W 12901 (see Fig. 25). These two variations show the flexibility in the manner in which oxygcn-atom transfer can be achieved. Possible ~ ~ c h a ~ i of sm s electron transfer, to reoxidize (or ro-reduce) the metal center to the level required for catalysis, have been discussed for some molybdenum oxotransferases for which crystallogTaphic information is available. In formate dehydrogenase, formate binds the oxidized enzyme and kinetic ~ e ~ r e m suggest e n ~that

the t ~ electrons o added to the ~ o l y b d e n ~center m exit in a p ~ i i ~ ~~ ~p ~ na ~n i t e ~i ~ ~ l i c in e We-48 c ~ ~ sbei ~ the ~ e el~ctron d t r a ~ f e ~r a t h ~ aFoy y. ase, r e ~ ~ ~of tLhi o o~( V > to the Mo(lV) state i s p ~ 0 ~ o to s eoccur ~ via ~ e ce ~~e c~~ r lo n~" t r a ~~s ~~etr ~ ~we taw ye e nthe ~ o l y b d ~ n zing [2663. In the oxidative half cycle of sul two e ~ e c t ~ o are n s transferred s e q ~ e ~ t ~from a ~ l the y ~0~~~ center to the ~ ~ - The h ch~racteris~~cs ~ ~ e ~ of the i n t r a ~ o l e c ueiec~ron ~~ transfer fro with a ~ e c ~ ~ a nthat i s ~ is n coupled to rot on ~ r a ~ ~ sfrom fer ~~~~~~

c inv~stigations,and the protein ~ s t ~ ~ o ~ now have the basis for a ~ e n ~ idiscussio ~lc ~ o l y b ~oxotra~~sferases s ~ u ~ and tungstoenzymes. owever, the present ~ ~ o r m a t ~ o n point, not the conc~us~or~, of our e n ~ u i ~ i eTs .~ u sfurther , high~ e p r e s ~a~starting ts s t a l l o ~ a ~ ~h n~ vc e s t ~ ~ a tlinked ~ o ~ sto, re lev^^ bioche~icaland spsctro, are EX ~ e c e s s ~a r~e ~r e q u i s i to t ~ properly define the s ~ , ~ c t ~ r e - f ~ n ted f each of these e n z ~ e ~~ ~. ~ h ~ rthe~ i on fro e~ ~, t i o~ n~ e s e ~in ~ a ~ 1~and~ 2eshow s that there are many of these enzymes for which no structural i ~ ~ ~ ~i s ~ ~t v i~ ol aThe n~ l rapid e . a d v a n ~ ein~ obtain in^ the ~ ~ s e ~ ~~ e ~ of co e s o ~ ~ will~~ n d~ o u ~ b t e ~sadd ~ y ~many~ more s molybdo- and t ~ n ~ s t o e n ~ to y ~the es ~oiisiderablec ~ ~ l e lie n ahead ~ e ~rtnd our ~ e ~ ~ oton these se ~ r ~ s lists. e n ~'~'~erefo~~e, to many ~ u r ~ ~ass Lhis e s i ~ ~ o topic r t ~int~ i ~~ ~ l o ~~~ a ~n c h e ~ i s at ~~v a ~ c e s .

the ~ o ~ t r i b u t ~of o nDrs, s S. the s u b j e ~ ~n ~ ~oft this e r chapter. Also, the ~ o ~ ~ assis~ d ~ iss Jo Jennings in the p r o d ~ c t ~ ofothis ~ ~ a n u ~ c r iis p t~ a t e ~ ~ l ~~l~no~lec~~e~.

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~ e ~ a ~of ~chemist^, e n t Imperial College of Science, ~ e c h n o l and o~ ~ ~ d ~ c i ~ South Kensington, London SW7 ZAY, UK

1092 1092 1094

1. INT~ODUCTION 1.1. A Half Century of Bioinorganic Chemistry 1.2. Some Emerging Themes

2. FOCUS ON THE PROTEIN: C ~ ~ F O ~ T C I O N ~ 2.1, M g A T P ~ eThe : World's Smallest Rotary Engine 2.2. €312-DependentMutases: A Molecular Nutcracker 2.3 Lessons for Enzymic Catalysis

~

1098E 1098 1099 1100

3. FOCUS ON THE METAL: METAL SPECIFICITY 3.1, CoGIf) + CH;: ~ e ~ l - L i g a n Covalency d and the Use af 4s Orbitals 3.2. Fe(I1) + NH3: Donor Ligands and C o ~ l o ~ b ~ c / ~ o l v aEffects tion 3.3. Fe(I1) + 0 2 : Acceptor Ligands 3.4. Fe(II) GO: ?? 3.5. Usage = Reactivity x Availability: Cr, Co, Cu

1103 1104 1104 1106 1107 1108

4. C O ~ C L ~ ~ I O N S

1111

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~ ~ ~ E ~ T I O N S

1113

~E~E~ENCES

1114

1091

1092

PRATT

1. INTRODUCTION 1.l.A Half Century of Bioinorganic Chemistry

Pioneering work on nwtalloeiizynles and proteins, stretching back to before 1900, was naturally concentrated on t he rcudily available and conspicuously co1ort.d heinopi-otcins, such as hemoglobin, myoglobin, peroxidase, and catalase. Thc study of a wider range of metalloproteins and enzymes began to merge under the banner of bioinorganic chemistry tor inorganic biochemistry) from the 195Os, in parallel with the d ~ ~ ~ r e l o p iof i i ~many n t of our current idcas of coordination chemistry. The new subject enjoyed ;1golden period of intellectual excitement in the 1960s. Structure determinations were rcported for myoglobin (as the very first protein) in 1960, the protein-free coenzyme form of R1, (with its unexpected and relatively stable Co-C bond^ in 1961, several fornis of hemoglobin (revealing the subtle conformation changes underlying the cooperative interaction between the four Fe atoms) over the period 1964-19638, arid lysozyme (as the first true but nonmetallic enzyme) in 1965. Cell-free fixation of nitrogen was achieved in 1960, methods for isolating and purifjing cytochrome oxidase developed during 1958-1961, and the membrane-bound knobs of the mitochondria (see Sec. 2 ) shown to h a w ATPase activity in 1960, followed by development of the c~ci~~i osmot theory ic for using the proton gradient to drive ATP synthesis in 1961-1962. For introductory refcrenccs, see the relevant chapters. The year 2000 (Y2K) marks the cnd of roughly a half century of robust growth and an obvious tirne to take stock ~ S a c i i i ~ v e m ~and n t sto attempt to identify emerging themes and pattwns. gaps, and opportunities. Y2K can also be taken as the start of a period of unusu;illy rapid change in both the commercial and scientific worlds, high1ightf:d by the dramatic ~ ~ ~ v e ~ o p in n icIot.com ~ ~ i t s and in uiiraveling the human and ~ ~ . is bound to inffuother gmomes and the increasing importance o f b i o t e c h n o ~ oThis enre tht: way in which science is organized and Sundcd. The next stage will focus on the functions of proteins in association with sinall molecules in catalytic and other processes as well as their in~eracti~)ns with other proteins, DNA, and RNA; b ~ o i n o r g ~ n ~ c chemistry will thercifore have a growing role in postgeiiomic research and understanding the coordination chemistry of metalloproteins will be “compulsory” [I]. It is well established that the tertiary structure of proteins i s better conserved than their primary scquence, and it is now possible to group the approximately 700 luiown folds into a limited number of‘fhmilies; the final number may possibly be little more Lhan 1000 difYereiit protein folds 121. The aim of biologists will be to explore the evolution of structure and function and the emergence of complex biochemical pathways. Metalloproteins represent a partnership between thc main group or transition metal ion, which may perform a catalytic, structural, sensory, or regulatory role, and the protein, and it is d i f ~ c uto ~ toveremphasize the potential vantage^ of‘s t ~ ~ proteins with metal ions, especially those with spectroscopically “visible” transition metal ions, over purely organic proteins. Metalloproteins and enzymes offer considerable ~ ~ ( ~ v ~ in n tstudying a ~ e s problems of interest to both bio~heniis~s and co(?rdiIi~t~o~ chemists.

~ E T A L L ~ P ~ ~ TEE~I E~ R S ~: I NTHEMES G

1093

Where the active site contains a transition metal with distinctive physical properties (e.g., LV-visible spectra), changes to the active site inay be identified, simple kinetics obtained (though the quaint and long-winded phrase “pre-steady-state kinetics” is still often heard), rapid changes at the act.ive site separated from slower conformation changes in the protein by challen~4ngwith Iight or electrons fe.g.,from -f-radi;it.ion)and, in soine cases (especially Co corrinoids and Fo porphyrins), the reactions of the cofactor compared in the absence of protein. The study of inetalloproteins and enzyrnts has been particularly important in breaking down the historical hut artificial distinctions between substrates and allosteric effectors, and hcncc also bctwccn nonallostwic enzymes on the one hand and protcins (receptors, allosteric proteins, etc.1 and allosteric enzymes on the other. The Blz-depc?ndent mutaaes and ribonucleotide reductases provide an excellent example where the binding of substrate and allosteric effector, respectively, trigger analogous enzymatic rnwhanisms (Chapt.cr 13). Thc illogical nature of such distinctions is also well illustrated where one can focus on a single shared step, such as coordination of 02,NO, or CO to an FetII1 porphyrin; the same ligand inay occur as the substrate or product of an enzymic reaction and in functions as diverse as storage, transport, scavenging, sensing, and signal transduction. H2 sensors similar to the hydrogenase enzymes occur in bactwia f3l and providc a further example. Since sequences similar to the Fe-only liydrog(?nasc?s(but with unknown function) rare present in the human and other eukaryotic genorncs and ddetion has been shown to be lethal in yeast. 141, could it be that Tf2,lilrc NO [Fil, can also act as a messenger molecule? Reactions uitalyzed by metalloenzymes may entail accepting, stabilizing, transforming, and/or donating entities ranging from electrons and protons, H and 0 atoms, and hydride ions, through simple molecules and ions such as H2, N2, 02,CO, CO?, K202?Me+, and NO’, up to large organic substrates such as amides and phosphate esters; such reactions frequently require the availability of more than one oxidation state or coordination numtier. The selection of a particular. iiietd (with a given oxidation and spin state and ligand environment) to catalyze a particular enzymatic reaction reflect.s the com~)inationof mailability (no platinum group metals, probably no zero-valent inelal ions or phosphine ligands) and fitness for t.hc job. In Inany cases, metals can replace each other in the saine enzyme, e.g., Mo by W (Chapter 221 and the numerous examples ofsubstitution between Mg, Mn, Zn, and Co (Chapters 8 and 19). In other cases, difyerent enzymes with ~ f f e r e nmetais t can catalyze analogous reactions; ci: the many parallels between Cu and Fe (Chapter 17) mid the superoxide ( ~ i s r n ~ t ~that. s e s may possess Mn, Fe, Ni, or Cu as the redox-active site (Chapters 8, 11, 14, and 18).However, some metals do seem to be preeminently suibed to catalyze particular reactions and to stabilize particular ~nterinediates;cf. the formation of Mo=O and W=O bonds in the transfer and insertion of 0 atoms (Chapter 22) and of’Co-CH: J. M. Pratt, Inorg. Persp. Biol. Med., 2, 357 (1979). 26. A. D. Mesecar and T. Nowak, Biochemistry, 36, 6792 (1997). 27. M. Gerstein, A. M. Lesk, and C. Chothia, Biochemistry, 33, 6739 (1994). 28. D. W. Uny, Angew. Chem. Int. Ed., 32, 819 (1993). 29. (a) D. A. Baldwin, V. M. Campbell, L. A. Carleo, H. M. Marques, and J. M. Pratt, J. Am. Chem. SOC.,103, 186 (1981); (a) D. A. Baldwin, V. M. Campbell, H. M. Marques and J. M. Pratt, FEBS Lett., 167, 339 (1984); (c) D. A. Baldwin, €3. M. Marques, and J. M. Pratt, S. Afr. J. Chem., 39, 189 (1986). 30. (a) D. A. Baldwin, H. M. Marques and J. M. Pratt, FEBS Lett., 183, 309 (1985); (b) D. A. Baldwin, H. M. Marques, and J. M. Pratt, J , Inorg. Biochem., 30, 203 (1987). 31. A. Kannt, C. R. D. Lancaster, and H. Michel, J. Bioenerg. Biom,embr., 30, 81 (1998). 32. M. S. A. Hamza and J. M. Pratt, J. Chem. SOC.Dalton Trans., 1996, 3721. 33. (a) H. Sigel and L. E. Kapinos, Coord. Chem. Reu., 200-202, 563 (2000); (b) C. P. Da Costa, B. Song, F. Gregafi and H. Sigel, J. Chem. Sue. Dalton Trans., 2000, 899. 34. P. €3. Armentrout and R. Georgiadis, Polyhedron, 7, 1573 (1988).

37.

LL

IN

1117

60.

1; (b) Z. Li and H. Taube, 1. o l a t ~ u ~ u Proc. ~ y , Natl. Acad. Sci.

62. 63.

64. 65. 66. 7.

uller, Nature, 392, 37 (1998).

68. 69. 70.

71

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A A.

awam,ori, see Aspergillus chroococcum, see Azotobacter cycloclasbs, see Achromobacter denitrificans, see Alcaligen,es eutrophus, see Alcaligenes faecalis, see Alcaligenes ficum, see Aspergillus hydrophila, see Aeromonas nidulans, see Aspergillus niger, see Aspergillus nodosum, see Ascophyllum oryzae, see Aspergillus paspali, see hotobacter proteolytica, see Aeromonas sojae, see Aspergillus thaliana, see Arabidopsis uariabilis, see Anabaena vinelandii, see Amtobacter xylosoxidana, see Alcaligenes Aceruloplasminemia, 800, 801 Acetogenin, 165 Acetase guanidino-, 239

Acetate kinase, 209 Acetyl coenzyme A, 408, 411, 634, 635, 699 Acetyl-coenzymeA synthase, 634, 635, 1057 active site, 684, 685 mechanism, 697-699 nickel, 683-685 synthesis, 684 Acetylene, 168, 169 hydratase, 1029, 1069 N-Aceiylglucosamine, 134, 136 Achromobacter cycloclastes, 774, 775, 831, 832 Acid phosphatase, 173 manganese, 237, 238 purple, see Purple acid phasphatase tartrate-resistant, 547, 548, 900 Aconitase, 365, 372, 421, 422 active site, 390, 423 cluster conversion, 422-424 properties, 389 sequence motif, 390 structure, 389-391, 423 Actinomycetes, 671 Active sites (of) (see also Cluster and Coordination spheres)

1119

11120 [Active sites (001 acety~-coenzy~e A synthase, 684, 685 aconitase, 390, 423 ~ - a d ~ n o s y l ~ e ~ h i oqmthetase, nine 25 amhie oxidase, 718, 722, 748 aniiriopeptidaseP, 213 x-amylase, 30, 31 wginase, 218 ascorbate peroxidase, 28, 306, 308 bac~erioferrit,~n~ 320 bro~operoxiaase~ 166, E67, 170, 171, 173 earbanioyl phosphate synthetase, 23, 24 carbon monoxide dehydrogenase, 1058 catalase, 303, 304 catcchol oxidase, 719, 727 chloroperoxidase, 160, 161, 370, 171, 173. 312 CooA, 303 coppcr proteins, 718, 719 c o ~ n o i d 624 , Cu/Zn superoxide dismutase, 865, 866, 871 cytochrome c, 28, 293 cytochrome c oxidase, 719 cytochrome c peroxidase, 305, 308 qtochrome P450, 24, 313, 334 A~ desaturase, 540 d e a c e ~ o x y c e ~ h ~ o s ~Gosynthase, rin 494, 495 a i a ~ k y l g ~decarboxylase, y~l~~ 16 2,3-d~hyaroxybi~~enyl dioxygenase, 506, 511 d i ~ e t h y ~ ~ reductase, ~ ~ ~ o x1041, ~ ~ ePO42 did ~ e h y ~ a t ~19, s e20 , enolase, 219 Fe-only hydrogenase, 386, 387, 426-428 RXL, 301,302 L-fucose isomerase, 223 galactose oxidase, 718, 720, 748 heat-shock cognate protein, 21 heme oxygenaise, 321 hemocyan~,718, 726, 748 horseradish peroxidase, 308, 310 hydrogenase, 676, 678-680 oxylamine oxidoreductase, 317 bypoxanthine phosphoribosyltransferase, 211 inorganic pyrophosphatase, 216 intradiol dioxygenases, 509-511 isocitrate dehydrogenase, 80 ~ s o p e ~ i c i lsynthase, ~ ~ n - ~ 498, 499, 501 b a s e , 74-77

JECT INB [Active sit.es (001 lignin peroxidase, 307, 308 lipoxygenase, 232, 474, 475, 478 manganese catalase, 207 manganese peroxidase, 308, 309 methane monooxy~enase,528, 539, 733 methionine arninopeptidase, 29 methyl-coenzyme M reductase, 681-683, 694, 695 MutY, 398 m y e l o ~ e r o x ~3a12 ~~, naphthalene 1,2-dioxygenase,512, 513, 516 nitric oxide synthase, 315, 316 nitrilc hydratam, 545 nitrite reductase, 318, 33 9 nitrophorin, 300, 301 oxygen evolving complex, 235 peptidylglycine ric-hydroxylating monooxygenase, 718, 724 phexiylalanine hydroxvllase, 486, 488, 489, 491 plastocyanin, 824

purple acid ~ h ~ s p ~238, d ~548 e , pyruvate kinase, 18, 19 Rieske dioxygenase, 513, 514 sulfite oxidase, 1052 sulfite reductase, 426 superoxide dismutase, 203, 205-207 thrombin, 30 tryptophan synthase, 25, 26 Lyrosine hydroxylase, 488 tyrosine phenol-lyase, 28 urease, 689, 672-674 xylosc isomerase, 222 Adamalysin IT, I311 Adenosine kinase, 66, 70 Adenosine deaminaw zinc in, 885 Adenosine diphosphate, see MI Adenosine ~ o n o p h o s ~ h a tsee e, Adenosine triphosphate, see ATP Adenosyl~balamin,19, 232 5’-aeoxy-, 607, 609, 611, 612, 619, 632, 627,628,632,638-646,653,654,658 S-Adenosylmet~onirie as cofaactor, 609, 627, 632, 636, 658 synthetase, 13, 24, 25, 372, 410-413, 425 Adenosyl transferase cob(l)alamin, 633

1121 [Adenosyl transferasel manganese in, 212 Adenovirus, 963 Adenylyl cyclase, 78, 322 manganese in, 200, 239 Adenylate kinase, 72, 75, 209, 892 ADP, 16,23,25, 65-69, 217, 224,226, 238, 240, 401-403 niagnesium, 20-22, 74, 208, 394, 396 manganese, 225 Adrenalin, see Epinephrine nor-, see Norepinephrine Adrenodoxin structure, 379-381 Aeromonas h.ydrophila, 901 proteolytytica, 212, 584, 939, 940 Aeropyrum pernix, 203 Agglutinin, see Lcctin Aglycon, 611 Agmatinase, 239 Agmatine hydrolysis, 239 Agricara bisporus, 732 Agrobacteriurn, 987 Albumin, 186 Mn2+,200 parv-, 100 serum, see Serum albumin Alcaligenes sp., 826 denitrificuns, 765, 803, 804 eutrophus, 223, 675, 687, 688 faecalis, 804, 819, 825, 831, 832 Zylosoxidans, 831, 832 Alcohol oxidation, 735 veratryl, 331 Alcohol dehydrogenase, 826, 925 classes of, 936 Go2' in, 890 mechanistic studies, 935-937 NAD+-dependence,855,935, 936 NADP-dependent, 885 zinc site, 891 Aldehyde ferredoxin oxidoreductase magnesium in, 1061 structure, 1060-1062 tungsten in, 1060-1062, 1070 Aldehyde oxidasc, 1051, 1057, 1069, 1071 Aldehyde oxidoi-eductase,381, 431, 437 m o l y ~ d e ~ uin, m 1025, 1034, 1055-1057, 1067 ~ t ~ c t u r1055, e , 1056

Aldolase, 22 fuculose, 890 Algae (see also individual names) blue-green, 631 bromoperoxidase, 157, 170 brown, 165, 166, 631 cytochrome, 287 green, 165, 200, 287, 631 H2 production, 675 lipoxygcnase, 471 marine, 156, 165 oxygen evolving complex, 233 plastoc;yanin, 820, 822 red, 165, 166, 170, 631 urease, 671 vitamin BIZ, 631 Alkali metal ions, see individual elcincnts Alkaline earth ions, see individual elements Alkaline phosphatasc, 74, 213, 237, 240, 930 cadmium in, 938 cobalt in, 938 magnesium in, 237, 937, 938 manganese in, 237, 238 niechanistic studies, 937, 938 zinc in, 237, 911, 1015 znmg, 900 Alkane hydroxylase, 525 hydroxylation, 744 Alkaptonuria, 503 Allergy, 472 Aluminum(IZI), 65-68, 75, 183 in transferrin, 186, 593 ionic radius, 185 Alzheimer's disease, 139 hicyanin, 803, 804, 818 amino acid sequence, 819 loop topology, 821 mutant, 838 redox potential, 825, 837 structure, 824, 825 Amidophosphoribosyltransferase,see Glutamine ~hosphoribosylp~uphosphate amidotransferase Amine oxidase active sites, 718, 722, 748 bacterial, 721-723 catalysis, 736 cofactors, 721, 736 copper in, 202, 713, 721-723, 736-758

1122 [Amine oxidase] function, 746, 748 humail, 721 inhibitors, 723, 736 mechanism, 736 Mn2 I ,202 pea, 716, 722 pig, 722, 736 plant, 721 reaction, 721 structure, 722 Amine oxidation by cytochronie P450, 313 Amino acid hydroxylase, see Hydroxylase Aminoacyi-tRNA synthetase magnesium in, 226 manganese in, 226 fbninolenilinate, 279, 632 Aminomutase, 609, 620-4322, 651, 658 lysine 2 3 , 413, 425, 657, 1110 2-Amino-6-oxopurine, see Guanine Aminopeptidase (see also Peptidam and individual names), 242, 884 A, 930 active site, 213 amino acid sequence, 910 aspartate, 200, 237 clostridial, 213 cobalt in, 213, 900, 939 Auoride, 940 glutamyl, 212 lens, 212 leucine, 212, 939 magnesium in, 939 manganese in, 237,244 mechanistic studies, 939, 940 methionine, see Methionine aminopeptidave P, 213, 237 pig, 212 zinc in, 909, 910 ZnjMg, 900 Amino transferase, 736 Ammonia, 167, 168, 224, 234, 335, 391, 393 heme coordination, 301 AMP, 23, 65, 69 cyclic, see CAMP a-Amylase, 13, 30, 31, 98 active site, 30, 31 Amyotrophic lateral sclerosis, 900 Anabama sp., 419, 822 variabilis, 167, 819

Anemia, 572 microcytic, 584 hgiotensin-converting enzyme, 922 Animal (see also individual species), 10 aniine oxidase, 721 cytochrome, 293 ferritin, 573, 588 lectin, see Lectin lipoxygena.se, 232 manganese metabolism, 200 2-oxoglutarate-dependent oxidase, 491 purple acid phosphatase, 547 tyrosinase, 726 Annexin, 114-121 I, 117-139,121 11, I l l , 114, 116-119 111, 120, 121 V, 117, 121, 125 VI, 114, 116, 121 XI, 114 XII, 119 biological properties, 115 electrostatic interactions, 119 lanthanum derivatives, 117 membrane binding, 119, 120 rat, 118 structure, 118, 119 Antagonistic action, see Interdependencies Anthranilate hydroxylase, 485 Antibiotics (see also individual names), 236, 415, 491, 494 biosynthesis, see Riosynthesis cephalosporin, 494 f%lactarn, 492-494 Antibodies, 134 monoclonal, 906 polyclonal, 157 Anticoagulanl activity, 115 Antifungal activity, 165 Antiinfiammatory activity, 165, 578 Antimicrobial activity, 165, 310, 547, 578 Antioxidant, 715 defense, 324 Antiproliferative activity, 324 Antitumor agents (see also Dmgs and individual names), 185, 519 Antiviral activity, 165, 324 agents, 619 Aplysia myoglobin, 298, 328 Apoptosis inhibition, 976 Aquacobalamin, 611

1123 IAquacobalaminl reductase, 633 Arabidopsis thaliana, 200, 819, 828, 834, 911, 914, 1051 D-Arabinose, 222 Arachidonic acid, 126, 232, 471-473, 475 Arachis hypogaea, 283 Archaebacteria(l), 818, 988 ferritin, 576 halocyanin, 834 hyperthermophilic, 1029, 1060, 3 062 iron-sulfur protein, 394, 397, 399, 416 magnesium in, 62 methanogenic, 680, 681, 683, 684, 1057 ~olybdo~terin , 1025 thermophilic, 391 Archaeoglobu,s fulgidus, 215, 373 Argnase, 242 active site, 218 mammalian, 217 manganese in, 217,238, 239, 244 related enzymes, 238, 239 Arginine N-a-L-acetyl-, 495.497 biosynthesis, see Biosynthesis conversion, 239 deaminase, 239 hydrolysis, 217 kinase, 66 Aromatic compounds degradation, 478 Arsenic methylation, 636 Arthritis, 472, 503, 572 rheumatoid, 922, 924 Arthrobacter sp., 232 glvbiformis, 231, 717, 722 PI, 723 xylose isomerase, 221 Artlzromyces rarnosus, 283, 309 Arthropodal hemocyanin, 725, 726 Arylalkyl transferase manganese in, 212 Arylsulfatase magnesium in, 237, 911 prokaryotic, 911 zinc-binding motif, 911-914 Asbestos, GO Ascaris suurn, 231 Ascidia nigru, 155 Ascidians, see individual names Ascvphylluin nodasum, 158, 166, 170-174

Ascorbate, 28, 182, 492, 771 oxygen oxidoreductase, see Ascorbate oxidase peroxidase, see Ascorbate peroxidase Ascorbate oxidase, 764, 766, 767, 770, 778, 830, 831 azide inhibition, 791, 793 biological role, 771 copper binding siles, 781 cupredoxin folds, 770-772, 817 disulfide bridge, 772 evolution, 802-805 hydrogen peroxide addition, 789, 790 hydroxyl bridge, 791 loop topology, 821 mechanism of oxygen reduction, 788-792 plant, 771 structure, 771 substrate binding, 787, 788, 796 trinuclear copper center, 785-791, 793, 841 Ascorbate peroxidase, 306, 331 active site, 28, 306, 308 bacterial, 306 calcium in, 307 compound I, 28, 29 cupredoxin fold, 770-772 pea (cytosolic), 13, 28, 283, 306 potassium in, 307 structure, 308 hparaginyl-tRNA synthetase, 492 Aspartate aminopeptidase manganese in, 200, 237 Aspartate carbamoyltransferase, 891, 894 Aspartyl-tRNA synthetase, 226 Aspergillus awamori, 586 ficum, 174 nidulans, 586, 716, 1029, 1069 niger, 200 oryzae, 773 sojae, 230 Assimilation of nitrate, 391 nitrogen, 391 sulfur, 391 Astacin, 892 Co(T1) in, 890 Cu(I1) in, 890 inhibitor, 890 Zn(II) in, 910, 911, 930 Asthma. 472

1124 Atherosclerosis, 198, 472 ATP, 10, 16, 19, 24, 44, 65, 66, 69, 72, 75-78, 199, 225,226, 240, 289,627, 631, 635, 656 -binding protein, 237 bound to kinase, 74, 75 ca2+,61 Co2+, 61 conversion, 208, 217, 224, 239 Cr3’, 186 formation, 634, 675 hydrolysis, 20 hf2’, 18, 20, 23, 61-63, 168, 186, 208, 226,393, 394, 396 Mn” , 61, 210, 226 synthesis, 681 ATPase, 20-22, 66, 67, 200, 1112 Ca2+,10,15, 199, 1098 CCC2, 715 manganese in, 67, 200 Menkes’ copper-transporting, see Menkes’ copper-transporting ATPase M$+, 61,63, 72, 1095, 1098, 1099, 1101 Na’,K+, 10, 15, 199, 1098 P-type, 63 Auracyanin, 818, 833, 834 amino acid sequence, 819, 834 loop topology, 821 Avidin, 646 Azide in ascorbate oxidase, 791, 793 carboxypeptidase A, 919, 920 ceruloplasmin, 791, 793, 794 cytochrome c oxidase, 729 A’ desaturase, 531 extx-adiol dioxygenase, 509 galactose oxidase, 734 hemerythria, 517 ribonucleotide reductase R2 protein, 525 superoxide disrnutase, 203-205, 699, 866 Azotobacter chroococcum, 167 paspali, 167 uinelandii, 167, 287, 361, 382-384, 394-396,424, 1030,1034-1037 Azurin, 317, 765, 766,818, 825,840 amino acid sequence, 819 cupredoxin fold, 767-769 evolution, 802-804 loop topology, 821 metal site, 820 mutants, 838, 839

SUBJECT INDEX [Azurin] pseudo-, see Pseudoazurin structure, 826

B. ammoniagenes, see Brevibacterium arnyloliquefaciens,see Bacillus breuis, see Bacillus caldouelox, see Bacillus cereus, see Bacillus fragilis, see Racterioides fascum, see Breuibacterium gingiualiu, see Bacterioides lichenifomis, see Bacillus macerans, see Bacillus pertussis, see Bordatella schlegelii, see Bacillus sphaericus, see Bacillus stearothernzophilus, see Bacillus subtilis, see Bacillus thermoproteolyticus, see Bacillus thetaiotamicron, see Bacterioides Bacillus sp., 240 amyloliquefaciens, 909, 930 brevis, 231 caldouelox, 217 cereus, 73, 901, 909, 912 licheniformis, 13, 30, 31, 415 macercms, 231 schlegelii, 382, 383 sphaericus, 412 stearotherrnophilus, 204,. 209, 221, 231, 240, 892, 909,930,971 subtilis, 215, 232, 233, 399-401, 415, 892,909, 911, 930 TB90, 687 thermoproteolyticus, 385, 909 Bacteria(1) (see also individual names) acetogenic, 631, 634, 636, 671, 675, 684, 1057 a g o - , 987 aldolase, 22 m i n e oxidase, 721-723 ammonia-oxidizing,733 anaerobic, 408, 607, 608, 631, 636, 654, 675, 684 archae-, see Archaebacteria blue copper protein, 816 bromoperoxidase, 1.70

S U B ~ E INDEX ~T [Bacteria(l)l catalase, 304 chemo~thotrophic,391, 1057 cyano-, see Cyanobackria cytochrome, 313 dehydratase, 19 denitrifying, 317, 387, 733, 766, 825 eu-, see Eubacteria fermentative, 680 ferredoxin reductase, 300 ferritin, 576 flavohemoprotein, 297 gram-negative, 240, 279, 318, 465, 581, 825, 826, 890 grrun-positive, 279, 415 heme oxygenase, 320 hyperlhermophilic, 675 iron metabolism, 464, 465 isomerase, 220 kinase, 16 Ilnallgas, 675 lactic acid, 197 lectin, 133 lyase, 26 magnetostatic, 465 manganese in, 199, 200 marine, 835 methanogenic, 631, 634, 636, 671, 675, 681 methanotrophic, 525, 733 methylot~ophic,824-826 myco-, see Mycobacteria nitrate-reducing, 675 nitrile hydratase, 545 nitrogen-furng, 287, 394 oxotransferase, 1039 pathogenic, 579, 581, 901, 917 photosynthetic, 72, 302, 303, 381, 387, 671, 675, 684,820 phototrophic, 387, 391, 833, 1039, 1057 plastocyanin, 820 potassium channel, 45-47 purple, 291, 387, 1039 purple acid phosphatase, 547 respiratory electron transfer, 387 RNase, 70 soil, 502, 511, 835 sdlfate-reducing, 373, 391, 402, 408, 541, 631, 634, 636, 675, 1048, 1055, 1057 thermohaloph~ic,387 thermophilic, 381, 383 toxins, 322

1125 [Racteria(l)J tyrosinase, 726 ureolytic, 688 vitamin B12, 631 Bacterioferritin (see also Cytochromes b), 285, 320, 543, 544, 576, 577, 588 active site, 320 axid ligation, 277 function, 277 heme type, 277 iron oxidation state, 277 iron spin state, 277 list, 574 structure, 320, 574, 576 Bactera'oides fragilis, 203, 901, 902, 912, 914, 917 gzngiualis, 203 thetaiotamicron, 203 Bacteriophage, 358 T4,411 Bacteriorhodopsin, 1095 Baculovirus, 976 expression system, 325 Barium ion, 125 Bean soy, see Soybean Jack, 227 kidney, see Phosphatases Benzendiol oxygen oxidoreductase, see Laccase Beryllium, 75 Bilayer lipid, 40 Bilirubin, 321, 834 oxidase, 834 transport, 859 Biliverdin, 276, 321, 335, 834 structure, 336 Biogenic amines (see also individual names), 794 oxidation, 798, 799 Biomethylation mercury, 636 metalloids, 636 metals, 636 Biomineralization (see also ~ i n e r ~ i z a t i o n ) ferritin, 465 Bioremediation, 502 by halogcnase, 609, 636 by methane monooxygenase, 525 of environmental pollutants, 478, 479 of groundwater, 636

1126 [Bioremediation] of nitrile-containing wastes, 544 of soil, 636 Biosynthesis (see also Synthesis) antibiotics, 491 arginine, 225 biotin, 412 carbon monoxide dehydrogenase, 688 cephalosporins, 492 clavulanic acid, 492 corrin, 610 cysteine, 392 fatty acids, 530 FeMoco, 394 grisans, 835 heme, 279 histidine, 73 iron-sulfur protein, 445 leucine, 201 methane, see Methanogenesis molybdopterin, 1028, 1029, 1051 nucleotides, 23 photosynthet~cpigments, 503, 506 purine nucleotides, 399, 401 pyrimidine, 225 sialic acid, 207 L-tryptophan, 25 vitamin Biz, 631, 632 Biotin, 646 biosynthesis, 412 sulfoxide reductase, 1069 Biotiii synthase cluster conversion, 425 sequence motif, 41 2 2,2'-Bipyridine, 424 Bism~th(111) in metallothionein, 1005 Bison heart, 23 1 Black smokers, 660 life forms around, 1094, 1097 Iood coagulation factors, see Coa,dation factors Blood-brain barrier manganese transfer, 200, 243 Blue copper oxidase (see also individual names), 821, 834, 835 copper types, 766 function, 766 list of, 766 sources, 766 Blue copper proleins (see also Type 1 copper), 816, 833, 834

[Blue copper proteins] bacterial, 816 coordination sphere of copper, 835 copper sites, 818, 820 disulfide bridges, 819, 828 electron transfer mechanisms, 840, 841 families, 818 list of, 837 loop topologies, 821 mutagenesis studies, 838, 839 plant, 816 redox potentials, 837, 839, 840 ruthenium-labeled, 840 sequence alignments, 819 spectroscopic studies, 836-838 Bohr effect, 289 Bombyxin-11, 1008 Bone manganese in, 200 mineralization, 547 Bordatella pertussis, 203 Bos taurus carboxypeptidase, 213 insulin, 1008 Botrocetin snake venom, 138 Bovine amine oxidase, 722, 736 calmodulin, 103, 112 heart, 378, 717, 719, 728, 729 heat-shock cognate protein, 20 lactoferrin, see Lactoferrin lens, 212 liver, 187, 304 mannose 6-phosphate receptor, 230 plasma, 723 Bradyrhiznbiurn japonzcum, 284, 301, 337, 687 Brain human, 201 manganese in, 200 mice, 201 rat, 212, 216, 237 Brevibacterium amrnorziagenes, 199 fuscurn, 231 Bromide in themolysin, 940 oxidation, 155, 156, 372 Bromoperoxidase active site, 166, 167, 170, 171, 173 bacterial, 170

1127

SUBJECT INDEX 1 Bromoperoxidase] catalytic cycle, 172 cobalt, 1110 Fe-heme-containing, 165 mechanism, 172, 173 occurrence, 165 structure, 166 vanadium, 156-158, 165-167, 170, 173, 174 BrGnsted plot p21'OS protein, 79 Bruton's tyrosine kinase (see also Zinc finger domains), 968, 969, 975 Bullfrog (see also Rana catesbeiana) ferritin, 574, 575, 577, 585, 588, 589 Butanase diguanidino-, 239 Butyrase guanidino-, 239

c C. acidi-urici, see Clostridium albicans, see Candida anzmoniagenes, see Corynebacterium be@erinckii, see Clostridium cochlearium, see Clostridium diphtheriae, see Corynebacterium elegans, see Caenorhabditis glutamicum, see Corynebacterium. pasteurianum, see Clostridiurn pilulifera, see Corallina, subterrninale, see Clostrzdiurri thermoaceticum, see Clostridium Cadmium(T1) (in), 215 '"Cd, 1007 calbincbn D9k, 110, 111 carboxypeptidase A, 934 diphtheria toxin repressor, 229 interdependency with other metal ions, see Interdependencies isocitrate dehydrogenase, 80 metallothionein, 1004-1007, 1014, 1015 phosphatase, 938 plastocyanin, 822 serum albumin, 862 transport protein, 584 Caenorhabditis elegans, 964, 983, 987 Galbindins Cd2+, 110, 111

[Galbindins]

108-312, 114 139 Mn2+, 110, 111 Calcineurin, 214, 215, 900 Calcium(I1) (in), 28, 30, 31, 40, 53, 62, 196, 215 affinity for, 96, 101, 108, 114, 115, 122, 126, 128, 129 as probe, 71 ascorbate peroxidase, 307 ATP, 61 ATPase, see A'1'Pase binding constants, 95 binding motifs, 97, 101, 106, 117, 128, 138 channel, see Ion channels chemistry, 95 coordination, 100, 134 coordination number, 127 cytochrome c reductase, 318 desulfoferrodoxin, 374, 376 dissociation constants, 115,122, 126, 130, 131, 133 enzymes, 93-141 formaldehyde ferredoxin oxidoreductase, 1063 guanylyl cyclase, 322 hemocyanin, 726 homeostasis, 95-97, 110 insulin, 1002 interdependency with other meld ions, see lnterdependencies isocitrate dehydrogenase, 80 lipoxygenase, 473 metabolism, see Metabolism myeloperoxidase, 3 10 nitrite reductase, 318 oxidases, 70 oxygen evolving complex, 233-235 peroxidase, 307, 338 psoriasin, 111, 116 pump, 200 release, 97 sulfatase, 237 Calcium-binding proteins, 97-99 binding to target molecules, 102, 110, 114, 123, 125, 131, 133, 134, 138 chicken, 98, 103 conformational changes, 96, 104, 110, 112-114, 121, 122, 125, 127, 129, 137 domain-domain interaction, 106, 137 DBk,

b8k,

1I2 [Calcium-binding proteins] electrostatic interactions, 106 function, 102, 110, 113-115, 122, 125, 133, 138 human, 98, 103, 112, 114, 117 internet resourceB, 97-100, 141 membrane binding, 115, 117, 119, 121, 122, 125-127 mutagenesis, see Mutagenesis PDB codes, 103, 117 S100, sec Sl00 proteins structural classification, 98, 99 Calcium-release channels voltage-operated, 97 Calcyclin, 109, 114, 115 Ca~darioin,ycesfurnago, 156, 207, 283, 312 Caldariomycin, 207, 312 Caldcsmon, 110, 114 Calgranulins, 115 Callinectes sapidus, 1003, 1006 Calmodulin, 53, 100, 102-109, 112, 140 binding to nitric oxide synthase, 315, 334 Ce3', 103 eukaryotic, 102 knock-out mutation, 109 structures, 102-105, 108 Calorimctric studies, 126 calmodulin, 107 c ~64, 239, ~ 302, -dependent protein kinase, 209 receptor protein, see Catabolite gene activator protein Camphor, 24 cytochrorne P450, 13, 24, 25, 313-315 Canavaliu ensiformis (see also Jack bean), 227 brasiliensis, 228 Cundida ulbicans, 891 Captopril, 909, 922 Carbamoyl phosphate synthetase, 208 active site, 23, 24 manganese, 226, 226, 242 potassium, 13, 23, 24 Ca~bamoyltr~sfcrase aspartate, 891, 894 Carbohydrate binding protein, see Lectin mctabolism, see Metabolism r e ~ o ~ i t i o134-139 n, Carbon cycle, 1025 C ~ b o n i canhydrase El, 927

[Carbonic anhydrasej cobalt in, 927, 928 copper in, 928 mechanism, 932, 933 properties, 925-928 scaffolding of zinc sites, 924-929 sources, 926 Carbonium ion, 619, 638, 648, 649, 653, 655 Carbon monoxide as inhibitor, 729, 731 as messenger, 321 as neurotran~m~tter, 302 binding to Fe-only hydrogenase, 426-428 copper@)in, 1107 chromium(111) in, 1108 dehyrogenase, see Carbon monoxide dehydrogenase formation by heme oxygenase, 335 gold(1) in, 1107 heme coordination, 274, 285, 288, 328 iridiurn(III), in, 1108 osmium(I1) in, 1108 oxidation system, 684 palladium(I1)in, 1107 platinum(I1) in, 1005 rhodium(II1) in, 1108 silver(1) in, 1107 Carbon monoxide dehydrogenase, 402, 634, 635,671 active site, 1058 biosynthesis, 688 carbon monoxide binding, 697 catalytic mechanism, 697-699 molybdenum, 1057-1059, 1071 nickel, 683-685, 688, 1057 redox potential, 697 selenide, 1058, 1071 structure, 1057-1059 Carboxykinasc manganese, 217-219 phosphoenolpy~~vate, see Phosphoenolpy~vatekinase Carboxylase, 69 manganese, 210 phenol, 230 phosphoenolpy~vat~, 210 pyruvate, 240 C ~ b o x y p e p t ~ d (see a s ~akso Peptidase) cadmium in, 934 manganese in, 213

SUBJ~CTINDEX Carboxypeptidase A, 890, 925, 930, 1015 azide, 919, 920 cadmium in, 934 cobalt in, 919, 923, 934 inhibition, 917-920, 922-924 lead in, 918-920 mechanism, 933-935 mutant, 890 properties, 929 sources, 933 Carcinogens, 182 Carotenoids (see also individual names), 233 Casein, 930 Caspase, 976 Catabolite gene activator protein, 414 Catalase, 541, 685 active site, 303, 304 axial ligation, 276 bacterial, 304 bovine liver, 304 catalytic cycle, see Catalytic cycles cyanide in, 206 fluoride in, 206 function, 276 fungal, 304 heme, 206, 276, 303, 304 heme type, 276 iron oxidation state, 276 iron spin state, 276 4 k e activity, 207 manganese, see Manganese catalase PDB codes, 283 proximal ligand, 337 structure, 304 structure-function relationships, 330 Catalytic cycle of catalase, 330 chloroperoxidase, 172, 332 cytochrome P450, 333 heme oxygenase, 335, 336 hydroxylamine oxidoreductase, 334 nitric oxide synthase, 333 nitrite reductase, 334, 335 Catalytic site, see Active site catalytic zinc sites, 884-891, 900-902, 915 list of, 886-889, 912 mechanistic studies, 931-937 Catechol(ates), 488, 489 4-chloro-, 505 4-methyl-, 505 oxidation, 743

1129 Catechol dioxygenase 1,2-, 479, 483 2,3-, 502, 505 chloro-, 231 Catechol oxidase, 717, 726, 727, 732 active site, 719, 727 catalysis, 741 dioxygen binding, 748, 749 plant, 726 reaction, 726, 727 CD, see Circular dichroism Cell calcium in, 95, 96, 100, 200 -cell interaction, 134 Chinese hamster ovary, 185 division, 102 endothelial, 137 growth, 115, 125 manganese in, 200 proliferation, 3 15, 125 recognition, 133 secretion, 115, 125 CeUulose, 307 Cephalosporins, 494 biosynthesis, see Biosynthesis Ceramide, 73 Cerium(l1l) calmoddin, 103 Cemloplasmin, 585, 764, 766, 767, 771, 780, 863 analogue, 715 and hemosiderosis, 800, 801 azide inhibition, 791, 793, 794 chaperone-type role, 800 CocII), 795, 796 copper binding sites, 781 cupredoxin fold, 770, 783, 799, 817 cyanide in, 791 disulfide bridge, 778 evolution, 800-805 Fe(II), 794-796 Fe(ITI), 794-796 ferroxidase activity, 776, 791, 794-797, 800, 801, 831 human, 770, 771, 776-780 loop topology, 821 manganese, 200 physiological function, 776, 805 redox potential, 831 structure, 776-779 substrate binding, 797-800 trinuclear copper center, 787

1130 Cesium ion, 25-28, 41 cGMP, 322 Chagas' disease, 300 Channels calcium, see Ion channel potassium, see Ion channel voltage-gated K", 906 Chaperones copper, see Copper chaperones insertase, 168 iron, 443 metallo-, 5 moleculm, 20 nickel, 687, 688 Chelation therapy removal of zinc, 922-924 Chickon calcium-binding protein, 98, 103 ovotramferrin, 578-580, 583 sulfite oxidase, 1051-1053, 1070 Chlamydomoas sp., 200, 631 Chlorella, 212 fisca, 380 Chloride in thermolysin, 940 oxidation, 155, 156, 172 oxygen evolving complex, 233,235 Chlorination by chloroperoxidase, 332 Chlorobium tepidurn, 373 uibrioforme, 72 Chloroctechof 1,2-dioxygenase, 231 Chlorocruorin, 297 2-Chloro-5,5-dimethyl-l,3-dimedone, see Monochlorodimedone Ghloroflexus aurantiacns, 819, 833, 834 Chloroheme, 273,274, 297 Chloromuconate eyeloisomerase manganese, 223 Cliloroperoxidase active site, 160, 161, 170, 171, 173, 312 PO-, 170 axial ligation, 276 catalytic cycle, 172, 332 coordination sphere, 159-165 distal site, 313 electrostatic interactions, 161 expression, 324 Fe-heme-containing, 156 function, 276 fun@, 156-162, 169

S U ~ J ~ CINDEX T [Chloroperoxida~el heme type, 276 iron oxidation state,276 iron spin state, 276 manganese, 207, 332 mechanism, 172, 173 mutants, 161-165, 170, 173 PDB codes, 283 proximal ligand, 313, 338 structure, 159-165, 311, 312 structure-function relationships, 332 tungstate-substituted, 161, 162 vanadium, 156-165, 169-173 Chlorophyll, 413 p680, 233 Chloroplasts, 233, 279 Choline PhoSphOryl-, 73 Chromatin zinc finger binding, 988 Chrontatium vI'rtosum,,382, 387, 388, 419, 420,437, 679 Chromium (oxidation state undefined) (in) ATP, 186 proteins, 181-188 oxidation states, 181, 182 rat, 182 toxicity, 181, 182 Chromium(II), 418 Chromium(II1) (in), 418 absorption, 184 ATP, 186 bioavailability, 185 carbon monoxide binding, 1108 collagen, 186 coordination sphere, 183 formation of carbon bonds, 613, 614, 655 glycylglycine, 186 hexahydrate, 182, 183 insulin, 186,187 low-moleculw weight chromium-binding substance, 186, 187 prolylglycine, 186 porphyrin complexes, 183 properties, 1104, 1110 Schiff base complexes, 183 transferrin, 186, 187 tris-picolinate, 184, 185 Chromium(VI) (or chromate), 181 lead, see Lead

SUBJECT INDEX Chromobacterium violaceurn, 485 Chymotrypsin, 933 Circular dichroism (studies 00 blue copper proteins, 836 clavaminate synthase, 497 galactose oxidase, 720 lactoferrin, 582 magnetic, see Magnetic circular dichroism rnethanc monooxygenase, 526 phenylalanine hydroxylase, 489, 491 Cirrhosis, 572 Cisplatin, 185 Citrate (or citric acid), 65, 183 cycle, 621 titanium, 693 CitruJline, 239 Clavaminate synthase, 492, 493 structure, 495-497 Clavulanic acid biosynthesis, 492 Clostridium acidi-urici, 382-382 beijerinckii, 885 cochlearium, 623 pasteurianum, 373, 381, 382, 385, 386, 394, 410, 416,424, 426, 427, 1030, 1036 subterminale, 413 thermoaceticum, 402, 631, 684, 697 Cluster conversion, 422-425 cubane, 168 FetIV)Fe(IV), 527,589 FeMoS, 1113 Fe3MoS3,1030 Fe3MoS4, 1030 Fe3WS4, 1031 Fe4, 402-405 Fe7MoSg (see also FeMoco), 1025, 1030, 1101 Fe20M06S30, 1030 H, see Iron-sulfur cluster hybrid, 402-405 iron-sulfur, see Iron-sulftir cluster “meatball”, 402-405 MgZt,Mn2’-, 76 M8+,Zn2’, 71 NiFe, 675, 676, 678, 692 NiFeS, 634 NiFeSe, 675 P, see Iron-sulfur cluster trhuclear copper, 782-790, 793

1131 CMP, 74 Coagulation cascade, 30 Coagulation factors V, 765, 766, 779 VII, 129, 131, 132 VTII, 765-767, 770, 779, 780 lX, 128, 779 X, 126, 128, 779 configuration, 779 disulfide bridges, 780 evolution, 800-805 model, 780 Cobalamin (see also Gominoids and Vitamin BIZ),605, 611 adenosyl-, see Adenosyl cobalamin alkyl-, 622 aqua-, 611, 633 cob(I)alamin, 611, 633, 637, 649 cob(II)alamin, 611,628,633, 642-644 hydroxo-, 611 methyl-, see Methylcobalamin trans-, 622, 633 Cobaloxime, 616, 637, 649, 650 alkyl-, 640, 647 Cobalt (different oxidation states) (in) carbonic anhydrase, 927, 928 carboxypeptidaseA, 919, 923,934 non-corrinoid nitrile hydratase, 544 phosphatase, 938 porphyrin, 610, 616, 656 transport protein, 584 vitamin Biz, see Vitamjn R12 CobalWI) (in), 29, 197, 242 alcohol dehydrogemse, 890 aminopeptidase, 213, 900, 939 astacin, 890 m p , 61 bromoperoxidase, I110 ceruloplasmin, 795, 796 diphtheria toxin repressor, 229 interdependency with other metal ions, see Interdependencies metallothionein, 1005, 1014 phosphatase, 214 prolidase, 1110 ribonucleotide reductase, 524 transferrin, 593 Cobalt(lI1) carbon bond formation, 655 hexammine, 80 in transferrin, 593 Cobaltochelatase, 632

1132 Cobester (see also Corrinoids), 611, 618 Cobinamide (see also Corrinoids and Vitamin BIZ),605, 611, 640 dicyano-, 616 methyl-, 612 CocaLalytic zinc sites, 884, 893, 900, 901, 912-914, 916, 920 heteronuclear, 900 list of, 896-899 mechanistic studies, 937-940 Cod insulin, 1013 Cofactor F430, 634 nickel, 671, 682, 683, 693-697 structure, 683 Cofiactors ~-adenosyImethionine,see S-Adenosylmethionine amine oxidase, 721, 736 Co-cominoid, 684 corphin, 683 FMN, 401 iron-molybdenum, see FeMoco molybdopterin, see Molybdopterin Collagen, 492, 730, 862 Cr(lll), 186 triple-helix repeat, 97, 133, 134, 137 Collagenase, 930 fibroblast, 910 Collectins, 133 Compound Z, 1028 +structure,1029 Concanav~linA manganese bond distances, 229 manganese, 227 Mii,Ca-, 228 Coo& 284 active site, 303 axial ligation, 276 binding of signalling molecule, 329 function, 276, 285 heme type, 276 iron oxidation state, 276 iron spin state, 276 proximal ligand, 337 secondary structure, 336 structure, 302, 303 Coordination spheres (or geometry) of (see also Active sites) cadmium, 1005 calcium, 124, 125 chromiumMI), 183

[Coordination spheres1 copper, 712, 718, 719, 735-747, 782-786, 789, 790, 793, 797, 820, 829, 833, 964, 865,872, 873 iron, 205, 474-476, 481, 487, 495, 504, 505, 507, 508, 513, 522, 528, 532, 534-536, 591, 678, 680, 747 magnesium, 74-77, 81, 210 manganese, 75, 76, 203, 205, 240, 241 molybdenum, 1027, 1028, 1046, 1047, 1050, 1053, 1056, 1059, 1070 nickel, 677, 680, 683, 685, 689, 696696 potassium, 11, 16, 17, 19, 21, 22, 24-27, 29 samarium, 593 sodium, 11, 16, 17, 21, 22, 25, 30, 31 tungsten, 1070 vanadium, 159, 162, 163,167 zinc, 929, 932, 974 Copper (different oxidation states) (in) bioinorganic role, 711-713 blue copper proteins, see Blue copper proteins and Type 1copper blue oxidase, 821, 834, 835 carbonic anhydrase, 928 ceruloplasmin, see Ceidoplasmin chaperone, see Copper chaperone classification of metal sites, 815 coordination chemistry, 710 CUA(see also Type 1 copper), 712, 715, 724, 731, 733, 738, 815, 816, 832, 833, 835, 840, 841 CUB(see also Type 2 copper), 713, 724, 728, 731, 738, 841 C U ~733, , 835 metallothionein, 1004 nitrite reductase, see Nitrite reductaso protein, see Copper proteins purple copper site, see CuA redox reactions, 711, 814 sites, 711 spectroscopic classification, 765, 767 transfer, 715 type 1, see Type 1copper type 2, see Type 2 copper type 3, see Type 3 copper Copper@) carbon monoxide coordination, 1107 in plastocyanin, 822, 824 Copper(l1) (in) affinity for sulfur, 240

1133 [Copper(II)I astacin, 890 blue oxidase, 70, 202, 821, 834, 835 Copper chaperoncs, 443, 714-716, 750, 814, 858, 873, 874 ATXl, 715, 716, 874 CCS, 715, 716, 874 COX17, 715, 874 LYS~,a74 Copper metallothionein, 858, 860, 861 &(I)-substituted, 872 structure, 860, 861, 872 Copper proteins (see also individual namcs) activation of dioxygen, 709-751 active site, 718, 719 blue, see Blue copper protein and ‘I’ype 1 copper list of, 716 PUB codes, 716, 717, 823 redox potentials, 326 reduction of inorganic molecules, 709-751 transport, 584 transport of dioxygen, 709-751 various functions, 857-874 Copper-zinc superoxide dismutase, 202, 685, 699, 767, 900, 963, 975 active site, 865, 866, 871 dimeric nature, 871 list of, 867-871 mechanism of action, 865 metal-binding sites, 863-865 PDB codes, 867-870 sources, 867471 structure, 860, 864-871 Coprinus cinereus, 309, 767, 773, 774, 786, 820, 830 macrorhizus, 309 Corallina officinalis, 158 pilulifera, 166, 170, 174 Corrinoids (see also individual names), 605, 610, 611 absorption, 632, 633 active site, 624 amide and nucleotide side chains, 618 as cofactor, 684 biosynthesis, 610 Co-6 bond, 607-613 conjugated chain, 616 coordination chemistry, 607, 611-613

LCorrinoidsl cyanide in, 612-614, 617 dielectric constant, 63 8 equilibrium constants, 640, 641 metal-free, 632 methane oxidation, 637 mutase, 605 nomenclature, 610, 611 oxidation states, 611, 612 PDB codes, 622 photolysis, 617, 647, 649, 650 properties, 615-619 protein-free, 615, 618, 626-628, 639-641, 647, 649, 650,652-655, 657 rate constants, 640, 641, 646, 653 redox potentials, 609, 613 stability constants, 633, 646 steric effects, 617, 618 substratc binding, 641-643 transport, 632, 633 Corynebactcriurn amrnoniagenes, 232, 233 diphtheriae, 229 glutarmicum,232 Crab blue, 1003, 1004 horseshoe, 725, 726 metallothionein, 1003 Creatinase, 213 Crustacean (see also individual names) hemoglobin, 299 Cryoelectron crystallography, 233 Crystal structure studies of (see also X-ray crystal structure studies) aldchydc oxidoreductasc, 1055, 1056 amidotransferase, 401 ascorbate oxidase, 771, 772 bacterioferrilin, 576 calcium-bindingproteins, 102, 110, 112, 119-122,127, 132, 134, 137, 138 carbon monoxidc dehydrogenase, 1057, 1058 catechol 1,2-dioxygenase7483 catechol oxidase, 727, 741 chloroperoxidase, 207 clavaminatc synthase, 495 copper amine oxidase, 722 copper proteins, 716-719 cycloisomerase, 223 cytochrome c oxidase, 728 dimethylsulfoxide redwtase, 1039-1042 dioxygenase, 231

CT INDEX

1134

I Crystal structure studies ofl diphtheria toxin repressor, 229 endonuclease 111, 397 enolase, 218 Fe-only hydrogenase, 385,426,427 forredoxin, 383,421,422 hmarate reductase, 406 hernocyanin, 726,748 isocitrate dehydrogenase, 80 lrinase, 210 laccase, 767,773,774, 786 lactoferrin, 579-581, 591-593 lipoxygenase, 477 magnesium-activated enzymes, 64-70,79 mandelate racemase, 224 manganese peroxidase, 309 metapyrocatechase, 505 methyl-coenzyme M reductase, 681,682,

694 monophosphatase, 215 mutase, 240 naphthalene 1,2-dioxygenase,514,515 rNiFe1-hydrogenase, 678,679 nitrogenase iron protein, 395,396 nitrogenases, 1035-1037 pept~dylg~ycin~ x-hydroxylating monooxygenase, 724 phenyldanine hydrox_Ylase, 487 potassium channel, 45,46 pyrophosphatase, 214 pyruvate synthase, 408410 ribonucleotide reductase, 411,534 rubrerythrin, 541 sulfite reductase, 1051-1054 superoxide dismutase, 205 synthetase, 227 transferrin, 579-581,584,591 trimethylamine dehydrogenase, 402 tyrosine lzydroxylase, 486,487 xylose isomerase, 220,221 Cubane cluster, see Cluster and Iron-sulfur

cluster Cucumber ascorbate oxidase, 771 blue basic protein, 803,819,828 plantacyanin, 820,828 stellacyanin, 819,820,829

Cucumis sattvus, 771 Cucurbita medullosa, 771 Cupredoxin fold (see also Blue copper protein), 765,767,780,816,817,860

[Cupredoxin fold] ascorbate oxidase, 770-772,817 azurin, 767-769 ceruloplasmin, 770,783,799,817 cytochrome c oxidase, 767,803,832 laccase, 773,782,817,830 multi-copper oxidase, 765,767,780,

817 nitrite reductase, 775,817 plasticyanin, 767-769,782,816 Curvularia inaequalis, 157-162,169-173 Cyanide (in) binding constant, 612 catalase, 206 ceruloplasmin, 791 corrinoid, 612-614,617 Fe-only hydrogenase, 426,427 heme coordination, 274,288,301 intradiol dioxygenase, 483 manganese catalase, 206 superoxide dismutase, 699 Cyanobacteria (see also individual names),

167,394 ascorbate peroxidase, 306 cytochromes, 287 heme oxygenase, 320 lipoxygenase, 471 oxygen evolving complex, 235 plastocyanin, 822,824 Gyanocobalamin, see Vitamin BI2 Cyclase adenylyl, see Adenylyl cyclase cytidylate, 239 guanylyl, see Guanylyl cyclase magnesium, 239 manganese, 239 sabinene hydrab, 239 C:yclohydrolase ~1-(5'-phosphoribosy1)adenosine~5'monophosphate, 73 Cycloisomerase chloromuconate, 223,224 dichloromuconate, 223 manganese, 223,224 muconate, see Muconate cycloisomerase Cyclooxygenase PDR codes, 283 Cystathionine P-synthase, 323 axial ligation, 278 function, 278 heme type, 278

1135 [Cystathionine B-synthasel iron oxidation state, 278 iron spin state, 278 Cysteine (and residues) biosynthesis, 397 homo-, 323 5'-selanyl-, 1058 seleno-, see Sclenocysteine Cytidine deaminase, 414 amino acid sequences, 912 zinc in, 911 Cytidine 5'-monophosphate, see CMP Cytidylate cyclase, 239 Cytochrome (see also individual names) axial ligation, 274, 275 electron transfer reactions, 326-328 fiavo-, see Flavocytochrome function, 275, 285 heme type, 275,286 invertebrate, 313 iron oxidation state, 275 iron spin state, 275 mitochondrial, 286 nomenclature, 286 oxidase (see also Cytochrome c oxidase), 715, 727, 733 prokaryotic, 287 proximal ligand, 337 redox potentials, 286, 288-291, 297, 326-328 secondary structure, 336 sequence motif, 288, 297 structure-function relationships, 326-328 Cytochronie b (see also BacterioTerritin), 293-296 apo-, 294 axial ligation, 275 hi, 320 bz, 275 br,, 275, 293, 327, 1051, 1072 h5 reductase, 293, 516 b5R71320 bR575, 320 b569, 223, 320 b,,, 275, 295 has, 832, 833 bcl, 297, 375-378, 387,430, 438,514 electron transfer, 293 function, 275 iron oxidation state, 275 iron spin state, 275 PDB codes, 281

[Cytochrome b] sequence motif, 297 structure, 294, 295 Cytochrome bet; 286, 297, 375, 376, 430, 438, 822 structure, 378 Cytochrome c, 236, 272, 286-293, 306, 733, 841, 1044, 1051, 1095, 1097 active site, 28, 293 apo-, 288 axial ligation, 274, 275 c ' , 274, 275, 288, 296 cI, 297 ~ 2 286, , 287 ~3~ 289, 290, 292, 319, 327, 391 c4, 826, 827 c&, 287 ~550,286, 287 ~ 5 5 1 824, , 825 5, 289 ~ 5 5 2 ,286 c553, 287 ~ 5 5 4 ,274, 276, 284, 291-298, 317, 319 ~ 5 5 6 288 , cfi, 286, 327, 822 CT, 289, 290, 319, 327 c a 3 , 387 c nitrite reductase, sep Nitrite reductasc cdl nitrite reductase, ,see Nitrite reductase class I, 275, 280, 286 class 11, 275, 280, 286, 288 class m, 275, 280, 286, 288-291 class IV,275, 280, 286, 291 complex formation, 327 expression, 324, 325 f'erro-, 305 function, 275 horse heart, 287 iron oxidation state, 275 iron spin state, 275 list of, 280 maturation, 279 mitochondrial, 286 PDB codes, 280 photosynthetic reaction center, 292 reductase, 318 sequence motif, 286, 288, 291, 292 structure, 287, 288, 290, 292, 293 Cytochrome c lyase, 285, 325 Cytochrome c oxidase, 717, 719, 727-729, 841, 874, 1097

1136 [Cytochrome c oxidase] active site, 719 axial ligation, 276 azide in, 729 copper site, 712, 832 cupredoxin fold, 767, 803, 832 eukaryotic, 727, 728 function, 2 76 heme type, 276, 728 heme-copper site, 728, 832 iron oxidation state, 276 iron spin state, 276 manganese in, 202 mechanism, 746 photolysis, 746, 747 proton transfer, 729 structure, 728, 823, 832, 833 Cytochrorne c peroxidase, 28, 29, 307, 334, 647 active site, 305, 308 complex formation, 327 compound I, 28 expression, 324, 325 mutant, 28, 29 PDB codes, 283 proximal lig-slid, 338 reaction mechanism, 331 structure, 305, 308 yeast, 305, 306 Cytochrome c reductase calcium in, 318 Cytochrome d l , 1095 Cytochrome J; 297, 822, 823 axial ligation, 274 function, 275 heme type, 275 iron oxidation state, 275 iron spin slate, 275 PDB codes, 281 sequence motif, 296 structure, 296 Cytochrome oxidase axial ligation, 276 bo quinol, 276 c, see Cytochrome c oxidase d, 414 function, 276 heme typc, 276 iron oxidation state, 276 iron spin state, 276 0,414

Cytochxome P450, 313-315, 379, 380, 382, 491,511, 533, 537,538, 551, 555 active site, 24, 313, 314 cam, 13, 24, 25, 313-315 catalytic reaction, 293, 333 class I, 313 class 11, 313 distal site, 313, 315, 337, 338 expression, 325 hydrophobic environment, 338 -like activity, 207 PDB codes, 283, 284 proximal ligand, 337, 338 reductasc, 313, 321 secondary structure, 336 structure, 314 structure-function relationships, 332, 333 Cytolcines, 137 Cytosol calcium in, 128 magnesium in, 63 pea, 13 phospholipase, 126, 128 potassium in, 10

D. acetoxidam, see Desulfuromonas africunus, see Desulfouibrio bacidaturw, see Desulfbmicmbium desulfuricarzs, see Desulfouibrio fructosouora.ns, see Desulfouibrio gigas, see Desulfovibrio uulgaris, see Desulfouibrw Data bases of proteins, 4, 82, 97-100, 141, 363,987 PDB, see Protein Data Rank codes DeacetoxycephalosporinC synthase, 492, 493, 497, 553 active site, 494, 496 structure, 494, 495 Deaminase adenosine, 886 arginine, 239 cytidine, see Gytidine deaminase Decarboxylase dialkylglycine, 12, 15, 16 Decarhoxylation by lithium, 15

SUBJECT INDEX Dehalogenase, 609 Dehydratase bacterial, 19 n-glucosaminate, 219 D-glutarate, 219, 242 did, see Diol dehydratase glycerol, 620 imidazoleglycerol, 219 manganese, 217-219 Dehydrogenase, 69 alcohol, see Alcohol dehydrogenase carbon monoxide, see Carbon monoxide dehydrogenase D-arabitol, 231 formaldehyde, 885, 936 formate, see Formate dehydrogenase formylmethanohran, 1029 ~lyceraldehyde-3-phosphate,114, 921 histidinol, 231 human, 231 isocitrate, 80, 81, 201 isopropylmalate, 201 list of, 231 manganese in, 201, 202, 231 methylamine, 824-826 2-oxoglutarate, 231 pymvate, 421 succinate, 382, 406 tartrate, 201, 231 threonine, 231 trimethykamine, 365, 401-403, 437 xanthine, 379, 1051 Denitrification, 774, 835 Density functional calculation hemerythrin, 5 19 methane monooxygenase, 537, 538 5 '-Deoxyadenosine in vitamin BIZ,606, 607 radical, see Radicals 5 '-Deoxyadenosylcobalamin,see Adenosylcobalamin Deoxyribonucleic acid, see DNA A9 Desaturase, 544, 555 %-labeled, 540 active site, 540 wide in 531 diferrous site, 532 hydrophobic pocket, 531 reaction cycle, 530 reaction mechanism, 540, 541 stearoyl-acyl carrier protein, 530, 532 structure, 531, 532, 534, 535

1137 Desulfoferredoxin, 373, 444 structure, 374-376 Desulforedoxin, 373, 444 hydrophobic core, 374 structure, 374, 375 Desulfomicrobium bmulatum, 676, 678-680 Demlfouibrio sp., 289 africanus, 408-410 desulfuricans, 290, 373, 376, 386, 387, 403, 426, 427, 636, 1048-1050 fructosovorans, 676, 678, 679, 692 gigas, 373-375,381,421,422,676-680, 690, 1055, 1056 vulgaris, 287, 373, 391, 402, 404, 405, 541, 676, 679, 680 Llesulfurornonas sp., 289 acetoxidans, 290 Dethiobiotin synthase, 226 Deuterolysin, 915 dGTP as allosteric effector, 642 Diabetes, 187, 198 mellitus, 572, 1007, 1016 Dialkylglycine decarboxylase, 12, 15, 16 active site, 16 catalytic cycle, 15 cis-Diammineplatinum(II), see Cisplatin Dictyosklium discoideum, 74 Dielectric constant, 11,49 corrinoid, 618 effective, 554 rubredoxin, 418 Diglycine, see Glycylglycine Dihydroacetone phosphate, 22 7&Dihydrobiopterin, 486489 3,4-Dihydrobenzoicacid, see Protocatechuate Dihydrogen as a messenger, 1093 production, 675 Dihydropteroale synthase, 212 2,3-DihydroxybiphenyIdioxygenase, 231, 502-506,511 active site, 506, 511 3,4-Dihydroxyphenylalanine,484, 524, 798, 799 5,6-Dimethylbenziminaole (see also Corrinoids and Vitamine BrL), 605, 611, 626-628, 640, 643 Dimethylmereury, 636 Dimethylsulfoxidereductase, 424 active site, 1041, 1042 list of, 1064

1138 [Dimethylsulfbxide reductase] molybdenum, 1027-1029, 1032, 1033, 1044-1046, 1048, 1049, 1064, 1069, 1071 structures, 1039-1043 tungsten, 1029 Di nitrogenase molybdenum, 168, 169 reductase-activating g-lycohydrolase,238 Dinitrogen oxide reductase, see Nitrous oxide reductase Dinitrogen reduction, 167, 168, 1031, 1036, 1037 Dioclea grandiflora, 228 Diol dehydratasc, 12, 19, 20, 605, 620, 621, 624, 626-628, 630, 640, 642, 643, 645-648, 651 active site, 19, 20 catalytic cycle, 19 structures, 623 Dioxygen (see also Oxygen) (in) 180,739, 741 activating enzymes, 587, 588 activation in larger diiron carboxylate proteins, 533-537 activation, 551, 553, 556, 557, 709-751 binding to hemerythrin, 519 carrier, 516 extradiol dioxygenase, 509 formation, 157, 234, 243 globin, 299, 328, 329 heme coordination, 274, 285, 297-299, 301, 328 intermediate adducts, 556 intradiol dioxygenase, 487 iron oxidation, 576 lipoxygenase, 478 metabolism, 685 nonheme iron protein, 461-557 6-oxoglutar.aLe-depeenClentenzyme, 499 reduction, 728, 746, 747, 830, 832, 1097 scavengers, 541 transport in copper proteins, 709-751 tyrosinase, 748 Dioxygenase catechol, see Catechol dioxygenase chlorocatechol 1,2-, 231 2,3-dihydroxybiphenyl 1,2-, 231, 502-506, 511 extradiol, see Extradiol dioxygenase gentisate 1,2-, 502 homogentisate 1,2-, 503

[Dioxygenasel homoprotocatechuate 2,3-, 231 hydroxylation steps, 515 4-hydi-oxyphenylpyruvate,503, 506, 507 indoleamine, see Indoleamine dioxygenase intradiol, see Intradiol dioxygenase iron, 231 list of, 502 mammalian, 323 manganese, 231 naphthalene-1,2-, see Naphtlialene dioxygenase phthalate, 379, 511, 514, 515 protocatechuate 4,5-, see Protocatechuate dioxygenase Rieske, see Rieslre-type dioxygenase tryptophan-2,3-, 278, 323, 324 2,3-Diphospl~oglycerate, 239, 240 Diphtheria toxin repressor cadmium, 229 cobalt, 229 manganese, 229, 230 nickel, 229 Disease (see ulso individual names) Chagas’, 300 inflammatory bowel, 472 neurodegencrative, 139 Parkinson’s, 139, 198, 484, 799 von Gierke, 173, 174 Wilson’s, 922, 924 Dissociation constants calcium binding, 115, 122, 126, 130, 131, 133 Disulfide bonds, 30,47, 134, 138, 582 bridge, 48, 127, 132, 166, 772, 778, 780, 819, 828, 841, 1007, 1015 links, 310 Dithiolene complexes, 1031-1033 Dithionite, 411414, 425 Dithiothreitol, 414 DNA -binding protein, see Protein cleavage by Cr(II), 185 Cr(II1) adducts, 182 damage, 202, 576,659 glycosylase, 396-398 interaction with zinc fingers, 961-989 lyase, 396 synthesis, 410, 519 virus, 520, 972

1139 DNA polymerase, 77, 78, 207 p, 98, 210 I, 210 bacteriophage, 77, 210 clamp loader complex, 963 human, 210 Kltrnow fragment, 68, 78, 210 Mil", 78, 210 rabbit, 210 rat, 210 Taq, 78 Zn2', 78 DNA-processing enzynic, 67, 68 DNA repair protein, 904 Fed&, 396398 ~equencemotif, 397 structures, 397, 398 Dog liver, 186, 187 Dolichos biflorus, 228 Dopa, see 3,4-Dihydroxyphenalanine Dopamine, 486, 488, '730, 731, 798, 799 hydroxylation, 739 Dopamine 8-monooxygenase, 713, 730, 731 l80isotope effect, 739 catalytic activity, 731 mechanism, 738-742 reaction, 730, 731 Drechslera biseptutu, 157 subpupedorjii, 157 Drosophda melanogaster, 42, 103 Drug (see also individual names) antibacterial, 581 design, 581 Duck ovotransfwrin, 5'79, 580 Dystonia, 484

E E. chvysanthemi, see Erwiniu coli, see &&el-ichia faecium, see Enterococcus Earth's cmst magnesium in, 60 vanadium in, 155 Eel, 42 EF-hand protein, 97, 98, 100-115, 117, 125, 130, 139 definition, 100, 101 rabbit, 98

[EF-hand protein1 zinc in, 906 EGF module, 95, 97, 128-133, 137 calcium affinity, 129-131 hydrophobic interaction, 131, 132 mutant, 132 properties, 128 protein-protein interactions, 128 structures, 129-131 Elastin, 730, 862 Electron density (in), 16, 22, 64, 167 amine oxidase, 723, 738 corrinoid, 618, 622, 647-652, 655 cytochrome c oxidase, 729 galactose oxidase, 720 maps, 11, 15, 48, 582, 592 Electron nuclear double resonance, see

ENDOR Electron paramagnetic resonance, see EPR Electron potentials, see Redox potentizls Electron spin echo envelope modulation, see ESEEM Electron spin resonance, see EPR Electron transfer in coppcr proteins, 813-842 cytochrome c , 28 heme, 274 iron-sulfur proteins, 416-420 plaslocyanin, 183 Electrophoresis gel, see Gel electrophoresis Electrophorus electricus, 4% Electrostatic effects (in) calcium-binding proteins, 101, 104, 106, 10'7, 119, 125-127, 132 long-range, 28, 107 membrane, 127 vanadium chtoroperoxidase, 161 Electrostatic surface potential, 319 Eliminase (see also individual names), 620, 621, 631,646 Embellesis didymosporu, 157 Endocytosis, 133, 578 receptor-mediated, 590, 591 Endoglycosidase, 773 Endonuclease Ill, 365, 396-399 Fe4S4, 437 human, 397 hydrophobic core, 397 magnesium, 210, 244 manganese, 244

1140 [Endonucleasel redox potential, 396 structure, 397, 398 Endopeptidase, 930 Endoprotease amino acid sequences, 910 zinc in, 909, 915 ENDOR (studies of3 I3C, 11067 1 7 0 , 1067 blue copper protein, 836 cytochrome c oxidase, 823 galactose oxidase, 720 methane monooxygenw, 534 Mn-oxalate-ATP complex, 210 oxygen evolving complex, 235 ribonucleotide reductase R2 protein, 520, 533 Endostatin, 892 Enolase active site, 219 manganese, 218, 219, 242 Enterococcus faecium, 917 Enterotoxin, 905 Cd-substituted, 905 staphylococcal, 904, 905 Enzymes (see also Proteins and individual names) calcium, 93-141, 197 cobalt, 197, 603-660, 1109, 1110 DNA-processing, 67, 68 magnesium, 59-82, 197, 1098 malolactic, 231 manganese, 193-244 metal specificity, 1103-1 111 molybdenum, see Molybdenum enzymes/ proteins MutT, 238 NAD-malic, 231 nickel, 197, 679-700 iioncorrinoid cobalt, 544 potassium, 9-31 pyridoxal phosphate-depended, 15, 22, 25, 26, 28 sodium, 9-31 tungsten, see Tungstoenzyme vanadium, 155-167 zinc, see Zinc enzyme Eosinopkil peroxidase, 156 Epidermal growth factor, 97, 311 -like module, see EGF module receptor kinase, 116

Epilepsy, 198 Epinephrine, 794, 798, 799 nor-, see Norepinephrine Epoxidation by chloroperoxidase, 312 cytochrome P450, 313 EPR studies of aminopeptidase, 213 arginase, 217 catalase, 206 channel proteins, 50, 51 chloroperoxidase, 207 concanavaliii A, 227 copper proteins, 712, 720,733, 836, 839 corrinoid, 630 cytochrome c oxidase, 833 dopamine p-monooxygenase, 731 enolase, 219 extradiol dioxygenase, 509 Fur repressor protein, 240 intradiol crlioxygenase, 479 kinase, 208 molybdenum oxotransferase, 1043, 1048, 1050, 1051, 1054, 1056 [NiFeJ-hydrogenases,690, 692 nitrile hydratase, 544 oxygen evolving complex, 235, 236 phosphatase, 214 pyruvate kinase, 77 ribonucleotide reductase, 233, 520 superoxide clismutase, 686 transferrin, 592 tungsten enzymes, 1063 Equilibrium constants of mutases, 609, 640,641,643 Equus caballus, see Horse Erwinia chrysanthemi, 230 Eryi!hriirucorullodendron, 228 Erythrocruorin 111, 281 Escherirhia coli (expression of) amidotransferase, 399, 401 amine oxidase, 716, 719, 722, 723, 736, 738 aminopeptidase, 212, 213 bacterioferritin, 576, 589 carboxykinase, 76, 217 corrinoid, 632 cucumber basic protein, 838 cycloisomerase, 223 cytidine deaminase, 911 cytochrome bsg2, 295 cytochrome c, 325

1141 [Escherichia coli (expression of)] cytochrome c peroxidase, 325 cytochrome P450, 325 dehydrogenase, 231 DNA polymerase, 210 endonuclease 111, 396, 397 ferritin, 574, 589 FixL, 301 Bavohemoglobin, 300 kcdose aidolase, 890 fumarate reductase, 406, 407 Fur repressor protein, 240 hemoglobin, 325 hydrogenase, 676 hydroperoxidase, 304 insulin, 1014 iron transporters, 584 isocitrate dehydrogenase, 80, 201 isomerase, 220, 222 isopropylmalate dehydrogenase, 801 kinase, 209, 892 ligase, 226 lingnin paroxidase, 325 magnesium-activated enzyme, 62, 67-69, 71, 72 manganese peroxidase, 325 metallothionein, 1013 methionine aminopeptidase, 13, 29, 213, 900 methionine syntfiase, 623 molybdenum oxotransferase, 1039, 1043, 1046, 1047 molybdcnum protein, 1034, 1069 molybdopterin, 1028 mutase, 240 MutY, 397 niyoglobin, 325 naphthalene dioxygenase, 512 nickel c~aperon~ns, 687, 688 oxidase, 202 phosphatase, 214, 237, 938, 939 yuinol oxidase, 767 rihonucleotide reductase R2 protein, 520-525, 533, 538 ribonucleotide recluctase, 232, 402, 411 RNase H, 67, 70, 71 sirohenie-containing reductase, 391 sulfite reductase, 392 superoxide dismutase, 202-205 synthetase, 224, 226, 412, 425 ESEEM studies of

LESEEM studies 00 oxygen evolving complex, 235 sullite reductase, 1054 ESR, see EPR Essentiality of chromium, 181, 185, 187 Esterase phosphodi-, 213 phosphomono-, 74, 237, 900 phosphotri-, 213, 916 thiol-, 911 Estrogen receptor, 977, 978, 986 synthesis, 231 Ethane, 169 Ethanolamine glycerophospho-, 119 phosphatidyl-, see Phosphatidylethanolamine Ethanolamine ammonia lyase, see Lyase Ethene, 168, 713 Ethylene, 169 Eubacteria ferritins, 576 gram-negative, 826 iron-sulfur proteins, 394, 416 magnesium in, 62 molybdopterin, 1025 queuosine, 637 zinc fingers, 987, 988 Euglena gracilis, 297, 631, 632 Eukaryotes (or eukamyotic) (see also individual names) amidotransferase, 399 calmodulin, 102 cytochrome c oxidase, 727, 728 ferritin, 575, 576 fumarate reductase, 406 iron-sulfur protein, 416 Krebs cycle in, 389 magnesium transport, 62, 64 metallothionein, 860 molybdopterin, 1025, 1029 mRNA, 212 nitrate reductase, 1048 nitric oxide production, 315 pyrophosphatase, 214 Rieske protein, 375 sulfatase, 911 superoxide dismutase, 686, 860, 866, 871 tmnsferrin, 579-581 zinc finger, 983, 986, 987

1142 Evolution of heme proteins, 338, 339 multi-copper oxidases, 800405 vitamin BIz-dependent enzymes, 627, 657-659 EXA.I% studies of a n ~ n oxidase, e 722 aminopeptidase, 213 catalase, 206 eytochrome c oxidase, 729 A9 desaturase, 680 dopamine p-monooxygenase, 731 extradiol dioxygenase, 509 FeMoco, 1030, 1036 galactose oxidase, 721 hemoeyanin, 726, 748 intradiol dioxygenase, 479 lipoxygenase, 477 methane inonooxygeiiase, 527, 534 molybdenum oxotransferase, 1042, 1047, 1048, 1050, 1053, 1056, 1070 molybdenum proteins, 1036 INiFeI-hydrogenase, 680 nitrile hydratase, 544 nitrogenase, 168 peptidylglycine a-hydroxylating monooxygenase, 724 ribonucleotide redudase, 521 superoxide dismutase, 205, 686, 687 tungstoenzymes, 1061 tyrosinase, 749 Exocytosis calcium-dependent, 115, 122 Exonuclease, 78 111, 197 3’,5’-,210 magnesium, 244 manganese, 210, 244 Exotoxin A, 905 Extended absorption fine structure spectroscopy, see EXAFS ExLradiol dioxygenase (see also individual names), 231, 236,478,479, 502-511 azide, 509 classification, 502 dioxygen in, 509 LigAB, 507-509 nitric oxide in, 509 reaction mechanism, 509-511 structures, 503-509 type I, 502407, 511 type 11, 502, 507-509, 511

CT INDEX

FAD, 291, 300, 313, 315, 402, 406-408, 526, 1057, 1058 Farnesyl transferase, 886 Fasciola hepatica, 212 Fatty acid, 471, 619 biosynthesis, 530 desaturages, see A9 Desaturase metabolism, 492 transport, 859 FeMoco, 1025, 1030, 1031, 1101, 1102 biosynthesis, 394 structure, 1026, 1038 Fenton reaction, 800 Fe-only hydrogenase, 365, 379, 382, 385-387,425-428, 675 active site, 386, 387, 426-428 carbon monoxide binding, 697 cyanide in, 426, 427 II cluster, 386, 425-427 hydrophobic channel, 427 sequence motif; 385, 426 structure, 427 Ferredoxin (see ulso individual names), 313, 362, 375, 394, 408, 410, 416, 421, 424, 429, 530, 1063 bacterioferritin-associated,576 cluster conversion, 421 cubane, 362, 381-384 dcsulfo-, 373-376, 444 r2Fe-2S1, 372, 379-381, 385, 394, 413, 414, 417,430,431, 439, 442-444, 576 [3Fe-4S], 360, 381-387, 417, 422, 444 14Fe-4S1, 360, 381-387, 401, 410, 417, 421,428, 435,439,442, 444 [7F&3S], 360, 382-385, 407, 417 [8Fe-8S],360, 382, 383, 409, 417, 435 hydrophobic core, 379 mutation, 419 plant, see Plant prokaryotic, 384 properties, 379, 381, 382 redox potential, 379, 382, 421, 442 sequence motif, 379, 382,409,410, 421 spinach, 378, 379 structures, 360, 361, 379-387 zinc in, see Zinc Ferredoxin oxidoreductase aldehyde, see Aldehyde ferredoxin oxidoreductase Ferredoxin reductase, 313

1143

SUBJECT INDEX [Ferredoxin reductase] bacterial, 300 fungal, 300 NADPH, 379, 382 Ferredoxin:thioredoxin reductase, 372 [E'e4S4l2+cluster, 413 sequence motif, 413 Ferrihydrite, 575, 586 Ferritin, 200, 372, 464, 541, 544 bacterio-, see Bacterioferritin biological role, 573, 586 biomineralization, 465 bullfrog, 574, 575, 577, 585, 588, 589 classification, 573, 574 eukaryotic, 575, 576 expression systems, 585, 586 ferroxidase site, 543, 575-577, 586-589 horse, 574, 577, 585 human, 574, 577, 585 iron release, 589, 590 list of, 574 mammalian, 589 Mg2' in, 577 PDU codes, 574 plant, 573, 377, 388 repressor protein, see lron regulatory protein structures, 575-577 subunits, 573, 577, 587 Ferrochelatase, 632 Ferroportin 1, 584 Ferroxidase (see also individual names) activity of ceruloplasniin, see Ceruloplasmin hephaestin, 585 site of ferritin, see Femitin Fet3 protein, 766, 767, 834 Fibrillin, 129-133 Fibrinogen, 30, 779 Fibrinolyssis, 128, 138 Fibronectin, 767 111, 374 FixL active site, 301, 302 axial ligation, 276 binding of signaling molecule, 329 conformational change, 330 dioxygen binding, 301 function, 276, 285 heme type, 276 heme-binding pocket, 337 iron oxidation state, 276

LFixLl iron spin state, 276 PDB codes, 284 proximal ligand, 337 secondaiy structure, 336 structure, 301, 302 Flavin adenine dinucleotide, see FAD Flavin-dependent hydroxylase, 490 Flavin mononucleotide, see FMN Flavocytochrome bz, 295 c3 fumarate reductase, 29 I Flavodoxin, 382, 394,408, 411, 676 Flavohemoglobin, 281 Flavohemoprotein, 300 axial ligation, 275 bacterial, 297 function, 275 fungal, 297 heme types, 275 iron oxidation state, 275 iron spin state, 275 Flavoprotein iron-sulfur, 403 Fluorescence studies of calcium-bindingproteins, 119-1 21, 125 Fluoride in aminopeptidase, 156 cataiase, 206 superoxide dismutase, 203 tliermolysin, 940 Fluoroperoxidase, 156 FMN, 313, 315, 402 cofactor, 401 Footprinting of cdmodulh, 107 Formaldehyde ferredoxin oxidoreductase calcium in, 1063 magnesium in, 1063 structure, 1062 tungsten in, 1062, 1063, 1070 Formaldehyde dehydrogenase glutathione-dependent, 885, 936 Fonnate acetyltransferase-activating enzyme, see Pyruvate formate-lyase activase pyruvate lyase, see Pyruvate formate lyase Forrnate dehydrogenase, 382, 437 active site, 1048 molybdenum, 1045-1048, 1050, 1071, 1072 structure, 1046-1048

SUBJECT INDEX

1144 Formiminoglutmase, 239 Formylmethanofuran dehydrogenase, 1029 Fosfomycin, 236 Fourier transform infrared spectroscopy, see FTIR Fructose, 81 l,bi-biphouphate,208 6-phosphate, 23 D-, 220 Fructose l,fi-biphosphatase, 12, 23 manganese, 216 Fructose 1,fi-biphosphatealdolase backrial, 22 class I, 22 class 11, 12, 22 FTIR studies of oxygen evolving complex, 235 Fucose, 134, 136 L-Fucose isomerase active site, 223 manganese, 222, 223, 239, 242 Fuculose aldolase, 890 Fueus distichus,166 Fumarate and nitrate reduction regulator, 37% cluster conversion, 425 conformational change, 424 mutagenesis, 414, 424 redox potential, 414 sequence motif', 414, 415 Fumarate reductase, 365, 379, 382, 424, 439 biological role, 406 eukaryotic, 406 flavocytochromec3, 291 prokaryotic, 406 sequence motif, 407 structure, 406-408 Fund (or fungal) (see also individual names) catalase, 304 chloroperoxidase, 156-162, 169 cytochromes, 293 ferredoxin redudase, 300 hvohemoprotein, 297 galactose oxidase, 720 laccase, 766, 773, 774, 782, 786, 830 lipoxygenase, 232, 471 manganese in, 200 metallothionein, 1003 nitrile hydratase, 544 oxotransferase, 1039

LFungi (or fungal11 peroxihse, 274, 283, 304, 305, 307 siroheme-containing proteins, 391 tyrosinase, 732 urease, 671 white-rot, 307 Fur repressor protein copper in, 240 manganese in, 240 Fusarium oxygporurn, 831 Fuscoredoxin, 365 biological role, 402, 403 redox poteiitial, 403 sequence motif', 404 structure, 404, 405

Galactose, 135, 136, 138 Galactose oxidase, 713, 716, 721 active site, 718, 720, 748 azide in, 734 function, 746, 748 fungal, 720 mechanisms, 73&736 reaction, 720 structure, 720 Galactosides, 134 Galactosyl transferase, 236 Galanthus nivnlis, 228 Galdiera sulphuraria, 231 Gallium(1II) ionic radius, 285 Gaumannonzyees graminis, 232 GDP, 68, 69, 78, 79, 217 -mannose pyrophosphorylase, 73 Gelatin, 930 Gelatinase, 910 A, 930 Gel electrophoresis sodium dodecyl sulfate polyaczrylamide, see SDS-PAGE %neb) coding for K channels, 42 CTR, 715 FRE, 715 HFE, 584 h,ox, 675 hup, 675 ras, 78 sox, 415

'

SUBJECT INDEX [GeneMl transcription, 63, 415 Genome C. elegans, 983 E. coli, 983 M . jannaschii, 983 sequencing, 2 yeast, 964, 983 Gentisate 1,2-dioxygenase, 502, 503 Gephyrin, 1029 Globin, 274,275, 29740,322 axial ligation, 275 cooperativity, 328, 329 dioxygen affinity, 328, 329 distal site, 337 function, 275, 285 heme type, 275 hemo- see Hemoglobin hydrophobic residues, 328 invertebrate, 297 iron oxidation state, 275, 295 iron spin state, 275 list of, 281, 282 myo-, see Myoglobin PDR codes?281,282 plant, 297 proximal ligand, 274, 337 secondary structure, 336 structure-function relationships, 328 Glucocorticoid receptor, 970, 977, 978, 986, 988

Gluconeogenesis, 22, 23 Glucose, 81 -6-phosphatase, 173 D-, 220 fermentation, 411 tolerance factor, 181, 183, 184-187 [3-Giucosidase, 230 Glutamate rnutase, 605, 619-622, 630, 642, 643, 645,647,650,653 radical formation, 648-651, 655 reaction mechanism, 627, 628 structure, 623, 626 Glutamnine, 23, 224 hydrolysis, 2 11 Glutamine phophoribosyli~~ophosphate amidotransferase, 211, 363, 372, 399401,439 biological role, 399 redox potengal, 399 sequence motif, 401 structure, 399401

1145 Glutaminc synthase manganese in, 200, 224, 225 ~/-L-Glutamyl-i-cysteinylglycine, see Glutathione Glutathione, 182, 184 reductase, see Reductases transferase, 236 Glycera dibranchiata,, 336, 337 Glyceraldehyde 3-phosphate, 22 dehydrogenase, 114,921 Glycerol dehydrogenase, 219, 620 Glycerol kiiiaue, 74, 209, 904 Glycine radical, see Radicals Glycine m a . (see also Soybean), 228 Glycogen phosphorylase, 208 Glycolysis, 16, 22, 208 Glycoprotein, 578, 579 trafficking, 134 Glycosidase endo-, 773 Glycosylase DNA, 396-398 Glycosyl transfer, 207 Glycosyl transferase, 236 Glycylglycine Cr(III), 186 Glyoxalase I, 671, 902 11, 911 cobalt in, 902 magnesium in, 902 zinc site, 902 zinc binding motif, 911-914 Glyoxal oxidase, 721 GMP, 65 7-methyl-, 212 Gold in carbon monoxide coordination, 1107 serum albumin, 862 Golgi apparatus, 230 @-protein, 44, 53, 61, 78, 97, 394, 982 receptor, 471 Grif[onia sirnplicirolia, 228 Grisans biosynthesis, 835 Growth factors epidermal, see Epidermal growth factor human neuronal growth inhibitory, 1003, 1012, 1013 insulin-like, 1008 GTP, 68, 69, 72, 78, 79,208, 212, 217, 322

~ U ~ J INDEX ~ C T

1146

TGTP] binding protein, 227,688,981 hydrolase, 981 hydrolysis, 79 GTPase, 68,69,241 p 2 P ,79 Guanidiixium chloride, 428,430 group, 19 hydrochloride, 582 &anidino acetasc, 239 Guanosine 5'-diphosphate, see GDP Guanosine 5'-monopliotjphate, see GMP cyclic, see cGMP Guanosine 5'-triphosphate, see GTP Guanylyl cyclase, 239,322,323 axial ligation, 277 binding of signaling molecule, 329 calcium in, 322 function, 277 heme type, 277 iron oxidation state, 277 iron spin state, 277 nitric oxide binding, 323 Guinea pig insulin, 1011

[Baloperoxidase] catalytic cycle, 172 expression systems, 169,170 function, 169-173 reaction scheme, 156,157 structures, 158 vanadium, 155-167 Hammerhead ribozyme, 79 Ilanster ovary cells, 185 Hansenula polymorpha, 202,717,722,723,

736 Haptocorrjn, 632,633,646 Heat-shock cognate protein, 3 2,20-22 active site, 21 bovine, 20 mutant, 20,21 Helicohacter pylori, 687,688 Helix-turn-helix motif, 672 Heme, 24,28,29,70 bioinorganic role, 285 biosynthesis, 279 ehloro-, 273,274,297 coordination chemistiy, 271-279 dioxygen coordination, 274,285,

297-299,301,328 electron transfer, 274 fine tuning, 336-339 hydrophobic interactions, 272,316

H. cutirubrum, see fialobacterium halobium, see Halobacterium pylori, see Helicobucter Iiaber-Weiss reaction, 800 Hnemophilus uzfluenzae, 399,411, 579-581,911,1034 Hairpin ribozyme, 79,80 Halides (see also individual names), 154-173 Haloarcula marisrnortui, 379 Halo bacterium cutirubrum, 237 halobium, 237 Halocyanin, 818,824 amino acid sequence, 819 loop topology, 821 redox potential, 834,837 I-Ialogenase for biorernediation, 609,636 Halopcroxidase (see also Broinoperoxidase atid Chloroperoxidase)

P460,274,316 PDB codes, 280-285 proteins, see Hemcproteins redox potential, 274,289 siro-, see Siroherne structures, 272,273 types, 271-278 Heme-based biosensor, 301-303,329,330 CooA, see CooA FixL, see FixL Heme oxygvnase, 320-322 axial ligation, 276 bacterial, 320 carbon monoxide Formation, 335 catalytic cycle, 335,336 distal site, 321,322 function, 276 heme type, 276 iron oxidation state, 276 iron spin state, 276 mammalian, 320 PDB codes, 284 plant, 320

SUBJ

x

[Heme oxygenaseI proxinial ligand, 321, 337 rat, 321 structure, 321 structure-function relationships, 335, 336 Hemeproteins (see also individual names ), 630 axial ligation, 275-278 distal interactions, 337, 338 evolution, 338, 339 expression systems, 324326 flavo-, see avohe he mop rote in function, 275-278 heme type, 275-278 iron oxidation state, 272-278 iron spin state, 275-278 list of, 275-278 proximal ligand, 337, 338 secondary structure, 336-338 Hemerythrin, 470, 516, 520, 530 azide in, 517 dioxygen binding, 519 myo-, 516, 517 reaction mechanism, 519 sti-ucture, 516-5 18 Hertiochromatosis, 572, 584 Hernocyanin, 516, 517, 732 active site, 718, 726, 748 deow-, 719, 726, 727 dioxygen binding, 748, 749 dioxygcn transport, 725 mollusk, 725-727 oxy-, 719, 726, 748 structure, 725 Hemoglobin, 297, 516, 573, 630, 654 annelid worms, 299 cooperativity, 328, 329 crustacean, 299 dioxygen binding, 299, 328, 329 expression, 325 Navo-, 281 function, 275 heme binding pocket, 337 heme type, 275 invertebrate, 299 iron oxidation state, 275 iron spin state, 275 leg-, 281, 299 list of, 281 mollusk, 299 oxy-, 746 PDB codes, 281

11147 [Hemoglobin] plant, 299 R state, 299, 329 secondary structure, 336 T state, 299, 329 Hemophilia, 133, 779 Hemosiderin, 465, 573 Hemosiderosis and ceruloplasmin, 800, 801 Hepatitis virus, 70, 891 Hepatocytes, 133 Herbicides, 544 Ileteronuclear single quantum coherence spectroscopy 'H,"N-, 123, 126 High-polential iron-sulfur protein, 363, 429,435, 439,442, 444 hydrophobic pocket, 388 mutation, 418, 419 properties, 387 redox potentials, 387, 416-420 sequence motif, 387 structure, 360, 387, 388 surface charges, 420 Hill coefficient, 102 HiPIPs, see High-potential iron-sulfur protein Rist arnine heme coordination, 301 release, 300 Histidine biosynthesis, 73 kinase, 301

HW integrase,972-974, 976 Holmium(II1) (in) mannose-binding protein, 134, 137 psoriasin, 111, 115 Homeostasis of (see also Metabolism) calcium, 95-97, 110 copper, 713-71 5, 1003 iron, 389,463465, 572, 584 magnesium, 64 manganese, 198-201 zinc, 988, 1003 Homocysteine, 323 Homolysis Go-6 bond, 19 Homo sappiens, see Human Hordeurn uulgare, 283 Hormones (see also individual names), 322 human growth, 915, 916

1148 FHormonesl plant, 473, 492, 713 steroid, 313 stress, 799 Norse ferritin, 574, 577, 585 heart cytochrome c, 287 lactoferrin, 466, 580, 592, 593 transferrin, 579, 580 I-torseradish peroxidase, 283, 310 active site, 308, 310 stnrcture, 305, 308 Hsc70, see Heat-shock cognate protein HSQC, see Hctoronuclear single quantum coherence spectroscopy Human (see also Mammal) amine oxidase, 721 brain, 201 calcium-bindingproteins, 98, 103, 112, 114, 117 ceruloplasmin, 770, 771, 776-780 chromium in, 182, 183 dehydrogenase, 231 DNA polymerase, 210 endonuclease, 397 erythrocytes, 214 ferritin, 574, 577, 585 insulin, 1008, 1013 kinase, 209 lactoferrin, 578, 586 magnesium deficiency, 62 mannosc-binding protein, 134-139 metallothionein, 1003-1005 molybdopterin, 1029 mutase, 619 oxidase, 202 oxygenase, 320, 321 pathogens, 199, 581 phosphatase, 214 prolidase deficiency, 237 sulfit,e oxidase, 1051 superoxide dismutase, 204 transferrin, 579, 583, 586, 591, 592 tumors, 78 tyrosinase, 727, 732 vitamin BIZ,632 Human growth hormone zinc in, 915, 916 Human immunodeficiencyvirus, see HW Human neuronal growth inhibitory factor, 1003, 1012, 1013 Hydratase

[Hydratase] acetylene, 1029 nitrile, see Nitrile hydratase Hydrazine, 169 Hydrocarbons volatile, 165 Hydrogenase, 291 active site, 676, 678-680 biological role, 675, 676 classes, 675 Fe-only, see Fe-only hydrogenase methylene-tetrahydroinethanopterindependent, 675 tNiFel-, see [ ~ i F e l - h y ~ r o ~ e n a s e s INiFeSel, 675, 676, 679,680, 691, 692 structure, 676-680 Hydrogenophaga pseudoflaua, 1058 Hydrogen peroxide (and anion), 154-157, 167, 172, 174, 202, 206, 207, 243, 305, 422, 720, 721, 736, 765, 860 activation, 313, 330, 331, 337, 338 -binding pocket, 306 decomposition, 241, 303, 312, 685 formation, 576, 588, 589, 738, 865 -generating system, 307 in ascorbate oxidase, 789, 790 scavenger, 28, 306 Hydrolase (see also Protease and individual names), 470 class 111, 885 cyclo-, see Cyclohydrolase GTP, 981 leukocyte &, 909, 910 manganese in, 212-217, 237-239, 241, 242 zinc in, 242 Hydrolysis o f =@nine, 217 glutamine, 211 nucleoside triphosphate, 20, 49, 238, 241 phosphodiesters, 79 Hydroperoxidase 11, 304 Hydroxyapatite, 95 5-Hydroxyindole, 794 2-J3ydroxyisonicotinic acid N-oxide, 481, 483 Hydroxylamine, 334, 335 reduction, 391, 392 Hydroxylamine oxidoreductase, 274, 284, 291,319 active site, 317 axial ligation, 277

SUBJECT INDEX [Hydroxylamine oxidoreductasel catalytic reaction, 334 function, 277 heme type, 277, 316 iron oxidation state, 277 iron spin state, 277 structure, 317 structure-function relationships, 334 Hydroxylase alkane, 525 amino acid, 484486, 490, 491, 551, 553, 555-557 anthranilate, 485 aspa&d p-, 492 flavin-dependent, 490 list of, 525 lysyl, 492 mandelate, 485 methane monooxygenase, see Methane monooxygenase molybdenum, see Xanthine oxidase phenol, 379, 525 phenylalanine, 484-491 pterin-dependent, 484491 prolyl, 492, 493 tryptophan, 481, 491 tyrosine, 484-486, 488 Hydroxyl radical, 685, 713, 800 scavcnger, 1057 6-Hydroxynicotinic acid N-oxide, 481, 483 4 - ~ y d r o x y p h ~ ~ y ~ p ~dioxygenase, uvate 503 structure, 506, 507 Kydroxytryptamine, see Serotonin Hypoxanthine phosphoribosyltransferase, 210,211 active site, 211

Imidazole stacking interactions, 916 Imidazoleglycerol dehydratase, 219 Immune response, 578 Immunoglobulin, 767, 860, 1052 Indole (see also Tryptophan) halogenated, 165, 166 Indoleamine 2,3-dioxygenase,323, 324 axial ligation, 278 function, 278 heme type, 278 iron oxidation state, 278

1149

IIndoleamine 2,3-dioxygenase] iron spin state, 278 Inflammation (or inflammatory), 115, 133, 472 bowel disease, 472 response, 471 Infrared spectroscopy (studies of) Fourier transform, see FTIR [NiFe] hydrogenase, 679, 690, 691 oxygen evolving complex, 234 Inorganic pyrophosphatase active site, 216 magnesium, 214 manganese, 213-215 Inositol phosphate, 125 1,4,5-triphosphate, 97, 125 Inositol monophosphatase, 215, 216 Insect blood-sucking, 300 cytochrome, 313 Insecticyanin, 300 Insertase, 168 Tnsulin, 198 action, 181, 186 biological role, 1007, 1015 calcium in, 1002 cod, 1013 conformations, 1009, 1010, 1015 Cr(III1, 186, 187 disulfide bridge, 1007, 1015 expression systems, 1013, 1014 human, 1008, 1013 -like proteins, 1008, 1013 list of, 1008 PDB codes, 1008 pig, 1008, 1009, 1013 sequence variations, 1011, 1012 structure, 1008-1012 zinc in, 1001-1016 Integrase avian sarcoma virus, 210, 230 HIV, 972-974, 976 magnesium, 210, 230 manganese, 230 retrovkal, 972 Tntegrin p-, 584 magnesium, 229 manganese, 228, 229 Interdependencies, 5 cadmium-manganese, 199

1150 [Interdependencies] calcium-manganese, 20 1 cobalt-manganese, 199 copper-iron, 200 iron-manganese, 200, 214 iron-zinc, 200 magnesium-manganese, 197, 199, 200, 201,208, 212, 213 manganese-zinc, 199, 212 molybdenum-tungsten, 1029 Interferon a-, 906 fi-, 906 y-, 324 zinc in, 906 Intradiol dioxygenase ( w e also individual names), 231, 470, 478-484, 510, 552, 554,557 active site, 509-51 1 cyanide in, 483 reaction mechanism, 483, 484 reaction, 479 role of dioxygen, 484 structure, 480-483 Intrinsic factor, 632, 633 Intron g o u p I, 79, 80 Invertehrate (see also individual names) cytochromes, 313 globin, 297, 299 Iodide oxidation, 155-157 Iodoperoxidase, 156 Ion channels, 39-53 bacterial, 45-47 calcium, 42, 97, 117, 119, 121, 128 definition, 40 inactivation domains, 47, 48 ligand-gated, 40 mammalian, 47,48 neuronal, 47 potassium, 39-53 sodium, 39-53 voltage-gated, 40-42, 44, 47, 50, 51, 53, 906 Ionophores (see also individual names) ferric, see Siderophores IR, see Infrared Iridium(~~~) carbon monoxide binding, 1108 Iron (different oxidation states) (in) chaperone, see Chaperone coordination sphere, see Coordination sphere

[Iron (different oxidation states) (in)] heme proteins, 269-340 interdependency with other metal ions, see Interdependencies manganese superoxide dismutase, 203, 205 metabolism, 463-465, 573, 590 methane monooxygenase, 733 purple acid phosphatase, 238, 244, 547-550, 900 storage proteins, 571-594 storage, 464 transport protein, 571-594 transport, 5, 464, 466 uptake, 767 IronfII) (in) affinity for sulfur, 240, 241 ceruhplasmin, 794-796 dioxygenases, 231 dioxygen oxidoreductase, see Ceruloplasmin lipoxygenase, 232 metallothionein, 1005 oxygen evolving complex, 233 Iron(II1) (in) ceruloplasmin, 794-796 dioxygenase, 231 ionic radius, 185 protoporphyrin IX, 28 transferrin, see Transferrin Iron(TV), 332, 520, 544 oxyrerryl intermediate, 830, 331, 334, 338,491, 499, 500, 533, 537, 556, 557 Iron-molybdenum cofactor, see FeMoco Iron proteins (see also individual names) heme, see Hemeprotein iron-molybdenum, see ~ o l y b d e n ~ ~ - i r o n protein iron-sulfur, see Iron-sulfur protein nonheme, see Nonheme protein Iron regulatory protein, 372 properties, 389-391 Iron-sulfur cluster bioinorganic role, 365, 370-372 cubane, 362, 365,372 Fe(S-Cys)4 (sep also Rubredoxin), 360, 417 FezSz (see also Ferredoxinj, 297, 360, 361, 363, 407, 408, 417, 425, 431, 432, 511, 514, 1055, 1057 Fe&, 360-362,372, 391, 407,408, 417, 423, 424, 431, 434, 435, 443, 678, 694 Yea, 365

SUBJECT INDEX [Iron-sulfur cluster] Fe4S4 (see also High-potential ironsulfur proteins), 168, 360, 361, 363, 387, 389-404, 407,411, 413415,417, 423, 425, 429, 431, 434, 435, 437, 443, 657, 678, 684, 685, 694,697-699, 1046, 1048-1050, 1057, 1060-1063, 1072 l?e7S8, 434 FesS7, 1036 Fe&, 168, 434 H cluster, 386, 425-428 hybrid, 365 interconversion of Fe3S4/Fe4S4,421-424, 439 list of, 364 P, 1030, 1036, 1037 [(RS)4Fe2S21"-!3--, 360, 362 l ( ~ ~ S ) ~ l ?/3e ~, 362 S*~~ spin distributions, 417 structures, 360, 361 Iron-suXftir proteins (see also individual names) bacterial, 394, 397, 399, 416 biosynthesis, 445 consensus sequences, 362-365 coordination chemistry, 359-362 electron transfer, 416-420 eukaryotic, 416 folding and stability, 428, 429 function, 370, 371 high-potential, see High-potential ironsulfur proteins list of, 364, 366-371 phylogenetic trees, 430-442 putative prismane, see Fuscoredoxin redox potential, 326,416-420 role of the cluster, 428, 429 sequence alignment, 430 structural ch&kation, 362-369 structural motifs, 431 topologies, 431 Isocitrate, 69, 80 Isocitrate dehydrogenase, 80, 81 active site, 80 Ca2', 80 Cd2', 80 manganese, 201 mutant, 80 Isomerase, 67 bacterial, 220 cyclo-, see Cycloisomerase

1151

IIsomerase I D-mannose, 239 u-ribose, 239 glucose, 1110 keto-aldo, see Keto-aldo isomerase L-arabinose, 239 I,-fucose,222, 223, 239, 242 L-rhamnose, 239 manganese, 219-224, 239 phosphomannose, 889,891 sugar, 219, 239 triosephosphate, see TIM barrel vitamin B12-dependent,see Vitamin Bizdependent mutase xylose, see Xylose isomerase zinc, 899 Isopenicillin-N synthase, 492-495, 553, 555, 556 active site, 498, 499, 501 reaction mechanism, 500-502 structure, 494, 497-499 Isopropylmalate dehydrogenase manganese in, 201 potassium in, 201 Isotope (see also individual elements) effects, 531, 533, 538, 540, 734, 739 labeling experiments, 477, 748 J Jack bean concanavalin A, 227 urease, 671

K. aerogenes, see Klebsiella pneurnoniae, see Klebsiellu Keto-aldo isomerase list of, 239 manganese, 219-222, 239, 242-244 a-Ketoglutarate, 80, 201 Kinase, 61, 65, 66, 186, 207, 965 acetate, 209 active site, 74-77 adenosine, 66, 70 adenylate, 72, 75, 209, 892 arginine, 66 ATP binding, 74, 75 Bruton's tyrosine, 968, 969, 975

1152 IKnase] MP-dependent protein, 209 cyclin-dependent, 209 EGF receptor, 116 glycerol, 74, 209, 904 liistidine, 301 human, 209 M$+-dependent, 71, 72, 74-77, 208 Mn2' "dependent, 75-77,208-210 myosin light chain, 103, 105 nucleoside diphosphate, 74, 75, 209 nucleoside monophosphate, 72, 74 phosphatidylinositol, 208 phosphoenolpyruvate carboxy-, 75-77, 217, 218 phosphofi-ucto-, 74 phosphoglycerate, 74, 209 phosphorylase, 208 pig, 209 protein, see Protein kinase pyruvate, see Pyruvate kinase tyrosine, 975 UMP/CMP, 74 Klebsiella aerogenes, 671, 688, 916 ptzeumoniae, 623, 1034, 1036 Klenow fragment DNA polymerase, 68, 78, 210 Kiebs cycle, 389,406

L.

lactis, see Lactococcus leichmannii, see Lactobacillus plantarum, see Lactobacillus sake, see Lactobacillus Laccase, 764, 766, 767, 772-774, 838, 839 copper binding sites, 781 cupredoxin fold, 773, 782, 817, 830 evolution, 802, 803 fungal, 766, 773, 774, 782, 786,830 loop topology, 821 metal site, 820 oxygen radical, 791 plant, 766, 772, 786 redox potential, 830, 840 structure, 773 substrate binding, 787 trinuclear copper center, 786 B-Laetamase amino acid sequences, 913

SUBJECT INDEX l~-Lactamase] serine, 492 zinc binding motifs, 911, 913, 914 zinc, 901, 902, 911-914, 916 Lactam, 496,498, 501 antibiotics, 492-494 Lactate bacterial, 197 I,-phospho-,16, 18 L~tobacillus leichmannii, 623, 633 plantarum, 197, 199, 206,207 sake, 237 xylose isomerase, 220 Lactococcus lactis, 231 Lactoferricin, 578 Lactoferrin, 578, 582, 585, 592 bovine, 578-580, 583 horse, 466, 580, 592, 593 human, 578, 580, 583, 586, 590 iron site, 466 samarium, 593 Lactoperoxidase, 156, 278 heme type, 274 Lanthanides in transferrin, 593 Lanthanum(II1) in annexin, 117 phospholipase, 125 Latex, 773 Lathyrus ochrus, 228 odoratus, 202 satiuus, 202 Lead in carboxypeptidase A, 918-920 metallothionein, 1005 rat, 183 Leather tanning, 186 Lectin, 133-139 animal, 133, 135, 136, 227 bacterial, 133 calcium in, 227 carbohydrate recognition, 133 endocytic, 133 manganese in, 133, 227, 228 occurrence, 133 plant, 133, 134, 227, 228 tunicate, 135, 136 viral, 133, 227 Leghemoglobin, 281, 295 Leishmania mexicana, 230 Leucine biosynthesis, 201

1153

SUBJECT INDEX Leucine aminopcptidase, 939 dinuclear zinc, 212 manganese in, 212 Leukemias, 114 promyelocytic, 972 Leukocyte myeloperoxidase, 310 trafficking, 133 Leukotrienes, 126, 471 & hydrolase, 909, 910 Ligand acceptor-, 1106, 1107 basicity and complex stability, 1104, 1105 donors, 1107, 1108 -metal covalency, 1104 Ligase manganese, 224-227, 240 UDP-~-acetylmuramoyl-L-alanine:Dglutanlate, 226 zinc, 895 Lignin, 307, 331 degradation, 331, 507, 766, 773, 830 synthesis, 773 Lignin peroxidase, 307, 309, 331 active site, 307, 308 expression, 324, 325 PDB codes, 283 structure, 308 Limulus polyphemus, 717, 719, 725, 726, 748 Linoleic acid, 232, 473 Linolenic acid, 232, 473 Lipase, 473, 475 phospho-, see Phospholipase Lipid bilayer, 40 metabolism, 1007 phospho-, see Phospholipid @-Lipoicacid synthase, 425 sequence motif, 413 Lipoprotein, 198 Upoxins, 471 Lipoxygenase, 470-478, 557 activating protein, 475 active site, 232, 474, 475, 478 bacterial, 471 Caz+ in, 473 dioxygen in, 478 Fe2+ in, 232 function, 471-473 fungal, 232,471 hydrophobic cm7ity, 476

LLipoxygcnasel linoleic acid, 232 mammalian, 232,471, 476 manganese in, 232 plant, 471, 473 rabbit, 473, 474 rat, 475 reaction mechanism, 476-478 redox potential, 476 soybean, 473-477 structure, 473-476 Listeria innocua, 574, 576, 585, 587, 589 Lithium ion, 15, 41 sulfide, 516 Lithostathine, 138 Liver, 133, 134 bovine, 187 dog, 187 mouse, 187 rabbit, 187 rat, 217 Lobster spiny, 725 LSD, 799 Lyase (see also individual names) bacterial, 26 cytochrome c, see Cytochrome c lyase DNA, 396 ethanolamine ammonia, 605, 620, 621, 627,639, 642,643,645-648, 651, 653 manganese, 217-219, 239 peptidyl amidoglycolate, 725 tyrosine phenol-, 13, 27, 28 UDP-Gal: f3-D-GlcNAc-fl-endopectate,230 ureidoglycollate, 239 zinc, see Zinc lyase Lymph nodes, 59 Lymphocytes, 133 Lysergic acid diethylamide, see LSD 1,ysine 2,3-aminomutase, 413, 425, 657, 1110 Lysyl hydroxylase, 492 oxidase, 730, 862

M. barkeri, see Methanosarcina capsulatus, see Methylococcus

1154

M.7 extorquens, see Methylobacterium frisia, see Methanosarcina jannaschii, see Methanococczcs lutens, see Micrococcus methylotrophus, see Methylophalus morganii, see Morganella radiodurans, see Micrococcus smegniatis, see Mycohactwium therrrioautotr~phicum,see ~ethanobacteriu~ thermophila, see Methanosarcina trichosporium, see Methylosinus tuberculosis, see Mycobacteriurn van,ielli, see Methanococcus xanlhus, see Myxococcus ixz-Macroglobulin, 200 Macrophages, 133 Magnesium(TI) (in), 16, 18, 20-22, 196, 215 -activated enzymes, 59-82 ADP, see AnP aldehyde €erredoxin oxidoreductase, 1061 aminopeptidase, 939 arylsulfatase, 237, 911 ATP, see ATP ATPase, see ATPase calbindin D9k, 110 carboxykinase, 217, 218 chelatase, 632 chemistry, 60, 61 cluster, see Cluster coordination sphere, see Coordination spheres dehydratase, 219 dehydrogenase, 231 Earth's crust, 60 endonuclease, 210 ferritin, 577 formaldehyde ferredoxin oxidoreductase, 1063 integrase, 210 intejyins, 229 interrelation with other metal ions, see Interdependencies ionic radius, 185 kinase, 71, 72, 74-77, 208 metabolism, 62-64 [Ni~el-hydrogenases,678 nucleotide complexes, 61 phosphatstse, 213-216,237, 900, 937, 938 polymerase, 210 racemase, 224

[Magnesium(IT)(in)] ribozyme, 210 sulfatase, 237 synthetase, 224 transport, 62-64 water exchange, 61 Magnetic circular dichroism studies of clavaminate synthase, 497 As desaturase, 531, 534 lipoxagenase, 476 methane monooxygenase, 526, 534, 540 molybdenum oxotransferase, 1043 nitrogenase, 168 nitrous oxide reductase, 733 phenylalanine hydroxylase, 489, 491 phthalate dioxygenase, 515 ribonucleotide reductase R2 protein, 520 Magnetic susceptibility measurements of ribonucleotide reductase, 520 Mammal(ian) (see also individual names and species) arginase, 217 dioxygenase, 323 fcrritin, 589 heme oxygenase, 320 iron metabolism, 464 lipoxygenase, 232, 471476 metallothimein, 1004-1007, 1012 mutase, 619 peroxidase, 156 phosphatase, 237, 238, 547-549 vitamin BIZ, 631, 632 Mandelate hydroxylase, 485 Mandebate racemase, 219, 223, 224, 242 Manganese (different oxidation states) (in) affinity for sulfur, 240 brain, 200 carbamoyl phosphate synthetasc, 225, 226, 242 chemistry, 196-198 coordination sphere, see Coordination spheres deficiency, 243 enzymes, 193-244 homeostasis, see Homeostasis interrelation with other metal ions, see Interdependencies metabolism, 198-200 oxidation states, 196 PDB codes of proteins, 221 redox potential, see Redox potentials toxicity, 198

1155 [Manganese (different oxidation states) (in11 transport proteins, 584 transport, 5 Mangmese(T1) (in), 16, 23, 61, 64, 65, 79 ADP, 225 albumin, 200 amine oxidase, 202 amino peptidase, 200, 237, 244 as probe, 71 ATP, 61, 210,226 ATPase, 67, 200 calbindin Dek, 110, 111 carboxykinase, 2 17-2 19 carboxylase, 210 ceruloplasmin, 200 chloroperoxidase, 332 cluster, see Cluster concanavalin A, 227, 229 coordination sphere, see Coordination spheres -dependent kinase, 71, 72, 74-77, 208 diphtheria toxin repressor, 229, 230 DNA polymerase, 78, 210 DNA-processing enzymes, 67, 68 extradial oxygemiase, 502 isopenicillin-iV synthase, 497 lectin, 133 mannose-binding protein, 134 phosphatase, 213-216,237, 238 ribonucleotide reductase, 524 synthetase, 69, 226 Manganese(III), 196, 198, 200, 241 transferrin, 243 Manganese(lV), 196 Manganese catalase active site, 207 cyanide, 206 dinuclear centers, 206, 207, 241 fluoride, 206 mechanism, 241 properties, 206 Manganese dehydratase, 2 17-219 Manganese ligase, 224-227, 240 Manganese peroxidase, 198, 207, 283, 307, 309, 331 active site, 308, 309 distal site, 309 expression, 324, 325 proximal ligand, 309 structure, 305, 308

Manganese superoxide dismutase, 198, 202-205, 243, 244, 685, 686, 699 active site, 203 bond distances, 204 Fe-substituted, 203, 205 fluoride, 203 mechanism, 241 redox potential, 205 Mannose, 136 I-phosphate, 73 -1-phosphotransferase, 230 6-pho~phatereceptor, 230 Mannose-binding protein, 133-137 carbohydrate binding, 134-139 Ho3+, 134, 137 human, 137, 138 hydrophobic packing, 134, 137 Mn2+, 134 rat, 134, 136, 138 structure, 135 Marchantia polymorphu, 233 Marfan syndrome, 130, 133 Marine animals, see individual names and species Markus theory, 327 Mass spectrometry studies of nitrile hydratase, 545 Matrilysin, 884 inhibition, 922 properties, 929, 930 Matrix metalloproteinase, 392, 910, 911, 925,930, 935, 940 Mavicyanin, 828 MGD, see Magnetic circular dichroism Melanomas, 114 Melanotransferrin, 578, 585 Melatonin, 484 Membrane channel proteins, 39-53 electrostatic interactions, 127 hydrophobic contacts, 127 phospholipids, 119, 121, 125-127 plasma, 40 potential, 40, 41, 44, 49, 63 trafficking, 115, 121 Menaquinone, 407, 408 Menkes' copper-transporting ATPases, 858, 374 cadmium in, 862 cobalt in, 862 silver in, 873

1 I56

[Mennkes' copper-transporting ATPases] structure, 862, 863 zinc in, 862 Menkes' disease, 862 Mental retardation, 485, 503 Meprin, 910 MercuryiII) (in) biometliylation, 636 cobalamin, 622 metallothionein, 1005 serum albumin, 862 Metabolism of (see also Homeostasis and Transport) calcium, 95-97 carbohydrate, 240, 1007 dioxygen, 685 fatty acid, 492 iron, 463-465, 573, 590 lipid, 1007 magnesium, 62-64 manganese, 198-200 pyrimidine, 492 Met~ochaperones,5 copper, see Copper chaperones iron, 443 nickel, 687, 688 Metalloprotease, see Protease Metalloproteins conformation changes, 1098-1103 emerging themes and patterns, 1091-11 13 metal specificity, 1103-1111 Metallothionein, 187, 713, 715, 814, 963, 975, 988 -3, see Human neuronal growth inhibitory factor cadmium in, 1004-1007, 1014, 1015 CdfZn, 872 cobalt in, 1005, 1014 copper, see Copper metallothionein crab, 1003 eukaryotic, 860 fungal, 1003 iron in, 1005 mammalian, 1004-1007, 1012 mice, 1003, 1004, 1013 nickel in, 1005 PDB codes, 1004 plant, 1003 platinum in, 1005 rabbit, 1003-1006, 1014, 1015 rat, 1003, 1004

SUBJECT INDEX CMetallothionein] technetium in, 1005 tin in, 1005 zinc, see Zinc metallothionein Metapyrocatechase, 502, 503, 505, 506 Metastasis, 133 Methane, 634, 635, 683 formation, see Metha~iogenesis oxidation, 609, 637 Methane monooxygenase, 542, 544, 551 active site, 528, 539, 733 diiron site, 528, 529 for bioremediation, 525 hydrophobic pocket, 539 hydroxylase, 525-530, 586-589 hydroqlation reactions, 538-4540 mechanism, 744, 745 membrane-bound particulate, 733, 744 protein B activator, 525, 526-528, 534, 540, 556 reaction cycle, 526 reaction, 732 reductase, 526 soluble, 733 structure, 527-530, 534537 ~ e t h a n o b ~ t e r ithermoaulotrophicum, ~m 373, 397, 403, 676, 681, 682, 693, 1029 Methanococcus jannaschii, 215,399,403,681,987,988 vanrelli, 73 Methanogenesis, 680, 681, 1111 Methanosarcina barkeri, 676 frisia, 402 thermophila, 209, 681 Methanotrix soehngenii, 402 Methionine S-adenosyl-, see S-Adenosylmethionine Methionine aminopeptidase, 13 active site, 29 cobalt, 213, 900, 1110 Methionine synthase, 607, 619, 628, 630, 632, 633, 657 reaction mechanism, 627 structure, 623, 625-627 Methylamine dehydrogenase, 824-826 Methylation (of') arsenite, 636 bio-, 636 selenate, 636 tellurate, 636

SUBJECT INDEX Methylcobalamin (see also Corrinoids), 614, 616, 619,625, 628, 632 Methylcobinamides (see also Corrinoids), 612 Methyl coenzyme M, 634 Methyl coenzyme M reductase, 671 active site, 681-683, 694, 695 biological role, 680, 681 catalytic mechanism, 693-697 structure, 681-683 Methylglutarate mutase, 619, 610, 646 Methylmalonyl-CoA mutase, 605, 619-622, 632, 641-643, 645, 646, 648,653, 654 cobalt in, 1110 radical formation, 651, 652 reaction mechanism, 627, 628, 630 structure, 622-627, 629 Methylmercury, 636 Methylobacterium extorquens, 824 Methylococcus capsulatus, 527-529, 538 Methylomonas sp., 826 Methy~op~iilus met~iylo~rophus, 401, 403 Methylosinus frichosporium, 527, 528, 538 ~ethyltetrahy~ofolate, 684 Melhyltransferase catalysis, 634 vitamin B12-dependent,605, 609, 610, 612,618, 626, 633-636, 654, 656,657, 684 zinc in, 609 Mice brain, 201 kinase, 209 liver, 187 metallothionein, 3 003, 1004, 1013 tyrosinase, 732 Micelles, 119, 126 Micrococcus lutens, 397 radiodurans, 197 Microfibrils, 130, 131, 133 Microorganisms, see individual names and species Microsomes cytochromes in, 293, 294 rat, 294 Microwave polarization, 206 Mineralization of bone, 547 organic matter, 680 Mitochondria bacterial, 279

1157

IMitochondria] calcium in, 95, 202 fungal, 285, 293 invertebrate, 285 magnesium in, 64, 201, 202 manganese in, 201, 202 plant, 279, 293 vertebrate, 285 zinc in, 202 Mobilferrin, 584 Molecular dynamics simulation (of), 132 insulin, 1010 myoglobin, 299 Molecular modeling of insulin, 1010 Molecular orbital calculations ab initio, 61 Mollusk hemocyanin, 725-727 hemoglobin, 299 Moloney murine leukemia virus, 210 Molybdenum (different oxidation states) biological role, 1024, 1025 coordination chemistry, 3 030-1033 coordination sphere, see Coordination sphere -iron cofactor, see FeMoco iron-sulfur cluster, see Clust,er reductase-activating glycohydrolase, 238 sulfur donors, 1031-1033 transport proteins, 1034, 1035 uptake, 1028 versus tungsten, 1029 Molybdenum iron protein, 394, 396 structure, 1036, 1037 Molybdenum enzymesiproteins (see also individual names), 3 023-1072 expression systems, 1069 reaction mechanism, 1071, 1072 structure, 1034 Molybdopterin, 1025-1072 bacterial, 1025 biosynthesis, 1028, 1029, 1051 cytosine dinucleotide, 1055-1059 eukaryotic, 1025, 1029 functions, 1027 guanine dinucleotide, 1039, 1040, 1043-1047, 1049, 1050, 1070, 1072 human, 1029 precursor, see Compound Z structure, 1026-1028 synthase, 1028, 1029 Monochlorodimedone, 156, 157

1158 Monooxygenase, 379 ammonia, 732, 733, 734 dopamine p-, see Dopamine pmonooxygenase glyceryl-ether, 485 heme-containing, 24 methane, see Methane monooxygenase peptidylglycine-~-amidating,723, 724, 731 peptidylglyciiie-~-hydroxylating, see Peptidylglycine-a-hydroxylating monooxygenase phenol, 727, 741 toluene 2-, 525 toluene 4-, 525, 533 xylene, 525 Monosaccharides (see also individual names), 133, 136 Morganella morganii, 403 Mijssbauer spectroscopy (studies of) 57Fe,1030 FNR protein, 415 methane monooxygenase, 527 nitrogenase, 168 ribonucleotide reductase, 520 Muconate chloro-, 223, 224 cis,cis-, 224 Muconate cycloisomerase magnesium in, 223 manganese in, 238, 223, 242 mutants, 223 Multi-copper oxidase (see also individual names), 713, 763-805 blue, 830 copper binding sites, 780-787 cupredoxin fold, 765, 767, 780, 817 evolutionary aspects, 800-805 PETS, 715 inhibitors, 791 overall architecture, 767-771 phylogenetic trees, 801, 802 structural relationships, 770, 803-805 Mussel manganese in, 197 Mutagenesis studies (of), 43, 45 blue copper proteins, 838, 839 calcium-binding proteins, 107, 114, 117, 135-137 ferritin, 577 fumarate and nitrate reduction regulator, 414, 424

[Mutagenesis studies (of)] glyoxalase I, 902 isopenicillin-N synthase, 497 site-directed, see Site-directed mutagenesis zinc enzymes, 915-917, 928-931, 936 Mutants of amicyanin, 838 amine oxidase, 736 calmodulin, 109 carboxypeptidase A, 890 chloroperoxidase, 161-165, 170, 173 EGF module, 132 enterotoxin, 905 hydrogenase, 678 insulin, 1011, 1012 isocitrate dehydrogenase, 80 lactoferrin, 590 lipoxygenase, 476, 477 phospholipase, 126 pyrophosphatase, 214 ribonucleotide reductase R2 protein, 520, 524, 525 rubrerythrin, 543 sphingomyelinase, 73 synthetase, 227 thrombin, 30 urease, 672, 673 xylose isomerase, 221 Mutase, 65 6-skeleton, 619-622, 631, 646, 652, 653 corrinoid-dependent, 605 CT complex, 647, 650-653, 655 glutamate, see Glutamate mutase human, 619 isobutyryl-CoA, 619, 620 lysine 2,3-amino-, 413, 425, 657 mammalian, 619 manganese, 239, 240 methyleneglutarate, 619, 620, 646 methylmalonyl-CoA, see MethylmalonylCoA mutase phosphoglycerate, 239 vitamin BjB-dependent,see Vitamin BIB-dependent mutase MutT enzyme, 238 MutY, 397, 398 active site, 398 Mycobacteriurn srnegrnatis, 73, 203 tuberculosis, 2 11

1159

SUBJECT INDEX Myeloperoxidase, 156, 278, 283, 310 active site, 312 calcium in, 310 disulfide linkage, 310 heme type, 274 leukocyte, 310 structure, 311, 312 Myoglobin, 297-299, 516, 630 axial ligation, 275 dioqgen binding, 298 distal site, 302, 337 expression, 325 function, 275 heme type, 275 iron oxidation state, 275 iron spin state, 275 list of, 281, 282 PDR codes, 281, 282 proximal ligand, 334, 338 redox potential, 327 sperm-whale, 298 structure, 298 Myohemerythrin, 516, 517 Myosin, 72, 1095 light-chain kinase, 103, 105 Myxococcus xanlhus, 209

N N. pharaonis, see Natronobactersum gonorrheae, see Neisseria meningitidis, see Neisseria europma, see Nitrosomonas NAD' -dependent alcohol dehydrogenase, 855, 935, 936 -dependent oxidoreductase, 201 NADH, 632, 732 NADP, 80, 201, 276, 391 NADPH, 80, 276, 304, 315, 321, 333, 336, 391, 392, 402, 511,525, 526, 530, 541, 544, 632 cytochrome c, 633 -cytochrome P450 reductase, 313 fen-edoxin reductase, 379, 382 reductase, 415, 511 Naphthalene l,a-dioxygenase, 376, 511 active site, 512, 513, 516 structure, 378, 512-514

Narcissus pseudonarcissus, 228 Natrorwhacteriurn pharaonis, 819, 834 Neelaredoxin, 374 Neisseria gonorrheae, 579, 580, 581 meningitidis, 581 Nernst potential, 40 Neurodegenerative diseases (see also individual names), 139 Neurogranin, 110 Neuroblastomas, 114 Neuronal sodium channel, 47 Neurospora crassa, 732 Ncurotransrnitters (see also individual names) biogenic amines, 799 carbon monoxide, 302 catecholamine, 484 serotonin, 484 Neutron diffraction studies, 227, 299 Neutron scattering studies of calmodulin Neutrophils, 137, 138 Nickel (oxidation state undefined) (in) "Ni, 692 bioinorganic role, 670 carbon monoxide dehydrogenase, 683-685, 688, 1057 cofactor F430, 671, 682, 683, 693-697 diphtheria toxin repressor, 229 hydrogenascs, see [NiFcI-hydrogenase metallothionein, 1005 properties, 670, 687 serum albumin, 862 transport, 687, 584 uptake, 687 Nickel chaperonins, 687, 688 Nickel-containingenzymes (see also individual names), 197, 669-700 Nickel superoxide dismutase, 202, 671 active site, 205 biological role, 685 inhibitors, 699 properties, 685 redox potential, 699 Nicotiana plumbagznifolia, 203 Nicotinamide adenine dinucleotide, see

NADf Nicotinamide adenine dinucleotide (reduced), see NADH Nicotinamide adenine dinucleotide phosphate, see NADP

N i c o t i n ~ i d eadenine dinucleotide phosphate (reduced), see N ~ P H Nicotinamide h ~ o x ~ t h i dinucleotide, ne 80 Nicotinic acid, 184, 187 nase, 424, 671, 688 15N-enriched,679 c a t ~ y t i cmechanism, 690-693 electron transfer pathway, 692 binding, 692 nickel redox states, 690 nickel site, 679 structure, 6 7 ~ 6 8 0 Xe derivative, 692 Nitrate re~uctase,387, 1025, 1052, 1069 assimilatory, 1048, 1051 dissim~atory,1048-1051 eukqotic, 1048 structures, 1048-105 1 Nitrate respiration, 286 Nitric oxide, 498, 501, 733, 900 as messenger, 321, 1093, 1094 binding to extradiol dioxygenase, 509 eduction, 391 from nitrite reductase, 334, 335 heme coordination, 274, 285, 288, 302, 322,323 in nitrile hydratase, 544, 545 transport, 300, 336 Nitric oxide synthase active site, 315, 316 axial ligation, 276 calmodulin binding, 315, 334 catalytic reaction, 333 endothelial, 904 expression, 316 function, 276 heme type, 276 iron spin state, 276

structure, 316 s t ~ c t u r ~ - f ~ n c trelations~ips, ion 333, 334 zinc in, 904 itr rile hydr~tase,552

fungal, 544

[Nitrile hydratase] iron site, 546 nitric oxide binding, 544, 545 plant, 545 photoactivation, 544 reaction mechanism, 544-4547 structure, 340, 545 Nitrite reduction, 379, 390-392, 831 respiration, 286 Nitrite reductase, 286, 317-319 active site, 318, 319 assimilatory, 295, 391 axial ligation, 277 catalytic reaction, 334, 335 copper binding sites, 781 copper-cont~ning,391, 765-767, 770, 774-776, 780, 825 cupredo~ndomain, 775, 817 cytochrome c, 277, 285, 318, 319,335 c~ochromecdl, 277,284, 317, 318, 334, 335,1095,1106 dissimilatory, 713 evolution, 801-805 function, 277, 774 green, 832, 836 heme type, 277, 317-319 iron oxidation state, 277 iron spin state, 277 loop topology, 821 PDB codes, 284, 285 p r o ~ a ~ o t i 1048 c, structure, 318, 319, 392, 393, 775 structure-function relationships, 334 Nitrogen I5N, 711 ~itro~enase di-, see ~initrogenase

s t ~ c t u ~ e1s0, 3 ~ 1 0 3 8 redox pot en ti^, 394, 39 tungsten in, 1029 ~ / F e /482 ~, vanadium in, 167-169 itrogenase iron protein, 167, 168, 363, 393-396, 411,412,424

1161

SUBJECT INDEX [Nitrogenase iron protein1 cluster conversion, 424 phosphate binding loop, 394 properties, 393, 394 redox potential, 394 structure, 394-396 Nitrogen fixation, 167, 299, 379, 382, 393, 394, 573, 1024, 1036, 1102 Nitrogen monoxide, see Nitric oxide Nitrophenylphosphate, 173 Nitrophorin, 274, 300, 301 active site, 300, 301 axial ligation, 275 disulfide bonds, 300 function, 275, 285 heme type, 275 heme-binding pocket, 337 iron oxidation state, 275 iron spin state, 275 PDB codes, 282 proximal ligand, 337 secondary structure, 336 structure, 300, 301 Nitrosornonas europaea, 284, 292, 317 Nitrous oxide reductase, 733, 734, 835 copper site, 712, 835 NMR ‘H, 419,487, 489, 491 ”N,130, 132 ‘lP, 61, 62 probes, 71 NMR structural studies of annexin, 119 ATPase, 873 blue copper protein, 836 calmodulin, 102-104, 107, 109, 110 carboxypeptidase, 934 corrinoid, 618 cytochrome c oxidase, 833 EGF modules, 129-132 ferredoxin, 382, 419 iron-sulfur proteins, 373, 387, 418, 419 insulin, 1030 metallothionein, 872, 1004, 1005, 1007, 1014 MgATP, 61,62 phenylalanine hydroxylase, 487, 489, 491 phosphatase, 938 plastocyanin, 822 probes, 71 protein kinase C, 127, 893 pymvate kinase, 77 rubredoxin, 373

[NMR structural studies 051 SlOO protein, 112-114 synaptotagmin, 125 zinc fingers, 975, 977-979 Noncorrinoid cobalt enzyme, 544 Nonheme iron protein charge distribution, 554, 555 classification, 468470 conformational flexibility, 555 effects of the coordination environment, 552-554 list of, 469 nitrogen coordination, 464-557 oxygen coordination, 464-557 properties, 465-467 structural properties, 470, 471 structure-function relationships, 550-557 Norepinephrine, 730, 731, 798, 799 Nuclear magnetic resonance, see NMR NQE spectroscopy, see Nuclear Overhauser effect spectroscopy Nuclear hormone receptors, see Zinc finger domains Nuclear Overhauser effect spectroscopy studies of calmodulin, 109 Nu c1ease 5’3’-DNA, 210 endo-, see Endonuclease exo-, see Exonuxlease manganese in, 210 ribo-, see RNase Nucleophilic attack of disulfide, 414 ferric superoxide, 509 hydroxide, 78, 213, 242, 546, 689, 690, 697, 900,932,934 Ni(I)-F430,695 thiolate, 699 water, 79, 213 Nucleoside diphosphates (see also individual names) kinase, 74, 75, 209 Nucleoside monophosphates (see also individual names) kinase, 72, 74 Nucleoside phosphates, see Nucleotides and individual names Nucleosides, see individual names Nucleoside triphosphates (see also individual names), 74, 75, 77 hydrolysis, 20, 79, 238, 241 5 ‘-Nucleotidase, 549

1I62 Nucleotides, see individual names Nucleotidyltransferases, 61

Octopus dofleini, 717, 726, 748 Oligosaccharides, 133, 134, 136, 230 Oligotropha carlxqdovorans, 1057-1059 Oncogenes ras, 78, 79 Ornithine formation, 217 Osmium(I1) carbon monoxide binding, 1108 Osteoporosis, 95 Ovotransferrin, 585, 590-592 chicken, 578-580, 583 duck, 579,580 structure, 582 Oxdoacetate, 210, 217, 240 Oxalosuccinate, 201 Oxidase (see also individual names) aldehyde, 1051, 1069, 1071 amiiie, see Axnine oxidase 1-aminocyclopropane-1-carboxylicacid, 492 bilirubin, 834 calcium, 70 cwtechol, see Catechol oxidase copper blue, 821, 834, 835 cytochrome c, see Cytochrome c oxidase dihydrogeodin, 835 glyoxal, 721 human, 202 hydrogen sulfide, 230 lysyl, 730, 862 manganese in, 202, 230 multi-copper, see Multi-copper oxidase 6-oxoglutarate-dependent,see 6Oxog~ut~ate-dependent oxidase quinol, 767, 803, 832 sulfite, see Sulfite oxidase sdockin, 835 ubiquinol, 727 xanthine, see Xanthine oxidase Oxidation-reduction potentials, see Redox potentials Oxidative damage, 305, 306,463, 573, 576, 860, 861 stress, 285, 382, 415, 425, 685

SUBJECT INDEX Oxidoreductase (see also Peroxidase and individual names) aldehyde ferredoxin, see Aldehyde ferredoxin oxidoreductase aldehyde oxidoreductase, see Aldehyde oxidoreductase L-ascorbate:dioxygen,see Ascorbate oxidase benzendiol oxygen, see Laccase bilirubin:dioxygen, 834 Fe(II):dioxygen, see Ceruloplasmin ferrocytochrome-cdioxygen, see Cytochrome c oxidase formaldehyde ferredoxin, 1062, 1063, 1070 heme type, 274 hydroxylarnine, see Hydroxylamine oxidoreductase manganese in, 201-207, 231-236 NADPN:ferredoxin, 530 p-diphenol:dioxygen, see Laccase pyruvate:ferredoxin, see Pyruvate:ferredoxin oxidoreductase ubiquino1:ferricytochrome c, 297 zinc, 886, 894, 896 6-Oxoglutarate dehydrogenase, 231 6-Oxog~utarate-depenaeiitoxidase, 491-502, 555, 556 iron site, 494 plant, 491 reaction mechanisms, 499-502 structures, 494.499 Oxotransferase (see also individual names) bacterial, 1039 classification, 1027, 1028 fungal, 1039 list of, 1064-1067 molybdenum, 1024-1033, 103%-1059, 1064-1067, 1070 structures, 1038-1059 tungsten, 1026-1033 Oxygen (different oxidation states) (see also Dioxygen) 180-labeling, 540, 1042 radical, see Radicals reduction, 202, 788-792 singlet, 156 toxicity, see Toxicity Oxygenase, 69 CYC~O-,283 heme, see Heme oxygenases human, 320, 321

S U ~ J ~ CINDEX T Oxygen evolving complex (see also Photosystem 11) active site, 235 bacterial, 235 calcium in, 233-235 chloride, 233, 235 iron(T1) in, 233 manganese, 233-236, 243,244 strontium in, 235

P.

abyssi, see Pyrococcus acetylin,icus, see Pelobacter acidovorans, see Pseudomonas aeruginosa, see Pseudomonas aureofaciens, see Pseudomonas cepacra, see Pseudomonas denitrificans, see Paracoccus diminuta, see Pseudomonas fluorescens, see Pseudomonas fieudenreichii, see Propionibacterium furiosus, see Pyrococcus haloden,itricans, see Paracoccus pulida, see Pseudomonas pyrrocina, see Pseudomonas shermanii, see Propionibacteriuin stutzeri, see Pseudomonas thermocarboxydouorans, see Pseudomonas versu?tus,see ParacoccrLs uiiale, see Penicillium ~ 2 1 protein, ' ~ 78, 79 P450 protein axial ligation, 276 function, 276 heme type, 276 iron oxidation state, 276 iron spin state, 276 Palladium(II), 185 carbon monoxide coordination, 1107 Panulirus interruptus, 717, 719, 725, 726 Paracoccus denitrificans, 202, 717, 728, 729, 733, 819, 824, 832 halodenitricans, 387 uersutus, 824 Paramagnetic resonance, see EPR and

NMR

1163 Parkinson's disease, 139, 198, 799 juvenile, 484 Parvalbumin, 100 Pathogens, 133, 134, 137, 139 bacterial, see Bacteria human, see Human plant, 157 Pea amine oxidase, 716, 722 ascorbate peroxidase, 13, 28, 283, 306 lectin, 228 Pelobacter ucetylinicus, 1069 D-Penicillamine, 185 removal o f zinc from proteases, 922-924 Penicillin iso-, 494 isopenicillin-N synthetase, see Isopenicillin-N synthetase Penicillium uitale, 303, 304 Pepsin, 933 Peptidase (see also individual names) amino-, see Aminopeptidase artificial, 2 carboxy, 213, 934 endo-, 930 manganese in, 212, 213 zinc in, 908, 917 Peplide deformylase, 671 Peptidyl amidoglycolate lyase, 725 Peptidylglycine cc-amidating monooxygenase, 723, 724, 731 Peptidylglycine a-hydroxylating monooxygenase, 713, 717, 723-725, 731 active site, 718, 724 copper sites, 724 inhibitor, 724 mechanism, 738-742 reaction, 723, 731 structure, 724 Peptostreptococcus asaccharolyticus, 382 Permease nickel in, 687, 688 Peroxidase animal, 304,310-312 ascorbate, see Rscorbate peroxidase axial ligation, 275 bacterial, 283, 304, 305 bromo-, see Bromoperoxidase calcium in, 307, 338 chloro-, see Chloroperoxidase class I, 305-207, 209

[Pero~dase] class IX, 307-310 class IXI, 309, 310 co~poun I, ~330, 331 c ~ o c h r o m ec, see ~ ~ o c h r o m c e pero~dase distal site, 310, 337, 338 eosinophil, 156 fluoro-, 156 ~ n c t i o n275 , 74,283, 304, 305, 307 halo-, see Haloperoxidase heme type, 274,275 heme type, 275 h o r s e r a ~ i s283, ~ , 305, 308, 310 hydro-, 304

[Phosphatase]

glucose-6-, 173 human, 214 inorganic pyro-, 2 inositol mono-, 21 kidney bean, 238,

p h o s p h o t ~ o s i ~187 e, rotein p h ~ s ~ h a t a s e serinekhreonine protein, 549 t ~ r a t e - r e s i s t acid, ~ t 547, 5 hosphati~icacid phosphatase, h o s ~ h a t i d y l e t h ~ o l a m i121 ~e, Phosp~atidylinositolb a s e , 20 Phosphati~ylserine,116, 119, 121, 12 127 hosp~odiesterase,2 13 P ~ o s ~ ~ ~ e n o 16, l ~ 208, ~ ~ 217 ~ a t e , hosphoenolpyruvate c h o s p h o e ~ o l ~ ~ vea t e 75-77 manganese in, 217, 21 s t ~ c t u r e 76, , 77

phosphoinositide"speei~cCF 1, 1

henol h y ~ r o ~ l a s379, e , 525 hospholipi~,10, 1 1 5 - 1 ~ 119, ~, 1 125,128 m e ~ b r a n e1, 1 9 , l ~osphomonoesterase, ~ o s p h o ~ r o t e transfer in system zinc in, 904 Phos~horibosyltrans€erase ~ l u t a m i ~211 e, h ~ o x a n t h i n e 210, , 211 h~o~anthine~anidine, m a n ~ ~ e in, s e210, 211 quinolinic acid, 210 hospho~lase ~ l y c o ~ e208 n,

SUBJECT INDEX Phosphorylation (00 auto-, 74 histidine, 74 proteins, 50, 52, 53 serine, 53 Phosphoryl transfer, 62, 74, 75, 77 Phosphotransferase rnannose-1-, 230 Phosphotriesterase, 213 cadmium in, 916 zinc in, 916 Pliosphotyrosine phosphatase, 187 Photoactivation of nitrile hydratase, 544 Photolysis (of) corrinoid, 617, 647, 649, 650 cytochrome c oxidase, 746, 747 flash, 640, 747 Photosynthesis, 413, 416, 463, 573, 766, 774 anoxic, 287 bacterial, see Bacteria Photosynthetic reaction center, 291, 387, 834 Photosystem I, 286, 379, 384, 424, 822, 823 Photosyskm IT (see also Oxygen evolving complex), 233-236 Phthalate dioxygenase, 379, 511, 514, 515 Phylogenetic trees (of), 1097 copper proteins, 802, 804 high-potential iron-sulFur proleins, 442 iron-sulfur proteins, 432435 Rieske ISP subunit of cytochrome bel or b,f complexes, 438, 439 Rieske ISP subunit of dioxygenasemonooxygenasc systems, 440, 441 rubredoxin-like proteins, 436,437 Phytase, 174 Phytochelatins, 814, 1013 Phytocyanin, 803, 818, 827, 828, 834 types of, 828 Pichin pastoris, 586 Pig aniinc oxidase, 722, 736 aminopeptidase, 212 insulin, 1008, 1009, 1013 kidney, 187, 216 kinase, 209 liver, 73 purple acid phosphatase, 238 Pisum satiuum, 228, 283 Plantacyanin, 818, 828 loop topology, 821

1165 [Planlacyanin] metal site, 820 structure, 829 Plant (see also individual names) arnine oxidase, 721 ascorbate oxidase, 771 blue copper proteins, 816 catechol oxidase, 726 cytochrome, 293, 313 dehydrogenase, 201 ferredoxin, 360, 379-381, 385, 391, 407, 416, 420, 432 ferritin, 573, 577, 588 globin, 297, 299 heme oxygenase, 320 hemoglobin, 299 hormones, 473, 492, 713 iron transporters, 584 isomerase, 220 laccase, 766, 772, 786 lectin, 133, 134, 227 lipoxygenase, 232, 471,473-476 manganese in, 199, 200 metallothionein, 1003 nitrile hydratase, 545 2-oxoglutarate-dependent oxidase, 49 1 oxygen evolving complex, 233, 235 pathogens, 157 plastocyanin, 820, 822 purple acid phosphate, 238 siroheme-containing protein, 391 superoxide disniutase, 686 urease, 671, 672 vitamin Biz, 632 Plasma (see nZso Serum) calcium in, 129, 131 human, 129 magnesium in, 64 membrane, 40 tetranectin, 138 Plasminogen, 138 Plastocyanin, 183, 287, 297, 766, 818, 840 active site, 824 amino acid sequence, 819 bacterial, 822, 824 Cd(T1)-substituted,822 Cu(1)-substituted, 822, 824 cupredoxin fold, 767-769, 782, 816 evolution, 802-804 loop topology, 821 metal site, 820 mutant, 839

1166 IPlastocyanin] occurrence, 820, 822 poplar, 817, 819, 820, 822, 825 redox potential, 822,823, 837, 840 structure, 817, 822-824 P1astoquinol:plastocyanin reductase, 297 Plastoquinone, 233 Platelets, 114, 133 aggregation, 300, 322 Phtinum(II), 185 carbon monoxide coordination, 1107 in metallothionein, 1005 Pneumococcal surface antigen adhesin A manganese in, 199, 230 zinc in, 199, 230 Polymerase DNA, see DNA polymerase magnesium in, 210 RNA, see RNA polymerase Polyporus pinsitis, 830 Polysaccharides (see also Carbohydrates and individual names), 30, 138 Populus niger, 523, 525, 822 Porphyrins (see also Heme and individual names), 615, 617,618, 630, 631 coball in, 610, 616, 656 Cr(1II) complexes, 183 proto-, see Protoporphyrin IX radical, see Radicals Rh(II), 637 Porphyromonas gingiualis, 203 Potassium ion (in) ascorbate peroxidase, 307 bioinosganic chemistry, 10 carbamoyl phosphate synthetase, 13, 23, 24

channel proteins, 39-53, 1095 channel, see Ion channel coordination chemistry, 10, 11 coordination sphere, see Coordination spheres interaction with proteins, 9-31 kinases, 209, 210 list of enzymes, 12-15 rnutases, 621 PDB codes of proteins, 11-15, 17, 3 8, 21, 22, 24-27, 29, 46 radii, 11 sodium pump, see ATPases Potatoe sweet, 238, 717, 719, 727 Potentials

[Potentials] membrane, 40,41, 44, 49, 63 redox, see Redox potentials Prenyltransferase, 2 12 Primase, 971 Prokaryotes (or prokaryotic) (see also individual names) amidotransferase, 399 anaerobic, 425 arylsulfatase, 911 cytochromes, 287 ferredoxin, 384 furnarate reductase, 406 Krebs cycle in, 389 magnesium transport, 62, 63 nitrate reductase, 1048 pyrophosphalase, 214 Rieske protein, 375 superoxide dismutase, 860, 866 zinc fingers, 987, 988 Prolactin receptor, 915, 916 Prolidase, 212 cobalt in, 1110 deficiency, 237 manganese in, 237 Prolinase manganese in, 237 Prolylglycine Cr(II1) in, 186 1,2-Propanediol,19, 20 Propionase guanidino-, 239 Propionibacterium fieudenreichii, 232 shermanii, 203-205, 622, 685 Prostaglandin, 126, 471 Prostaglandin H synthase, 311, 312 active site, 311, 312 PDB codes, 283 structure, 312 Protease (see also Peptidase, Proteinase, and individual names) alkaline, 890, 911 B, 910 C , 132 endo-, 909, 910, 915 inhibition, 921, 922 new members, 914, 915 NS3,891 S2P, 915 serine, 30, 132, 780, 921, 930 snake venom, 910,911

1167 [Proteasel sterol-regulatory element binding proteins, 915 zinc, 908-911, 929-931, 933-935,963 Protein(s) (see aEso Enzymes and individual names) ACE, 714 actin-binding, 110 ADRl, 964 ALR, 64 ATP-binding, 237 B, see Methane monooxygenase bilin-binding, 300 blue copper, see Blue copper proteins and Type 1 copper C, 130, 779, 780 C2 domain, 121-128 calcium-binding, see Calcium-binding proteins CAMPreceptor, 414 carbon monoxide-sensing transcription regulator, 684 catabolic gene activator, 414 CheY, 72 CooC, 688 CooJ, 688 copper, see Copper protein CorA, 62-64 CTR, 714 divalent metal transporter-1, 584 DNA repair, 396-398, 904 DNA-binding, 576, 585, 908, 963 Drcll, 516 EF-hand, see EF-hand protein epidermal growth factor-like modules, see EGF module feoB, 584 Fepr, see Fuscoredoxin Fet3, 766, 767, 834 flavo-, 403 FNR, see Fumarate and nitrate reduclion regulator FRE1, 714 FTR1, 584 Fur repressor, 240 G, see 6-protein ~ ~ Y c o134, - , 578, 579 GTP-binding, 237 heat-shock cognate, 12, 20-22 heme, see Hemeproteins HoxN, 688

IProtein(s)l HypB, 688 IREGI, 584, 585 iron in, 269-340 iron storage, 571-594 iron transporter, 571-594 IRTI, 584 lipo-, 198 MACl, 714, 715 mannose-binding, see M~nose-bind in^ protein membrane-bound transferrin-like, 585 molecular chaperones, 20 molybdenum, see Molybdenum enzymes/ proteins NiE, 415 NikA, 687 NikC, 688 nitrogenase iron, see Nitrogenase iron protein NixA, 688 nonheme iron, see Nonheme iron proteins Nramp2, 584 p l l , 116 P450, 276 p53, 110, 113 pancreatic stone, 138 PAS, 301 phosphorylation, 50, 52, 53 -protein interactions, 132, 908, 963, 965, 970-972, 975, 977, 980 Rab, 78 retinal-binding, 300 S, 130 S100, 109-116 sea raven antifreeze, 138 SFT, 584 signal-transducing, 904 SoxR, 372, 415 spingolipid Ca2 release-mediating protein from endoplasmic reticwlum, 97 superantigens, ,904906 trans-membrane, 10, 15 TroA, 892 tumor suppressor p53, 963 tungsten, 1023-1072 UreE, 688 vitamin B12-binding,606-660 X I I , 892

'

1168 Proteinase (see also Protease an,d individual names), 890 K, 95, 582 matrix metallo-, see Matrix metalloprotein Protein Data Bank codes (of), 2, 11 calcium proteins, 103, 111, 112, 117 copper proteins, 716, 717, 820, 823, 875-877 chloroperoxidase, 283 copper-zinc superoxide dismutase, 867-870 corrinoids, 622 cyclooxygenase, 283 cytochromes, 280,281,283, 284 E'ixL, 284 globins, 281, 282 ferritins, 574 hemes, 280-285 insulin, 1008 iron-sulfur proteins, 366-369, 399, 418, 419,426 magnesium proteins, 65-70 manganese proteins, 204, 209, 221 metallothioneins, 1004 nonheme iron proteins, 469 molybdenum proteins, 1067 multi-copper oxidases, 767 mutascls, 622, 623 nickel enzymes, 671-673, 676 potassium proteins, 11-15, 17, 18, 21, 22, 24-27, 29,46 sodium proteins, 11-15, 17, 21, 22, 30, 46 transferrins, 580 urease, 671-673 vanadium proteins, 158 zinc finger proteins, 966, 969, 973 Protein interface zinc site, 884, 902-906 list of, 903 Protein kinase C, 48, 97, 110, 116, 122, 126, 127, 893, 981 Ca'+/ca~mod~i~~-dependent, 103, 104, 106 CAMP-dependent,209 eukqotic, 97 insulin receptor, 187 zinc binding, 127, 971, 972, 981 Protein phosphatase, 213, 244 2B, see Calcineurin active site, 215 bacteriophage 1, 214

SUBJECT INDEX [Protein phosphatase] human, 214 iron in, 214, 215 manganese in, 214 serinelthreonine, 549, 900 tyrosine, 214 zinc in, 214, 215 Proteus rnirabilis, 1039 uulgaris, 1039 Protocatechuate, 481, 508, 509 3,4-dihydro~henylacetate,480, 483 Protocatechuate dioxygenase 3,4-, 479-483 4,5-, 502, 503, 507 coordination sphere of iron, 481 cyanide binding, 483 inhibitor complexes, 482, 483 LigAB, 507-509 structure, 480-482, 507, 508 Proton magnetic resonance, see NMR Protoporphyrin IX, 28, 272, 279, 576 chelatase, 72 Pseudoazurin, 317, 733, 775, 776, 803, 818, 832 amino acid sequence, 819 loop topology, 821 mutant, 839 structure, 825 Pseudomonas sp., 239 acidouorans, 323 aeruginosa, 203, 284, 318, 803, 804, 819, 820,825, 890, 909, 911 aureofaciens, 831, 832 cepacia, 15, 231 diminuta, 916 fluorescens, 219 K172, 231 putida, 219, 223, 224,231, 379, 505, 826 pyrrocina, 156 stutzeri, 733, 835 thermocarboxydovorans, 1058 Psoriasin, 111, 114116 calcium in, 111, 11615 ?303-, 111, 1 structure, 116 zinc in, 111, 115, 116, 906 Psoriasis, 115, 472 I? t erins molybdo-, see Molybdopterin P, 1039-1041, 1043-1045 pyrano-, see Molybdopterin

1169 [Pterins I Q, 1039-1045 tetrahydrobio-, 315, 334, 485, 488-490, 904 Pterin-dependent hydroxylase, 484-491 bacterial, 485 human, 485 reaction mechanism, 489-491 structure, 486-489 Purple acid phosphatase, 213, 552, 553 active site, 238, 548 bacterial, 547 Fe(I~~)/Fe(II), 900 Fe(III)/Zn, 900 iron in, 238, 244, 547-550 kidney bean, 537, 547449 mammalian, 237, 238, 547449 manganese in, 237, 238, 244 pig, 238 reaction mechanism, 549, 550 soybean, 238 zinc in, 238, 547-549 Putidaredoxin, 379, 380 Putrescine, 239 Ryranopterin, see Molybdopterin 3-Pyridine carboxylic acid, see Nicotinic acid P,yridoxal 5’-phosphate, 323 phosphate-dependent enzymes, 15, 22, 25, 26, 28 J?y rimidine biosynthesis, 225 metabolism, 492 Pyrococcus abyssi, 403 furiosus, 213, 373, 374, 900, 1025, 1060-1063, 1067 kodakaraensis, 226 Pyrophosphatase, 214 Pyruvate, 15, 16, 19, 26, 28, 65 carboxylase, 240 dehydrogenase, 421 kinase, Pyruvate kinase phosphoenol, 16,208, 217, 218 synthase, see Pyruvate:ferredoxin oxidoreductase Pyruvate:ferredoxin oxidoreductase, 383, 365,373 biological role, 408 conformational change, 410 sequence motifs, 409, 410

[Pyruvate:ferredoxin oxidoreductasel structure, 408-410 Pyruvate formate-lyase activase, 425 sequence motif, 411, 412 Pyruvate:flavodoxin reductase, 382 Pyruvate kinase, 11, 12, 16, 18, 19, 76, 77 active site, 18, 19 bacterial, 16 manganese in, 208-210 potassium in, 210 rabbit muscle, 16, 19, 77

Queuosine, 637 Quinolinic acid phosphoribosyltransferase, 210 Quinone (see also individual names) in fumarate reductase, 406 lysine tyrosyl, 730 trihydroxyphenylalanine, 721-723

R. capsulaius, see Rhodobacter erythropoli, see Rhodococcus &obi formis, see Rhodopila marinus, see Rhodothermus meliloti, see Rhizobi urn rubrum, see Rhodospirillum sphaeroides, see Rhodobacter and R hodopseudomonas viridis, see Rhodopseudomonus Rabbit aldehyde oxidase, 1057, 1069 DNA polymerase, 210 EF-hand protein, 98 lipoxygenase, 473, 474 liver, 187 metallothionein, 1003-1006, 1014, 1015 muscle, 16, 19, 77, 210, 214 transferrin, 578-580 Racemase mandelate, 219, 223, 242 magnesium in, 224 manganese in, 224 Radical(s1 (see also individual names) adenosyl, 608, 609, 619, 630, 639, 657, 658, 1101 “clock”, 538

1170 [Radical(s)J cyclopropyl, 509 cysteine, 410, 721 5’-deoxyadenosyl,19, 232,411-413, 425 glycyl, 232, 413 , 520, 652, 658 heterodisulfide, 697 hydroxyl, see Hydroxyl radical in ribonucleotide reductase 132 protein, 537, 538 ketyl, 734 methane, 538 methyl, 614, 617, 697 oxygen, 539, 791 peroxy, 478 porphyrin cation, 28, 330, 332-334 protein-bound free, 410, 609, 612, 627, 638, 1101 scavenger, 525 substrate, 608, 609, 619, 628, 630, 637, 638, 646-652, 655 superoxide, see Superoxide thiyl, 621, 646, 658, 592, 697 trap, 647 tryptophan, 28, 307,331, 521, 524, 537, 555 tyI*osyl,232, 520-522, 524, 525, 534, 538. 540,555,658, 720, 721, 734, 746, 748 tyrosylicysleinyl, 410 Ramachandran coordinates of iron-sulfur proteins, 437, 439, 444 Raman spectroscopy studies of cytochrome c oxidase, 746 dimethylsulfoxide reductase, 1042, 1043 galactose oxidase, 720 hemerythrin, 516 hemocyanin, 748 oxygen evolving complex, 235 ribonucleotide rductase, 520 sulfite oxidase, 1053 tyrosinase, 749

Rana catesbeiana, 574 nigromaculata, 732 Rat brain, 201, 216, 237 calcium-bindingproteins, 103, 104, 112, 117, 118, 134, 136-138 chromium in, 182 copper proteing, 717, 719 DNA polymerase, 210 ferritin, 585 fluorine in, 183

Bat1 heme oxygenase, 323 lead in, 183 lipoxygenase, 475 liver, 217 mannose-binding proteins, 134, 136, 138 metallothionein, 1003, 1004 pancreas, 213 purple acid phosphatase, 238 silicon in, 183 sulfite oxidase, 1051, 1069 tin in, 183 vanadium in, 183 Reactive oxygen species, 685, 814 Redox potentials (in) micyanin, 825, 837 carbon monoxide dehydrogenase, 697 ceruloplasmin, 831 Co(1) corrinoid, 609, 613 Co(II)/Co(l), 635, 657, 683 copper proteins, 326 Gu(II)/Cu(I),711, 839 cytochromes, 286, 288-291, 297, 326-328 endonuclease 111, 396 I~e(1I)/Fe(IH), 326, 327 Fe(III)/~e(II), 1054 Fe,S4 cluster, 396, 399, 402, 403, 421 ferredoxin, 379, 382, 421, 442 fuscoredoxin, 403 halocyanin, 834, 837 hemes, 274, 289 high-potential iron-sulfur protein, 387, 416-420 iron, 467 iron-sulfbr protein, 326, 416420 laccase, 830, 840 lipoxygenase, 476 ~ n ~ I I ) ~ n ( l 197 II), Mo(V)/Mo(IV), 1043, 1051, 1054, 1057 Mo(VI)/Mo(V), 1043, 1051, 1054, 1057 myoglobin, 327 Ni(lI)/Ni(I),683, 697, 699 Ni(III)/Ni(ll), 686, 692, 699 nitrogenase, 394, 396 Oz/Oz ,684 02 /H202, 685 peroxidase, 330 plastocyanin, 822, 823, 837, 840 Rieske proteins, 419, 514 rubrerythrin, 541 rusticyanin, 826, 827, 837, 840 siroheme, 392

S U ~ J INDEX ~ ~ T [Redox potentials (in11 SoxR protein, 415 stellacyanin, 829, 837, 839, 840 sulfite reductase, 392 superoxide disn~utase,205, 699 trimethylamine dehydrogenase, 402 vanadium, 154 Reductase, 168 aquacobdamin, 633 biotin sulfoxide, 1069 cob(Il)alamin, 633 cyanocobalamin, 633 cytochrome bg, 293, 512 cytochrome c, 318 cytochrome P450, 313, 321 dihydropterin, 485, 626 dimethylsulfoxide, see Dimetliylsulfoxide reductase ferredoxin, 300, 313, 379, 382 ferredoxin:thioredoxin, 372, 413 flavin-containing, 511 flavocytochrome c3 fumarate, 291 fumarate, see Fumarate reductase glutathione, 402 heterodisulfide, 681 methane monooxygenase, 526 methyl coenzyme M, see Methyl coenzyme M reductase NADPH,415, 511 NADPH-cytochrome c, 633 NADPH-cytochrome P450, 313 nitrate, see Nitrate reductase nitrite, see Nitrite reductase nitrous ocide, see Nitrous oxide reductase oxido-, see Oxidoreductases plastoquinol:plastocyanin, 297 pymTvate:flavodoxin, 382 trimethylamine oxide, see Trimethylamine oxide reductase Reduction potentials, see Redox potentials Relaxin, 1008 Resonance Raman spectroscopy, see Raman spectroscopy Retroviral integrase, see Integraae ribonuelease, 70, 71 zinc finger domain, see Zinc finger domains Reverse transcriptase, 210 HIV-1, 70, 71 Rheuniatoid arthritis, 922, 924

1171 Rhrzobium sp., 988 meliloti, 284, 301 Rhodium(I1) in porphyrins, 637 Rhodium(I11) carbon monoxide binding, 1108 Rhodrbius prolixus nitrophorin, 300, 301, 337 R hodobacter capsulatus, 675, 1028, 1034, 1039-1043, 1049, 1069 sphaeroides, 831, 1039-1044, 1049, 1069, 1071 Rhodococcus erythropoli, 231 R312, 545 Rhodopila globiformis, 418 Rhodospirillum rubnsm, 69, 238, 284, 302, 303, 402, 676, 684, 688, 697 Rhodopseudomori as sphaeroides, 231 viridis, 291, 292 Rhodotherinus marinas, 387 Rhus uernieifera, 828, 830 Riboflavin 5'-monophosphate, see FMN Ribonuclease, see RNase Ribonucleic acid, see RNA Ribonucleotide reductase, 372, 586-589 anaerobic, 411, 412, 425 class 111, 410-412 classes, 232, 410, 519, 520 cobalt in, 524 Fe4S4,411 iron in, 232, 244, 519-525 manganese in, 232, 233, 244 radical formation, 658 vitamin B12-dependent, 605, 609, 619-623, 632, 639, 642, 643, 645, 646, 651 Ribonucleotide reductase R2 protein, 519-525,527, 530, 531, 533, 540, 542, 544, 551, 553 azide in, 525 Co(l1)-substituted, 524 hydrophobic patch, 522, 524 iron site, 522, 523 Mn (11)-substituted, 524 radical generation, 537, 538, 555 reaction cycle, 520, 521 structure, 520-525, 533-536 Ribosyltransferase manganese in, 210, 211, 236, 237 phospho-, see Phosphoribosyltraiisferase

1172 Ribozyme, 70, 79, 80 group I intron, see Introns hairpin, 79, 80 hammerhead, 79 magnesium in, 210 n-Ribulose, 222 Rieske center, 297, 362, 511 Rieske protein, 373, 430 consensus sequence, 377 eukaryotic, 375 Fe&, 373, 431, 443, 444 mutation, 419 properties, 375 redox potential, 419, 514 structure, 360, 376-378 Rieske-type dioxygcnase, 478, 511-516, 553,555-557 active site, 513, 514 reaction mechanism, 514-516 structure, 512-514 RNA synthase, 226, 242 RNA polymerase, 207, 415 11, 971, 972 manganese, 210 RNase 13, 70, 71 Rubidium ion, 41, 48 tryptophanase activation, 26 Rubredoxin (see also Iron-sulfur proteins), 359, 360, 362, 428, 430, 431, 436, 437, 443, 444, 543, 970, 979 dielectric constant, 418 properties, 372-375 redox potential, 373, 417, 418, 541 sequence motifs, 373 structure, 360, 361, 373, 374 Rubrerythrin diiron site, 542, 543 ferroxidase activity, 541 redox potential, 541 structure, 541-544 zinc in, 543 Rusticyanin, 803, 804, 818, 826 amino acid sequence, 819 loop topology, 821 mutant, 839 redox potential, 826, 827, 837, 840 structure, 827 Ruthenium(11) carbon monoxide binding, 3 108 -labeled blue copper proteins, 840 Ryanodine receptor, 97

SUBJ~~ INDEX T

s S. aureus, see Staphylococcus caespitosus, see Streptomyces cerevisiae, see Sachcharomayces coelicolor, see Strep foornyces cricetus, see Streptococcus elongatus, see Synechpococcus fiigidimarine, see Shewanella gordonii, see Streptococcus griseus, see Streptomyces lividms, see Streptomyces maltophilia, see S t e ~ o t r o p h o ~ n a ~ ~ marcescens, see Serratia rnassilia, see Shewane~la meliloti, see Sinorhizobium rnutans, see Streptococcus olivochromogertes, see Streptomyces pneumoniae, see Streptococcus pombe, see Schizosaccharom.yces ruhiginosus, see Streptornyces seoulensis, see S ~ r e p t ~ m y ~ e s sobrinus, see Streptococcus typhamurium, see Salmonella S100 protein (see also individual names), 109-116 SlOOfi, 110-113 list of, 111, 112 structure, 113 zinc binding, 115 Saccharornyces cerevtsiae (see also Yeast), 64, 169, 199, 200, 214, 219, 397, 419, 713-718 copper chaperone, 874 cytochrome c peroxidase, 280, 283, 305 cytochrome c, 324 metdlothionein, 1003, 1014 zinc finger, 964 Salmon,ella typhim~~riu?n, 62-64, 69, 201, 212, 224,232, 525, 538, 632, 1034 Sam~~um(I11), 125 as probe, 71 in lactoferrin, 593 Scanning tunneling microxopy, 233 Schiff bases (see also individual names) Cr(II1) complexes, 183 Schizosaccharomyces pombe, 231 Scorpion toxin, 43 SDS-PAGE of bromoperoxidase, 166 chloropcroxidase, 158

1173 S€!dS q U k k S , 155 Sea urchin metallothionein, 1003, 1004, 1007, 1013 Seawater chromium in, 182, 1109 copper in, 1109 magnesium in, 60 manganese in, 1109 molybdenum in, 1034, I109 nickel in, 1109 tungsten in, 1109 vanadium in, 155, 1109 zinc in, 1109 Selectin, 133, 134 E, 133, 134, 137, 138 L, 133 P, 133 SeJenium in carbon monoxide dehydrogenase, 1058, 1071 methylation, 636 Selenocytmate, 1058 Selenocysteine (and residues), 1046, 1058, 1070 Serine glycerophospho-, 119 p-lactamase, 492 phosphatidyl-, 116, 119, 121, 125, 127 protease, 30, 132, 780, 921, 930 threonine protein phosphatase, 549, 900 Serotonin, 484, 798, 799 Serum albumin (binding of), 858, 859 cadmium, 862 copper sites, 862, 863 disulfide bridges, 859, 862 hnction, 858, 859 goid, 862 mercury, 862 nickel, 862 silver, 862 structure, 859 Serum transferrin, 578, 582, 585, 590, 592 Serralysin, 890, 911 Serratia marcescms, 890 Seryl-tRNA synthetase, 226 Shewanella ~ r ~ g i ~ ~ 291 ~ a r i ~ e , massilia, 1043-1045 Sialic acid, 134, 778 biosynt~esis,207 Siderophores (see also individual names), 154, 464, 465, 1036

[Siderophoresl synthesis, 229 Signal-transducing protein IIA, 904 Signal transduction (in) bacterial, 63, 72 calcium-bindingproteins, 96, 97, 102, 121, 122, 125 FixL, 330 mammalian, 173, 322 zinc fingers, 981 Silicate magnesium, 60 Singlet oxygen, 157 Silver(1) (in) carbon monoxide coordination, 1107 Menkes’ copper-transporting ATPase, 873 metallothionein, 872, 1004 semm albumin, 862 Sinorhizobiurn rneliloti, 203 Sirohcme, 273, 274, 277, 279, 425 in enzymes, 391-393 redox potential, 392 Site-directed mutagenesis (of) cytochrome c peroxidase, 28, 29 cytochrome P450, 333 ferritin, 587 hemes, 324 inducing cluster conversion, 424 lactoferrin, 583 molybdenum enzymes, 1069 MUtY, 398 pyruvate formate-lyase activase, 412 Itieske iron-sulfur proteins, 419 transfcrrin, 592 vanadium chloroperoxidase, 170, 173 zinc enzyme, 915-917 Small-angle X-ray scattering studies of calmodulin, 107 EGF module, 131 Snake venom, 138 protease, 910, 911 Sodium ion, 64, 65, 77 bioinorganic chemistry, 10 channel proteins, 39-53 channel, see Ion channel coordination Chemistry, 10, 11 coordination sphere, see Coordination spheres interaction with proteins, 9-31 list of enzymes, 12-15 PDB codes of enzymes, 11-15, 17, 21, 22, 30,46

1174 [Sodium ion] -potassium pump (see also ATPases), 10. 15 radii, 1 1 Sodium dodeql sulfate polyacrylamide gel electrophoresis, see SDS-PAGE So& protein, 372 redox potential, 415 sequence motif, 415 Soybean lectin, 228 lipoxygenase, 473-477 purple acid phosphatase, 238 Sperm whale myoglobin, 298 Sphingomonas paucimobilis, 507 Sphingomyelin, 73 Sphingomyelinase, 73 Spinach cytochrome b6f, 378 ferredoxin, 378, 379 ferredoxin:thioredoxin reductase, 413 ~ructose~1,6-~isphosphatase, 216 nitrate reductase, 1051 nitrite reductase, 391 oxygen evolving complex, 236 photosystem 11, 233 Spin-labeling Bite-directed, 50, 51 Spirodela oligorrhiza, 238 Standard potentials, spe Redox potentials Staphylococcus aureus, 198, 905, 909 Steady-state kinetic studies of extradiol dioxygenase, 509 peptidylglycine a-hydroxylating monooxygenase, 739 protocatechuate 3,4-dioxygenase, 479 Rieske-type dioxygenase, 514 Stellacyanin, 803, 818, 819, 828, 834, 839 loop topology, 821 m e t d site, 820, 829 redox potential, 829, 837, 839, 840 Stenotrophomon,as maltophilia, 901, 914 Steroid hormone, 313 Streptococcus cricetus, 199 gordunii, 199, 215 mutans, 203, 215, 411, 685 pneumoniae, 199, 200 sobrinus, 199 Streptomyees BP., 732 caespitosus, 9 15 coelicolor, 686

LStreptotnyces sp.] griseus, 382 lividans, 45, 686 olivochrornogenes, 81, 221 rubiginosus, 221 seoulensis, 686 Stress hormone, 799 Stromelysin, 910 Strontium ion, 215 in oxygen evolving complex, 235 Struclural zinc sites, 884, 891-893, 900 list of, 894, 895 Subtilisin, 95 Succinate oxalo-, 201 Succinate dehydrogenase, 382 biological role, 406 Succinyl coenzyme A, 279, 621, 624, 648 Sugars (see also Carbohydrates and individual names), 10, 136 a&, 219 dolichol phosphate, 236 isomerase, see Isomerase keto, 219 transferase, see Transferase Sulktase, 207 ayl-, 237, 911-914 bacterial, 237 calcium in, 237 eukaryotic, 911 magnesium in, 237 mammalian cytosolic, 237 manganese, 237 Sulfide oxidation by cytochrome P450, 313 Sulfite reduction, 379, 391, 392 Sulfite oxidase, 295, 1032 active site, 1052 chicken, 1051-1053, 1070 human, 1051 list of, 1065 molybdenum in, 1027, 1065, 1069, 1071, 1072 rat, 1051, 1069 structures, 1051-1054 tungcrten in, 1029 Sulfite reductase, 365, 425, 437 active site, 426 axial ligation, 277 function, 277 heme type, 277 iron oxidation state, 277

1175

SUBJECT INDEX [Sulfite reductasel iron spin state, 277 properties, 391 redox potential, 392 structure, 392, 393 Sulfolobus SP., 382-385 acidocaldarius, 2 14 Sulfonium, 658 Sulfoxides, 174 Sulfur "S, 711 cycle, 1025, 1039 Sulfurospirillum deleyiarium, 285, 319 Superantigens (see also individual names) zinc sites, 904-906 Superoxide, 202, 241, 422, 425, 490, 715, 739-741,860 disproportionation, 685 Superoxide dismutase, 5, 541 active site, 203, 205-207 azide in, 203-205, 699, 866 cambidistic, 686 copper, 715 CulZn, see Copper-zinc superoxide dismutase eukaryotic, 860 FeiZn, 686 fluoride in, 203 human, 204 iron, 202, 203, 244, 685, 686 manganese, see Manganese superoxide dismutase mechanism of dismutation, 865-871 nickel, see Nickel superoxide dismutase prokaryotic, 860, 866 redox potential, 205, 699 structure, 203, 204, 207, 860 Synaptotagmin, 122-125 dissociation consiants, 122 electrostatic switch, 106, 124, 128 I, 122-128 stmcture, 123 S.ynechococcus elorgatus, 233 Synechocystis PCC6803, 199, 236 Syntaxin, 123, 124, 128 Synth(et)ase acetyl coenzyme A, see Acetyl coenzyme A synthatase S-adenosylmethionine, see SAdenosylmethionine aminoacyl-tRNA, 226 biotin, 412, 425

[Synth(et)ase] carbamoyl phosphate, see Carbamoyl phosphate synthetase clavaminate, 492-497 cystathionine p-, 278, 323 deacetoxycephalosporin C, 492-495, 497, 553 dethiobiotin, 226 dihydropteroate, 212 glutamine, 200, 224, 225 isopenicillin-N, see Isopenicillin-N synthetase p-lipoic acid, 413, 425 magneisum in, 224 methionine, see Methionine synthase molybdopierin, 1028, 1029 nitric oxide, see Nitric oxide synthase phenoxazinone, 835 prostglandin H, 283, 311, 312 tRNA, see tRNA synthase tryptophan, see Tryptophan synthase Synthesis (of) acetyl-coenzyme A, 684 ATP, 681 bio-, see Biosynthesis DNA, 410, 519 estrogen, 231 glutamate, 379 lignin, 773 siderophore, 229 Syphilis, 197

T. album, see Therrnoleophilurn aquaticus, see Thernius ferrooxidans, see Thiobacillus litoralis, see Thermococcus thermophila, see Tetrahym,ena thernzophilus, see ThJermus uersutus, see Thiobacillus Tapeworm, 631 Tartrate dehydrogenase, 201, 231 Technetium in metallothionein, 1005 Tellurate methylation, 636 Terpenes, 165, 166 Terpredoxin, 379, 380 Tetrahydrobiopterin, 315, 334, 4R5, 488-490, 904

1176 Tetrahymena thermophila, 70, 80 Tetranectin, 134, 138, 139 structure, 139 Thallium(I), 23 in transferrins, 593 Thermcoccus litoralis, 1062 Therrnoleophilum album, 206 Thermolysin, 95, 884, 904, 909, 925, 940 amino acid sequence, 910 bromide, 940 chloride, 940 fluoride, 940 inhibition, 920-922 mechanistic studies, 933, 935 properties, 929, 930 Thermotoga maritima, 386, 403 Thermws aquaticus, 68, 210 thermophilus, 201, 204, 206, 207, 214, 226, 241,832 YS&13,206 Thiamine pyrophosphate, 408410 Thiobacillus ferrooxidans, 387, 403, 819, 826, 827 versutus, 236 Thiocapsn pfennigii, 291 Thionein, 921, 924 metallo-, see Metallothionein Thioredoxin, 414, 415 Thiosphera pantotropha, 284, 318 Thiosulfate oxidizing enzyme, 236 Thiyl radml, see Radicals Threonine dehydrogenase, 23 1 Threoninciserine protein phosphatase, 549, 900 Thrombin, 13, 30, 779, 780 active site, 30 human, 30 mutant, 30 Thromboxane, 471 Thyroid hormone receptor, 988 TIM barrel, 98, 125, 624-631, 642, 654, 1099,1100 Tin(I1) in metallothionein, 1005 Tissue factor, 131 Titanium citrate, 693 Tomato, 216 aminopeptidase, 212 Tonin, 905 Toxicity of chromium, 181, 182

[Toxicity of manganese, 198 metals, 1,5 oxygen, 202 Toxins (see also individual names) bacterial, 322 diphtheria, 229 scorpion, 43 toxic shock syndrom, 906 Transamination reaction, 15 Transcobalamin, 632, 633 Transcriptases HIv-1 reverse, 70, 71 reverse, 210 Transcription activator, 415 Transcription factors IIB, 971, 972 IIS, see Zinc finger domains IILA, see Zinc finger domains Acel, 975 Amtl, 975 copper-dependent, 975 GAL4, see Zinc finger domains GATA, see Zinc finger domains Transferase (see also individual names) adenosyl-, 212. 633 amino-, 736 arylalkyl-, 212 aspartatc carbamoyl, 891, 894 farnesyl-, 886 galactosyl, 236 glutamine phosphori~osylpyrophosphate amido-, 211, 263, 372, 399401, 439 glutathione, 236 glyCoSyl-, 236 manganese, 207-212,236, 237 methyl-, see ~ethyltransferase nucleotidyl-, 61 oxo-, see Oxotransferases phospho-, 230 prenyl-, 212 ribosyl-, see Ribosyltransferase sugar, 210, 211, 236, 237, 244 sulfo-, see Sulfatase zinc, see Zinc transferase Transferrin, 183, 464, 466 Al(III), 186, 593 apo-, 796, 800 bacterial, 579-581 biological role, 578, 590 classification, 578, 579 cobalt in, 593

SUBJECT INDEX

I Transferrin] cooperativity for iron binding, 590 Cr(III),186, 187 dilysine trigger, 583 eukaryotic, 579-581 expression systems, 585 F'e(III), 186 horse, 579, 580 human, 579, 583, 586, 591,592 hydrophobic interactions, 581 iron binding sites, 581-584 iron uptake and release, 590-593 lacto-, see Lactoferrin list of, 580 manganese, 200, 243 melano-, 578, 585 m e ~ - s u b s ~ ~ t u t593 ed, ovo-, see Ovotransferrin rabbit, 578-580 PDB codes, 580 receptor, 372, 464, 573, 584, 590 receptor-independent pathway, 573 serum, 578, 582,585,590, 592 site-directed mutagenesis, 592 structures, 579, 580, 582, 583 thallium(l), 593 vanadium(II1) in, 593 vertebrate, 578 Transkctolase, 409 Transmembrane domains, 42-45,50,51 Transport o f (see also ~etabolism) copper, 5 fatty acid, 859 iron, 5, 464, 466 magnesium, 62-64 manganese, 5 nitrite oxide, 300, 336 potassium, 10, proteins, see individual names sodium, 10 tungstate, 1034, 1035 zinc, 5 Tree oriental lacquer, 772 Ti-eponema palladurn, 197, 892 Trimethylamine dehydrogenase, 365,437 properties, 401, 402 redox potential, 402 structure, 402, 403 T r i m e ~ h y l ~ oxide ~ n e reductase, 1029 structures, 1043-1045 T r i o s e p h o s p h ~isomerase, ~ see TIM barrel

1177 Trithionate, 391 tRNA synthetase, 242 aminoacyl, 226 asparaginyl-, 226 aspartyl, 226 seryl-, 226 Troponin C, 98, 100, 104 2 , 4 , 5 - T r i h y ~ o ~ p h e n y l a l ~quinone, ine 721-723, 736, 738, 748 Trypanosoma cruzi, 210 Trypsin, 933 inhibition, 921, 922 Tryptophan biosynthesis, 25 degradation, 26 fluorescence, 63, 119-121 fi-hydrov-, 484 hydroxylase, 481, 491 radical, see Radicals Tryptophanase, 13, 26-28 ammonium activation, 26 rubidium activation, 26 Tryptophan 2,3-dioxygenase, 323, 324 axial ligation, 278 function, 278 heme type, 278 iron oxidation state, 278 iron spin state, 278 Tryptophan synthase, 13 complex, 25, 26 active site, 25, 26 Tumor, 78, 114 suppressor protein p53, 963 Tunicate lectins, 135, 136 Tungstate, 161, 547, 548 in chloroperoxidase, 161, 162 transport, 1034, 1035 Tungsten (different oxidation states) (in) aldehyde ferredoxin oxidoreductase, 1060-1062, 1070 chloroperoxidase, 161, 162 coordination chemistry, 1030-1033 dimethyl sulfoxide reductase, 424 enzyme, see Tungstoenzyme formaldehyde ferrcdoxin oxidoreductase, 1062, 1063, 3070 iron-sulfur cluster, see Cluster sulfite oxidase, 1029 sulfur donors, 1031-1033 versus molybdenum, 1029 Tungsten proteins, 1023-1072

117 Tungstoenzymes (see also individual names), 1059-1063 list of, 1068 reaction mechanisms, 1071, 1072 structures, 1060-1063 Tunicates (see also Ascidians and individual names), 155 lectin, see Lectin Tunichromes, 155 Turkey EF-hand protein, 98 Type 1 copper (see also Blue copper proteins), 765, 766, 805, 815, 816, 818, 838 ascorbate oxidase, 770, 784, 787 azurin, 782 bond lengths, 823 cerdoplasmin, 770, 776, 782, 831 coordination geometry, 818 copper sites, 711, 712, 780-783 lacease, 774, 782, 787, 839 list of proteins, 823 nitrite reductase, 771, 775, 776, 783, 831, 832 plastocyanin, 782 rusticyanin, 826 Type 2 copper, 713, 765-767, 786,815,816, 838, 839 ascorbate oxidase, 770, 784, 793 binding sites, 781, 783, 784 blue oxidase, 830 ceruloplasmin, 784, 794 coagulation factor VTII, 780 cytochrome c oxidase, 728 laccase, 773, 774, 783, 786 methane monooxygenase, 733 nitrite reductase, 771, 776, 783, 784, 831, 832 peptidylglycine a-hydroxylating monooxygenase, 724 Type 3 copper, 713, 765-767, 786, 815, 816 ascorbate oxidase, 770, 784, 787, 793, 841 binding sites, 781, 784-787 blue oxidases, 830 ceruloplasmin, 794 nitrous oxide reductase, 733 tyrosinase, 732, 741 Tyrosine active site, 488 degradation, 503, 506 hydroxylase, 484486, 488

SUBJECT INDEX [Tyrosinel kinase, 975 phenol-lyase, 13, 27, 28 phosphorylation, 116 phosphatase, 214 radical, see Radicals Tyrosinase, 727, 731, 732 bacterial, 726 copper center, 73’2, 741, 749 dioxygen binding, 748 fungal, 732 human, 727, 732 mouse, 732 mechanism, 741, 743, 744 reactions, 731, 732 Tyrosinemia, 503 Tyrosine phenol-lyase, 13, 27, 28 active site, 28

u Ubiquino1:ferricytochrome c oxidoreduetase, 297 Ubiquitin, 980 Uclacyanin, 818, 828, 834 amino acid sequence, 819 loop topology, 821 Ulex europaeus, 228 Ulocladium chartarum, 157 Umecyanin amino acid sequence, 819 UMP, 74 Urea, 238, 239 hydrolysis, 689 Urease, 5, 688, 916 active site, 689, 672-674 biological role, 671 dinuclear nickel center, 674 fungal, 671 inhibitors, 673, 674, 689, 690 jack bean, 671 mechanism, 689, 690 operon, 688 structure, 671-674 Ureidoglycohte lyase, 239 Uridine 5 ‘-monophosphate, see UMP Urushiols polymerization, 773 UV-Vis spectrophotometry studies of corrinoid, 612, 615, 616, 634, 644, 645 cytochrome c oxidase, 746

1179

S U ~ J ~ CINDEX T 1UV-Vis spectrophotometry studies of] molybdenum oxotransferase, 1043 tyrosinase, 749

V Vanadium (different oxidation states) chemistry, 154, 155 haloperoxidases, see Haloperoxidase in Earth’s crust, 155 in rat, 183 oxidation states, 154 PDR codes of proteins, 158 properties, 154 proteins, 153-174 redox potential, 154 Vanadium(II1) in transferrin, 593 Vanadium(V1 andlor vanadate, 158, 159, 161, 162, 166, 171, 172,174 oxodiperoxo-, 154, 172 oxoperoxo-, 151, 172 properties, 154 Vancomycin, 917 Van der Waals contacts, 49, 51, 327, 517, 787 Vasodilatation, 300, 322 Veratryl dcohol, 331 Verdoheme, 335 structure, 336 Vertebrates (spe also individual names) calmodulin, 104 cytochrome P450, 313 ferritin, 573, 574, 576, 577 globin, 297-299 transferrin, 578 Vicia villosa, 228 Virus (or viral) (see also individual names) adeno-, 963 avian sarcoma, 210 baculo-, 325, 976 DNA, 520, 972 equine herpes, 980 hepatitis, 70, 891 lectin, 133, 227 Moloney murine leukemia, 210 PBCV-1, 212 retro-, see Retroviral Viscum album, 228 Vitamin($ (see also individual names) Biz, see Vitamin B12

LVitamin(s)] C, see Ascorbate D, 971 Vitamin B12, 19, 646 bacterial, 631 biosynthesis, 631, 632 corrin ring, 606 5 ‘-deoxyadenosinein, 606, 607 human, 632 in plants, 632 structure, 606 Vitamin Blz-dependent mutase (see also individual names), 608-616, 618-631, 633, 1099, 1100-1 102 C skeleton, 619-621, 631 Co-C bond fimion, 638-646, 654 evolution, 627, 657, 659 K’ in, 621 list of, 620 radical formation, 646-652, 655 rate constants, 653 reaction mechanism, 627-630 reactions catalyzed by, 619-622 stability constants, 642, 646, 653 structures, 623-631 substrate binding, 641-643 Vitamin Blz-dependent ribonucleotide reductase, see Ribonucleotide reductase Von Gierke disease, 3 73

Walker A consensus sequence, 72 Water oxidation, 233, 234 sea-, see Seawater Water buffalo lactoferrin, 579, 580, 582 Wilson’s disease, 922, 924 Wood degradation, 207 Worm annelid, 299 cytochrome P450, 313 marine, 156, 165, 516 tape-, 631

x XAFS studies of carboxypeptidase, 934

1180 [XAFS studies ofl protein kinase C , 893 XANES studies of aminopeptidase 213 catalase, 206 galactose oxidase, 734 lipoxygenase, 476 oxygen evolving complex, 234, 235 Xanthine oxidase (see also individual names) list of, 1066 molybdenum, 1024, 1028, 1032, 1054-1057, 1065-1067 XAS, see X-ray absorption spectroscopy Xenobiotics, 313 Xen,opus calcium-bindingprotein, 103 laevis, 963, 972 oocytes, 584 5'-XMP, 65 X-ray absorption near-edge structure, see XANES X-ray absorption fine-structure spectroscopy, see XAFS X-ray absorption spectroscopy studies of catalase, 206 enolase, 219 ferritin, 588 molybdenum oxotransferase, 1033, 1042 molybdenum proteins, 1034, 1035 I Nib'el-hydrogenase, 679, 691, 692 nitrile hydratase, 544 oxygen evolving complex, 235 superoxide dismutase, 686 urease, 671, 672 X-ray crystal structure studies of (see uEso Crystal structures) alkaline phosphatase, 938 amidotransferase, 399 ascorbate oxidase, 771, 772 ascorbate peroxidase, 306 astacin, 910 calcium-bindingproteins, 102, 110, 112, 125, 126, 137 carboxypeptidase, 934, 935 ceruloplasmin, 776-778 corrinoid, 630, 64(j1647 cytochromes, 296, 378 dioxygenase, 378 ferredoxh, 419 FixL, 301 haloperoxidase, 158-165, 171, 172

1X-ray crystal structure studies ofl heme oxygenase, 321 human serum albumin, 859 insulin, 1010, 1011 iron-sulfur proteins, 376, 388-390, 394396 isomerase, 222, 242 laccase, 767, 773, 774 p-lactamase, 901, 916 nitric oxide synthase, 315 nitrophorin, 300 nonheme iron proteins, 473 plastocyanin, 822 Rieske cluster, 378 rubredoxin, 373 superoxide dismutase, 203, 204, 207, 860 synthetase, 224 transferrin, 582 X-ray diffraction spectroscopy studies of bacterioferritin, 574 cytochrome c, 292 ferritin, 574, 589 insulin, 1008 metallothionein, 1004 NlgKI'Y, 61 myoglobin, 299 transferrin, 580 X-ray fluorescence studies of chloroperoxidase, 207 X-ray solution scattering experiments of transferrin, 582, 583 Xylitol, 220, 221 Xylose isomerase, 81, 220-222 active sit,e,222 co2+, 220 hydride shift mechanism, 242 list of crystal structures, 221 manganese, 220-222, 242 M 2 ' , 220

Yeast (see also Saccharomyces cerevisiae) (expression 00,64, 161, 169, 170, 200, 713 amine oxidase, 721 brewer's, 183-185 calmodulin, 109 cytochrome c peroxidase, 305, 306 endonuclease, 397 Fet3, 766, 767

SUBJECT INDEX [Yeast] genes, 64 genome, 964 iron transporter, 584 iron-sulfur protein, 399 kinase, 209 manganese in, 199, 200 metallothionein, 1003, 1004, 1014 mutase, 240 phosphatase, 216 pyophosphatase, 214

Zinc(I1) (in), 22, 71, 73, 74, 196, 197, 199, 203, 242 "Zn, 941, 1015 7Fe-8S ferredoxin, 383, 384 adenosine dcaminase, 885 alkaline phosphatase, 74 ATPases, 67 binding motifs, 908, 909, 911-915, 917 biological role, 882, 883 chemistry, 883, 884 clusters, see Clusters coordination sphere, see Coordination sphere detection of zinc sites, 915-917 DNA polymerase, 78 DNA-processing enzymes, 68 EF hand protein, 906 enzymes, see Zinc enzymes function, 883, 884 homeostasis, 988 insulin, 186 interdependency with other metal ions, see Interdependencies kinase, 66 methyltransferase, 609 peptidylam~doglycolate lyase, 724 phosphatase, 214, 215, 237, 900, 911, 2015 pneumococcal surface antigen adhesin A, 199, 230 protein kinase C, 127, 971, 972, 981 psoriasin, 111, 115, 116, 906 purple acid phosphatase, 238, 547-549 rubrerythrin, 543 S l O O protein, 115 superantigen, 904-906

1181 [Zinc(II) (in)] superoxide dismutase, see Superoxidedismutases transport proteins, 584 transport, 5 Zinc enzymes, 881-942 binding motifs, 908, 909, 911-915, 927 catalytic zinc site, see Catalytic zinc site classification of zinc sites, 884-906 cocatalytic zinc site, see Cocatalytic zinc site effect of scaffolding, 924,931 inhibition, 917-924 list of, 886-889, 894-899, 903, 910, 921 mechanistic studies, 932-940 mutagenesis, 915-917,928-931,936 number of, 907, 908 protein interface zinc site, 884, 902-906 structural zinc site, see Structural zinc site Zinc finger domains amino acid sequences, 973 B box, 968, 969,972,980 BIR, 968,969, 976 Bruton's tyrosine kinase, 968, 969, 975 C2H2 or retroviral, 965, 968-970, 975, 983 C2HC or TFIIIA, 963-965, 968-970, 975, 976,980, 986, 987 C4 nuclear hormone receptors, 968-970, 975-978,987 GI, 969, 981 containing one zinc, 964-976 containing two zinc, 976-982 coordination sphere of zinc, 974 copper fist, 975, 976 definition, 962, 963 FYW, 969, 981,982 GAL,968,969 GAL4,976,978,979 GATA, 968-971, 979 LIM, 968, 969,979,980 list of, 966, 967, 973, 984986 membrane binding double, 981, 982 rabphilin, 969, 981, 982 RING or C3HC4, 969, 979, 980 structures, 968, 969 XPA, 968,969 zinc ribbon or TFIIS, 968, 969, 971, 972, 976 Zinc finger proteins, 230, 373, 435, 892, 908

1182 LZinc finger proteins! bacterial, 987, 988 chromatin binding, 988 definition of domains, 962, 963 DNA interaction, 396-398 eukaryotic, 983, 986, 987 evolutionary aspects, 983, 986 nucleotide excision repair protein, 975 PDB codes, 969 prokaryotic, 987, 988 signal transduction, 981 statistics, 987 yeast, 964 Zinc hydrolase (see also individual names), 242,885, 900,908,909,911 inhibitors, 909 leukotriene 4,909, 910 list of, 886, 895-899 Zinc isomerase (see also individual names), 899 Zinc lyase, 895 list of,888, 889 Zinc metallothionein, 1001-1016 Ag(1) in, 1004 Bi(I1I) in, 1005 biological role, 1002, 1003 blue crab, 1003, 1006

S~BJECTINDEX [Zinc metallothioneinl Cd(1T) in, 10041007, 1014, 1015 Co(11) in, 1005, 1014 Cu(II) in, 1004 expression systems, 1013 Fe(1.I) in, 1005 Hg(I1) in, 1005 interaction with metals, 1014 list of, 1004 metal clusters, 1005 metal exchange, 1015 Ni(I1) in, 1005 Pb(I1) in, 1005 Pt(I1) in, 1005 sea urchin, 1003, 1004, 1007 Sn(1l) in, 1005 sources, 1003 structural classification, 1003 structures, 1003-1007 Tc in, 1005 Zinc oxidoreductase list of, 886, 894, 896 Zinc transferase (see also individual names) list of; 894, 895 Zucchini ascorbate oxidase, 771, 772, 830 mavicyanin, 828

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