Cellular Implications of Redox Signaling

Cellular Implications of Redox Signaling Editors Carlos Gitler Avihai Danon Imperial College Press Cellular Implica...

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Cellular Implications of Redox Signaling

Editors

Carlos Gitler Avihai Danon

Imperial College Press


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Editors

Carlos Gitler Avihai Danon Weizmann Institute of Science, Israel

Imperial College Press

Published by Imperial College Press 57 Shelton Street Covent Garden London WC2H 9HE Distributed by World Scientific Publishing Co. Pte. Ltd. 5 Toh Tuck Link, Singapore 596224 USA office: Suite 202, 1060 Main Street, River Edge, NJ 07661 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

CELLULAR IMPLICATIONS OF REDOX SIGNALING Copyright © 2003 by Imperial College Press All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.

For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from the publisher.

ISBN

1-86094-331-4

Typeset by Stallion Press.

This book is printed on acid-free paper. Printed in Singapore by Mainland Press

Preface

Electron transfer between proteins is an essential cellular process. It underlies primary metabolic reactions such as in photosynthesis and respiratory oxidative phosphorylation. Recently, electron transfer reactions between specific proteins have also been found to play a key regulatory role in many fundamental cellular phenomena, including cell proliferation, regu lation of specific gene expression, programmed cell death, and cell responses to oxygen levels, free radicals, and oxidants. Increasing number of genetic, molecular and biochemical studies in bacteria, plants, and animals point to the importance and the ubiquity of redox signaling. These redox-regulated phenomena are most likely controlled by specific factors that use intrinsic redox changes to regulate their biological activity. The intrinsic redox changes of the regulatory proteins, typically involve, similarly to protein phosphorylation, covalent modifications that allosterically modulate the protein catalytic activity or its capacity to bind other proteins in a selective manner. The covalent modifications that the regulatory proteins undergo derive from electron transfer between the regulatory protein and specific electron donors or acceptors. In the majority of cases, the regulatory redox reactions involve transfer of two electrons. The most common regulatory chemical groups involved are two proximal cysteinyl moieties (vicinal thiols) that alter nate between the oxidized intraprotein disulfide and the reduced dithiol configurations. Another type of two electron-redox reaction involves a protein monothiol that reacts with glutathione to form a proteinglutathione mixed disulfide and an acceptor intraprotein disulfide (Chapter Thomas and col.). Historically, the electron donor proteins were discovered in reactions that involved the catalytic turnover of the enzyme ribonucleotide reductase. The active site disulfhydryl form was found to be oxidized to a disulfide during one round of conversion of a ribonucleotide to a deoxyribonucleotide. Reichart and Holmgren discovered that subsequent reduc tion of the active site disulfide, necessary for a new round of catalysis, was mediated by the reactive dithiol of reduced thioredoxin acting as an elec tron donor. The thioredoxin disulfide was reduced in turn by thioredoxin reductase utilizing NADPH as the electron source. Holmgren furthermore

V

vi Preface

showed that another protein, glutaredoxin, can use glutathione to function as an alternative reductase of the active site intraprotein disulfide of ribonucleotide reductase (Chapter Holmgren). Subsequent studies have shown that cells contain many proteins with surface localized vicinal thiols. In many cases, the dithiols oxidized to disulfides are not involved directly in the catalytic activity of the proteins. Rather, oxidation of these dithiols to the disulfides functions as an allosteric site regulating the activity of enzymes (Chapter Schurmann). The intraprotein disulfides formed on oxidation are typically reduced to the dithiols by the thioredoxin-NADPH (or ferredoxin) — thioredoxin reductase systems (Chapter Holmgren, Buchanan, Gitler and col.). Selenium has been found to be present as selenocysteine in thioredoxin reductase (Chapter Holmgren, Arner). The glutaredoxin-glutathione pair can also participate in selected reactions as an electron donor or acceptor (Chapter Carmel-Harel and Storz). Thioredoxin is the main cellular intraprotein disulfide reductase. Coupled with NADPH- thioredoxin reductase it maintains the majority of the cell dithiol proteins in the reduced state. It also functions as a regula tory subunit in a growing number of protein complexes (Chapter Yodoi and col.). In addition, thioredoxin migrates to the nucleus and has a cen tral function in DNA repair and in regulation of transcription factors activity. As mentioned above, reduced thioredoxin is essential for the synthesis of deoxyribonucleotides, which because of their toxicity do not accumulate in cells. Thus, every round of DNA synthesis or repair requires the nuclear presence of reduced thioredoxin. Nuclear-localized reduced thioredoxin may thus become available for its known 2-electron transfer to Ref-1. The Ref-1 protein, in addition to its apurine nuclease activity, may act as a key nuclear protein disulfide reductase regulating activity of many transcription factors that must be in the reduced state to bind to DNA. Thioredoxin is also secreted from cells by an unknown mechanism. Thioredoxin has been shown to play a role as an autocrine growth factor, as a cytokine and as a cytokine modulator. (Chapter Yodoi and col.) Redox-dependent regulatory conformational changes in proteins can also result from alteration in the stability or in the valence of prosthetic iron-sulfur centers. Protein thiols participate in the formation of a diverse set of iron-sulfur centers that are sensitive to electron transfer (Chapter Beinert). In most cases, a change in the oxidation of the iron results in a loss of the stability of the iron-sulfur center leading to an altered activity of the regulatory protein (Chapters Beinert, Kaplan and col., and Kuhn).

Preface vii

Thus, the catalytic activity or the specific binding of a protein may be modified in cells by this type of mechanism. Noteworthy, reversible changes in redox state of iron in the regulatory iron-sulfur center of SoxR, and not iron-sulfur center stability, were found to modulate SoxR transcriptional activity. In addition, recent data suggest that valence changes in metalophosphatases could also modulate activity of these enzymes (Chapter Gitler and col.). Because the above redox reactions involve electron transfer between two proteins as a redox donor/acceptor pair, the reacting moieties have to be in or close to the protein surface (Chapter Gitler and col.). Furthermore, selective site recognition is possible by specific proteinprotein interaction. This is best exemplified by the selective interaction of plant thioredoxin isoforms with different enzymes (Chapters Buchanan, Schurmann). However, because the redox reactions can also occur with small oxidants, the target proteins are highly sensitive to oxidants such as diamide, alkylhydroperoxides and hydrogen peroxide. For this reason, redox changes in selected transcription factors function as sensitive cellu lar sensors of peroxide (Chapter Carmel-Harel and Storz), superoxide (Chapter Beinert and col.) or oxygen (Chapter Kaplan and col.). Changes in cellular iron levels are detected by alterations in the stability of the iron-sulfur center of cytoplasmic aconitase. The loss of the iron sulfur cluster activates the RNA-binding activity of the protein which then acts simultaneously as regulator of translation of ferritin mRNA and stability of transferrin receptor mRNA (Chapter Kuhn). Selective protein dithiol oxidation plays a key role in the maturation of nascent polypeptides to form the native disulfide-linked proteins. Recent work has illustrated the electron pathway in the Dsb system that forms disulfides in nascent polypeptides in the bacterial periplasmic space (Chapter Beckwith). In protein disulfide-bond formation in the endoplasmic reticulum (ER) of eukaryotes, oxidizing equivalents are transferred from a conserved ER-membrane protein, Erolp, to substrate proteins via protein disulfide isomerase (Chapter Gilbert and col.). Recently, the ERV1/ALR family of proteins has been found to function as protein dithiol oxidases in the cellular cytoplasm and to interact with glutaredoxins. In growth initiation, and probably in other cellular reactions involving activation of receptor phosphotyrosine kinases, a dual initial calciummediated burst in hydrogen peroxide formation and inhibition of thioredoxin reductase occurs (Chapter Gitler and col.). Thus, early in these ligand-receptor mediated reactions, an oxidizing cellular milieu is required for activation of the phosphorylation cascades. A central role is

viii Preface

suggested for thioredoxin-dependent peroxidases or peroxiredoxins (Chapters Gitler and col., Yodoi and col.). Furthermore, the ensuing oxida tion of cellular thioredoxin probably leads to the activation of ASK1. This protein is negatively regulated by its selective binding to reduced thiore doxin. ASK1 activation by its dissociation from the oxidized thioredoxin may be critical for its role in the normal activation of the so-called stress pathway of MAP kinases and for cellular commitment to apoptosis (Chapters Gitler and col., and Yodoi and col.). The required inhibition of excess phosphotyrosine phosphatases to allow regulation by kinases could also require the redox oxidizing environment that ensues in cells on ligand-binding to receptor phosphotyrosine kinases. The regulation of gene expression requires the transduction of specific redox signals via unique signaling pathways. These regulatory redox reactions must occur in an otherwise highly reductive intracellular milieu, suggesting that specific oxidation of regulatory factors must occur. Dissection of the reactions that regulate light-mediated translation of chloroplast mRNAs shows that illumination directs the unique oxidation of a protein disulfide isomerase-like protein, acting as a translational activator. Both the reduction and oxidation reactions that govern the redox state of the regulatory PDI-like protein were found to be specific, resulting in coupling of redox regulated translation and light. The mani fested selective redox regulation of this process is not transient and occurs throughout the day, demonstrating that the redox state of regulators of gene expression could be uniquely controlled according to their biological function (Chapter Danon and col.). Mitochondria are key organelles in the cell redox status. They have a basic role in defining the cellular levels of reduced pyridine nucleotides by means of electron-transfer and of transhydrogenases. In addition, mitochondria are a constant cellular source of significant levels of superoxide and hydrogen peroxide. The redox regulation of the permeability transition pore, a cyclosporin A-sensitive mitochondrial channel, is a good example of the complex redox interactions that occur in this organelle (Chapter Bernardi). The basic bioenergetic aspects of pore modulation are discussed, with some emphasis on the links between oxidative stress and pore regulation as a potential cause of mitochondrial dysfunction that may be relevant to a variety of forms of cell death. One electron transfer to protein vicinal thiols can occur making redox regulatory proteins highly sensitive to cellular radical reactions. Mounting evidence suggests that ascorbic acid may play a key role in reducing tocopheryl radicals and thus may be essential for radical termination in cell membranes. Dismutation of the ascorbyl radical may be linked to

Preface ix

thioredoxin reductase while dehydroascorbate reduction may be carried out by the cell glutaredoxins. Furthermore, a general mechanism of radicalchain termination may use vicinal thiol proteins to convert radicals to superoxide. Thus, vicinal thiol proteins, superoxide and superoxide dismutase could function as a general cell radical chain-termination system (Chapter Winterbourn). In plant cells, the interplay between radicals and H 2 O z on the one hand and the antioxidants ascorbic acid and glutathione varies in different cellular compartments. The ensuing changes in the lev els of ascorbic acid and/or glutathione (and their oxidized forms) can function as a signal for cells to activate different systems that guard against cell damage and disease (Chapter Foyer). Cells contain two different systems involved in reduction of dioxygen and disulfides. One is thioredoxin-based and the other is glutathionebased. The thioredoxin systems regulate protein disulfide reduction by direct reaction of reduced thioredoxin with intraprotein disulfides. Thioredoxin peroxidases or peroxiredoxins are a large family of proteins involved in the reduction of cellular peroxides (dioxygen). The selenoprotein thioredoxin reductase links this system to NADPH as an electron source. On the other hand, glutaredoxins use reduced glutathione to reduce intraprotein disulfides while the selenoprotein glutathione peroxidase functions in the reduction of cellular peroxides. Thus, cells have evolved two different systems — both selenium-dependent — to regulate the redox state of cellular proteins. Genetic links can be found in, for example, hydroperoxide reductases that utilize a thioredoxin-like domain in their function. Processing of peroxides and disulfides evolved presum ably in parallel to transfer electrons to reduce the products of radicalchain termination and to reduce disulfides and iron-sulfur centers for redox regulation. The purpose of this book is to present an overall picture of the current state of redox regulation. Not all facets could be covered because of the extensive nature of the subject matter. Rather, we hope that this joint effort will serve as a timely and integrated presentation describing the underlying principles of this key regulatory mechanism. The Editors would like to acknowledge the support of the Weizmann Institute of Science through the Aharon Katzir-Katchalski Center, The Goldshlager Fund, The Dr. Josef Cohn Minerva Center for Biomembrane Research and the Dean of the Faculty of Biology. CG would like to thank Ana and Pablo Brener for their continued support of his research. Our personal thanks also to Beky Gitler and Tami Danon for their active part and total commitment over the long and trying times that were needed to bring this book to fruition.


Contents

Preface 1

v

The Role of Thioredoxin and Glutaredoxin Systems in Disulfide Reduction and Thiol Redox Control

1

2 Selenocysteine Insertion and Reactivity: Mammalian Thioredoxin Reductases in Relation to Cellular Redox Signaling

27

3

Iron-Sulfur Proteins: Properties and Functions

47

4

The Ferredoxin Ferredoxin/Thioredoxin Thioredoxin System. A light-Dependent Redox Regulatory System in Oxygenic Photosynthetic Cells

73

Thioredoxin and Redox Regulation: Beginnings in Photosynthesis Lead to a Role in Germination and Improvement of Cereals

99

5

6

The Role of Thioredoxin in Regulatory Cellular Functions

115

7

Protein S-Thiolation, S-Nitrosylation, and Irreversible Sulfhydryl Oxidation: Roles in Redox Regulation

141

Radical Scavenging by Thiols: Biological Significance and Implications for Redox Signaling and Antioxidant Defense

175

Ascorbate and Glutathione Metabolism in Plants: H 2 0 2 -Processing and Signalling

191

8

9

10 Disulfide Bond Formation in the Periplasm and Cytoplasm of Escherichia Coli 11 The Thiol Redox Paradox in the Requirement for Disulfide Isomerization in the Eukaryotic Endoplasmic Reticulum

xi

213

233

xii

Cellular Implications of Redox Signalling

12 Mechanisms Controlling Redox Balance in Cells. Inhibition of Thioredoxin and of Thioredoxin Reductase

257

13 Regulatory Disulfides Controlling Transcription Factor Activity in the Bacterial and Yeast Responses to Oxidative Stress

287

14 Redox Signaling During Light-Regulated Translation in Chloroplasts

311

15 Regulation of mRNA Translation and Stability in Iron Metabolism: Is there a Redox Switch?

327

16 Redox Flow as an Instrument for Gene Regulation

361

17 The Permeability Transition Pore as Source and Target of Oxidative Stress Author Index

393 421

Subject Index

423

Chapter 1 The Role of Thioredoxin and Glutaredoxin Systems in Disulfide Reduction and Thiol Redox Control Arne Holmgren Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77, Stockholm, Sweden [email protected]

Keywords: Selenium, thioredoxin reductase, thioredoxin, glutaredoxin

1. Summary The intracellular redox environment in Escherichia coli and mammalian cells is reducing with a high level (1-10 mM) of the tripeptide thiol glutathione (GSH) and the proteins contain free sulfhydryl groups and disulfides are very rare. This is in contrast to the outer cell surface or the extracellular environment where oxidizing conditions prevail due to the presence of oxygen and proteins have stabilizing disulfides and no or few free sulfhydryl groups. The thioredoxin (thioredoxin reductase and thioredoxin) and the glutaredoxin (glutathione reductase, GSH and glutaredoxin) systems are responsible for maintaining the low intracellu lar redox potential using electrons from NADPH. Thioredoxin and glutaredoxin are required also in essential metabolic reactions like the synthesis of deoxyribonucleotides for DNA synthesis by ribonucleotide reductase, one of several enzymes which requires disulfide reduction for each catalytic turnover. Other such enzymes are the family of thioredoxin peroxidases or peroxiredoxins, which use cysteine sulfur residues to reduce hydrogen peroxide with a mechanism-derived disulfide inter mediate. Thioredoxin, via its classical active site Cys-Gly-Pro-Cys dithiol, is used to maintain protein SH-groups reduced, but can also make disulfides

l

2

Cellular Implications ofRedox Signalling

via its disulfide form. Thioredoxins are regulating the activity of enzymes, transcription factors and receptors by reversible disulfide bond formation (thiol redox control) oxidized thioredoxin is made following rapid and temporal generation of superpoxide and hydrogen peroxide. Analogous reactions for the glutaredoxins are to also catalyze reversible S-glutathionylation of protein SH-groups, from glutathione disulfide (GSSG) another mechanism of thiol redox control of protein activity. Recent studies of the FAD-containing mammalian thioredoxin reductase has resulted in the determination of the structure and mechanism. This has shown surprisingly large differences to the conserved family of thioredoxin reductases from bacteria, fungi and plants. The larger mam malian thioredoxin reductases are structurally built from a glutathione reductase scaffold with a 16-residue elongation containing the conserved active site sequence: -Gly-Cys-SeCys-Gly, where the penultimate SeCys is selenocysteine. In its oxidized form, each active site in the dimeric enzyme contains a selenenylsulfide which is reduced to a selenolthiol in the reduced enzyme with electrons from the active site disulfide in the second subunit. The reductive half-reaction is identical to that of glu tathione reductase leading to reduction of the identical disulfide in thiore doxin reductase. A 3 A resolution X-ray structure of the rat enzyme demonstrates the close similarity to glutathione reductase including conserved residues involved in GSSG binding. However the C-terminal 16-residue swinging arm blocks GSSG binding, but enables electron trans port to the SeCys-Cys selenenylsulfide and the enzyme surface. The open active site enables docking of oxidized thioredoxin without any large conformational changes in sharp contrast to the bacterial thioredoxin reduc tases. The selenium is essential for enzyme activity, since without selenium, the truncated polypeptide, lacking the terminal SeCys-Gly dipeptide arising from the UGA in the mRNA acting as a stop codon is folded, but lacks all enzymatic activity. Replacement of selenium by sulfur yields an active enzyme but with a 100-fold major loss in Kcat but with a lower Km-value for thioredoxin. The selenium is also essential for the inher ent NADPH-dependent lipid hydroperoxide and hydrogen peroxide reductase activities of mammalian thioredoxin reductase. The selenazol drug ebselen, known to act as a glutathione peroxidase mimick, is a direct efficient substrate for mammalian thioredoxin reductase strongly enhanc ing its hydrogen peroxide reducing activity particularly with thioredoxin present. Future studies of the use of drugs that affect the thioredoxin sys tem will be useful for developing treatments of diseases either involving damage caused by overproduction of reactive oxygen species.

The Role of Thioredoxin and Glutaredoxin Systems

3

Glutaredoxins operate as disulfide reductases and have a CysPro-Tyr-Cys active site and a GSH-binding site which is used in binding GSH for reduction of the active site disulfide to a dithiol via a glutathione mixed disulfide intermediate. Glutaredoxins are species specific electron donors for ribonucleotide reductase. Recently, the structure of the large unusual £. coli glutaredoxin-2 (23 kDa), which is a powerful GSH-disulfide oxidoreductase and an electron donor for arsenate reductase was deter mined in solution and shown to be similar to glutathione-S-transferases. This structure also defines a novel family of mammalian large monothiol glutaredoxins, which have only the N-terminal nucleophilic Cys-residue and catalyze GSH-disulfide oxidoreductions. Future research should define how thiol redox control via thioredoxin and glutaredoxin systems is integrated with phosphorylation. Also the control of thioredoxin activity and expresssion in cells by specific binding proteins remains to be classified. Almost nothing is yet known about the mechanism by which thioredoxin is secreted without a leader sequence, how it is located on the cell surface and how it can move between different compartments within the cell like the cytoplasm and the nucleus. Future goals will also be to utilize specific drugs targeted to induce thioredoxin or thioredoxin reductase and also to develop gene therapy vectors with appli cation to prevent degenerative diseases in e.g. the brain. Thus, both thiore doxin and glutaredoxin have in preliminary experiments been shown to protect nerve cells from apoptosis. The redoxin electron donor identity for ribonucleotide reductase in tumor cells of different tissues is another area where more knowledge is required, also for rational use of current chemotherapy.

2. Introduction and Historical Perspective Thioredoxin was discovered 1964 by Peter Reichard and coworkers in E. coli1 as a small heatstable protein cofactor containing a dithiol required to enable the synthesis of dCDP from CDP by a partially purified enzyme today known as ribonucleotide reductase. This essential enzyme catalyzes the reduction of all four ribonucleotides to deoxyribonucleotides by replacing the 2'-OH-group in the ribose of the nucleotide by a hydrogen using a free radical mechanism. 2 The enzymatic reduction of CDP to dCDP required a hydrogen donor and the dithiol of dihydrolipoic acid gave activity, whereas monothiols like glutathione (GSH) or mercaptoethanol were inactive. NADPH was active as a hydrogen donor when coupled with

4


an enzyme activity, thioredoxin reductase, required to regenerate a dithiol from the disulfide in the oxidized thioredoxin. E. coli thioredoxin (12 kDa) contained a single cystine disulfide group and after improve ment of the purification procedure to get homogeneous protein cleavage with cyanogen bromide at the single methionine residue yielded two peptide fragments, where the N-terminal 37-residues contained the disulfide group. 3 The complete amino acid sequence of thioredoxin with its now classical active site sequence: -Cys-Gly-Pro-Cys- was published in 1968. After 2 years of fruitless attempts to crystallize thioredoxin, useful single crystals were obtained in 1970 from the oxidized protein by addition of cupric ions 5 and in, 1975, the three-dimensional structure of thioredoxinS2 was solved. 6 The active site was located in a protrusion of the thiore doxin molecule, which was described as a first example of a male protein. 6 Thioredoxin consists of a central core of 5 B-strands surrounded by 4 a-helices with more than 75% of the residues in well defined secondary structures explaining the high stability of the structure (the thioredoxin fold). The structure of reduced thioredoxin remain elucive for many years although a localized conformational change was early observed from the three-fold increase in tryptophan fluorescence following reduction of oxi dized thioredoxin. 7 This unusual large increase in tryptophan fluorescence unique to the bacterial thioredoxin with its Trp-28 apart from the con served Trp-31 has proven to be of great importance to enable direct mea surements of the kinetics of thiol-disulfide exchange for thioredoxin.8,9 For a long time up to the mid-1970s, thioredoxin was almost exclu sively connected to ribonucleotide reductase and DNA synthesis as well as sulfate reduction or methionine sulfoxide reduction. The isolation of viable E. coli cells, which lacked thioredoxin, called into question its role in ribonucleotide reduction and lead to the discovery of glutaredoxin as a glutathione-dependent hydrogen donor for ribonucleotide reductase. 1011 Studies of thioredoxin and thioredoxin reductase in mammalian cells by purification and characterization of the proteins was initiated around 1970 based on use of ribonucleotide reductase as an assay system. This complicated assay and the fact that mammalian thioredoxins now known to contain additional sulfhydryl groups, which upon air oxidation lead to aggregation and inactivation made progress slow. A major break-through was the real ization that only the reduced form obtained after incubation with a thiol like dithiothreithol could be purified as a single peak component from liver or thymus extracts.12 Oxidized form show multiple artefactial peaks involving aggregation by different mechanisms. Since E. coli thioredoxin reductase shows no cross-reactivity with the human, rat or bovine thioredoxin it was of


5

no use for coupling to NADPH using reduction of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB). Reduction of the latter is an easily used assay for thiore doxin from E. coli or yeast.3 In contrast, the mammalian thioredoxin reductase of calf thymus showed completely different properties with a higher molecular weight and a wide substrate specificity, which involved direct reduction of DTNB13 and rather complete inhibition in reduction of DTNB by the addition of bovine thioredoxin. In order to avoid ribonucleotide reductase as an assay system, the use of insulin in disulfide reduc tion was developed. 1213 Furthermore, the general role of thioredoxin and thioredoxin reductase as main disulfide reductase system of cells was shown e.g. by selective reduction of 5 out of 28 disulfide bonds in human fibrinogen by the thioredoxin system.14 This was later followed by selective disulfide reduction in a number of different proteins and has been used as a selective tool to probe disulfides and get wild selective reduction. The speci ficity is impressive since 3 of 5 disulfides in trypsin react, but none of 17 disulfides in albumin. A review of the method is found in Ref. 15 The wide distribution of thioredoxin in mammalian cells and its pres ence irrespective of DNA synthesis and ribonucleotide reductase activity was proven by studies of the distribution of calf thioredoxin. 16 This demonstrated thioredoxin in the nucleus, the microsomal fraction and mitochondrial fraction using radioimmunoassays. The first evidence of human thioredoxin was found in extracts from human platelets 14 and later in extracts from cultured fibroblasts.16 The localization of thioredoxin and thioredoxin reductase in adult rats 17 demonstrated a general cytoplasmic staining with prominent expression in epithelial cells including large amounts of thioredoxin in the nervous system, 18 axoplasmic transport of both thioredoxin and thioredoxin reductase in nerves and functional related changes in pancreatic B-cells and the gastric mucosa.19,20 A new area in thiredoxin research was initiated when Yodoi and coworkers identified adult T-cell leukemia derived factor (ADF) as a thiore doxin present in conditioned medium from lymphocytes and involved as a growth factor in upregulation of IL-2 recptor.21 This started a novel field in thioredoxin research regarding redox regulation of extracellular phenomena and growth control.22 Furthermore, thioredoxin released from B-cells infected with the EBV-virus is shown to be involved in lymphocyte immortalization. 23 Also from CD-4 T-cells a secreted factor, growthpromoting for normal and leukemic B-cells was identified as thioredoxin.24 Thus, in the last decade there has been an intense search for new functions of thioredoxin in redox control of cell growth, transcription factors and apoptosis.

6


The concept of thiol redox control of redox regulation of cellular phenomena by changes in the structure of SH-groups on proteins has a long history and was suggested early to involve thioredoxin. 8 Only in the last decade it has been realized that there is an oxidizing mechanism con trolling the disulfide status in cells, via the generation of reactive oxygen species, which are converted into a disulfide signal by glutathione peroxidases generating GSSG or thioredoxin peroxidases (peroxiredoxins) generating disulfide forms of thioredoxins. The latter can then be trans formed to generation of disulfides in proteins as part of thiol redox con trol. Thus, the discovery of the peroxiredoxins 25 is a major concept in thioredoxin-dependent regulation of cellular activation. A human thiore doxin was first purified to homogeneity from placenta by A. Ernberg in Stockholm (1979) (cited in Ref. 26) and the antibodies against this protein was used by Sitia and coworkers to demonstrate a leaderless secretory pathway for thioredoxin 27 . Escherichia coli glutaredoxin was purified to homogeneity and its activity with ribonucleotide reductase, showed a higher turnover number as seen by the ten-fold lower Kra-value (0.13 uM) compared with thiore doxin. It was also discovered that pure glutaredoxin had inherent glutathione-disulfide oxidoreductase activity or was a member of glutathione disulfide transhydrogenase. This rapid spectrophotometric assay and showed that bacteria were indeed a very rich source of such activity. In fact, the activity in E. coli crude extracts was a 100-fold higher than what was specifically measured as glutaredoxin assayed with ribonucleotide reductase.28,29 The additional activities were found to be due to two new glutaredoxins called glutaredoxin-2 and -3, 30 which have no or some activity with ribonucleotide reductase, but represent major proteins in E. coli. Homogeneous preparations of calf thymus glutaredoxin, 31 which acts as a species-specific electron donor for calf thymus ribonucleotide reductase 32 contained the same conserved active site sequence Cys-Pro-TyrCys33 as E. coli glutaredoxin. 34 However, it was not clear at that time, as discussed in Ref. 31, if glutaredoxin and a GSH-homocystine transhydro genase from rat liver renamed thioltransferase 35 were identical proteins. The latter was reported to contain 8.6% carbohydrate. 35,36 However, the carbohydrate content was not confirmed and sequencing of proteins including a revised sequence of calf thymus glutaredoxin 37 showed the identity of the two proteins. Earlier work on thioredoxin and glutaredoxin have been summarized in review articles such as Refs. 26 and, 38—40. The proceedings of a Nobel

The Role of Thioredoxin and Glutaredoxin Systems 7

conference in 1985 on thioredoxin and glutaredoxin systems has been published. 41 Recent reviews with particular foccus on mammalian thiore doxin and thioredoxin reductase may be consulted42,43 for details. In this article some novel data on the structure and function of mammalian thioredoxin reductase will be described. General aspects of thiol redox control will be discussed.

3. The Thioredoxin System Thioredoxin reductase (TrxR) will reduce oxidized thioredoxin (Trx-S2) at the expense of NADPH [Reaction (1)] and reduced thioredoxin (Trx-(SH)2) is reoxidized by disulfides in proteins generating sulfhydryl groups [Reaction (2)]:

Trx-S2

+

Trx-(SH)2

NADPH +

Protein-S2

+

H+

TrxR ► Trx-(SH)2 ► Trx-S2

+ +

NADP+

(1)

Protein-(SH)2.

(2)

The Km-value for Trx-S2 is typically from 1 to 3 uM. Thioredoxin is an effi cient reductant with a low redox potential of - 270 mV.44 Today we know that there are some major differences between the thioredoxin systems of prokaryotes like E. coli and that of mammalian organisms. Thus, E. coli and mammalian cytosolic thioredoxins are very similar proteins in term of substrate specificity and reactivity with a con served -Cys-Gly-Pro-Cys-active site. However, mammalian thioredoxin must be purified and stored in the fully reduced form since they contain structural SH-groups which form additional disulfides upon oxida tion.13,45 This may have autoregulatory function of thioredoxin activity in resting cells or upon oxidative stress yet incompletely known in vivo. Thioredoxin reductases from mammalian cells have very different prop erties when compared with the enzymes from E. coli, yeast or plants; review in Ref. 46. The mammalian cytosolic enzyme has subunits with 55 kDa or larger instead of the 35 kDa in the E. coli enzyme with known three-dimensional structure. 46 As will be described below, the mam malian enzyme has an unusually broad substrate specificity entirely different from the species-specific enzymes only reducing Trx-S2 present in prokaryotes, yeast and plant cytosol.

8 Cellular Implications ofRedox Signalling

3.1 Thioredoxin Reductase and Selenium The fact that administration of selenium compounds like selenite (Se032~) cause inhibition of tumor cell proliferation in vivo and the knowledge that thioredoxin reductase appeared to be more highly expressed in malignant cells prompted us to start investigations on the reactivity of selenium compounds with pure mammalian thioredoxin reductase and thiore doxin. Contrary to expections, we discovered that selenite is a direct sub strate for thioredoxin reductase as well as an efficient oxidant of Trx-(SH)2.47'48 With 200 uM NADPH and 50 nM calf thymus thioredoxin reductase, addition of 10 uM selenite caused oxidation of 40 uM NADPH in 12 min and 100 uM NADPH after 30 min demonstrating a direct reduc tion of selenite with redox cycling by oxygen.47'48 This was demonstrated by incubation under anaerobic conditions where only 3 mol of NADPH was oxidized per mol of selenite according to Reaction (3):

Se032" + 3 NADPH

+

3 H+

TrxR ►

Se2- +

3NADP+

+ 3H 2 0.

(3)

Addition of thioredoxin stimulated the reaction further since selenite rapidly reacts with Trx-(SH)2 to oxidize it to Trx-S2.47-49 Since glutathione reductase will not react with selenite, Reaction (3) should provide cells with selenide, a required precursor for selenophosphate and selenocysteine synthesis. 50 Selenite and glutathione react to form selenodiglutathione (GS-Se-SG) which has been suggested to be a major metabolite of inor ganic selenium salts in mammalian tissues. 51 Reaction of selenodiglu tathione by NADPH and glutathione reductase was demonstrated by Ganther 51 and it has been proposed to be a source of selenide in cells as well as an inhibitor of neoplastic growth. We synthesized GS-Se-SG48 and discovered that is a direct efficient substrate for mammalian thioredoxin reductase and a highly efficient oxidant of reduced thioredoxin. Since GSSG is not a substrate for mammalian thioredoxin reductase,13,52 the insertion of the selenium atom in the GSSG molecule to form GS-Se-SG makes this molecule highly reactive with the enzyme. Reduction of GS-Se-SG to yield selenide by glutathione reductase requires 2 mol of NADPH. We found only the first stoichiometric reduction to be fast with GS-Se- as a product. 48 The second reaction was slow and inefficient. These results strongly suggest that the major selenide generation in cells is via thioredoxin reductase and thioredoxin. Thus, in mammalian cells the selenoenzyme thioredoxin reductase is also responsible for the synthesis


of selenide required for its own synthesis. An oxygen dependent non-stoichiometric consumption of NADPH is given by the thioredoxin system in the presence of selenite, selenodiglutathione and selenocystine.47"49 The latter is an efficient substrate for mammalian thioredoxin reductase with a Km of 6 uM. 49 The mechanism may be that the XSe" reacts with a dithiol (or selenolthiol) to catalyze oxidation according to Reaction (4): XSe"

+

R-(SH)2

+

02

►

XSe"

R-S2

+

H 2 0.

(4)

The effect will be 0 2 -dependent consumption of NADPH and provides an explanation for the lack of an autooxidizable free pool of selenocysteine as well as the acute toxic effects of selenium compounds on cells, for example, leading to apoptosis. Mammalian thioredoxin reductases dis play a surprisingly very wide substrate specificity as first observed dur ing purification.13,52 This is in contrast to the smaller prokaryotic enzymes, which do not react with mammalian thioredoxins despite the identical active sites and closely related three-dimensional structures of the thiore doxins. As summarized in Table 2, a truly wide range of direct reductions are catalyzed by the mammalian cytosolic thioredoxin reductases. Thiore doxin from E. coli is a substrate with a similar Kcat, but with a 15-fold higher Km value (35 uM) compared with the rat liver protein. 52 The mam malian cytosolic thioredoxins generally show full crossreactivity with the enzymes from different sources and vice versa.

3.2 Structure of Mammalian Thioredoxin Reductase Recent biochemical studies, sequencing and cloning of mammalian thioredoxin reductases has revealed that the enzymes are selenoproteins and entirely different from the corresponding enzymes in bacteria, yeast and plants (review in Ref. 46). Stadtman and coworkers serendipitously discovered that a human tumor cell thioredoxin reductase is a selenoprotein using labeling of selenoproteins with radioactive selenite.63 This also explained 64 why a putative clone of the human enzyme, 65 where the TGA codon for selenocysteine (SeCys) was interpreted as the stop codon (Fig. 1) gave no enzyme activity. The TGA acts as a stop codon in E. coli due to the fact that the species-specific machinery for synthesis of seleno proteins is different in bacteria and mammalian cells.66 By sequencing large parts of the cytosolic bovine enzyme, we directly identified the C-terminal peptide as containing selenocysteine. The bovine peptides were used to identify a rat cDNA clone which was

10

Cellular Implications ofRedox Signalling CVNVGC

GCUG

,\ / H,N-|

V FAD

NADPH

Inierrace

]-COOH

-Gln-Ala-Gly-Cys-Sec-Gly-Ter (human TrxR) CAG GCT GGC TGC TGA GGT TAA GCC CCA . . . CAG TCT GGC TGC TQA GGT TAA GCC CCA . . . -Gln-Ser-Gly-Cys-Sec-Gly-Ter (rat TrxR)

Fig. 1. Structure of the subunit of human and rat cytosolic thioredoxin reductase. The N-terminal glutathione reductase-like active site disulfide (CVNVGC) is shown in the upper portion of the figure as well as the FAD, NADPH and interface domains. The active site is shown in the C-terminus with GCUG denoting Gly-Cys-SeCys-Gly. Below, the region of that the part of the human and rat cytosolic genes with the TGA codon encoding selenocysteine (Sec) is shown. sequenced 67 and showed a polypeptide chain with a high homology to glutathione reductase including an identical active site disulfide (CVNVGC) (Fig. 1), but with a 16-residue elongation containing the con served C-terminal sequence -Gly-Cys-SeCys-Gly. A selenocysteine inser tion sequence (SECIS) was identified in the 3'untranslated region. 67 Furthermore, digestion of thioredoxin reductase by carboxypeptidase after reduction by NADPH released selenocysteine with loss of activity; the oxidized form of the enzyme was resistant to carboxypeptidase digestion.67 Redox titrations with dithionite and NADPH demonstrated that the mechanism of the human placenta enzyme is similar to that of lipoamide dehydrogenase and glutathione reductase and distinct from the mechanism of thioredoxin reductase from E. coli.68 The results also demon strated that the SeCys residue of human thioredoxin reductase is redox active and communicates with the redox active disulfide, since more than 4 electrons per subunit are required to completely reduce the FAD of the oxidized enzyme. Furthermore, the SeCys residue is alkylated with loss of activity only after reduction by NADPH. 67 The SeCys residue is also the target of the irreversible inhibitor l-chloro-2,4-dinitrobenzene only after reduction by NADPH 69 as shown by peptide analysis.70 The essential role of selenium in the catalytic activities of mammalian thioredoxin reductase was revealed by characterization of recombinant enzymes with selenocysteine mutations. 56 This was done by removing the selenocysteine insertion sequence in the rat gene and changing the SeCys49s encoded by TGA to Cys or Ser codons by mutagenesis. The trun cated protein having the C-terminal dipeptide deleted, which is expected


NADPH domain

Interface domain

11

16 aa elongation with Cys-SeCys

FAD domain

FAD domain 16 aa elongation with Cys-SeCys

Thioredoxin

Interface domain

NADPH domain

Reductase

Fig. 2. Structure model of mammalian TrxR (71). The 16-residue C-terminal extension with the active site is displayed as well as the head to tail arrangement of the subunits in the dimer as in glutathione reductase. The FAD, NADPH and interface domains are shown (see also Fig. 1). to mimic selenium deficiency, was also engineered. All three mutants were successfully overexpressed in E. coli and purified to homogeneity with 1 mol of FAD per monomeric subunit. All three mutant proteins rapidly gen erated the AJJO absorbance resulting from the thiolate-flavin charge transfer complex characteristic of mammalian TrxR.56 Only the SeCys498 Cys enzyme showed catalytic activity with thioredoxin, with a 100-fold lower Kcat, but also a 10-fold lower Km compared to the wild type rat enzyme. The pH-optimum of the SeCys-containing wild type enzyme was 7 whereas the SeCys498 Cys enzyme showed a pH optimum of 9. This strongly suggested the involvement of the low pKa SeCys selenol in the enzyme mechanism. Also selenium was required for hydrogen peroxide reductase activity.56 Thus, selenium is required for the catalytic activities of thioredoxin reduc tase explaining the essential role of this trace element in cell growth. Based on the homology to glutathione reductase, we proposed a schematic structure of mammalian thioredoxin reductase (Fig. 2). The active enzyme is a head to tail dimer with the 16-residue elonga tion in principle taking the place of GSSG in glutathione reductase. The catalytic site of the enzyme is a selenolthiol in its reduced form and a

12


selenenylsulfide formed from the conserved cysteine-selenocysteine sequence in the oxidized form.71 The selenenylsulfide was isolated by peptide sequencing and also confirmed by mass spectrometry. 71 The reductive half-reaction is similar to that of glutathione reductase leading to reduction of the active site disulfide (Figs. 1 and 2). Electrons are thereafter transferred from the redox-active dithiols to the selenenylsulfide of the other subunit generating the selenolthiol (see below). Characteriza tion of the Cys mutant enzyme revealed that the selenium atom with its larger radius is critical for the formation of the unique selenenylsulfide,71 since the C-terminal dithiol remains reduced in the Cys mutant. 71 The presence of selenocysteine in the mammalian enzyme precludes direct recombinant expression of the enzyme in E. coli, although, engi neered constructs have overcome this and given promising results.72 Attempts to crystallize the native enzyme to get X-ray quality crystals from regular preparations have not been successful probably due to microheterogeneity in the selenium content also reflected in varying specific activities. The active SeCys498Cys mutant enzyme in contrast can be pre pared in large quantities and has been crystallized 73 in three different forms. Recently, the X-ray crystal structure of the rat SeCys498Cys mutant enzyme in complex with NADP + was solved to 3 A resolution respresenting the first structure of this unique class of selenoenzymes. 74 The most impressive result (Fig. 3) is the close similarity overall to the structure of glutathione reductase, including conserved amino acid residues binding the cofactors FAD and NADPH. Surprisingly, all residues interacting directly with the substrate GSSG in glutathione reductase are conserved despite the fact that GSSG is not acting as a substrate for thioredoxin reductase. The 16-residue C-terminal tail, which is unique to mammalian thioredoxin reductase and carries the SeCys residue, folds in such a way that it can approach the active site disulfide of the other subunit in the dimer. A model of the complex of rat thioredoxin reductase with human thioredoxin-S 2 (Fig. 4) suggests that electron transfer from NADPH to the disulfide of the substrate is possible without large conformational changes. Thus, the C-terminal extension to glutathione reductase scaffold typical of mammalian thioredoxin reductase has two main func tions. First, it extends the electron transport chain from the catalytic disul fide to the enzyme surface, where it can react with thioredoxin and a range of other substrates. Second, the C-terminal extension prevents the enzyme from acting as a glutathione reductase by blocking acess of GSSG to the redox active disulfide. The structure of the enzyme is also compatible with


Fig. 3. Ribbon representation of the dimer of rat TrxR. The two subunits are shown in light or dark colors respectively. Red, FAD-binding domain; yellows, NADP-binding domain, blue interface domain. Bound FAD and NADP are shown as ball and sticks models. Also the positions of SeCys498 and His 472 are shown as ball and sticks model. Taken from Ref. 74. The figure was kindly made by Dr Tarjana Sandalova.

evolution of mammalian thioredoxin reductase from a glutathione reductase scaffold rather than from the prokaryotic counterpart. Such an evolu tionary switch also rendered cell growth dependent upon selenium.


Fig. 4. Model of complex of mutant rat TrxR with human thioredoxin. The

structure of rat TrxR is shown as a ribbon model with red, FAD-binding domain and FAD shown as a ball and sticks model; yellow NADPH-binding domain and the blue interface domain from the second subunit with His 472, Cys 479 and Cys 498. Thioredoxin is shown in green with active site disulfide C32-C35 and other residues indicated.

3.3 Mechanism of Mammalian Thioredoxin Reductase A mechanism of reduction of thioredoxin is shown in Fig. 5. The low pKa value and the high nucleophilicity of the selenium atom in the selenol makes this an excellent reducing agent, but also the likely target of drugs

The Role of Thioredoxin and Glutaredoxin Systems 15 (A) -FAD +

64 - S 59

J V

2NADPH+H1'-

497' 498' 2NADP^

(D) -FAD

(E) -FAD

-S

NADPH+H

(B) l-FAD

^ ^

« - * ^

^ i

59 -SH

HS- 497' "Se- 498'

Fig. 5. Mechanism of mammalian TrxR in reduction of thioredoxin-S2. A, oxidized enzyme; B, reduced enzyme; B with charge transfer complex and the selenenylsulfide being reduced to the selenolate anion. This attacks the disulfide in Trx-S2 and forms a Trx-TrxR mixed selenenyl sulfide as shown in C. As Trx(SH)2 is released the selenenyl sulfide is formed (D), which will be reduced by the active site thiolate from the other subunit to give E. like goldthioglucose 75 known to inhibit mammalian thioredoxin reductase. For reduction of hydrogen peroxide and lipid hydroperoxides the selenol which is available at the enzyme surface will take up the oxygen from hydrogen peroxide. 71 To the large group of substrates for mammalian thioredoxin reductase can also be added the selenazol drug ebselen (2-phenyl-l,2-benzoisoselenazol-3(2H)-one) an antioxidant and anti-inflammatory agent, which is a known glutathione peroxidase mimic.76 We have recently shown 77 that ebselen is an excellent substrate for mammalian thioredoxin reductase and stimulates its hydrogen peroxide reductase activity quite dramatically. Ebselen is also an efficient oxidant of reduced thioredoxin. 77

4. The Glutaredoxin System The glutaredoxin system is comprised of GSH, NADPH, glutathione reductase and glutaredoxin.40,78 The level of GSH in cells are generally

16


/ GSH YmonothlolV I mechanism I Sll * \

/^~\

/

y^tJ] I mductes* j

V GSH *

«Nol mechanism

* NADP* / '

Fig. 6. Mechanism of glutaredoxin catalyzed monothiol or dithiol reactions. The monothiol mechanism involves formation and cleavage of GSH-mixed disulfides. The dithiol mechanism as in ribonucleotide reductase involves reduction of a disulfide. h i g h (1-10 m M ) a n d it is kept r e d u c e d b y N A D P H a n d glutathione r e d u c t a s e (GR) a s s h o w n in Reaction (5): GSSG +

NADPH

H+

GS

2GSH

NADP+.

(5)

Via g l u t a r e d o x i n (Grx) electrons from G S H are u s e d to r e d u c e disulfides [Reactions (6) a n d (7)] Grx-S2 Grx-S,

2 GSH Protein-S,

Grx-(SH)2 Grx-S,

GSSG Protein-(SH)2.

(6) (7)

Grx also catalyzes formation a n d cleavage of glutathionylated p r o t e i n s [(Reaction (8)]: Protein-SH

GSSG

Protein-S-SG

GSH

(8)

A s s h o w n in Fig. 6, glutaredoxins can either catalyze m o n o t h i o l or dithiol reactions. T o d a y glutaredoxins a r e a multifunctional family of G S H disulfide oxidoreductases w h i c h b e l o n g t o t h e thioredoxin fold s u p e r family. 79 ' 80 T h e y h a v e a GSH-binding site a n d a redoxactive disulfide w i t h the concensus sequence -Cys-Pro-Tyr-Cys-. O n l y t h e N-terminal nucleophilic Cys-residue 8 1 is required for catalyzing reversible glutathiorylation


Fig. 7. Left: NMR solution structure of E. coli Grx 1 in mixed disulfide with GSH (Glygs) via Cys 11. Right: Molecular surface with residues interacting with the GSH molecule; GSH shown as a sticks model.

reaction (monothiol mechanism) whereas both Cys residue are required for disulfide reduction (dithiol mechanism) (Fig. 6). Structural studies by NMR have solved the solution structure of glutaredoxin (Fig. 7).82 As molecular machines catalyzing thiol-disulfide oxidoreductions by cysteine thiols glutaredoxins are particularly interesting. A recent superb study of the structure, dynamics and electrostatics of the active site in glutaredoxin-3 from £. coli has recently been published. 83 This gives a unifying theme for the chemistry of the active site cysteine residues in the whole thioredoxin superfamily explaining why the pKa value of the N-terminal CXXC Cys-residue varies. This is primarily due to direct hydrogen bonding with the thiol proton of the other C-terminal Cys residue 84 and amide protons of the other residues inside the CXXC loop. 83 Glutaredoxins in mammalian cells have a growing list of functions such as reduction of dehydroascorbic acid,85 cellular differentiation or regulation of transcription factor activity.86 A new class of monothiol glutaredoxins in yeast and many other organisms appear to be particu larly important in defense against oxidative stress.87 Glutaredoxin also pro tects cerebellar granulae neurons from dopamine induced oxidative stress by activating NF-kappaB via Ref l.88 Recently, a novel human glutaredoxin

18


with both mitochondrial and nuclear isoforms has been cloned.89 The structure of E. colt Grx2 in solution demonstrates a similarity in structure to glutathione-S-transferases and defines a novel family of large monothiol glutaredoxins. 90

5. Redox Regulation of Cellular Function Control of the activity of proteins by the reversible oxidation of SH-groups or thiol redox control26,40 is now recognized as a major mecha nism for signal transduction. Oxidants generated upon cell activation or exposure to oxidative stress are converted to a disulfide signal via GSHperoxidases or thioredoxin peroxidases and are balanced by antioxidants from the thioredoxin and glutaredoxin systems. Transcription factor binding to DNA is particularly sensitive to the redox state of critical SHgroups. 91 The outside of cells is an oxidizing environment dominated by disulfides whereas the cytosol is rich in SH-groups. Changes in the levels of GSH and GSSG will be an important global parameter in determining the intracellular redox potential since glutathione is the major redox buffer of mammalian cells.92

6. Future Perspectives Today we have a relatively good idea about the catalytic activity of various forms of thioredoxins and glutaredoxins in the cytosol. Obvi ously, it will take a long time to understand the flux through these cat alytic proteins and the dynamics in the regulation via thiol redox control. Of particular interest in the future will be to understand phenomena at the cell surface and mechanisms of global regulation of secretion and movement of thioredoxin and glutaredoxin isoforms within cells. The use of specific inhibitors and pharmacological agents inducing or supressing the activities of thioredoxins and glutaredoxins will be future goals. Such potential use of the antiapoptotic effects of thioredoxins may be to rescue cells from tissues undergoing degenerative cell death. In other situations, like in cancer therapy, directed inhibitors or use of gene therapy would be possible to selectively block the growth promoting and growth advantage of cells expressing high thioredoxin or glutaredoxin. Clearly, the complexity of SH-groups and their interplay in a cellular environment will keep us busy for the forseen future.


19

Acknowledgments Research support by the Swedish Medical Research Council, the Swedish Cancer Society and the K.A. Wallenberg Foundation is gratefully acknowledged.

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51. Ganther HE. 1971. Reduction of the selenotrisulfide derivative of glutathione to a persulfide analog by glutathione reductase. Bio chemistry 10: 4089-4098 52. Luthman M, Holmgren A. 1982. Rat liver thioredoxin thiore doxin reductase: Purification characterization. Biochemistry 21: 6628-6633 53. Lundstrom J, Holmgren A. 1990. Protein disulfide-isomerase is a sub strate for thioredoxin reductase has thioredoxin-like activity. /. Biol. Chem. 265: 9114-9120 54. Nikitovic D, Holmgren A. 1996. S-nitrosoglutathione is cleaved by the thioredoxin system with liberation of glutathione redox regulat ing nitric oxide. /. Biol. Chem. 271:19180-19185 55. Bjornstedt M, Xue J, Huang W, Akesson B, Holmgren A. 1994. The thioredoxin glutaredoxin systems are efficient electron donors to human plasma glutathione peroxidase. /. Biol. Chem. 269: 29382-29384 56. Zhong L, Holmgren A. 2000. Essential role of selenium in the cat alytic activities of mammalian thioredoxin reductase revealed by characterization of recombinant enzymes with selenocysteine muta tions. /. Biol. Chem. 275:18121-18128 57. Bjornstedt M, Hamberg M, Kumar S, Xue J, Holmgren A. 1995. Human thioredoxin reductase directly reduces lipid hydroperoxides by NADPH selenocystine strongly stimulates the reaction via catalytically generated selenols. /. Biol. Chem. 270:11761-11764 58. Holmgren A, Lyckeborg C. 1980. Enzymatic reduction of alloxan by thioredoxin NADPH-thioredoxin reductase. Proc. Natl. Acad. Sci. USA 77: 5149-5152 59. Andersson M, Holmgren A, Spyrou G. 1996. NK-lysin a disulfide containing effector peptide of T-lymphocytes is reduced inactivated by thioredoxin reductase. Implication for a protective mechanism against NK-ysin cytotoxicity. /. Biol. Chem. 271: 10116-10120 60. Arner ESJ, Nordberg J. Holmgren A. 1996. Efficient reduction of lipoamide lipoic acid by mammalian thioredoxin reductase. Biochem. Biophys. Res. Commun. 225y: 268-274 61. May JM, Mendiratta S, Hill KE, Burk RF. 1997. Reduction of dehydroascorbate to ascorbate by the selenoenzyme thioredoxin reduc tase. /. Biol. Chem. 272: 22607-22610 62. May JM, Cobb CE, Mendiratta S, Hill KE, Burk RF. 1998. Reduction of the ascorbyl free radical to ascorbate by thioredoxin reductase. /. Biol. Chem. 27: 23039-23045

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63. Tamura T, Stadtman TC. 1996. A new selenoprotein from human lung adenocarcinoma cells: Purification properties thioredoxin reductase activity. Proc. Natl. Acad. Sci USA 9: 1006-1011 64. Gladyshev VN, Jeang K-T, Stadtman TC. 1996. Selenocysteine identi fied as the penultimate C-terminal residue in human T-cell thiore doxin reductase corresponds to TGA in the human placental gene. Proc. Natl. Acad. Sci. USA 9: 6146-6151 65. Gasdaska PY, Gasdaska JR, Cochran S, Powis G. 1995. Cloning sequencing of a human thioredoxin reductase FEBS Lett. 37: 5-9 66. Bock A, Forchhammer K, Heider L, Leinfelder W, Sawers G, Veprek B, Zinoni F. 1991. Selenocysteine: The 21st amino acid. Mol. Microbiol. 5: 515-520 67. Zhong L, Arner ESJ, Ljung J, Aslund F, Holmgren A. 1998. Rat calf thioredoxin reductase are homologous to glutathione reductase with a carboxyterminal elongation containing a conserved catalytically active penultimate selenocysteine residue. /. Biol. Chem. 273: 8581-8591 68. Arscott LD, Gromer S, Schirmer RH, Becker Williams CH. 1997. The mechanism of thioredoxin reductase from human placenta is similar to the mechanism of lipoamide dehydrogenase glutathione reductase is distinct from the mechanism of thioredoxin reductase from Escherichia coll Proc. Natl Acad. Sci. USA 94: 3621-3626 69. Arner ESJ, Bjornstedt M, Holmgren A. 1995. l-chloro-24-dinitrobenzene DNCB. is an irreversible inhibitor of human thioredoxin reductase: Loss of thioredoxin disulfide reductase activity is accom panied by a large increase in NADPH oxidase activity. /. Biol. Chem. 270: 3479-3482 70. Nordberg J, Zhong L, Holmgren A, Arner ESJ 1998. Mammalian thioredoxin reductase is irreversibly inhibited by dinitrohalobenzenes by alkylation of both the redox active selenocysteine its neigh bouring cysteine residue. /. Biol. Chem. 27:10835-10842 71. Zhong L, Arner ESJ, Holmgren A. 2000. Structure mechanism of mammalian thioredoxin reductase: The active site is a redoxactive selenolthiol/selenenylsulfide formed from the conserved cysteineselenocysteine sequence. Proc. Natl. Acad. Sci. USA 9: 5854-5859 72. Arner ESJ, Sarioglu H, Lottspeich F, Holmgren A, Bock A. 1999. High level expression in Escherichia coli of selenocysteine-containing rat thioredoxin reductase utilizing gene fusions with engineered bacterialtype SECIS elements co-expression with the selA, selB and selC genes. /. Mol. Biol. 29: 1003-1016


25

73. Zhong L, Persson K, Sandalova T, Schneider G, Holmgren A. 2000. Purification crystallization preliminary crystallographic data for rat cytosolic selenocysteine-498 to cysteine mutant thioredoxin reductase. Acta Cryst. D5:1191-1193 74. Sandalova T, Zhong L, Lindqvist Y, Holmgren A, Schneider G. 2001. Three-dimensional structure of a mammalian thioredoxin reductase: Implications for mechanism evolution of a selenocysteine-dependent enzyme. Proc. Natl. Acad. Sci. USA, 98: 9533-9538 75. Gromer S, Arscott LD, Williams CH Jr, Schirmer RH, Becker K. 1998. Human placenta thioredoxin reductase. Isolation of the selenoenzyme steady state kinetics inhibition by therapeutic gold com pounds. /. Biol. Chem. 273: 20096-20101 76. Schewe T. 1995. Molecular actions of ebselen — An antiinflammatory antioxidant, Gen. Pharmacol. 26:1153-1169 77. Zhao R, Masayasu H, Holmgren A. 2002. Ebselen: a substrate for human thioredoxin reductase strongly stimulating its hydroperoxide reductase activity and a superfast thioredoxin oxidaut Proc. Natl. Acad. Sci. USA 99: 8579-8584 78. Holmgren A, Aslund F. 1995. Glutaredoxin. Meth. Enzymol. 252: 283-292 79. Martin JL. 1995. Thioredoxin — A fold for all reason. Structure 3: 245-250 80. Holmgren A. 1995. Thioredoxin structure mechanism: Conformational changes on oxidation of the active site sulfhydryls to a disulfide. Structure 3: 239-243 81. Bushweller JH, Aslund F, Wuthrich K, Holmgren A. 1992. Structural functional characterization of the mutant Escherichia coli glutaredoxin C14-»S. Its mixed disulfide with glutathione. Biochemistry 31:9288-9293 82. Bushweller JH, Billeter M, Holmgren A, Wuthrich K. 1994. The nuclear magnetic resonance solution structure of the mixed disulfide between Escherichia coli glutaredoxin C14S. Glutathione. /. Mol. Biol. 235:1585-1597 83. Foloppe N, Sagemark J, Nordstrand K, Berndt KD, Nilsson L, 2001. Structure dynamics electrostatics of the active site of glutaredoxin-3 from Escherichia coli: Comparison with functionally related proteins. /. Mol. Biol. 310: 449-470 84. Jeng M-F, Holmgren A, Dyson HJ. 1995. Proton sharing between cysteine thiols in Escherichia coli thioredoxin: Implications for the mechanism of protein disulfide reduction. Biochemistry 34: 10101-10105

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85. Wells WW, Xu DP, Washburn MP. 1995. Glutathione: Dehydroascorbate oxidoreductases. Meth. Enzymol. 252: 30-38 86. Nakamura T, Ohno T, Hirota K, Nishiyama A, Nakamura H, Wada H, Yodoi J. 1999. Mouse glutaredoxin — cDNA cloning high level expression in £ coli. Its possible implication in redox regulation of the DNA binding activity in transcription factor PEBP2. Free Radio. Res. 4: 357-365 87. Rodriguez-Mazaneque MT, Ros J, Cabiscol E, Sorribas A, Herrero E. 1999. Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae. Mol. Cell. Biol. 12: 8180-8190 88. Daily D, Vlamis-Gardikas A, Offen D, Mittelman L, Melamed E, Holmgren A, Barzilai A. 2001. Glutaredoxin protects cerebellar gran ule neurons from dopamine induced apoptosis by activating N F - K B via Ref-1. /. Biol. Chem. 276:1335-1344 89. Lundberg M, Johansson C, Chandra J, Enoksson M, Jacobsson G, Ljung J, Johansson M, Holmgren A. 2001. Cloning expression of a novel h u m a n glutaredoxin Grx2 with mitochondrial nuclear isoforms. /. Biol. Chem. 276: 26269-26275 90. Xia B, Vlamis-Gardikas A, Holmgren A, Wright PE, Dyson HJ. 2001. Solution structure of Escherichia coli glutaredoxin-2 shows similarity to mammalian glutathione-S-transferases. /. Mol. Biol, 310: 907-918 91. Zheng M, Aslund F, Storz G. 1998. Activation of the OxyR transcrip tion factor by reversible disulfide bond formation. Science 279: 1718-1721 92. Gilbert HF. 1990. Molecular cellular aspects of thiol-disulfide exchange. Adv. Enzymol. Relat. Areas Mol. Biol. 63: 69-172

Chapter 2

Selenocysteine Insertion and Reactivity: Mammalian Thioredoxin Reductases in Relation to Cellular Redox Signaling Elias S.J. Arner Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77, Stockholm, Sweden [email protected]

Keywords: Selenocysteine, selenoprotein, thiol redox control, reactive oxygen species, intracellular signaling

1. Summary Selenocysteine (Sec) — the 21st amino acid — is incorporated into selenoproteins at the position of specific TGA codons (UGA in the mRNA), normally conferring termination of translation. Stretching the genetic code by insertion of Sec at the Sec-specific UGA involves a highly intricate trans lation machinery, which differs significantly between species and which is dependent upon a secondary structure in the selenoprotein mRNA; a SECIS (Selenocysteine Insertion Sequence) element. In man, at least 20 selenoproteins are known, including the glutathione peroxidases and thyroid hormone deiodinases. Since the mid-1990s, the mammalian thio redoxin reductase (TrxR) isoenzymes are also known to be selenoproteins, in contrast to the smaller non-selenoprotein thioredoxin reductases from bacteria, plants or yeast. In E. coli, the three formate dehydrogenase H, O and N isoenzymes are the only natural selenoproteins. In most instances, selenoproteins are oxidoreductases dependent upon the high reactivity of the selenocysteine residue. Mammalian thioredoxin reductases (the cytosolic, mitochondrial and testis specific TrxR iso enzymes) all carry a Sec residue within a rather unique carboxyterminal motif being -Gly-Cys-Sec-Gly-COOH. A high reactivity of cytosolic 27


TrxR with diverse electrophilic agents, including dinitrohalobenzenes (e.g. DNCB), iodoacetic acid, 4-vinylpyridine or platinum drugs, has been demonstrated. All these compounds irreversibly inactivates the enzyme, but only when in a reduced form. This is explained by derivatization of the reactive Sec residue being exposed when the enzyme is reduced by NADPH. An additional unique effect in inhibition of TrxR with dinitro halobenzenes is a pronounced induction of an NADPH oxidase activity in the dinitrophenyl-derivatized enzyme. This can be mechanistically explained by a functional half-reaction with subsequent interaction of the enzyme-bound FAD and/or disulfide/dithiol motif in the N-terminal domain, with the nitro groups of the dinitrophenyl moieties at the derivatized C-terminus. Moreover, this reaction may be proposed to mediate some of the strong inflammatory components of the immunostimulatory effects seen upon topical treatment with dinitrohalobenzenes. This model for inflammation is based upon the induced intracellular oxidative stress due to the inactivated TrxR with a superoxide-producing NADPH oxidase activity, in combination with an increased synthesis of thioredoxin with secretion to the extracellular space where it is known to have cytokine-like activity. Recent studies of the human promoter for cytosolic TrxR reveal that the gene seems to be the first known to have a housekeeping-type promoter with regulation of mRNA levels through AUUUA motifs in the 3'-untranslated region. Such AU-rich elements are otherwise known to be present in cytokines or proto-oncogenes regulated in response to intracel lular redox signaling. The reactivity, function and regulation of cytosolic TrxR indicates that this selenoprotein plays a central role in cellular redox signaling, which shall be discussed in this chapter.

2. The Mammalian Thioredoxin System The mammalian thioredoxin system consists of thioredoxin (Trx), thio redoxin reductase (TrxR) and NADPH. Thioredoxin is reduced by TrxR and participates in many different types of reactions, including synthesis of deoxyribonucleotides, redox control of transcription factors, reduction of peroxides and redox regulation of apoptosis. Extracellularly thiore doxin has immunoregulatory activities as co-cytokine or chemokine. These functions are reviewed elsewhere 1 " 3 and will not be discussed at length in this chapter. It is of importance, however, to note that the redox status of thioredoxin is essential for most, if not all of its many vital cellular functions. Consequently, perturbations of the TrxR activity

Selenocysteine Insertion and Reactivity

29

are implicated in a number of cell proliferative or immunological diseases and the enzyme is increasingly being recognized as an important pharmacological target in a number of medical conditions, as reviewed in Ref. 4. In addition to reduction of the active site disulfide in thioredoxin, mammalian TrxR also reduces disulfides in other proteins like protein disulfide isomerase or NK-lysin, low molecular weight disulfides like 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent) or lipoic acid, low molecular weight non-disulfide substrates like selenite or alloxan, or even lipid hydroperoxides (see Ref. 5). The mammalian TrxR enzymes are implied to play central roles in cell proliferation, redox regulation and protection against oxidative damage, but conclusive experimental insights regarding the cellular functions of these seleno proteins are yet, however, quite scarce. Here we shall discuss the selenoprotein nature and characteristics of mammalian TrxR with a focus on the possible functional roles this enzyme may indeed play in cellular redox control.

3. Selenocysteine in Thioredoxin Reductase 3.1 Co-Translational Insertion of Selenocysteine In 1996, Theresa Stadtman and coworkers reported that human cytosolic TrxR contains the rare amino acid selenocysteine at its penultimate carboxyterminal position. 6,7 It was subsequently shown that bovine, rat and human cytosolic TrxR all contain a conserved carboxy-terminal tetrapeptide motif-Gly-Cys-Sec-Gly-COOH (where Sec is selenocysteine) and that the selenocysteine residue is essential for the enzymatic activity.8 More over, the overall amino acid sequence of the enzyme is not homologous to that of TrxR from lower organisms but instead closely similar to the sequence of glutathione reductase, with the addition of a 16-residue carboxy-terminal elongation carrying the selenocysteine-containing amino acid motif.8 Selenoproteins containing selenocysteine residues are found in most bacteria, archaea as well as eukarya. Selenocysteine is the selenium analogue of cysteine and is due to the electrophilicity of selenium nor mally ionized at physiological p H to a selenolate, in contrast to cysteine which is most often present in the protonated sulfhydryl form (Fig. 1). This difference between selenocysteine and cysteine usually lead to a higher reactivity of selenoproteins in comparison to their cysteine mutants and known selenoproteins are most often oxidoreductases with

30

Cellular Implications ofRedox Signalling H

H

I

I

'HgH-O-COO"

*H3N-C-COO"

Se~

SH

Selenocysteine

Cysteine

(U, Stc)

(C. Cy>)

Fig. 1. Selenocysteine versus cysteine. The figure depicts the difference between selenocysteine (U, Sec) and cysteine (C, Cys) with a selenium atom taking the place of the sulfur in cysteine, and with selenocysteine usually being present in the ionized selenolate form at physiological pH (free Sec pKa = 5.2) in comparison to the usually protonated thiol of cysteine (pKa = 8.3). a catalytic selenocysteine residue in their active site. In bacteria and archaea these selenoproteins include formate dehydrogenases, hydrogenases or glycine reductase, whereas in mammals other known seleno proteins apart from thioredoxin reductases constitute the glutathione peroxidase family and the thyroid hormone deiodinases. In addition, mammalian selenoproteins have been described with yet unknown func tion such as selenoprotein P or W.9"11 Recent database searches indicate that the list of mammalian selenoproteins will continue to grow. 1213 Selenocysteine is in all organisms cotranslationally inserted at the position of an opal (UGA) codon, normally conferring termination of translation. The UGA codon is encoded as selenocysteine by a highly complex translation machinery, characterized in detail for E. coli by August Bock and coworkers, using formate dehydrogenase H as a model system — for reviews, see Refs. 10, 11 and 14. In short, mRNA for E. coli selenoproteins contain an about 40 nucleotides long selenocysteine inser tion sequence (SECIS) positioned immediately 3 ' of the UGA codon. These nucleotides have dual functions; they provide codons for the trans lation of amino acids following the selenocysteine residue, and they fold into a stem-loop type secondary structure — a SECIS element. The SECIS element binds the SELB protein, the selB gene product. SELB is homolo gous to elongation factor EF-Tu but, in addition, carries a carboxy-terminal domain binding the loop region of the SECIS element. SELB also binds to a selenocysteine specific tRNA (tRNA 3 "), the selC gene product, in its selenocysteinylated form. Thereafter, in analogy with EF-Tu, SELB is at the ribosome catalyzing selenocysteine insertion at the specific position of the selenocysteine UGA codon. The tRNA560 is originally charged with a seryl-residue which by utilization of selenophosphate is converted to

Selenocysteine Insertion and Reactizrity 31

Fig. 2. The selenocysteine insertion machinery in E. coli. In translation of a non-selenoprotein mRNA, the elongation factor Ef-Tu catalyzes insertion of amino acids into the elongating polypeptide chain utilizing tRNA's for any of the common 20 amino acids and if a UGA codon is encountered, the release factor RF2 terminates the elongation. If a bacterial-type SECIS element structure is present in the mRNA next to the UGA, however, this will be recognized by the selenocysteine-specific elongation factor SELB. The SELB only utilizes the selenocysteine-specific tRNA8*0 (the SelC gene product) in its selenocysteinylated form, thereby elongating the polypeptide chain with selenocysteine insertion at the correct UGA codon. The tRNA 5 " is originally charged with a seryl moiety, which is converted to selenocysteinyl while bound to the tRNA. See text for further details and references to reviews on this subject. selenocysteinyl b y selenocysteine synthase, a n oligomer of the selA g e n e p r o d u c t . The s e l e n o p h o s p h a t e , in t u r n , is p r o v i d e d b y s e l e n o p h o s p h a t e synthetase, the selD gene p r o d u c t . T a k e n together, selenocysteine inser tion in E. coli involves: an E. co/z'-type SECIS e l e m e n t at the right position after the U G A c o d o n in the selenoprotein m R N A , a n d the selA, selB, selC a n d selD g e n e p r o d u c t s . This E. coli selenoprotein translation m a c h i n e r y is schematically s u m m a r i z e d in Fig. 2. A SECIS e l e m e n t is found also in the m R N A of m a m m a l i a n selenop r o t e i n s b u t this h a s other s e c o n d a r y structures a n d conserved features

32


than found in E. coli and, moreover, is situated in the 3'-untranslated region several hundred nucleotides downstream of the UGA codon. 915 Thereby mammalian selenoprotein genes are generally incompatible with direct recombinant expression in E. coli. A technique, however, to by-pass the barriers to heterologous expression of selenoproteins in E. coli16 enabling bacterial production of recombinant mammalian TrxR was developed utilizing engineered variants of the bacterial SECIS element, encoding the C-terminal motif of TrxR.17 Use of this recombinant method ology is likely to facilitate further studies of mammalian TrxRs as well as other selenoproteins.

3.2 Selenocysteine in TrxR as a Drug Target The catalytic mechanism of mammalian TrxR shall not be described in detail here but can be concluded to be similar to that of glutathione reductase 18 but in addition involving a reversible selenolthiol/selenenylsulfide formed by the penultimate selenocysteine and its neighboring cysteine, constituting a second non-flavin redox active center.19 The oxidized selenenylsulfide-containing form of the enzyme is highly resistant to modi fication with electrophilic agents or to digestion with carboxypeptidase. 8 However, when the enzyme is reduced by NADPH the Cys-Sec site becomes susceptible to modification by the above treatments, which thereby easily inactivate the enzyme.8,20-22 This molecular mechanism hence generally suggests how the many inhibitors of TrxR act. These elec trophilic inhibitors include antitumor quinones, 23 doxorubicin, 24 antitumor nitrosourea drugs, 25 retinoic acid,26 anti-rheumatic gold compounds such as gold thioglucose.27,28 Molecular modeling of the C-terminal tetrapeptide of TrxR in both the oxidized and the reduced state may illus trate how the selenenylsulfide must induce a beta-turn like bend at the C-terminus protecting this redox active center, and, alternatively, when reduced, how the selenol(ate) of the selenocysteine residue becomes exposed and hence highly susceptible to reactions with either substrates or inhibitors of the enzyme (Fig. 3). It is probable that the inhibition of thioredoxin reductase by electro philic drugs in clinical use should contribute to their therapeutic effects, or side effects, which is a notion that has also recently been reviewed elsewhere. 4 Dinitrohalobenzenes are unique in their inactivation of TrxR by derivatizing the enzyme concomitant with an induction of an NADPH oxidase activity in the derivatized enzyme.20'21 The reactivity with


33

Fig. 3. Molecular modelling of the C-terminal tetrapeptide of TrxR in reduced and oxidized form. In (A), a stereo view of a modelled reduced C-terminal Gly-Cys-Sec-Gly tetrapeptide is given, illustrating the highly exposed Sec and Cys residues on opposite sides of the polypeptide backbone. In (B), the same tetra peptide has been modelled with a selenenylsulfide bridge between the Cys and Sec residues, as has been experimentally demonstrated to be present in oxidized TrxR, using Edman degradation and mass spectrometry. 19 In order to make possi ble this selenenylsulfide, a beta-turn like bend must be imposed on the structure to place the two side chains of Cys and Sec on the same side of the peptide back bone. This unique structure may explain why the oxidized enzyme is resistant to carboxypeptidase treatment 8 or derivatization with electrophilic agents (see text). It is also possible that the larger atom radius of the selenium atom may help to form the bridge in the oxidized motif, as the cysteine mutant seem not to be able to easily form a corresponding disulfide but leaves a dithiol motif in the oxidized mutant holoenzyme. 70 Modelling was performed using the CORINA algorithm (see http://www2.ccc.uni-erlangen.de/software/corina/corina.html). The sulfur of cysteine is shown in yellow and the selenium of selenocysteine is purple.

dinitrohalobenzenes will b e discussed b e l o w in m o r e detail, also being the basis for a discussion o n the relation b e t w e e n the activities of cytosolic TrxR a n d diverse intra- as well as extra-cellular signaling systems. T w o additional TrxR i s o e n z y m e s h a v e b e e n identified, one m i t o chondrial 29 " 32 a n d one testis specific, 33 w i t h b o t h h a v i n g the s a m e overall d o m a i n structure as T r x R l . Interestingly, the testis specific isoenzyme, h o w e v e r , also contains a n N - t e r m i n a l m o n o t h i o l g l u t a r e d o x i n d o m a i n w h i c h seems to give this e n z y m e a n a d d i t i o n a l g l u t a t h i o n e r e d u c t a s e

34


activity which the other TrxR isoenzymes lack, and was therefore recently named TGR for its thioredoxin and glutathione reductase. 34 The possible role of the cytosolic TrxRl isoenzyme in relation to intracellular redox signaling shall now be discussed in some further detail.

4. Regulation of Cytosolic Thioredoxin Reductase in Relation to Cellular Redox Signaling The 3 ' untranslated region of the mRNA for cytosolic TrxRl contains in addition to the SECIS element also AU-rich elements, AREs, which in untreated cells lead to a rapid TrxRl mRNA turnover.35,36 In fact, TrxRl was independently cloned as KDRF in a study specifically set out to identify genes being regulated through AREs.35 The presence of func tional AREs are otherwise typically found in mRNAs of cytokines, protooncogenes, transcription factors and other transiently expressed genes. 37 Post-transcriptional regulation via AREs enables quick expression responses to various stimuli, by a block in the rapid mRNA degradation through specific ARE-interacting proteins responding to intracellular signaling. 37 It is interesting that TrxRl contains functional AREs35,36 since this enzyme is not transiently expressed only under specific growth conditions, but is widely expressed in many diverse tissues and cells.30,38-40 TrxRl is nonetheless known to display significant and fast (within hours) increase of protein as well as mRNA upon treatment of cells with a number of different exogenous agents. Examples of this include human epidermoid carcinoma A431 cells treated with epidermal growth factor, H 2 0 2 or l-chloro-2,4-dinitrobenzene 33 or thyrocytes given calcium ionophore (A23187) and PMA.41 The latter was also seen in human umbili cal vein endothelial cells, although less pronounced much due to more than 10-fold higher basal TrxR levels in these cells compared to thyrocytes.42 In human bone marrow-derived stromal cells (KM102) both PMA in com bination with A23187 or, alternatively, treatment with interleukin-lp or lipopolysaccharide significantly increased the TrxRl mRNA levels within 4 hrs, being the KDRF study referred to above. 35 In peripheral blood monocytes and myeloid leukaemia cells43 as well as osteoblasts 44 TrxRl mRNA levels were shown to be increased above basal levels in a fast but transient manner by vitamin D3 treatment. How is the increase of TrxRl levels upon diverse exogenous stimuli transmitted and what may this regulation tell us about the cellular func tion of TrxRl? We recently found that human TrxRl has an Octl- and

Selenoq/steine Insertion and Reactivity

35

Spl-driven TATA- and CCAAT-less typical housekeeping-type core promoter, with expression in many different cell types. 45 Considering this functional organization with a housekeeping-type promoter in combina tion with ARE-mediated post-transcriptional regulation, being quite unique, we propose a novel type of regulation of the enzyme in relation to intracellular redox signaling. The presence of 3' untranslated region AREs may generally enable a quick stabilization of mRNA and can thereby upregulate protein levels in fast response to various signals.37,46 Upon many different exogenous stimuli, reactive oxygen species (ROS) such as superoxide or hydrogen peroxide are also produced as common mediators for intracellular signaling.47 One regulatory protein which is rapidly upregulated upon formation of ROS is the p38 mitogen-activated protein (MAP) kinase, as reviewed in Ref. 48. The stress-activated p38 MAP kinase in turn upregulates the MAP kinase-activated protein kinase-2 (MK2) and, interestingly, MK2 was shown to induce stabilization of ARE-containing mRNAs, thereby exe cuting their stabilization under intracellular formation of ROS.49 Since TrxRl contains functional AREs and is also known to be upregulated by many exogenous agents (see above) which in turn are known to mediate intracellular ROS formation as a common denominator, 50 it becomes possible that the AREs participate in mediating a fast response in increased expression of TrxR upon intracellular ROS formation. In addi tion, in cells, the TrxRl enzyme has been reported to be rapidly inacti vated by ROS, targeting the selenocysteine residue. 28 This chain of events makes it possible to propose the following model for TrxRl regulation and function. With a strong constitutive transcription, suggested from the initial characterizations of the promoter, 45 combined with ARE-regulated mRNA turnover and generally a short mRNA half-life in non-stimulated cells,36 TrxRl thereby has the inherent capacity for a fast response to an increase of intracellular ROS in their role of stabilizing the mRNA via MK2 and the ARE motifs; this would occur concomitant with a momen tary inactivation of the enzyme.28 Once more TrxRl rapidly has been syn thesized as a result of the stabilized mRNA, the antioxidant properties of the newly produced enzyme would then possibly be able to carry the cells back to a correct basal balance of the intracellular redox status, yet having allowed the transient burst of ROS being a necessary component for the many diverse systems of intracellular signaling. This proposed model for TrxRl activity and regulation in relation to intracellular redox signaling has yet, however, to be experimentally scrutinized. Still the model can indicate how the thioredoxin system may interrelate to intracellular signaling

36


systems via a fast regulated activity of TrxR. The reactivity of TrxR with dinitrohalobenzenes is another example which may illustrate this interrelationship.

5. Effects of Dinitrohalobenzenes by Interactions with Thioredoxin Reductase l-chloro-2,4-dinitrobenzene (DNCB, CDNB) is an electrophilic compound used as a substrate in assays to determine glutathione S-transferases, being involved in elimination of DNCB in vivo.52 DNCB is therefore also used in cell culture experiments as a GSH-depleting agent. 53 Furthermore, DNCB has an established use as an immunomodulatory agent to provoke delayed-type hypersensitivity reactions.54 Although proposed to function as a hapten, the mechanism of DNCB immunomodulation is however not clear, especially regarding the pro-inflammatory properties of the compound. 55 In 1995, we found that DNCB irreversibly inhibited NADPH-reduced mammalian thioredoxin reductase, with a concomitant induction of an NADPH oxidase activity20 and we later demonstrated that the selenocysteine residue in thioredoxin reductase indeed was the target for derivatization. 21 In addition, the neighboring cysteine residue in the carboxyterminal tetrapeptide of the enzyme (-Gly-Cys-Sec-Gly-COOH), was also derivatized. Also incubation with other electrophilic compounds, like iodoacetic acid or 4-vinylpyridine, inhibited TrxRl irreversibly but in this case no induction of an NADPH oxidase activity was seen.21 This indi cated that an inherent property of dinitrohalobenzenes was necessary for the NADPH oxidase activity to be induced and this property was most likely carried by the nitro groups. The induced NADPH oxidase activity was found to produce superoxide anions 21 and since superoxide-producing NAD(P)H-dependent redox cycling of aromatic nitro compounds with flavoenzymes is a known phenomenon,56-59 a model for the interaction between mammalian thioredoxin reductase and dinitrohalobenzenes became possible to propose. 60 In this model, NADPH is proposed to reduce the enzyme-bound oxidized FAD even if the C-teminal Sec-containing redox active motif has been derivatized with dinitrophenyl groups. Of importance for the reac tivity of TrxR with dinitrohalobenzenes is that only upon reduction with NADPH is the carboxyterminal motif known to be accessible for alkylation20,21 or digestion with carboxypeptidase Y,8 most likely being


37

PADH2 S — ^ X

Fig. 4. Derivatization of TrxR with DNCB and model for the NADPH oxdiase activity. The selenenylsulfide at the C-terminus of one subunit of the oxidized holoenzyme TrxR must first be reduced to a selenolthiol for derivatization to occur. The reduction to a selenolthiol is NADPH dependent (A) and occurs via the FAD (B) and redox active dithiol (C) of the other subunit, as described in Ref. 19. When the selenolthiol has been exposed (see Fig. 3), two DNCB molecules easily derivatize both the cysteine and the selenocysteine at the C-terminus (E). Possibly the selenolate is first derivatized, which may induce a thiolate at the cysteine by resonance effect, hence leading to alkylation also of the neighboring cysteine residue. The experimentally observed superoxide-producing NADPH oxidase activity by the dinitrophenyl-derivatized enzyme 21 may be explained by a functional half reaction and NADPH-dependent reduction of the FAD in the derivatized enzyme (F) and consecutive one-electron transfers to the nitro groups of the dinitrophenyl moieties. First, a flavin semiquinone and a nitro anion radical is formed (G). The nitro anion radical readily reacts with molecu lar oxygen to form one molecule of superoxide (H). The semiquinone subse quently forms a second nitro anion radical (I) which also reacts with oxygen to produce another molecule of superoxide (J), returning the enzyme to a fully oxi dized dinitrophenyl-derivatized form, which again may go through a cycle of NADPH oxidase acitivity (F-J). This model has been published in a somewhat simplified form in Ref. 60.

38


explained by the selenenylsulfide of the oxidized enzyme 19 being highly inert. Once reduced, however, the free selenolthiol motif should be highly susceptible to derivatization with dinitrohalobenzenes, explaining the experimentally found 10,000-fold higher reactivity of reduced TrxRl with DNCB compared to derivatization of reduced GSH under the same conditions at pH 7.5.20 See also Fig. 3 illustrating the difference between the reduced (susceptible) and oxidized (protected) C-terminal motif of TrxR. To explain the induced NADPH oxidase activity in the dinitrophenylderivatized TrxR, it is hence proposed that the FAD of the alkylated enzyme still can be reduced by NADPH but that one (or two) of the nitro groups in the dinitrophenyl moieties of the alkylated enzyme in two con secutive one-electron transfers are converted to nitro anion radicals that in turn react with oxygen to form superoxide. This would regain dinitrophenyl-derivatized TrxR having oxidized FAD, that again can be reduced with NADPH to give the observed superoxide producing NADPH oxidase activity (Fig. 4). Does the specific and high reactivity with mammalian TrxR of dinitrohalobenzenes like DNCB play a role in the molecular mechanism behind the immunomodulatory properties of these compounds? In dis cussing this question, it is of importance to note that a number of DNCB analogs which failed to inhibit TrxR or to induce any NADPH oxidase activity21 previously had been tested in vivo for induction of hypersensitivity reactions in mice and shown to provoke no reaction.55 In that same study, 0 2 utilization, H 2 0 2 production and NADPH consumption in skin or liver microsomes was also measured upon addition of dinitrohaloben zenes or the DNCB analogs. All of these properties correlated well to mouse ear swelling upon application of the compounds, whereas changes in levels of GSH or GSSG did not.55 The enzyme(s) responsible for the NADPH consumption and superoxide (or H 2 0 2 ) production were not identified, but it should be safe to conclude that TrxR is a strong candi date. How would the interaction with TrxR by dinitrohalobenzenes take part in the mechanism of immunostimulation by these compounds? Two mechanisms are possible. First, thioredoxin is known to play a central role in redox regulation of cell function1'3'61 and an irreversible inhibition of TrxR with concomitant superoxide production would therefore with cer tainty affect thioredoxin-related functions in the immune system, possibly mimicking the natural intracellular signaling conveyed by increased lev els of intracellular ROS.3'47,48 Second, it may be proposed that secretion of thioredoxin a n d / o r its shorter truncated variant Trx-80 is stimulated upon oxidative stress, resulting in immunostimulation through co-cytokine or

Selenocysteine Insertion and Reactimty

39

chemokine activities of full-length thioredoxin62"65 or Trx80,66 the latter also involving stimulation of interleukin-12 production from monocytes thereby favouring a Thl response. 67 It should also be noted that if excessive, increased oxidative stress is a known initiatiator of cell death by either apoptosis or necrosis. This may also be exaggerated through inhibition of the thioredoxin system; e.g. by the facts that reduced thioredoxin binds to and thereby inhibits the proapoptotic apoptosis signaling kinase-1 (ASK-1)68 or that peroxiredoxins, being dependent upon thioredoxin activity, seem to counteract apoptosis at an early stage upstream of bcl-2.69 The actual difference between mimicking intracellular signaling through induction of ROS formation and thereby stimulating cells, or inducing cell death by excessive ROS forma tion through the inactivation of TrxR, may therefore possibly be dose dependent with regard to the TrxR inhibitor being utilized. The interrelationships between the thioredoxin system and cellular signaling are certainly intimate but also complex. It has been the aim of this chapter to focus on the reactivity of the selenocysteine residue in TrxR and the relation of this enzyme to the intracellular redox signaling pathways as well as to the inflammatory response seen upon use of dinitrohalobenzenes. The idea of TrxR as a cellular redox sensor33 is intriguing and certainly deserves further functional studies on the cellular or organism level.

Acknowledgements The research of the author is supported by the Karolinska Institute and the Swedish Cancer Society (projects 3775-B00-05XAC and 4056-B99-02PBD).

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5. Arner ESJ, Zhong L, Holmgren A. 1999. Preparation and assay of mammalian thioredoxin and thioredoxin reductase. Meth. Enzymol. 300: 226-239 6. Gladyshev VN, Jeang K-T, Stadtman TC. 1996. Selenocysteine, iden tified as the penultimate C-terminal residue in human T-cell thio redoxin reductase, corresponds to TGA in the human placental gene. Proc. Natl. Acad. Sci. USA 93: 6146-6151 7. Tamura T, Stadtman TC. 1996. A new selenoprotein from human lung adenocarcinoma cells: Purification, properties, and thioredoxin reductase activity. Proc. Natl. Acad. Sci. USA 93: 1006-1011 8. Zhong L, Arner ESJ, Ljung J, Aslund F, Holmgren A. 1998. Rat and calf thioredoxin reductase are homologous to glutathione reductase with a carboxyl-terminal elongation containing a conserved catalytically active penultimate selenocysteine residue. /. Biol. Chetn. 273: 8581-8591 9. Low SC, Berry MJ. 1996. Knowing when not to stop: Selenocysteine incorporation in eukaryotes. TIBS 21: 203-208 10. Bock A, Forchhammer K, Heider J, Leinfelder W, Sawers G, Veprek B, Zinoni F. 1991. Selenocysteine: The 21st amino acid. Mol. Microbiol. 5: 515-520 11. Stadtman TC. 1996. Selenocysteine. Ann. Rev. Biochem. 65: 83-100 12. Lescure A, Gautheret D, Carbon P, Krol A. 1999. Novel selenoproteins identified in silico and in vivo by using a conserved RNA structural motif. /. Biol. Chem. 274: 38147-38154 13. Kryukov GV, Kryukov VM, Gladyshev VN. 1999. New mammalian selenocysteine-containing proteins identified with an algorithm that searches for selenocysteine insertion sequence elements. /. Biol. Chem. 274: 33888-33897 14. Hiittenhofer A, Bock A. 1998. RNA structures involved in selenoprotein synthesis. In RNA Structure and Function, eds. Simons RW. Grunberg-Manago M, Cold Spring Harbor Laboratory Press, New York 15. Walczak R, Westhof E, Carbon P, Krol A. 1996. A novel RNA struc tural motif in the selenocysteine insertion element of eukaryotic selenoprotein mRNAs. RNA 2: 367-379 16. Tormay P, Bock A. 1997. Barriers to heterologous expression of a selenoprotein gene in bacteria. /. Bacteriol. 179: 576-582 17. Arner ESJ, Sarioglu H, Lottspeich F, Holmgren A, Bock A. 1999. High-level expression in Escherichia coli of selenocysteine-containing rat thioredoxin reductase utilizing gene fusions with engineered


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38. Rozell B, Hansson HA, Luthman M, Holmgren A. 1985. Immunohistochemical localization of thioredoxin and thioredoxin reductase in adult rats. Eur. }. Cell. Biol. 38: 79-86 39. Gasdaska JR, Gasdaska PY, Gallegos A, Powis G. 1996. Human thioredoxin reductase gene localization to chromosomal position 12q23-q24.1 and mRNA distribution in human tissue. Genomics 37: 257-259 40. Rundlof A-K, Carlsten M, Giacobini MMJ, Arner ESJ. 2000. Prominent expression of the selenoprotein thioredoxin reductase in the medullary rays of the rat kidney and thioredoxin reductase mRNA variants dif fering at the 5' untranslated region. Biochem. ]. 347: 661-668 41. Howie AF, Arthur JR, Nicol F, Walker SW, Beech SG, Beckett GJ. 1998. Identification of a 57 kDA selenoprotein in human thyrocytes as thioredoxin reductase and evidence that its expression is regulated through the calcium-phosphoinositol signaling pathway. /. Clin. Endocrinol. Metab. 83: 2052-2058 42. Anema SM, Walker SW, Howie AF, Arthur JR, Nicol F, Beckett GJ. 1999. Thioredoxin reductase is the major selenoprotein expressed in human umbilical-vein endothelial cells and is regulated by protein kinase C. Biochem. }. 342:111-117 43. Schutze N, Fritsche J, Ebert-Dumig R, Schneider D, Kohrle J, Andreesen R, Kreutz M, Jakob F. 1999. The selenoprotein thio redoxin reductase is expressed in peripheral blood monocytes and THP1 human myeloid leukemia cells-regulation by 1,25-dihydroxyvitamin D3 and selenite. Biofactors 10: 329-338 44. Schutze N, Bachthaler M, Lechner A, Kohrle J, Jakob F. 1998. Identi fication by differential display PCR of the selenoprotein thioredoxin reductase as a lalpha,25(OH)2-vitamin D3-responsive gene in human osteoblasts—regulation by selenite. Biofactors 7: 299-310 45. Rundlof A-K, Carlsten M, Arner ESJ. 2001. The core promoter of human thioredoxin reductase 1: Cloning, transcriptional activity and Octl, Spl and Sp3 binding reveal a housekeeping-type promoter for the ARE-regulated gene. /. Biol. Chem., epub ahead of print 46. Chen CY, Shyu AB. 1995. AU-rich elements: Characterization and importance in mRNA degradation. Trends. Biochem. Sci. 20: 465-470 47. Dalton TP, Shertzer HG, Puga A. 1999. Regulation of gene expression by reactive oxygen. Ann. Rev. Pharmacol. Toxicol. 39: 67-101 48. Allen RG, Tresini M. 2000. Oxidative stress and gene regulation. Free Radic. Biol. Med. 28: 463-499

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49. Winzen R, Kracht M, Ritter B, Wilhelm A, Chen Y, Shyu AB, Muller M, Gaestel M. Resch K, Holtmann H. 1999. The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase-2 and an AU-rich region-targeted mechanism. EMBO J. 18: 4969-4980 50. Finkel T. 2000. Redox-dependent signal transduction. FEBS Lett. 476: 52-54 51. Hori K, Katayama M, Sato N, Ishii K, Waga S, Yodoi J. 1994. Neuroprotection by glial cells, through adult T cell leukemia-derived factor/human thioredoxin (ADF/Trx). Brain Res. 652: 304-310 52. Habig WH, Pabst MJ, Jakoby WB. 1974. Glutathione S-transferases. The first enzymatic step in mercaptopuric acid formation. /. Biol. Chem. 249: 7130-7139 53. Meister A, Anderson ME. 1983. Glutathione. Ann. Rev. Biochem. 52: 711-760 54. Ahmed AR, Blose DA. 1983. Delayed-type hypersensitivity skin testing. A review. Arch. Dermatol. 119: 934-945 55. Schmidt RJ, Chung LY. 1992. Biochemical responses of skin to allergenic and non-allergenic nitrohalobenzenes. Evidence that an NADPH-dependent reductase in skin may act as a prohaptenactivating enzyme. Arch. Dermatol. Res. 284: 400^408 56. Sreider CM, Grinblat L, Stoppani AOM. 1990. Catalysis of nitrofuran redox-cycling and superoxide anion production by heart lipoamide dehydrogenase. Biochem. Pharmacol. 40:1849-1857 57. Sreider CM, Grinblat L, Stoppani AOM. 1992. Reduction of nitrofuran compounds by heart lipoamide dehydrogenase: Role of flavin and the reactive disulfide groups. Biochem. Int. 28: 323-334 58. Mason RP, Josephy PD. 1985. Free radical mechanism of nitroreductase. In Toxicity of Nitroaromatic Compounds, ed. Rickert, DE, Hemisphere, New York, pp. 121-140 59. Mason RP, Holtzman JL. 1975. The role of catalytic superoxide formation in the 0 2 inhibition of nitroreductase. Biochem. Biophys. Res. Commun. 67: 1267-1274 60. Arner ESJ. 1999. Superoxide production by dinitrophenyl-derivatized thioredoxin reductase—A model for the mechanism and correlation to immunostimulation by dinitrohalobenzenes. Biofactors 10:219-226 61. Holmgren A, Arner E, Aslund F, Bjornstedt M, Liangwei Z, Ljung J, Nakamura H, Nikitovic D. 1998. Redox regulation by the thioredoxin and glutaredoxin systems. In Oxidative Stress, Cancer, AIDS and


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Neurodegenerative Diseases, eds. Montagnier L, Olivier R, Pasquier C, Marcel Dekker, Inc., New York, pp. 229-246 Wakasugi N, Tagaya Y, Wakasugi H, Mitsui A, Maeda M, Yodoi J, Tursz T. 1990. Adult T-cell leukemia-derived factor/thioredoxin pro duced by both human T-lymphotropic virus type I- and Epstein-Barr virus-transformed lymphocytes, acts as an autocrine growth factor and synergizes with interleukin-1 and interleukin-2. Proc. Natl. Acad. Sci. USA 87: 8282-8286 Blum H, Rollirighoff M, Gessner A. 1996. Expression and co-cytokine function of murine thioredoxin/adult T cell leukaemia-derived factor (ADF). Cytokine 8: 6-13 Bertini R, Howard OM, Dong HF, Oppenheim JL Bizzarri C, Sergi R, Caselli G, Pagliei S, Romines B, Wilshire JA, Mengozzi M, Nakamura H, Yodoi J, Pekkari K, Gurunath R, Holmgren A, Herzenberg LA, Herzenberg LA, Ghezzi P. 1999. Thioredoxin, a redox enzyme released in infection and inflammation, is a unique chemoattractant for neutrophils, monocytes, and T cells. /. Exp. Med. 189: 1783-1789 Schenk H, Vogt M, Droge W, Schulze-Osthof K. 1996. Thioredoxin as a potent costimulus of cytokine expression. /. Immunol. 156: 765-771 Pekkari K, Gurunath R, Arner ESJ, Holmgren A. 2000. Truncated thioredoxin is a mitogenic cytokine for resting human peripheral blood mononuclear cells and is present in human plasma. /. Biol. Chem. 275: 37474-37480 Pekkari K, Avila-Carino J, Bengtsson A, Gurunath R, Scheynius A, Holmgren A. 2001. Truncated thioredoxin (Trx80) induces production of interleukin-12 and enhances CD-14 expression in human mono cytes. Blood 97: 3184-3190 Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K, Sawada Y, Kawabata M, Miyazono K, Ichijo H. 1998. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase-1 (ASK-2). EMBO }. 17: 2596-2606 Zhang P, Liu B, Kang SW, Seo MS, Rhee SG, Obeid LM. 1997. Thio redoxin peroxidase is a novel inhibitor of apoptosis with a mechanism distinct from that of bcl-2. /. Biol. Chem. 272: 30615-30618 Zhong L, Holmgren A. 2000. Essential role of selenium in the catalytic activities of mammalian thioredoxin reductase revealed by characterization of recombinant enzymes with selenocysteine muta tions. /. Biol. Chem. 275:18121-18128


Chapter 3 Iron-Sulfur Proteins: Properties and Functions Helmut Beinert Institute for Enzyme Research, University of Wisconsin, 1710 Univ Ave. Madison WI53726-4087 USA, hbeinert@facstaff. wise, edu

Keywords: Iron-sulfur (Fe-S) clusters, cluster stability, cluster interconversions, redox potentials, electron transfer

1. Summary Many biologically active proteins contain clusters of high-spin iron ions in complexation with sulfide (S2~) and thiolate (RS~), called Fe-S clusters. The basic chemical structures and their electronic structure and stability are discussed. There are three basic cluster types: [2Fe-2S], [3Fe-4S], and [4Fe-4S], which can undergo a number of interconversions or even destruction depending on external conditions, such as pH, presence of oxidants or reductants, or of N O and other compounds. Their principal function is in electron transfer, e.g. in biological oxidations. Depending on their environment, Fe-S clusters may have a wide range of redoxpotentials (~1V). Their structural flexibility and sensitivity to external influences has also been exploited by nature in signaling and regulatory pathways. Examples of their use in such functions and their structural flexibility are discussed.

2. Historical Comments Fe-S clusters are most likely among the very first cofactors that were available as life started, because, under suitable conditions, they may be

47

48


formed spontaneously. If we consider this, it may seem surprising that we do not know them for more than about 40 years. One of the reasons is that most cofactors, vitamins and prosthetic groups such as heme, flavin, pyridoxal, carotenes, and others were discovered because of their strong color; and the pale yellow, with a reddish or greenish tinge, of Fe-S proteins is not very conspicuous. Another reason is that the rather robust methods that could be used for the purification of known cofac tors did not work for Fe-S proteins, but only led to their decomposition. They were eventually found by their catalytic action, as brown bands on columns during protein purification and as EPR signals, which are unique markers of most of them. 10 However, there was no EPR until 1943 and it was not introduced into biology for at least another 10 years.

3. Properties 3.1 Structure Redox regulation, the main theme of this volume, is actually not what applies for Fe-S proteins in most instances of which we know; it is rather regulation by oxidative destruction and reconstitution. This makes the process more complicated than simple one- or two-electron oxidation, followed by reduction, or vice-versa; namely, in addition to a reductant and the simple building blocks, other components such as enzymes, cofactors, chaperones or transport proteins are required for reconstitu tion, as far as we know today. In order to understand what we are faced with, we have to take a closer look at Fe-S clusters, Fe-S proteins and their properties. Figure 1 shows the most common structures that we have to deal with.33 The corresponding compounds are formed in mixtures of ferric chloride, thiol and sulfide combined in the indicated proportions in an anaerobic solution of an organic solvent such as ethanol or acetonitrile: a mono-nuclear complex, and the di- and tetra-nuclear clusters and the cage complex (upper right). The 4Fe cluster is the "sink" toward which every thing goes, if all ingredients are present in sufficient quantity. The cluster charge is given here as 2-. There is a difference between coordination chemists' and biochemists' nomenclature, which can be confusing to the outsider. In this presentation the charge of the iron-sulfur core only is


49

FeCI< 2>5RS-

3.5 RS2-

SR

S-^ F %/ R

2RS

SR

n ?FeN RS—Fe-f—S^ SR

R-HT

Fe /

RS

\

/

SR

R-

\

R'S 4S

SR RS

2 RS\

...s-..

/SR

RS^ ^ S ^

^SR

MeOH

S p /?—£* Fe-j—S

RS--b Fe -T7 5 S—Fe \

SR

Fig. 1. Depiction of the course of reactions resulting in assembly of [Fe4S4(SR)4]2" clusters via the intermediates [Fe(SR)4]2", [Fe2S2(SR)4]2~, and [Fe4(SR)10]2~. Note that the symbols shown here are those that are used when the whole complex, including the ligands, is considered (adapted with permission from Hagen et al.,33 copyright 1981, American Chemical Society).

m e a n t , as it is s h o w n in the formulae, i.e. [xFe-yS], w i t h o u t the four negatively charged Cys Kgands; the charge is then 2+ instead of 2 - for the clusters s h o w n in Fig. 1. With the basic m o d u l a r units s h o w n , m o r e complex structures can t h e n b e built u p , as seen in Fig. 2: on the left a n Fe-S-heme c o m b i n a t i o n is s h o w n , o n the right, t w o 4Fe m o d u l e s c o m b i n e d , as in the so-called P- (for protein) clusters of nitrogenase.

50


Fig. 2. Schematic structures of native assemblies, in which a cubane-type or cuboidal Fe-S cluster is bridged to the other component of the active site; left: Escherichia coli sulfite reductase; right: the P-cluster of nitrogenase. Irons and carbons are in black, sulfurs white (reprinted with permission from Beinert et al.,n copyright 1997, American Association for the Advancement of Science). 3.2 Electronic Structure Sulfur is considered as a weak ligand; and iron with all-sulfur ligation assumes a state with the maximal number of unpaired electrons allowed by the Pauli exclusion principle; as one says, it is "high-spin", and highspin complexes are less stable than low-spin ones with strong ligands such as, e.g. cyanide. The electronic structure is schematically illustrated in Fig. 3. 1112 The formal charges on the iron atoms (large circles) for the different oxidation states are indicated by shading: Fe34" black, Fe2+ white, and "mixed valence", Fe25+ gray; and the system spins and net charges are given under the structures. The cluster irons are magnetically coupled in pairs: in 2Fe clusters the electron spins are coupled antiparallel (antiferromagnetically) to a spin of zero in the 2+ (the "oxidized") state, and to a spin of 1/2 in the 1+ ("the "reduced") state, in 4Fe clusters, two pairs are formed by coupling of two Fe, in this case each pair with spins parallel (ferromagnetically); the spins of these primary pairs are then coupled antiparallel to the system spin. It has been ascertained by NMR that there may be shifts of electrons between Fe atoms of the pairs within 4Fe clusters as schematically shown in Fig. 46 Usually the formal charges are attributed to and written down for the Fe atoms. However, calculations of the distribution of valence electron

Iron-Sulfur Proteins: Properties and Functions 51

Fig. 3. Localization and delocalization patterns in Fe-S clusters, showing localized Fe3+ (black) and localized Fe2+ (white) sites, delocalized Fe25+Fe25+ (gray), sulfur (white). Indicated are also the spin S of the cluster and its core oxidation state (reprinted with permission from Beinert et al.,n copyright 1997, American Society for the Advancement of Science). density, carried out by density functional methods 61 clearly show that, on oxidation-reduction, by far the greater changes in electron density occur on all sulfur atoms, bridging sulfides and Cys-sulfurs, not on the iron atoms. For instance, for every electron's worth of charge density on a 4Fe cluster, the sulfurs account for about 0.1 electron per sulfur and the irons for slightly more than one half of this. Figure 5 shows the electron density change on reduction of the [4Fe-4S]2+ to the [4Fe-4S]+ state. The gray shaded

52


CysS

^rf

SCys

SCys CysS

CysS

Fig. 4. Shift of mixed valence pair of irons (filled squares) in high-potential Fe-S protein between cluster faces, here from irons 3 and 4 to irons 3 and 1. As a result, iron 3 is Fe 25+ and iron 2 is Fe3+, whereas irons 1 and 4 have oxidation numbers between 2.5+ and 3+ (reprinted with permission from Banci et al.,6 copyright 1993, American Chemical Society).

Fig. 5. Total valence electron density difference between 1+ and 2+ states of a [4Fe-4S] cluster. Dark shading shows increased electron density on reduction and white decreased density (reprinted with permission from Noodleman and Case,61 copyright 1992, Academic Press).


53

areas are those that experience increased electron density, while the white areas show depletion of electron density. Similar changes in the opposite direction occur on oxidation of the cluster from the 2+ to the 3+ state.

3.3 Stability All these properties, such as electron delocalization and spin coupling, are, of course, not unrelated to the stability of Fe-S clusters. Can they be considered as cofactors, such as flavins, hemes or pyridine nucleotides, which can be isolated and put into a bottle? According to the electronic structure, schematically indicated in Fig. 3, and the magnetic coupling between the iron atoms, the 2- and 4-Fe clusters with their thiol ligands are quite stable and self-contained; however they are vulnerable through outside influences: water protons, oxygen, reduction products of oxygen, or other oxidants, N O and also high concentrations of thiols and sulfide and, under some conditions, also chelators. However, reaction with chelators cannot a priori be expected, i.e. failure to observe an effect of iron chelators is no proof of the absence of Fe-S clusters. Fe-S clusters are most stable when embedded into proteins, or better yet membrane proteins, which may prevent access of deleterious compounds, such as solvent which carries oxidants or chelators. 3.4 Complex Fe-S Proteins In addition to these intrinsic properties, nature often introduces modula tions, as we have seen in Fig. 2, by welding together various cluster modules or fragments, or by juxtaposing two or more clusters in a protein or other cofactors such as heme, flavin, or by forming heterometal clus ters, such as Fe/Ni clusters as in hydrogenases or CO dehydrogenase, 29 or by use of unusual ligands other than cysteines.66 Thus, given the different cluster types to start with, the possibilities of subtle modulation are almost unlimited, and so is also the variety of uses that FeS clusters can be put to.

3.5 Cluster Ligands The availability and location of the ligating Cys residues determine much of the behavior of Fe-S clusters. There are some characteristic patterns of


16

Leu

26

/

_

Ser 24 Cys

21

Gln ^ — ^

F e lll

14

Cys

^>*—o -S^ JJCys_^

^ S ^ 5*" ^

( 6 0

Felll,

\ Cys-^

II

X

^S o — ^ v,

56Cys-—^

Fig. 6. Tentative scheme of the Fe-S active site environment of the C. ■pasteurianum 2 Fe ferredoxin. Cys56 and Cys60 are indicated as ligands of the reducible Fe. Those residues that may become ligands of the cluster upon muta tion into cysteines are shown (reprinted with permission from Golinelli et a/.,31 copyright 1998, American Chemical Society).

Cys distribution in proteins which allow one to make predictions whether an Fe-S cluster is most likely present and also what type it may be;40,57 and from amino acid sequence similarities, conclusions can often be drawn as to what the biological function of the respective proteins is likely to be. In the following we will see a few instructive examples of choice of ligands and cluster interconversions. In a 2Fe Fd from Clostridium pasteurianum it seemed impossible to find out by systematic mutation of residues, which Cys residues of the five present in the molecule were the iron ligands. 31 It turned out that only two of the four were definitely required, and that the other two could be replaced either by the fifth cysteine present or when other neighboring residues in a certain region of the structure were changed to cysteines. This particular region was a flexible loop located on the outside of the protein, whereas the two clearly required cysteines were located in a more structured region toward the core of the protein (Fig. 6, residues 11 and 56). Another example documenting the plasticity of Fe-S clusters in proteins became apparent when the enzyme aconitase, in its inactive 3Fe form, was exposed to elevated p H (>9 ).45 The cubane type 3Fe cluster was stretched out to a linear cluster as shown in Fig. 7, by detaching one of the Cys residues and recruiting two new ones from a helix lying 15-17 A


OH

s

Cys-S 358

/

+Fe +e-

^r •Fe -e

424Cys-S

424Cys-S

£ - * »

S-Cys421

S-Cys421

S ^Fe

S-Cys Fe

cys-s^ \ / W

Fe

\ S-Cys

Fig. 7. Schematic description of the interconversion between cubane-type and linear Fe-S clusters (reprinted with permission from Beinert and Kennedy,13 copyright 1989, Blackwell Science).

away in the crystal structure (Fig. 8). The linear cluster is more stable than the cubane type 3Fe cluster; however, on lowering the p H under reduc ing conditions, the 4Fe cluster of the active enzyme was formed again in good yield, indicating that the protein bearing the cluster has regained its original structure. Another oddity is a unique "loosened-up" cluster (Fig. 9) discovered in Desulfovibrio species. 3 This so-called hybrid cluster has sulfide- and oxo-bridges simultaneously and a persulfide group, and may appear as a Fe-S cluster which is either in a precursor form or on the way to destruc tion. This seems, however, barely compatible with the fact that it can be crystallized. 3 The function of the respective protein is yet unknown.

3.6 Heterometal Clusters The 3Fe cluster was mentioned and shown repeatedly above (Fig. 3). Obviously this is the ideal starting material for heterometal 4Fe clusters,28,37

56


Fig 8. Close-up view of part of the structure of mitochondrial aconitase, showing the active site with substrate bound. When the linear cluster is formed, the two Cys ligands on the right side are maintained and two cysteines (indicated by small arrows at the lower right) from a distant helix are used to complete the ligation. As shown in [11] from unpublished work of SJ Loyd, GS Prasad and CD Stout and reprinted with permission, copyright 2000, Society for Biological Inorganic Chemistry. a n d this h a s b e e n exploited in in vitro work. Yet, such clusters are not found in n a t u r e ; the only exceptions are the Mo-Fe or V-Fe proteins of nitrogenase 6 9 a n d t h e Ni-Fe clusters of h y d r o g e n a s e s a n d C O d e h y d r o g e n a s e s . 2 9


►(His 244) (Cys 459}S

(Gfu 494)

(Glu268)

%e

g

\

1

(Cys406)S \ ^ S^

1

Fe6« k

(Cys 434)8

S(Cys312)

^

Fig. 9. A schematic view of cluster 2, the "hybrid" cluster from Desulfovibrio vulgaris which contains both S and O bridges between the iron atoms. X represents a putative substrate-binding site, which may be partially occupied in the present structure (reprinted with permission from [3], copyright 1998, Society for Biological Inorganic Chemistry).

However, these are unusual, specialized structures. Artificially, though, a whole series of metals have been incorporated into man-made or naturally occurring 3Fe clusters.37,41 These turned out to be useful materials for explor ing the electronic structure of Fe-S clusters, because it was possible to incor porate non-magnetic metals. Why are such clusters not found in nature? An answer was proposed by Armstrong and Williams:5 They argued that heavy metals are known not to float around freely in tissue; they are carefully guided and chaperoned by special proteins. Thus, metals like Zn, for instance, or Cd, which are known to have, in vitro, a higher affinity for the 3Fe cluster than Fe,22 are bound so tightly to their chaperones — that is, in this example, metallothionein for Zn and Cd — that they cannot compete with Fe for the 3Fe cluster. For instance, it was determined that the 3Fe cluster of aconitase competes for Fe2+ with some success even with EDTA: the formation constant for the iron complex is 10 for 3Fe aconitase and 14 for EDTA.27

58


Active

Inactive S-1/2

PRJ-4S]1*

^ *k

(^Fe^f*)

S-1/2

V

-e*

+6"

Fe2*

+©"

-©"

>

S =9

faPo.MIRIO

*_£* >

[4Fe-4S]2+ +0

S-0

.0

I4F©-4SJ1+

S-1/2

Fig. 10. A model showing the relationship between the various cluster forms of aconitase. For each cluster type, its oxidation state (i.e. the charge balance of the core) is presented as a superscript and its spin state at the outer sides. In the model, aconitase is inactive when it contains a 3Fe cluster and active when it contains a 4Fe cluster. Fe2+ plays the role of converting the 3Fe clusters to 4Fe clus ters. The existence of a [4Fe-4S]3+ cluster is uncertain and, therefore, the corresponding symbol is placed within parentheses (from [27] with the permis sion of the American Society for Biochemistry and Molecular Biology).

3.7 Self-activation of 3Fe-enzymes The high affinity of the 3Fe cluster for iron is the basis for the pheno menon of self-activation under reducing conditions that has been observed repeatedly for enzymes that require a 4Fe cluster for activity, but are obtained in the 3Fe form on purification. What happens is that, under reducing conditions, some 3Fe clusters are spontaneously disassembled and the iron that becomes available is used to build up 4Fe clusters, which are, under reducing conditions, more stable. This has repeatedly misled investigators, when they ascribed activity to enzymes containing 3Fe clus ters, while in fact 4Fe clusters were being made by self-activation during the assay or during preparations for it.2046 Figure 10 describes the situation encountered with aconitase, which is obtained in its 3Fe form on


59

routine purification. All these observations show again the enormous flexibility and plasticity of Fe-S clusters. In this context we should also consider the reuse of sulfide, not only of the iron of a cluster. What happens to the sulfide when clusters are dis mantled? When oxygen is excluded — oxygen would either lead to for mation of disulfides or sulfenate (SO") — it has been found that the sulfide sulfur can also be reused for formation of more stable clusters, such as the 4Fe type. There are indications that iron sulfides may be attached to pro tein in some fashion and may be reused as such for rebuilding clusters. 63 All this has a bearing on the transport of Fe-S clusters or their precursors. There is evidence that Fe-S clusters have to cross membranes during Fe-S cluster biosynthesis in eukaryotes.48,52 Such iron-sulfides on proteins, pos sibly on specific chaperones, or even whole 2Fe- or, maybe, linear 3Fe clusters might be the forms that can be transported more easily than the cubic 4Fe-clusters. We may also recall here the hybrid cluster shown above (Fig. 9), which could be looked at as a model for such a loosenedu p cluster form.

3.8 Degradation and Biosynthesis of Fe-S Clusters While it is likely that originally Fe-S clusters may have been formed spon taneously, their building blocks, iron and sulfide, are much too dangerous materials to be allowed in living tissues unguarded. Thus, a whole set of proteins has evolved that has the task of bringing about the uptake and transport of iron, and the generation and transport of sulfide to the place of synthesis. Iron, of course, has to be imported from the outside or taken from internal stores previously formed; and sulfide is produced from cysteine by pyridoxal-containing enzymes51,75 and safeguarded and trans ported in the form of a hydrodisulfide (RSS*H, also called persulfide) attached to protein 75 or a cysteine.51 Under reducing conditions, it can then be released from its carrier as sulfide. The iron, on the other hand, is assembled into a cluster precursor on three cysteines of a different protein, 74 IscU, in E. coli.1 NifU (or IscU) is able to form a complex with the sulfane carrrier NifS (or IscS), in which the transfer of sulfide takes place with the formation of a 2Fe cluster, from which eventually a [4Fe-4S] cluster may be formed. 1 In in vitro work with proteins purified from the cyanobacterium Synechocystis it has been possible to observe transfer of a 2Fe cluster from the IscU homolog of this organism to the apoprotein of a

60


Fd isolated from Synechocystis; in this way the specific electron transfer activity of the holo-Fd was restored to the inactive apoFd.60 The last few years have witnessed enormous progress in the area of Fe-S cluster biosynthesis, both in pro- and eukaryotes. As one would expect, the mechanism of synthesis in multicellular organisms is considerably more complex than in unicellular ones and requires a number of additional fac tors or proteins. The respective original or review literature26,39'52'59 will have to be consulted about details of the status in this field, which is rapidly moving at present. On the contrary, little detail is available on the degradation of Fe-S clusters. Thorough studies of the decomposition of Fe-S clusters by acid or base are available,56 but this has not as much relevance to biological con ditions than degradation by oxygen, its reduction products or by nitric oxide. We know that Fe-S clusters are destroyed by these agents, but little detail is known about the mechanisms involved. The sulfides and cysteine ligands are assumed to be the primary targets and as products proteinbound di- or tri-sulfides have been identified.43'65 As it is not possible to quantitatively account for the originally present sulfide as disulfide or sulfane sulfur (also called sulfur zero, S°), it can be assumed that some sul fur oxides are formed such as sulfenate (SO "), sulfinate (SO2-), or yet more oxidized forms. On oxidation of 4Fe clusters the first iron, at least, is released as Fe2"1".7'44 This is clearly the case on oxidation of the 4Fe to the 3Fe cluster; it has also been observed, when the end product is the 2Fe cluster,7 which may suggest, but does not prove, that the 3Fe cluster is an interme diate in this process. In the reaction of N O with Fe-S clusters protein-bound DiNitrosyl-Iron Complexes (DNIC), Fe(Cys)2(NO)2, are formed with destruction of the original Fe-S clusters .42'50,71 All these destructive reactions, except for the simple 4Fe to 3Fe conversion, are not readily reversed in vitro by adding reductants with or without iron and sulfide, but can, apparently be repaired in cellular preparations.1819,62'64 The factors, probably proteins, required for such repair, have not been identified. They may or may not68 be identical to the enzymes used in the original synthesis.

4. Functions of Fe-S Proteins 4.1 Electron Transfer Foremost is their use in electron transfer, for which the "respiratory chain" of mitochondria is the prime example (Fig. 11). There are as many

Iron-Sulfur Proteins: Properties and Functions 61 Succinate

1 FAD [2Fe-2S]S-1 [4Fe-4S]S-2 [3Fe-4S]S-3 Cytb Qs Complex I NADH-

I

Complex

a

Complex m

Cytt%66 FMN I - l QM ™ N 6[4Fe-4S] ^ -♦•Q-pool—*• Cytb562 f2Fe-2S]R,este Cytc, 2 2Fe 2S

t

\

[4Fe-4S] FAD

Cytc

t

Complex IV

JoA —♦■ AcylCoA —*• Electron dehydrogenase transferring flavoprotein

1

Cyta Cu-Cu

Cyta3 Cue

\ 02

Fig. 11. Mitochondrial respiratory chain. Qs and QN are protein-associated pools of ubiquinone that can be distinguished from the bulk ubiquinone pool (adapted from Johnson40 with permission from Wiley).

Fe-S clusters in NADH dehydrogenase alone as there are hemes and flavins in the components of the whole system: there are a total of 13-14 Fe-S clusters altogether! Note also that, in order to reach ubiquinone in any pathway, electrons have to pass through Fe-S proteins, more pre cisely, Fe-S flavoproteins. There are many variants of such electron transfer systems in different organisms or even within individual macromolecules, which, almost without exception, make use of Fe-S proteins, if the range of low redox potentials is involved.

4.2 Oxidation-Reduction Potentials The function of Fe-S clusters in electron transfer leads to a consideration of redox potentials. As we can see in Fig. 12,23 the redox potentials that are possible with Fe-S proteins cover a wide range, even exceeding 1 volt. On the low side we have the most negative potentials with 7Fe Fds ([4Fe-4S2+/+], [3Fe-4S]+/0) of -650mV, and at the high end the HiPIP clusters


4Fe cluster in [8Fe-8S] Ferredoxins 4Fe cluster in HiPIP

4Fe cluster in [7Fe-8S] Ferredoxins 4Fe cluster in [4Fe-4S] Ferredoxins 3Fe cluster in (7Fe-8S] Ferredoxins ■ 3Fe cluster in [3Fe-4S] Ferredoxins 2Fe cluster in r2Fe-2S] Ferredoxins

Rubredoxins —I

[

!

1

1

1

j

-700 -600-500-400-300-200-100 0 E°'{mV)

1

1

1

1

100 200 300 400 500

Fig. 12. Experimental ranges of reduction potentials (versus NHE) of various subclasses of Fe-S proteins (from Capozzi et alP with permission from Springer Verlag).

([4Fe-4S3+/2+]) with up to +450 mV; with 4Fe, 3Fe, 2Fe clusters, rubre doxins and Rieske clusters (not shown) in between. Rieske clusters, are [2Fe-2S] clusters that have two Cys and two His ligands, asymmetrically disposed. The potentials of these clusters fall into the range of 100 to 320 mV for the bc1 type, and those of the related dioxygenase type Rieske clusters fall between -100 and -150 mV.53 It is easy to understand that the net charge of the clusters is one of the primary determining features of these potentials, but what are the deter minants of the enormous spread of values? One might think that with hundreds of Fe-S proteins, and often derivatives of them available that were generated by mutations, one should have been able to come to some answers on this point. However, there is still a lively debate about this subject and there are many opinions represented. 14 Clearly, it must be the environment in which the cluster finds itself that modulates the intrinsic potential of the cluster, and this environment is one of the most com plex ones that nature can supply, namely protein in water. Most success in predicting or rationalizing differences in redox potentials of Fe-S proteins has been achieved, when a series of homologous proteins of relatively simple structure were available, such as the rubredoxins, plant


63

2Fe Fds, HiPIPs, or Rieske proteins; however, it remains very difficult to predict potentials by comparing more complex, less or unrelated Fe-S proteins. A number of factors that influence, or are most likely to influ ence the potentials of redox proteins, have been identified, but the extent to which each one of these can influence the values of the potentials in any one case is subject to great variations in an aqueous protein environment. The following factors must be considered: first, the identity of the cluster ligands, hydrogen bonds from protein constituents to cluster sulfides or its Cys ligands, charges or dipoles on adjacent peptide chains, water molecules in the protein as shields or as dipoles; further, of course, other clusters or redox groups such as hemes or flavins; second, the distance of any of the interacting species from the Fe-S cluster, which, when increas ing, will attenuate any of these effects; and, for dipoles also their orienta tion: if a dipole points toward the cluster, it will increase the redox potential, if it points away it decreases it. Then, it has to be considered that this environment is not static, but is subject to continuous dynamic fluctuations. As the value of the dielectric of the medium, in which all these interactions occur, is of importance, it is a questionable simplification, if a uniform continuum is assumed as the dielectric, instead of a microscopic, heterogeneous one.72 It has also been pointed out that, in addition to the expected random fluctuations, the very process of oxidation-reduction may bring about distinct, reversible conformational changes in the protein; these could influence the redox potential transiently during a reaction, but may not be expressed in the statically measured potential. 4 Thus, it could be misleading to assume that the spatial sequence of redox components in a protein can be simply deduced from their statically measured potentials or vice-versa. Surface charges on the protein may have some influence; and it has been possible, within a series of similar HiPIPs, to relate the electrostatic contributions of such charges to the observed potentials. 15 On the other hand, it has been observed that net charges fully exposed to solvent have little effect on the potential, whereas buried charges have to be considered as impor tant factors. In the Rieske (bCj type) protein series it has been possible to relate a sizeable increase in redox potential (+150mV) to the presence of a serine residue, which forms a hydrogen bond to one of the bridging sulfide groups of the cluster in those proteins that use ubiquinone as elec tron donor versus those that use menaquinone as donor. 53 An instructive example as to how subtle structural details can influence redox potentials can be found in a recent paper on the comparison of the structures of a Rieske and a Rieske-type Fe-S protein. 24 A peculiar property of Rieske

64


proteins is also that their redox potentials are pH dependent, which presumably has to do with the ligation of His residues to the reducible iron. This p H dependence of the redox potentials of bcx proteins is an important feature in their ability to generate a proton gradient. It has also been learned that in 2Fe Fds that have all-Cys ligation, the presence of solvent plays a major role in determining the iron of the cluster that becomes ferrous on reduction of the protein. 16 Most recently a thorough study of the thermodynamics of reduction of a variety of Fe-S clusters by variable temperature electrochemistry has been published, 9 which draws particular attention to entropic contributions and should be consulted in connection with the matter discussed above. After these considerations that are more germane to classical one- or two-electron oxidation-reductions, we should also consider other effects of the electronic makeup of Fe-S clusters, which do not necessarily lead to net electron transfer, such as polarization of adjacent structures or elec tron storage; in other words, we can look at Fe-S clusters as reservoirs of electrons that can be drawn on in reactions carried out by neighboring cofactors, such as, e.g. flavins, or adenosyl-methionine. 11 ' 25 For instance in the shuffle that leads to oxidative phosphorylation in NADH dehydrogenase, Fe-S clusters are bound to play a crucial role of this kind. By the use of the reducing power of a [4Fe-4S]+ cluster in conjunction with ATP hydrolysis, very low redox potentials can be achieved as required, e.g. for reduction of N 2 to NH 3 by the Fe-protein — Mo-Fe-protein complex of nitrogenase,38 in the anaerobic microbial reduction of aromatic compounds, 17 and in dehydration reactions, when a hydrogen is to be removed from an unactivated carbon.21

4.3 Non-Redox Functions of Fe-S Proteins These functions have to do with the integrity of Fe-S proteins. Thus, Fe-S proteins have the ability to serve as what has been called "circuit breaker"; 30 i.e. there are oxygen sensitive Fe-S proteins that catalyze vital reactions, which require an intact 4Fe cluster for their function. If oxygen, superoxide or hydrogen peroxide are present, these clusters are con verted to the 3Fe form, which is more stable toward oxygen, but can be easily reconverted to the 4Fe form, when anaerobicity is restored. Thus the enzymes can be preserved in a quasi-intact state, only temporarily shut off, but readily reconverted to the active form. This, for example, occurs with aconitase, an enzyme indispensable for the functioning of the


65

Krebs cycle,55 and with anaerobic ribonucleotide reductase of Lactococcus lactis.5* One of the simplest uses of Fe-S clusters is observed if a certain function requires a distinct oxidation state, such as the alarm against oxidative damage. An example is the SoxR-SoxS system of E. coli, which simply depends on the oxidation state of a [2Fe-2S] cluster.36 The SoxR protein sounds the alarm against oxidative stress brought about by the superoxide anion, O". The SoxR protein occurs as a dimer with each monomer bearing a [2Fe-2S] cluster. SoxR is inactive when the cluster is in the 1+ state and becomes activated on oxidation to the 2+ state. SoxR is able to bind to DNA in both oxidation states and apparently even in the apoform, but only the oxidized form activates the gene coding for the SoxS protein, which in turn induces formation of a whole series of pro tective proteins and enzymes. A more drastic modification is used in other systems, namely complete dismantling of Fe-S clusters. This was first observed with glutamine phosphoribosylpyrophosphate amidotransferase of Bacillus subtilis, a key enzyme in the formation of pyrimidine nucleotides. 32 This enzyme has an oxygen sensitive 4Fe cluster, which is not used in any oxidation-reduction reaction. However, it stabilizes the protein against proteolytic attack: when the 4Fe cluster is destroyed by oxygen, the protein is then also rapidly degraded. One of the most interesting examples in this category is probably the bifunctional protein cytoplasmic aconitase, which is con verted to the "iron regulatory protein" (IRP) on complete removal of its [4Fe-4S] cluster; in this case, the protein is preserved34,35 and acquires a new function! Another well-studied example of a similar nature is the global transcription factor FNR of E. coli, which regulates the conversion of anaerobic to aerobic metabolism in this organism. Closely related proteins with similar functions occur in a great number of other micro organisms. 70 In the presence of oxygen, the 4Fe cluster of FNR is rapidly (within seconds) converted to a more stable 2Fe cluster, which then, within minutes or hours, depending on conditions, is converted to apoprotein. 47 Only the presence of the 4Fe cluster allows induction of the enzymes and transport proteins required for anaerobic metabolism. As with many tran scription regulators, FNR acts in its dimeric form of ~60kD. The monomeric form is inactive. The inactive 2Fe form is monomeric; how ever, it has been observed that in some mutants of FNR the monomeric form can have a 4Fe cluster, but such monomers are not transcriptionally active.58 Information so far available indicates that the Fe-S cluster of FNR is necessary for effective dimerization. 49 The influence of the cluster seems to be transmitted through the protein structure, as the cluster is not likely

66


Active FNR dimer OOH

NH 3

DNA Binding and Transcription Activation

jo,

Fe

S

4Fo cluster

XK=2F« cluster

Inactive 2FeFNR monomer

Fig. 13. Scheme describing the pathway of FNR inactivation under aerobic conditions. (Courtesy of PJ Kiley, University of Wisconsin, Madison).

to be in the vicinity of the dimerization helix according to a synopsis of secondary structure data and the crystal structure of the closely related CAP protein. 73 It has been possible to follow the synthesis and conversion of the 4Fe cluster of FNR into the 2Fe cluster and its reconstitution in whole cells of £. coli by Mossbauer spectroscopy, which can easily dis criminate between the two cluster forms.67 By this technique and by the use of an oxygen-stable mutant it could also be ascertained that FNR con taining a 4Fe cluster is synthesized even in the presence of oxygen, but in the wild type protein the cluster is then obviously rapidly destroyed by oxygen. 8 Figure 13 shows a scheme that encompasses present ideas about the involvement of the Fe-S cluster in the function of FNR.

5. Conclusion and Outlook All these functions can be understood on the basis of the properties of Fe-S clusters, which were described at the outset. Of course, in the frame of this chapter it was only possible to give a glimpse of the iron-sulfur world, of which, just at the present time, we keep learning more practically every day that passes. It seems though that, while the biological aspects and implications are expanding in an unforeseen way, some of the basic chemical information is still lacking. We know preciously little about the


67

mechanisms of the reaction of Fe-S clusters with oxygen and with its reduction products, or with nitric oxide, while all clearly share the natural environment with each other. To a large extent this seems not so much for lack of trying, than for the complicated and multifaceted chemistry of not only iron and sulfur but also of oxygen and nitric oxide. The number of species that may be formed from each single one of these reactants, and consequently the number possible with all combined, is discouragingly complex, even if one only considers end products and does not attempt to follow the time course of the interactions. To me, as one who largely came from the (bio)chemical side into this field, progress on this front seems an impor tant goal for the near future. It clearly would benefit progress on the biological front considerably.

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Agar JN, Krebs C, Frazzon J, Huynh BH, Dean DR, et al. 2000. IscU as a scaffold for iron-sulfur cluster biosynthesis: Sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU. Biochemistry 39: 7856-7862 2. Agar JN, Yuvaniyama P, Jack RF, Cash VL, Smith AD, et al. 2000. Modular organization and identification of a labile mononuclear ironbinding site within the NifU protein. /. Inorg. Biol. Chem. 5:167-177 3. Arendsen AF, Hadden J, Card G, McAlpine AS, Bailey S, et al. 1997. The "prismane" protein resolved: X-ray structure at 1.7 A and multi ple spectroscopy of two novel 4Fe clusters. /. Biol. Inorg. Chem. 3: 81-95 4. Armstrong FA. 1997. Evaluations of reduction potential data in rela tion to coupling, kinetics and function. /. Biol. Inorg. Chem. 2:139-142 5. Armstrong FA, Williams RJP. 1999. Thermodynamic influences on the fidelity of iron-sulphur cluster formation in proteins. FEBS Lett. 451: 91-94 6. Band L, Bertini I, Ciurli S, Ferretti S, Luchinat C, et al. 1993. The elec tronic structure of [Fe4S4]3+ clusters in proteins. An investigation of the oxidized high-potential iron-sulfur protein II from Ectothiorhodospira vacuolata. Biochemistry 32: 9387-9397 7. Bates DM. 1999. Role of iron-sulfur cluster conversion in the oxygensensing mechanism of the Escherichia coli transcription factor FNR. Dissertation, University of Wisconsin, Madison 8. Bates DM, Popescu CV, Khoroshilova N, Vogt K, Beinert H, et al. 2000. Substitution of leucine 28 with histidine in the Escherichia coli

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18. 19.

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transcription factor FNR results in increased stability of the [4Fe-4S]2+ cluster to oxygen. /. Biol. Chem. 275: 6234-6240. Battistuzzi G, D'Onofrio M, Borsari M, Sola M, Macedo AL, et al. 2000. Redox thermodynamics of low-potential iron-sulfur proteins. /. Biol. Inorg. Chem. 5: 748-760. Beinert H. 1973. Development of the field and nomenclature. In IronSulfur Proteins, ed. W Lovenberg, Academic Press New York, I: 1-36 Beinert H. 2000. Iron sulfur clusters: Ancient structures, still full of surprises. /. Biol. Inorg. Chem. 5: 2-15 Beinert H, Holm RH, Miinck E. 1997. Iron-sulfur clusters: Nature's modular, multipurpose structures. Science 277: 653-659 Beinert H, Kennedy MC. 1989. Engineering of protein bound ironsulfur clusters. 1989. Eur. /. Biochem. 186: 5-15 Bertini I. 1997. Determinants of reduction potentials in metalloproteins. /. Biol. Inorg. Chem. 2:108 Bertini I, Gori-Savellini G, Luchinat C. 1997. Are unit charges always negligible? /. Biol. Inorg. Chem. 2:114-118 Bertini I, Luchinat C, Rosato A. 1999. NMR spectra of iron-sulfur proteins. Adv. Inorg. Chem. ed. Sykes AG, Cammack R, 47: 251-282 Boll M, Albracht SPJ, Fuchs G. 1997. Benzoyl-CoA reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism. A study of adenosine triphosphatase activity, ATP stoichiometry of the reaction and EPR properties of the enzyme. Eur. }. Biochem. 244: 840-851 Bouton C, Raveau M, Drapier J-C. 1996. Modulation of iron regula tory protein function. /. Biol. Chem. 271: 2300-2306 Brazzolotto X, Gaillard J, Pantopoulos K, Hentze MW, Moulis J-M. 1999. Human cytoplasmic aconitase (iron regulatory protein-1) is converted into its [3Fe-4S] form by hydrogen peroxide in vitro but is not activated for iron-responsive element binding. /. Biol. Chem. 274: 21625-21630 Broderick JB, Henshaw TF, Cheek J, Wojtuszewski K, Smith SR, et al. 2000. Pyruvate formate-lyase-activating enzyme: Strictly anaerobic isolation yields active enzyme containing a [3Fe-4S]+ cluster. Biochem. Biophys. Res. Commun. 269: 451^56 Buckel W, Golding BT. 1998. Radical species in the catalytic path ways of enzymes from anaerobes. FEMS Microbiol. Rev. 22: 523-541 Butt JN, Fawcett SEJ, Breton J, Thomson AJ, Armstrong FA. 1997. Electrochemical potential and p H dependences of [3Fe-4S] [M3Fe-4S] cluster transformations (M=Fe, Zn, Co, and Cd) in


23.

24.

25.

26. 27.

28.

29.

30. 31.

32.

33.

34.

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Ferredoxin III from Desulfovibrio africanus and detection of a cluster with M = Pb. /. Am. Chem. Soc. 119: 9729-9737 Capozzi F, Ciurli S, Luchinat C. 1998. Determinants of electronic and functional properties of iron-sulfur proteins. In Structure and Bonding, Springer Verlag, Berlin, Heidelberg, 90: 127-160 Colbert CL, Couture MMJ, Eltis LD, Bolin JT. 2000. A cluster exposed: Structure of the Rieske ferredoxin from biphenyl dioxygenase and the redox properties of Rieske Fe-S proteins. Structure 8:1267-1278 Cosper NJ, Booker SJ, Ruzicka F, Frey PA, Scott RA. 2000. Direct FeS cluster involvement in generation of a radical in lysine 2,3aminomutase. Biochemistry 39:15668-15673 Craig EA, Voisine C, Schilke B. 1999. Mitochondrial iron metabolism in the yeast Saccharomyces cerevisiae. Biol. Chem. 380:1167-1173 Emptage MH, Dreyer J-L, Kennedy MC, Beinert H. 1983. Optical and EPR characterization of different species of active and inactive aconitase. /. Biol. Chem. 258:11106-11111 Fawcett SEJ, Davis D, Breton JL, Thomson AJ, Armstrong FA. 1998. Voltammetric studies of the reactions of iron-sulphur clusters [3Fe-4S] or [M3Fe-4S]) formed in Pyrococcus furiosus ferredoxin. Biochem. f. 335: 357-368 Fontecilla-Camps JC, Ragsdale SW. 1999. Nickel-iron-sulfur active sites: hydrogenase and CO dehydrogenase. Adv. Inorg. Chem., eds. Sykes AG, Cammack R, 47: 283-333 Gardner PR, Fridovich I. 1991. Superoxide sensitivity of the Escherichia coli aconitase. /. Biol. Chem. 266:19328-19333 Golinelli M-P, Chatelet C, Duin EC, Johnson MK, Meyer J. 1998. Extensive ligand rearrangements around the [2Fe-2S] cluster of Clostridium pasteurianum ferredoxin. Biochemistry 37: 10429-10437 Grandoni JA, Switzer RL, Makaroff CA, Zalkin H. 1989. Evidence that the iron-sulfur cluster of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase determines stability of the enzyme to degradation in vivo. ]. Biol. Chem. 264: 6058-6064 Hagen KS, Reynolds JG, Holm RH. 1981. Definition of reaction sequences resulting in self-assembly of [Fe4S4(SR)4]2" clusters from simple reactants. /. Am. Chem. Soc. 103: 4054-4063 Haile DJ, Rouault TA, Harford JB, Kennedy MC, Blondin GA, et al. 1992. Cellular regulation of the iron-responsive element binding protein: Disassembly of the cubane iron-sulfur cluster results in highaffinity RNA binding. Proc. Natl. Acad. Sci. USA 89:11735-11739

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35. Hentze MW, Kiihn LC. 1996. Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc. Natl. Acad. Sci. USA 93: 8175-8182 36. Hidalgo E, Ding H, Demple B. 1997. Redox signal transduction via iron-sulfur clusters in the SoxR transcription activator. Trends Biochem. Sci. 22: 207-210 37. Holm RH. 1992. Trinuclear cuboidal and heterometallic cubane-type iron-sulfur clusters: New structural and reactivity themes in chem istry and biology. Adv. Inorg. Chem., eds. Cammack R, Sykes AG, 38: 1-17 38. Howard JB, Rees DC. 1996. Structural basis of biological nitrogen fixation. Chem. Rev. 96: 2965-2982 39. Jensen LT, Culotta VC. 2000. Role of Saccharomyces cerevisiae ISA1 and ISA2 in iron homeostasis. Mol. Cell. Biol. 20: 3918-3927 40. Johnson MK. 1994. Iron-sulfur proteins. In Encyclopedia of Inorganic Chemistry, ed. King RB, John Wiley & Sons, England, 4: 1896-1915 41. Johnson MK, Duderstadt RE, Duin EC. 1999. Biological and synthetic [Fe3SJ clusters. Adv. Inorg. Chem., eds. Sykes AG, Cammack R, 47: 1-82 42. Kennedy MC, Antholine WE, Beinert H. 1997. An EPR investigation of the products of the reaction of cytosolic and mitochondrial aconitases with nitric oxide. /. Biol. Chem. 272: 20340-20347 43. Kennedy MC, Beinert H. 1988. The state of cluster SH and S2" of aconitase during cluster interconversions and removal. /. Biol. Chem. 263: 8194-8198 44. Kennedy MC, Emptage MH, Dreyer J-L, Beinert H. 1983. The role of iron in the activation-inactivation of aconitase. /. Biol. Chem. 258: 11098-11105 45. Kennedy MC, Kent TA, Emptage M, Merkle H, Beinert H, et al. 1984. Evidence for the formation of a linear [3Fe-4S] cluster in partially unfolded aconitase. /. Biol. Chem. 259: 14463-14471 46. Kent TA, Emptage MH, Merkle H, Kennedy, MC, Beinert H, et al. 1985. Mossbauer studies of aconitase. /. Biol. Chem 260: 6871-6881 47. Kiley PJ, Beinert H. 1999. Oxygen sensing by the global regulator, FNR: The role of the iron-sulfur cluster. FEMS Microbiol. Rev. 22: 341-352 48. Kispal G, Csere P, Prohl C, Lill R. 1999. The mitochondrial proteins A t m l p and Nfslp are essential for biogenesis of cytosolic Fe/S proteins. EMBO. J. 18: 3981-3989


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49. Lazazzera BA, Bates DM, Kiley PJ. 1993. The activity of the Escherichia coli transcription factor FNR is regulated by a change in oligomeric state. Genes Dev. 7: 1993-2005 50. Lee M, Arosio P, Cozzi A, Chasteen ND. 1994. Identification of the EPR-active iron-nitrosyl complexes in mammalian ferritins. Biochemistry 33: 3679-3687 51. Leibrecht I, Kessler D. 1997. A novel L-cysteine/cystine C-S-lyase directing [2Fe-2S] cluster formation of Synechocystis ferredoxin. /. Biol. Chem. 272:10442-10447 52. Lill R, Kispal G. 2000. Maturation of cellular Fe-S proteins: An essen tial function of mitochondria. Trends Biochem. Sci. 25: 352-356 53. Link TA. 1999. The structures of Rieske and Rieske-type proteins. Adv. Inorg. Chem., eds. Sykes AG, Cammack R, 47: 83-157 54. Liu A, Graslund A. 2000. Electron paramagnetic resonance evidence for a novel interconversion of [3Fe-4S]+ and [4Fe-4S]+ clusters with endogenous iron and sulfide in anaerobic ribonucleotide reductase activase in vitro. J. Biol. Chem. 275:12367-12373 55. Martius C, Lynen F. 1950. Probleme des Citronen-saurecyklus. Adv. Enzymol. 20: 167-222 56. Maskiewicz R, Bruice TC. 1977. Kinetic study of the dissolution of Fe4S^" cluster core ions of ferredoxins and high potential iron protein. Biochemistry 16: 3024-3029 57. Matsubara H, Saeki K. 1992. Structural and functional diversity of ferredoxins and related proteins. Adv. Inorg. Chem., eds. Cammack R, Sykes AG, 38: 223-280 58. Moore LJ, Kiley PJ. 2001. Characterization of the dimerization domain in the FNR transcription factor. / Biol. Chem., 276:45744-45750 59. Miihlenhoff U, Lill R. 2000. Biogenesis of iron-sulfur proteins in eukaryotes: A novel task of mitochondria that is inherited from bac teria. Biochim. Biophys. Ada 1459: 370-382 60. Nishio K, Nakai M. 2000. Transfer of iron-sulfur cluster from NifU to apoferredoxin. /. Biol. Chem. 275: 22615-22618 61. Noodleman L, Case DA. 1992. Density-functional theory of spin polarization and spin coupling in iron-sulfur clusters. Adv. Inorg. Chem., eds. Cammack R, Sykes AG, 38: 423^170 62. Oliveira L, Bouton C, Drapier J-C. 1999. Thioredoxin activation of iron regulatory proteins. /. Biol. Chem. 274: 516-521 63. Ollagnier-de Choudens S, Sanakis Y, Hewitson KS, Roach P, Baldwin JE, et al. 2000. Iron-sulfur center of biotin synthase and lipoate synthase. Biochemistry 39: 4165-4173.

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64. Pantopoulos K, Mueller S, Atzberger, A, Ansorge W, Stremmel W, et al. 1997. Differences in the regulation of iron regulatory protein-1 (IRP-1) by extra- and intra-cellular oxidative stress. /. Biol. Chem. 272: 9802-9808 65. Petering D, Fee JA, Palmer G. 1971. The oxygen sensitivity of spinach ferredoxin and other iron-sulfur proteins. /. Biol. Chem. 246: 643-653 66. Pierik AJ, Roseboom W, Happe RP, Bagley KA, Albracht SPJ. 1999. Carbon monoxide and cyanide as intrinsic ligands to iron in the active site of [NiFe]-hydrogenases. /. Biol. Chem. 274: 3331-3337 67. Popescu CV, Bates DM, Beinert H, Miinck E, Kiley PJ. 1998. Mossbauer spectroscopy as a tool for the study of activation/inactivation of the transcription regulator FNR in whole cells of Escherichia coli. Proc. Natl. Acad. Sci. USA 95:13431-13435 68. Schwartz CJ, Djaman O, Imlay JA, Kiley PJ. 2000. The cysteine desulfurase, IscS, has a major role in in vivo Fe-S cluster formation in Escherichia coli. Proc. Natl. Acad. Sci. USA 97: 9009-9014 69. Smith BE. 1999. Structure, function and biosynthesis of the metallosulfur clusters in nitrogenases. Adv. Inorg. Chem., eds. Sykes AG, Cammack R, 47:159-218 70. Van Spanning RJ, De Boer APN, Reijnders WNM, Westerhoff HV, Stouthamer AH, et al. 1997. FnrP and NNR of Paracoccus denitrificans are both members of the FNR family of transcriptional activators but have distinct roles in respiratory adaptation in response to oxygen limitation. Mol. Microbiol. 23: 893-907 71. Vanin AF, Stukan RA, Manukhina EB. 1996. Physical properties of dinitrosyl iron complexes with thiol-containing ligands in relation with their vasodilator activity. Biochim. Biophy. Ada 1295: 5-12 72. Warshel A, Papazyan A, Muegge I. 1997. Microscopic and semimacroscopic redox calculations: What can and cannot be learned from continuum models. /. Biol. Inorg. Chem. 2: 143-152 73. Weber IT, Steitz TA. 1987. Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 A resolution. /. Mol. Biol. 198: 311-326 74. Yuvaniyama P, Agar JN, Cash VL, Johnson MK, Dean DR. 2000. NifSdirected assembly of a transient [2Fe-2S] cluster within the NifU protein. Proc. Natl. Acad. Sci. USA 97: 599-604 75. Zheng L, Dean DR. 1994. Catalytic formation of a nitrogenase ironsulfur cluster. /. Biol. Chem. 269:18723-18726

Chapter 4 The Ferredoxin Ferredoxin/Thioredoxin Thioredoxin System. A light-Dependent Redox Regulatory System in Oxygenic Photosynthetic Cells Peter Schurmann Laboratoire de Biochemie Vegetale, Universite de Neuchdtel, CH-2007 Neuchatel Switzerland [email protected]

Keywords: ferredoxin/thioredoxin system, ferredoxin, ferredoxinithioredoxin reductase (FTR), thioredoxin m, thioredoxin/, site-directed mutagenesis, signal transfer, target enzymes, fructose 1,6-bisphosphatase, NADPmalate dehydrogenase, redox potential, regulatory disulfides

1. Summary Redox signaling and the regulation via disulfide interchange reactions was first described for the activation of chloroplast enzymes by light. In recent years this type of regulation has gained a lot of interest since it appears to be involved not only in regulation of photosynthetic enzymes, but also in light harvesting, germination, transcription, translation, apoptosis and detoxification. The redox regulatory system of oxygenic photo synthetic organisms, known as the ferredoxin/thioredoxin system, links the activity of key enzymes to light, thereby regulating the carbon flow. Catalysts involved in carbon assimilation are activated by reduction in the light and deactivated by oxidation in the dark. In contrast an enzyme tunneling carbon intermediates into degradation is turned off by reduc tion in the light, but activated by oxidation in the dark. This lightdependent redox regulation avoids the concomitant operation of carbon assimilatory and degradative pathways and might also regulate the carbon flux depending on the light intensity. It operates as regulatory 73

74


cascade involving several proteins: ferredoxin, ferredoxinrthioredoxin reductase (FTR) and thioredoxins modifying the activity of target enzymes by reduction of their regulatory disulfides. The electrons needed for the reductions are provided by the photosynthetic light reactions and transmitted by ferredoxin to FTR. This unique enzyme transforms the light signal, received in the form of electrons, into a thiol signal which is then transmitted through disulfide-dithiol interchanges involving thioredoxins to the target proteins. Several recent reviews have discussed various aspects of the ferredoxin/ thioredoxin system.8,15'26,39,56'65,69"71 This chapter will describe recent struc tural information obtained on the participating components of the system and advances in the understanding of its mechanism.

2. The Components of the Ferredoxin/Thioredoxin System All components of this regulatory system are rather small, soluble proteins containing either a Fe-S cluster or a redox-active disulfide bridge or both. They have been found in the chloroplasts of higher plants and algae as well as in cyanobacteria. The components have been purified and charac terized from different sources and their genes have been cloned and the proteins overexpressed. The availability of recombinant proteins finally enabled their structural analysis by X-ray crystallography. 2.1 Ferredoxin The first member of the system accepting the electron signal from the thylakoids is ferredoxin. The plant-type ferredoxins, involved in oxygenic photosynthesis, are small, acidic 2Fe-2S proteins of about 12 KDa. The Fe-S cluster is attached to the protein by 4 cysteine ligands, has a redox potential of around -400 mV and can carry one electron. The ferredoxins are well studied proteins and for many the primary structures are known showing that the positions of the four cluster liganding cysteines are present in a highly conserved cluster-binding motif. Several three- dimensional structures have also been obtained either by NMR or crystallography and they exhibit large similarities sharing the same fold.15 The structures reveal two patches of negative surface charges on either side of the Fe-S cluster, which have been shown to be essential for the interaction with other pro teins. Differential chemical modification of free and target bound ferredoxin indicates that interaction with positively charged FTR involves essentially only one such negative domain and that Glu92 in spinach ferredoxin is one of the important residues.19 This conclusion is supported by mutagenesis

The Ferredoxin/Thioredoxin System 75

Fig. 1. Sequence comparison of spinach and Synechocystis ferredoxin:thioredoxin reductase. The sequences were aligned using CLUSTALX77 and formatted with BOXSHADE (http://www.Ch.Embnet.org/software/BOX_form.html). The cysteine containing motifs are in bold type.

experiments in which the replacement of this C-terminal glutamate residue resulted in a protein incapable of reducing FTR.42 2.2 Ferredoxin:Thioredoxin Reductase FTR is the key enzyme of this regulatory system. It transmits the redox signal from ferredoxin to thioredoxins. FTR is a unique photosynthetic enzyme, different from the well-known NADP-dependent thioredoxin reductase, which is a flavoprotein, present also in the cytoplasm of plants. Purified FTR is a yellowish-brown protein with an apparent molecular mass of 20 to 25 KDa. It is composed of two dissimilar subunits, a catalytic subunit and a variable subunit. The catalytic subunit contains a 4Fe-4S cluster and a redox-active disulfide bridge, both essential for catalysis, whereas the variable subunit appears to have only structural function. The FTR has been isolated and characterized from different sources and a number of gene and protein sequences are known.15,70 In higher plants and green algae, the FTR is nucleus encoded and both subunits carry transit peptides which guide them after synthesis in the cytoplasm into the chloroplast. Interestingly, in the red alga Porphyra purpurea63 and the cryptomonad Guillardia theta21 the catalytic subunit is coded by the chloroplast genome suggesting that at least this subunit is of bacterial endosymbiotic origin. The catalytic subunits of FTR from different organisms have a constant size of about 13 kDa and a highly conserved primary structure. Among the strictly conserved residues are seven Cys, six of them organized in two CPC and one CHC motifs (Fig. 1). These six Cys are the functionally essential

76


residues constituting the redox active disulfide bridge and ligating the Fe-S cluster. Cluster ligation does not follow the usual consensus motifs for 4Fe-4S centers,36 but shows a new arrangement with the following finger print: CPCX16CPCX8CHC (cluster ligands are in bold). In spinach FTR Cys54 and Cys84 form the active site disulfide. Cys54 is accessible to the solvent whereas Cys84 is protected. The four remaining cysteines, Cys52, Cys71, Cys73 and Cys82 are ligands to the iron center. This arrangement positions the redox-active disulfide bridge adjacent to the cluster.13 Two archaebacteria, Archaeoglobus fulgidusi6 and Methanobacterium thermoautotrophicum™ contain a gene coding for a protein with some strik ing resemblances to the catalytic subunit of FTR. The overall identities between the archaebacterial proteins and the photosynthetic FTRs are rather low (25-35%), but the CXC motifs, essential for the function of the FTR, are conserved at about identical positions. No functions are reported for those proteins in the archaea. However, the striking structural simi larities suggest that the catalytic subunit of photosynthetic FTR might be derived from such an ancient precursor protein whose function has been adapted during evolution. The variable subunits range in size from 8 to 13 kDa and show pro nounced sequence variability with only 46 to 60% identity within the eukaryotes and 33 to 40% between eukaryotes and prokaryotes. The size variability stems from a variable extension of the N-terminus present in all three known eukaryotic enzymes, but absent from the prokaryotic counterparts (Fig. 1). In spinach FTR, this N-terminal extension was found to be unstable, being degraded to discrete shorter peptides 78 which exhibit no functional differences. The FTRs from spinach and Synechocystis sp PCC6803 have been cloned and expressed in E. coif7,73 using a dicistronic construct containing the genes for both subunits in series in the same expression vector. Both recombinant proteins were perfectly active and produced in amounts large enough to initialize structural studies. Recombinant Synechocystis FTR crystallized as dark brown crystals,16 which diffracted very well and permitted structural resolution to 1.6 A (pdb ldj7). 1517 The FTR is a rather flat, disk-like molecule. The variable subunit is heart-shaped with a /J-barrel constituting the main body and with two loops forming the upper, outer part of the heart. The catalytic subunit, which sits on top of the variable subunit is an overall a-helical structure containing five helices. The Fe-S center and the active site disul fide bridge are both located in the catalytic subunit, in the center of the heterodimer, where the molecule is only 10 A across. The cubane 4Fe-4S cluster is situated on one side of the flat molecule close to the surface,

The Ferredoxin/Thioredoxin System 77

Fig. 2. Modeling of the interaction between ferredoxin (red, to the left), ferredoxin: thioredoxin reductase (variable subunit in green and catalytic subunit in blue, in the middle) and thioredoxin (yellow, to the right). The thin, disk-like structure of the FTR allows simultaneous docking of ferredoxin and thioredoxin on opposite sides of the molecule. The Fe-S centers and the disulfide bridges are shown in ball and stick representation. (Reproduced with permission from Ref. 15. Copyright Cambridge University Press).

which contains three positive charges. The redox active disulfide bridge is on the opposite surface, which has a more hydrophobic character. This arrangement with a positively charged docking site for the negatively charged ferredoxin on one side and a rather hydrophobic docking site for thioredoxins on the opposite side of the flat molecule is perfectly adapted for the transfer of electrons from ferredoxin to thioredoxin across its center (Fig. 2). These properties make the FTR a versatile thioredoxin reductase, capable of accepting electrons from diverse ferredoxins and reducing the disulfides of various thioredoxins.

2.3 Thioredoxins Plant cells contain at least four different types of thioredoxins/ 1 which display a certain specificity in their interaction with other proteins. Two types

78


of thioredoxins, the/-type and the m-type, are located in the chloroplasts, one type, thioredoxin h, is present in the cytoplasm and mitochondria appear to house still another type. The thioredoxins involved in the ferredoxin/thioredoxin system transmitting the light-generated redox signal to target enzymes are the /- and m-type thioredoxins from the chloroplasts. These two types of thioredoxins can be clearly distinguished by their primary sequence and phylogenetic background. Both chloroplast thioredoxins are nucleus encoded with the exception of the m-type in red algae, where its gene was found on the chloroplast genome. 63

2.3.1 Thioredoxin m Thioredoxin m was originally described as activator protein for the NADPdependent malate dehydrogenase (NADP-MDH) of chloroplasts in C3 and C4 plants. This type of thioredoxin is found in chloroplasts of dicots, monocots and algae as well as in cyanobacteria and resembles strongly the thioredoxin from anoxygenic prokaryotes, both hetero-trophic and photosynthetic. Thioredoxins of the m-type are, therefore, also known as bacterialtype thioredoxins. Due to their structural relatedness the bacterial and m-type thioredoxins are functionally similar and can be used interchange ably. A comparison of thioredoxin m sequences from prokaryotes, eukaryotic algae and higher plants clearly demonstrates that they are related, however they display less sequence similarity than their /-type counter parts known to date. The greater diversity of the m-type thioredoxins even within higher plants can be seen in Fig. 3, where thioredoxins m and / sequences from the same five organisms have been aligned. The thiore doxin m sequences contain half as many conserved residues (23%) compared to the corresponding thioredoxin/sequences (46%).

2.3.2 Thioredoxin f Thioredoxin / has been discovered as the specific activator protein of chloroplast fructose 1,6-bisphosphatase (FBPase). In contrast to the m-type this thioredoxin is restricted to eukaryotic organisms. There are fewer sequences known for thioredoxin / but display a significantly higher homology than the m-type thioredoxins. The / thioredoxins are slightly longer than other types due to additional amino acids at their N-termini, and the C-terminal part of the sequences resemble classical animal thioredoxin in containing a third, strictly conserved Cys (Fig. 3).

The Ferredoxin/Thioredoxin System

79

Fig. 3. Sequence comparison between chloroplast thioredoxins m and/from the five same organisms with thioredoxin from E coli. The sequences were aligned from the redox active cysteines, which are given in bold type as is also the third cysteine in thioredoxin/. 2.3.3 Three-Dimensional

Structures of Thioredoxins

While extensive structural data has been available for thioredoxins from nonphotosynthetic organisms, such information has only recently been provided for plant thioredoxins. Crystal structures have been determined for a somewhat unusual thioredoxin from Anabaena (pdb ltxh) 66 and for the chloroplast thioredoxins / and m from spinach 9 and solution struc tures for thioredoxin m (pdb ldby) 5 1 and the cytosolic thioredoxin h (pdb ltof)59 from the green alga Chlamydomonas reinhardtii. The structure of spinach thioredoxin m has been solved for the oxidized and reduced protein at 2.1 and 2.3 A resolution, respectively (pdb lfb6, lfbO).9 The structure is very similar to that of E. coli thioredoxin,45 which corroborates the biochemical evidence showing the proteins are functionally interchangeable. The secondary structures of the m-type and E. coli thioredoxins are nearly identical and the surfaces around the active site Cys are largely similar. There is also no large conformational change between oxidized and reduced protein, thus confirming and extending

80


observations reported for E. coli and human thioredoxins.43,80 However, some slight structural differences in the main chain conformation of the active site render the solvent-exposed Cys37 more accessible in the reduced protein. The structure of spinach thioredoxin/ 9 has been solved for two forms of recombinant protein, a "long form" (pdb lf9m) resembling closely the in vivo form1 and a N-terminus truncated "short form" (pdb lfaa). 20 Both structures are essentially identical aside from the N-terminus, which con tains an additional a-helix in the long form, and a difference in the con formation of their active site regions. Whereas the overall structure of thioredoxin/ does not differ markedly from a typical thioredoxin, its sur face topography is distinct from that of others. Thioredoxin/is more posi tively charged and some of these charges surround the active site where they must be instrumental in orienting thioredoxin/correctly with target proteins. The hydrophobic residues, also prominent in the contact area, may be more important in the less specific interaction with FTR, which reduces various thioredoxins efficiently. A striking difference is the pres ence of the third Cys exposed on the surface (Cys73 in spinach), 9.7 A away from the accessible Cys46 of the active site. As already mentioned, this third Cys is conserved in all/-type thioredoxins. The structural analy sis also shows that the active site Cys with the lower sequence number (Cys46 in spinach) is exposed whereas its partner is buried, confirming biochemical experiments which showed that Cys46 is the attacking nucleophile in the reduction of target disulfides. 6 Another, maybe important feature is the apparent flexibility of the active site region of thioredoxin/as evidenced by different conformations observed in its long and short forms. Trp45, which is part of the active site sequence (WCGPC), can flip its indole ring away from the active site. This is possible due to the absence of a hydrogen bond between the indole ring and the carboxyl group of a neighboring aspartate observed in thiore doxin m and E. coli thioredoxin. Trp45 of thioredoxin/cannot make such a hydrogen bond, because the residue corresponding to aspartate is Asn74, whose side chain points in the opposite direction and receives a hydrogen bond from the main chain nitrogen of Asn77. Asn74 in thiore doxin / is followed by the insertion of a Gln75 with respect to the other types of thioredoxins and this insertion appears to be a distinctive feature of /-type thioredoxins. It modifies the loop conformation (residues 74 to 77) keeping the Asn74 side chain away from Trp45. This deviating local conformation may represent an important structural factor contributing to the specificity of thioredoxin/ 9


2.3.4 Specificity of

81

Thioredoxins.

One of the puzzling facts is that chloroplasts contain two types of thioredoxins with practically identical redox potential 34 and catalyzing identical redox reactions. However, they display a certain selectivity in their interaction with target enzymes when tested under conditions approaching their in vivo situation. The Calvin cycle enzymes FBPase,29 sedoheptulose 1,7-bisphosphatase (SBPase),83 phosphoribulokinase (PRK)82 as well as Rubisco activase 84 and ATP synthase (CFj)72 are exclu sively or very efficiently activated by thioredoxin /. NADP-MDH, origi nally thought to be specifically light-regulated through thioredoxin m, was shown to be even more efficiently activated by thioredoxin f.29,35 Glucose 6-phosphate dehydrogenase (G6PDH), on the contrary, is modu lated specifically by thioredoxin m81 and appears to be so far the only enzyme responding exclusively to thioredoxin m. Recent reexamination of the interaction between the two chloroplast thioredoxins and PRK suggests that thioredoxin m might be somewhat more efficient in activating PRK than thioredoxin /. 28 In general these observations indi cate that, at least as far as carbohydrate metabolism is concerned, thio redoxin / functions primarily in enzyme activation (i.e. enhancing the rate of biosynthesis) whereas thioredoxin m acts mainly in enzyme deactivation (i.e. enhancing the rate of degradation). Thioredoxin / has also been reported to be an efficient activator of acetyl CoA carboxylase catalyzing the first committed step in fatty acid biosynthesis in chloroplasts68 as well as for ADP-glucose pyrophosphorylase catalyzing the first committed step in starch biosynthesis. 4 Thioredoxin m, however, has been suggested to be involved in processes like translation, 18,52 removal of reactive oxygen species 3 and N-metabolism activating ferredoxin: glutamate synthase. 53 The observed specificity of thioredoxins in their interaction with target proteins raises the question of which structural features could be respon sible for it. Answers have been sought by applying site-directed mutagenesis to thioredoxins. A sequence comparison based on the threedimensional structure of E. coli thioredoxin 24 revealed several residues, which in thioredoxin / are different from the consensus or from thiore doxin m. Such residues, especially if a change of charge is involved, could be, at least in part, responsible for the specificity. In spinach thioredoxin / s o m e of these residues have been replaced to make the protein more sim ilar to thioredoxin m and in E. coli thioredoxin50,60 and pea thioredoxin m54 residues typical for thioredoxin / have been inserted (see Fig. 4 and


Fig. 4. Structure-based alignment9 of the sequences of the spinach chloroplast and E. coli thioredoxins. The positions of residues, which have been mutated, are indicated by T. Table 1. Summary of the various residues which have been mutated and their respective nonmutated counterparts in the three thioredoxins used to probe the interaction specificity. References are given in parentheses A = deletion mutant. Thioredoxin/ Q44 K58E (28, 29) C73S, C73A, C73G (20) N74A, N74D (28, 29) Q75D (28, 29) N77A (28, 29) E83 V89I/T105I (28) T105I (28, 29) K108

Thioredoxin E. coli

Thioredoxin m

E30K (60) E44 160 D61N (50) — N63 K69 I75/V91 V91 L94K (60)

P35 E49 T65 D66 — A68 K70E (pea (54) = Q74 spinach) 180/196 196 V100

Table 1). In general the analyses of the properties of the modified proteins point into the expected direction, i.e. conversion of thioredoxin / to a more m-like thioredoxin and, on the other hand, improvement of acti vation of FBPase by E. coli thioredoxin. Thioredoxin/contains more pos itively charged residues on its surface than E. coli thioredoxin or thioredoxin m. Replacement of positively charged or neutral amino acids by negatively charged residues reduces the affinity of thioredoxin/mutants for FBPase or PRK.28'29 Furthermore the replacement of the surface exposed


83

third Cys reduces the affinity for FBPase.20 In contrast introduction of positive charges in E. coli thioredoxin improves its capacity to activate FBPase, which however still requires thioredoxin concentrations well beyond any physiological level. It appears that electrostatic components play a crucial role in the interaction with the target proteins but are not the only important factors. Any mutation so far done on the thioredoxin/ has been counterproductive with respect to activation of FBPase, however beneficial with respect to activation of NADP-MDH where this has been tested.29 These results show that the interplay of several factors is respon sible for the specificity in the interaction of thioredoxins with their target proteins.

3. The Redox Signal Transfer through the Ferredoxin/Thioredoxin System The redox signal, originating in the thylakoid membranes in the form of electrons, has to be transmitted via ferredoxin, FTR and thioredoxins to the target proteins where it is received as a thiol signal. The transforma tion of the electron signal into a thiol signal is accomplished by FTR. That FTR is indeed capable of making this conversion has been clearly demon strated with isolated chloroplasts 14 and purified components. 23 Ferredoxin, the first soluble electron acceptor, carries the electrons, one at a time, from photosystem I to the FTR. Ferredoxin is a negatively charged protein which has been shown to form an electrostatically stabilized 1:1 complex with FTR.19,32 Although FTR can accept electrons from heterologous ferredoxins, as has been demonstrated by the use of spinach ferredoxin for the reduction of FTR from corn and Nostoc,22 Chlamydomonas,37 Synechocystis73 and soja (P. Schurmann, unpublished), the best electron donor appears to be the homologous ferredoxin, forming the most stable complex. This must be due to the arrangement of the neg ative charges on ferredoxin and the complementary positive charges on the FTR surface. The FTR contains a Fe-S center and a redox-active disulfide bridge. While all known disulfide reductions except the one by FTR are catalyzed by flavoproteins or by thiol/disulfide exchange reactions, the FTR uses its Fe-S center to cleave the active site disulfide. This appears to be possible not as a result of an unusual geometry of the Fe-S center, which is a normal cubane 4Fe-4S cluster, but due to the close proximity of active site disulfide and cluster. Of the two active site cysteines the one with the

84 Cellular Implications ofRedox Signalling [4Fe-4S]3*

[4Fe-4S]2* S(Cys) 2-

S

(Cys)s,

(Cy5)S

Fe°

(His)

(cys)

t2_V ^ ^ V....J S(Cys)

2-

Fd

red

w

S(Cys) Fe"' (His)

S

(Cys)S,

(Cys)slp?

S(Cys) &

J // "

( C y s ) s " ^* b (CVS)S

[Pro)

•(Pro)

^(Cys)

2-

(Cys)s,

[4Fe-4S]3* S(Cys) (His) -Fe'

(Cys)sjp? 1 / , e

\

(Cys)S—S—I

(Pro) \>(Cys)-^

Fd

" J red

2- (Cys)S,

[4Fe-4S]2* S(Cys) v ,S Fe' (His)

2- (Cys)S,5

[4Fe-4S]2* S(Cys) N N S Fe(His) Fe~pS I Fe-I

I rs(Cys) S V

(Cys)S \ 'S(Cys)'

_- NTR -> Thioredoxin hred + NADP

(3)

Early cell fractionation experiments revealed that thioredoxin h was extraplastidic and occurred in the cytosol, endoplasmic reticulum and mitochondrion 44 (Fig. 4). Current evidence suggests that the N A D P / thioredoxin system is present in all plant cells, photosynthetic as well as nonphotosynthetic. As with i t s / a n d m counterparts, thioredoxin h has its own evolutionary history. 49 Following a description of the system, we set out to determine the func tion of thioredoxin h. Good fortune led us to cereals and to ask


Fig. 5. Role of NADP/thioredoxin system in seed germination. A contemporary view.

several questions including, (1) whether the disulfide groups of seed storage proteins undergo redox change during grain formation, development and germination, and (2) whether thioredoxin was involved in germination and seedling development. Working with wheat in collaboration with K. Kobrehel, we found the answer to both questions to be yes.29,40 This and subsequent work provided evidence that thioredoxin h acts as an early wakeup call in germination and seedling development by facilitating the (a) mobilization of nitrogen and carbon through the reduction of storage proteins with disulfide groups (gliadins and glutenins); (b) inactivation of low-molecular-weight disulfide proteins that inhibit starch-degrading enzymes, and (c) activation of individual enzymes as occurs in chloroplasts4,12,33'34'35'41'42'53 (Fig. 5). A key feature turned out to be the specificity of thioredoxin in the reduction of mframolecular versus, mtermolecular disulfide bonds of the proteins studied.34,35'38'53'55 The observed proclivity of thioredoxin to reduce intramolecular disulfide bonds, confirmed in recent experiments, 63 has influenced our subsequent work.

Thioredoxin and Redox Regulation

105

3. Applications of Thioredoxin 3.1 Alleviation of Allergies The experiments discussed above revealed that four changes accompany the thioredoxin-linked reduction of low-molecular-weight proteins from plant as well as animal sources. • • • •

Loss (or gain) of biochemical activity. Increased susceptibility to proteolysis. Increased susceptibility to heat. Decrease in allergenicity.

The first three changes appear to be general features of low-molecularweight proteins containing intramolecular disulfide bonds. For example, on reduction by thioredoxin, the soybean Kunitz and Bowman-Birk trypsin inhibitors lose their ability to inhibit trypsin and show increased susceptibility to proteolysis and increased heat.34 The fourth change, decrease in allergenicity, may not be universal but has been observed with a number of allergens containing intramolecular disulfide bonds. We have studied the allergy problem using a colony of high IgE-producing dogs sensitized to specific foods.24 In our initial study with differentially soluble proteins from wheat (Osborne fractions),8 we used the hyper sensitive skin test response to (1) identify and rank the allergens according to their allergenicity (gliadins > glutenins > albumins > globulins); (2) show that the effect of the major allergens, gliadins and glutenins, was dimini shed on reduction by thioredoxin; (3) document that the results were statistically significant; and (4) show that reduced glutathione had no effect on allergenicity. The more we learn the better the dog seems to be as an allergy model for humans. 7 After the wheat work, we carried out a study on milk.23 We found that, as in humans, the major allergen is beta-lactoglobulin — a protein with a known human epitope 2 and with two disulfide bonds 47 — was reduced actively by thioredoxin. 23 In this case, the allergic response was shown to be mitigated by thioredoxin as measured by both skin tests and feeding challenges. In the latter experiments, we monitored allergenicity by both the immediate (vomit) and delayed (diarrhea/constipation) response of dogs fed untreated versus thioredoxin-treated beta-lactoglobulin. Thioredoxin is believed to mitigate the allergic response manifested by a wheal in skin tests8,23 and by the vomit response in feeding challenges, 23

106


through changing the structure of the allergen (beta-lactoglobulin) so that it is less well recognized by the IgE of mast cells in the majority of animals and is much more readily digested in the stomach. These changes are reflected in the observed decrease in the immediate and delayed gastrointestinal responses. It appears that increased sensitivity to pepsin is a general feature accompanying the reduction of low-molecular-weight disulfide proteins by thioredoxin. Current data thus indicate that thioredoxin disarms food allergens in two ways: (1) by decreasing epitope accessibility to the IgE immune sys tem, thereby lowering the immediate vomit response, and (2) by increas ing sensitivity to pepsin, thereby facilitating digestion in the stomach and lowering the delayed gastrointestinal (diarrhea/constipation) response. As an extension of this work, we are currently testing the capability of thioredoxin to improve desensitization (immunotherapy) to ragweed pollen by shifting the immune response from IgE to IgG. Such a shift could make the desensitization process (immunotolerance) both safer and more effective. We have observed that a disulfide allergen of ragweed pollen (Amb t5) is largely inactivated on reduction by thioredoxin, thus making such a shift in the immune response conceptually feasible.22

3.2 Improved Dough Quality A second problem we are actively pursuing is the effect of thioredoxin on dough strength. Early studies in collaboration with K. Kobrehel showed that, when added to poor quality flour, components of the N A D P / thioredoxin system (NADPH, NTR and thioredoxin) strengthened dough products as determined by Farinograph measurements 61 and increased loaf volume and viscoelasticity.39 As seen below, these findings are currently being extended in experiments with transformed grain overexpressing thioredoxin h.

4. Thioredoxin-Enriched Grain 4.1 Cereal Transformations To make the application of thioredoxin economically feasible, we have transformed cereals to overexpress thioredoxin h. The overall goal is to determine whether the improvement in dough quality effected by thiore doxin in vitro can be obtained with thioredoxin h overexpressed in vivo. To


107

Fig. 6. DNA construct for transformation of cereals with wheat thioredoxin h Gene (from Ref. 18).

this end, in collaboration with P. G. Lemaux, we have developed a gene expression system designed to express proteins of interest specifically in the grain endosperm. 18 Using a barley Bj-hordein promoter, we have obtained maximal expression of thioredoxin h using a DNA construct with which the gene is linked to a signal sequence for targeting to the endosperm protein body. Here the wheat thioredoxin h gene (kindly pro vided by Dr Philippe Joudrier) is linked to the B a -hordein promoter and a protein body signal sequence (Fig. 6). Homozygous barley lines trans formed with this construct showed up to 30-fold enrichment in the con tent of thioredoxin h relative to null segregants. 19 A similar pattern of overexpression has recently been obtained with transformed wheat (unpublished findings).

4.2 Properties of Transgenic Cereals We have only recently begun to analyze transgenic cereals with increased levels of thioredoxin h. Our studies have corroborated earlier in vitro results and shown that transgenic grain grown either in the greenhouse or the field is enriched in starch debranching enzyme (also called limit dextrinase or pullulanase). 19 Based on spectrophotometric and gel assays, extracts from the homozygous transgenic lines showed up to 3-fold more starch debranching enzyme activity than corresponding null segregants. More recent analyses indicate that the transgenic barley also shows an acceleration in germination (radicle emergence) and oc-amylase biosyn thesis, both by up to a day.62 Other properties of the transgenic cereals are under investigation.


Strategy

Target Protein

Chance

Chloroplast FBPase

Light activation

Chloroplast PRK + others

Dithiothreitol activation

Chloroplast NADP-MDH

mBBr / 1 D SDS-PAGE

Multiple suspected target proteins from seeds + other sources

Gene overexpression

Seed a-amylase

Cassette mutagenesis

Yeast periredoxin

mBBr/ 2D Electrophoresis

> 20 unknown proteins: 3 allergens + 2 proteins new to peanut

Fig. 7. Identification of new proteins targeted by plant thioredoxins.

5. A New Development One of the challenges of thioredoxin research is knowledge of its target pro teins. In the original studies on photosynthesis, target enzymes were identi fied by chance (e.g. fructose bisphosphatase) or by showing that an enzyme activated either by light or DTT in vitro (e.g. NADP-malate dehydrogenase) could be similarly activated by reduced thioredoxin. 6 (Fig. 7). The opportunity to label the sulfhydryl groups newly generated by thioredoxin in either known individual proteins34,35'38'41'53'55 or protein families40 with mBBr, in combination with one-dimensional gel elec trophoresis, led to the identification of a number of new targets. The proteins identified are extraplastidic and primarily serve a storage or pro tective function in seeds.3'4'40 In ongoing work with seeds, we have devel oped a new strategy for the identification of thioredoxin target proteins. 63 The approach is based on the application of mBBr to tag target proteins reduced in vitro by thioredoxin. The labeled proteins are isolated by electrophoresis [2D-isoelectric focusing/reducing SDS-PAGE or 2D-nonreducing/reducing SDS-PAGE] and identified by amino acid sequencing. When applied to extracts of peanut seeds we isolated at least 20 thioredoxin targets revealed by the fluorescent spots (Fig. 8) and identified 5, all with intramolecular disulfide bonds: 3 allergens (Ara h2, Ara h3, Ara h6) and 2 proteins not known to occur in peanut (desiccation-related and seed


109

Ul

o

Fig. 8. Strategy for identifying thioredoxin target proteins (from Ref. 63). maturation protein). 63 These findings open the door to the identification of proteins targeted by thioredoxin in a wide range of systems, thereby enhancing our understanding of its function and extending its technologi cal and medical applications. The present studies show how research initiated in the early 1960s on carbon dioxide fixation in fermentative bacteria led to the discovery of a carbon cycle in photosynthetic bacteria and then, sequentially to regulatory systems functional in oxygenic photosynthesis and seed germination. The seed research, in turn, has opened the door to emerging new technologies, including ones applicable to the improvement of major foods. Three lessons have been learned as this work has unfolded during the past four decades. The development of technologies and products from basic research — that is (1) the movement of results from the laboratory into the research and development pipeline — requires multiple talents and thus collabo ration with colleagues from diverse disciplines; (2) the trail from discovery to application is long; and (3) the trail is uncharted so that once a discovery is made, there is no way to predict whether it will result in a useful product or technology.

110


Note added in proof The importance of the reverse citric acid cycle in chlorobium was recently confirmed in the determination of the complete genome sequence (J.A. Eisen et al. 2002. Proc. Natl. Acad. Sci. USA 99: 9509-9514).

References 1. Bachofen R, Buchanan BB, Arnon DI. 1964. Ferredoxin as a reductant in pyruvate synthesis by a bacterial extract. Proc. Natl. Acad. Sci. USA 51: 690-694 2. Ball G, Shelton MJ, Walsh BJ, Hill D, Hosking CS, Howden MEH. 1994. A major continuous allergenic epitope of bovine /J-lactoglobulin recognized by human IgE binding. Clin. Exp. Allergy 24: 758-764 3. Besse I, Buchanan BB. 1997. Thioredoxin-linked plant and animal processes: The new generation. Bot. Bull. Acad. Sin. (Taipei) 38: 1-11 4. Besse I, Wong JH, Kobrehel K, Buchanan BB. 1996. Thiocalsin: A thioredoxin-linked substrate-specific protease dependent on calcium. Proc. Natl. Acad. Sci. USA 93: 3169-3175 5. Berstermann A, Vogt K, Follman H. 1983. Plant seeds contain several thioredoxins of regular size. Eur. }. Biochem. 131: 339-344 6. Buchanan BB. 1980. Role of light in the regulation of chloroplast enzymes. Ann. Rev. Plant Physiol. 31: 341-374 7. Buchanan BB. 2001. Genetic engineering and the allergy issue. Plant Physiol. 126: 5-7 8. Buchanan BB, Adamidi C, Lozano RM, Yee BC, Momma M, Kobrehel K, Ermel RW, Frick OL. 1997. Thioredoxin-linked mitigation of aller gic responses to wheat. Proc. Natl. Acad. Sci. USA 94: 5372-5377 9. Buchanan BB, Arnon DI. 1990. A reverse KREBS cycle in photo synthesis: Consensus at last. Photosyn. Res. 24: 47-53 10. Buchanan BB, Evans MCW, Arnon DI. 1967. Ferredoxin-dependent carbon assimilation in Rhodospirillum rubrum. Arch. Microbiol. 59:32-40 11. Buchanan BB, Kalberer PP, Arnon DI. 1967. Ferredoxin-activated fructose diphophatase in isolated chloroplasts. Biochem. Biophys. Res. Commun. 29: 74-79 12. Buchanan BB, Lozano RM, Wong JH, Jiao J, Yee BC, Kobrehel K, Nimbona C. 1994. Thioredoxin linked reduction of wheat storage proteins. I. Physiological consequences. In Gluten Proteins, Associa tion of Cereal Research, Detmold, Germany, pp. 369-380 13. Buchanan BB, Schurmann P, Decottignies P, Lozano RM. 1994. Thioredoxin: A multi-functional regulatory protein with a bright


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future in technology and medicine. Arch. Biochem. Biophys. 314: 257-260 Buchanan BB, Schurmann P, Kalberer PP. 1971. Ferredoxin-activated fructose diphosphatase of spinach chloroplasts: Resolution of the system, properties of the alkaline fructose diphosphatase component, and physiological significance of the ferredoxin-linked activation. /. Biol. Chem. 246: 5952-5959 Buchanan BB, Wolosiuk RA. 1976. Photosynthetic regulatory protein found in animal and bacterial cells. Nature 264: 669-670 Carr PD, Verger D, Ashton AR, Ollis DL. 1999. Chloroplast NADPmalate dehydrogenase: Structural basis of light-dependent regula tion of activity by thiol oxidation and reduction. Structure 7: 461^75 Chiadimi M, Navaza A, Miginiac-Maslow M, Jacquot JP, Cherfils J. 1999. Redox signaling in the chloroplast structure of the oxidized pea fructose-1, 6-biphosphatase. EMBO }. 18: 6809-6816 Cho M-J, Choi HW, Buchanan BB, Lemaux PG. 1999. Inheritance of tissue-specific expression of hordien promotor-uicLA fusions in transgenic barley plants. Theor. Appl. Genet. 98:1253-1262 Cho M-J, Wong JH, Marx C, Jiang W, Lemaux PG, Buchanan BB. 1999. Overexpression of thioredoxin h leads to enhanced activity of starch debranching enzyme (pullulanase) in barley grain. Proc. Natl. Acad. Sci. USA 96:14641-14646 Dai SD, Schwendtmayer C, Johansson K, Ramaswamy S, Schurmann P, Eklund H. 2000. How does light regulate chloroplast enzymes? Structure-function studies of the ferredoxin/thioredoxin system. Q. Rev. Biophys. 33: 67-108 Dai SD, Schwendtmayer C, Schurmann P, Ramaswamy S, Eklund H. 2000. Redox signaling in chloroplasts: Cleavage of disulfides by an iron-sulfur cluster. Science 287: 655-658 del Val G, Yee BC, Buchanan BB, Frick OL. 1999. Disulfide bond reduction by thioredoxin alleviates the allergenicity of ragweed pollen. /. Aller. Clin. Immunol. 103: S139 del Val G, Yee BC, Lozano RM, Buchanan BB, Ermel RW, Lee YM, Frick OL. 1999. Thioredoxin treatment increase digestibility and lowers allergenicity of milk. /. Aller. Clin. Immunol. 103: 690-697 Ermel EW, Kock M, Griffey SM, Reinhart GA, Frick OL. 1997. The atopic dog: A model for food allergy. Lab. Animal Sci. 47: 40^49 Evans MCW, Buchanan BB and Arnon DI. 1966. A new ferredoxindependent carbon reduction cycle in a photosynthetic bacterium. Proc. Natl. Acad. Sci. USA 55: 928-934

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26. Florencio FT, Yee BC, Buchanan BB. 1988. A NADP/thioredoxin system in green leaves: Purification and characterization of NADPthioredoxin reductase and thioredoxin h from spinach. Arch. Biochem. Biophys. 266: 491-507 27. Follmann H, Haberlein I. 1996. Thioredoxin: Universal, yet specific thiol-disulfide redox cofactors. BioFactors 5: 147-56 28. Fridlyand LE, Scheibe R. 1999. Regulation of the Calvin cycle for C 0 2 fixation as an example for general control mechanisms in metabolic cycles. Biosystems 51: 79-93 29. Gobin P, Ng PKW, Buchanan BB, Kobrehel K. 1997. Sulfhydryldisulfide changes in proteins of developing wheat grain. Plant Physiol. Biochem. 35: 777-783 30. Hartman H, Syvanen M, Buchanan BB. 1990. Contrasting evolution ary histories of cholorplast thioredoxins / and m. Mol. Biol. Evol. 7: 247-254 31. Jacquot JP, Lancelin JM, Meyer Y. 1997. Thioredoxins: Structure and function in plant cells. New Phyto. 136: 543-570 32. Jacquot J-P, Rivera-Madrid R, Marinho P, Kollarova M, Le Marechal P, Miginiac-Maslow M, Meyer Y. 1994. Arabidopsis thaliana NAPHP thioredoxin reductase. cDNA characterization and expression of the recombinant protein in Escherichia coli. }. Mol. Biol. 235:1357-1363 33. Jiao J, Yee BC, Buchanan BB. 1992. Thioredoxin-linked changes in properties of protease inhibitors of seed. Plant Physiol. 99S: 57 34. Jiao J, Yee BC, Kobrehel K, Buchanan BB. 1992. Effect of thioredoxinlinked reduction on the activity and stability of the Kunitz and Bowman-Birk soybean trypsin inhibitor proteins. /. Agric. Food Chem. 40: 2333-2336 35. Jiao J, Yee BC, Wong JH, Kobrehel K, Buchanan BB. 1993. Thioredoxinlinked changes in regulatory properties of barley a-amylase/subtilisin inhibitor protein. Plant Physiol. Biochem. 31: 799-804 36. Johansson K, Ramaswamy S, Saarinen M, Lemaire-Chamley M, Issakidis-Bourguet E, Miginiac-Maslow M, Eklund H. 1999. Structural basis for light activation of a chloroplast enzyme. The structure of Sorghum NADP-malate dehydrogenase in its oxidized form. Biochemistry 38: 4319-4326 37. Johnson TC, Cao RQ, Kung JE, Buchanan BB. 1987. Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells. Planta 171: 321-331 38. Johnson TC, Wada K, Buchanan BB, Holmgren A. 1987. Reduction of purothionin by the wheat seed thioredoxin system and potential


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function as a secondary thiol messenger in redox control. Plant Physiol. 85: 446-451 Kobrehel K, Buchanan BB, Bergmann CJ, Wong JH, Yee BC. 1994. Thioredoxin-linked reduction of wheat storage proteins II. Techno logical consequences. In Gluten Proteins 1993, Association of Cereal Research, Detmold, Germany, pp. 381-392 Kobrehel K, Wong JH, Balough A, Kiss F, Yee BC, Buchanan BB. 1992. Specific reduction of wheat storage proteins by thioredoxin h. Plant Physiol. 99: 919-924 Kobrehel K, Yee BC, Buchanan BB. 1991. Role of the N A D P / thioredoxin system in the reduction of a-amylase and trypsin inhibitor proteins. /. Biol. Chem. 266: 16135-16140 Lozano RM, Wong JH, Yee BC, Peters A, Kobrehel K, Buchanan BB. 1996. New evidence for a role for thioredoxin h in germination and seedling development. Planta 200:100-106 Madigan MT, Martinko JM, Parker J. 1997. Brock Biology of Micro organisms, 8th edn., Prentice Hall, Upper Saddle River, New Jersey, pp.650-651 Marcus F, Chamberlin SH, Chu C, Masiarz FR, Shin S, Yee BC, Buchanan BB. 1991. Plant thioredoxin h: An animal like thioredoxin occurring in multiple cell compartments. Arch. Biochem. Biophys. 287: 195-198 Meyer Y, Verdoucq L, Vignols F. 1999. Plant thioredoxins and glutaredoxins: Identity and putative roles. Trends Plant Sci. 4: 388-394 Mittard V, Blackledge MJ, Stein M, Jacquot J-P, Marion D, Lancelin JM. 1997. NMR solution structure of an oxidised thioredoxin h from the eukaryotic green alga Chamydomonas reinhardtii. Eur. ]. Biochem. 243: 374-383 Papiz MZ, Sawyer L, Eliopoulos EE, North AC, Findlay JB, Sivaprasadarao R, Jones TA, Newcomer ME, Kraulis PJ. 1986. The structure of (3-lactoglobulin and its similarity to plasma retinolbinding protein. Nature 324: 383-385 Ruelland E, Miginiac-Maslow M. 1999. Regulation of chloroplast enzyme activities by thioredoxins: Activition or relief from inhibi tion? Trends Plant Sci. 4: 136-141 Sahrawy M, Hecht V, Lopez-Jaramillo J, Chueca A, Meyer Y. 1996. Intron position as an evolutionary marker of thioredoxin and thiore doxin domains. /. Mol. Evol. 42: 422^31 Schurmann P, Buchanan BB. 2001. The structure and function of the ferredoxin/thioredoxin system in photosynthesis. In Regulation of

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51. 52.

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59.

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Photosynthesis. Advances in Photosynthesis, eds. Andersson B, Aro E-M, Kluwer Academic Publishers. Dordrecht, The Netherlands, 11:331-361 Schurmann P, Jacquot J-P, 2000. Plant thioredoxin systems revisited. Ann. Rev. Plant Physiol. Plant Mol. Biol. 51: 371-400 Schurmann P, Wolosiuk RA, Breazeale VD, Buchanan BB. 1976. Two proteins function in the regulation of photosynthetic C 0 2 assimila tion in chloroplasts. Nature 263: 257-258 Shin S, Wong JH, Kobrehel K, Buchanan BB. 1993. Reduction of castor seed 2S albumin protein by thioredoxin. Planta 189: 557-560 Villeret V, Huang S, Zhang Y, Xue Y, Lipscomb WN. 1995. Crystal structure of spinach chloroplast fructose-1, 6-biphosphatase at 2.8 A resolution. Biochemistry 34: 4299^306 Wada K, Buchanan BB. 1981. Purothionin. A seed protein with thiore doxin activity. FEBS Lett. 124: 237-240 Wagner W, Follmann H. 1977. A thioredoxin from green algae. Biochem. Biophys. Res. Commun. 77:1044-1051 Wagner W, Follmann H, Schmidt A. 1978. Multiple forms of thioredoxins. Z Naturforsch Teil C. 33: 517-520 Wahlund TM, Tabita FR. 1997. The reductive tricarboxylic acid cycle of carbon dioxide assimilation: Initial studies and purification of ATP-citrate lyase from the green sulfur bacterium Chlorobium tepidum. J. Bacteriol. 179: 4859-4867 Wolosiuk RA, Ballicora MA, Hagelin K. 1993. The reductive pentose phosphate cycle for photosynthesis C 0 2 assimilation: Enzyme modu lation. FASEB J. 7: 622-37 Wolosiuk RA, Buchanan BB. 1977. Thioredoxin and glutathione reg ulate photosynthesis in chloroplasts. Nature 266: 565-567 Wong JH, Kobrehel K, Nimbona C, Yee BC, Balough A, Kiss F, Buchanan BB. 1993. Thioredoxin and bread wheat. Cereal Chem. 70: 113-114 Wong JH, Ren P-H, Cai N, Cho M-J, Lemaux PG, Buchanan BB. 2000. Transgenic barley grain over-expressing wheat thioredoxin h shows improved germination properties. Ann. Meeting Amer. Soc. Plant Physiol, Abstract No. 187 Yano H, Wong JH, Lee YM, Cho M-J, Buchanan BB. 2001. A strategy strategy for the identification of proteins targeted by thioredoxin. Proc. Natl. Acad. Sci. USA 98: 4794^799

Chapter 6 The Role of Thioredoxin in Regulatory Cellular Functions Junji Yodoi/ Hajime Nakamura, Hiroshi Masutani, Yumiko Nishinaka, and Itaro Hattori Department ofNeurosurgery, Graduate School of Medicine, Kyoto University, Kyoto, 606-8507 Japan '[email protected]

Keywords: thioredoxin, oxidative stress, thioredoxin transgenic mice

1. Summary Increasing evidence has indicated that oxidative stress mediates various cellular responses, although continuous and excessive stress is threaten ing life on the earth. Regulation of reduction/oxidation (redox) is funda mentally important to maintain homeostasis of life. Thioredoxin (Trx) is a 12 kD protein with redox-active dithiol in the active site. Human thiore doxin has been cloned as adult T cell leukemia derived factor produced by HTLV-I transformed cells. Thioredoxin is one of the major components of the thiol-reducing system and plays multiple roles in cellular processes such as proliferation, apoptosis and gene expression. Thioredoxin is induced by a variety of stresses including viral infection.1,2 The promoter sequences of the Trx gene contain a series of stress-responsive elements except for heat shock element. Thioredoxin promotes DNA binding of transcription factors such as NF-kappaB, AP-1 and p53.3,4 Thioredoxin has been already demonstrated to be directly associated with target proteins and activate those proteins by dithiol-dependent reduction. The impor tance of the Trx catalytic site has also been shown in the interaction between Trx and Trx-binding proteins such as Trx-binding protein-2/ vitamin D3 up-regulated protein-1 (TBP-2/VDUP1) 5 and apoptosis signal-regulating kinase-1 (ASK-1).6 We have identified Trx-binding 115

116


protein-2 (TBP-2), which was identical to vitamin D3 up-regulated protein-1 (VDUPl). TBP-2/VDUP1 suppressed the reducing activity of Trx. Treatment of HL-60 cells with vitamin D3 caused an increase of TBP2/VDUP1 expression, suggesting that the Trx-TBP-2/VDUPl interaction may be an important redox regulatory mechanism in cellular processes, including differentiation of myeloid/macrophage lineage. Potential action of TBP-2/VDUP1 as a redox-sensitive tumor suppressor will be discussed. The biological functions of thioredoxin may be strictly regu lated by its enzymatic reaction and/or structure-dependent interaction with the target. 7 We will also discuss our recent data on the anti-apoptotic activity of mitochondoria-specific thioredoxin-2 (Trx-2) based on in vitro knock out system (in cooperation with Spyrou G.). Redox regulation by thioredoxin plays a crucial role in biological responses against oxidative stress. Transgenic mice overexpressing thioredoxin show resistance against ischemic and excitotoxic neuronal injury.8,9 In addition, thiore doxin transgenic mice exhibit u p to 30% extension of median life span and one-third of maximum life span. Overexpressing thioredoxin may have protected mice from oxidative stress-induced tissue damage during aging process. Thioredoxin-Tg mice are useful to investigate the biological functions of thioredoxin in vivo. Thioredoxin is also secreted from the activated cells as a redox-sensitive cytokine with cytokine-like and chemokine-like activities.10 Understanding of thioredoxin-dependent redox regulation will give us a new strategy for preventing diseases related to oxidative stress. Reactive oxygen species (ROS) are generated in eukaryotic cells from oxygen during respiration for energy metabolism, or in response to vari ous stimuli, such as UV irradiation, X-ray, ischemia/reperfusion, inflam matory cytokines and chemical carcinogens. ROS can alter or disrupt the balance of redox potential in cells, which may cause various cellular dysfunction and diseases. 1112 Redox regulation is fundamentally impor tant to maintain homeostasis of life. Eukaryotic cells have acquired sev eral regulatory systems to maintain intracellular redox status by scavenging ROS in evolution. Those systems basically include the glutathione (GSH)13 and the thioredoxin systems 14 based on mono- and di-thiol reaction respectively. In addition to this basic function to cope against oxidative stress, recent evidence has accumulated indicating that reducing molecules such as thioredoxin play important roles in cellular signaling through not only the reduction of cysteine residues of, but rather the interaction with, various important components of signal transduction pathways. Thioredoxin physiologically has cytoprotective

The Role of Thioredoxin in Regulatory Cellular Functions

117

effects against oxidative stress by scavenging ROS together with peroxiredoxin (thioredoxin-dependent peroxidases) system and is induced by various oxidative stresses through the activation of responsive elements in its promoter sequence. In addition, thioredoxin is quickly translocated from the cytoplasm into the nucleus upon oxidative stress and various stimuli, physically interacting with Ref-1 (redox factor 1)/APEX, an endoexonuclease located in the nucleus. Several reports showed that thioredoxin and/or Ref-1 enhance the DNA binding activity of AP-1, polyoma enhancer binding protein-2 (PEBP2), NF-kappaB, p53, and other transcription factors. Thioredoxin is considered as a unique redoxsensitive regulator/modulator of cellular signaling. In this chapter we focus on thioredoxin and its associated molecules and discuss the role of thiore doxin-dependent redox regulation in cellular functions.

2. Cytoprotective Effects of Thioredoxin Thioredoxin has been shown to play crucial roles in cytoprotection against a variety of oxidative stress. Recombinant thioredoxin can pro tect cells from anti-Fas antibody-induced apoptosis and cytotoxicity induced by TNF-alpha, hydrogen peroxide and activated neutrophils. 1516 Thioredoxin is also a potent costimulator of various cytokine expression. 1718 Recently, Nilsson et al. reported that thioredoxin induces the secretion of TNF-alpha and maintains the expression of Bcl-2, whereby prolongs survival of B-LCL.19 Overexpression of thiore doxin has been observed in a wide variety of oxidative conditions such as viral infection, diabetes, ischemic/reperfusion and malignant tis sues.20"22 During viral infection, considerable amount of ROS is gener ated, causing tissue damage and DNA breaks. As thioredoxin was first purified from HTLV-1 transformed cells,21 thioredoxin is induced and/or secreted from transformed cells related to infection of viruses such as HTLV-1, EBV,23 hepatitis C virus and papilloma virus. Elevated thioredoxin level in serum was also reported in late stage HIV patients. 24 Recently, Sono et al. have reported that thioredoxin suppresses lytic replication of EBV induced by 12-0-tetradecanoylphorbol-13-acetate (TPA) and prevented the cell death evoked by the lytic induction. 1 These observations suggest that thioredoxin is closely involved in both the process of virus infection and the prognosis of the infected patients. In vivo study showed that recombinant thioredoxin attenuated ischemia/ reperfusion lung injury in rat.25

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NADPH + H+ 4 TRX-R-S2

TRX-(SH)2

oxidized protein

NADP*

TRX-S2

reduced protein

TRX-R-(SH)2

TRX-R: thioredoxin reductase TRX: thioredoxin Fig. 1. Reducing cycle of thioredoxin.

3. Thioredoxin and its Related Molecules Thioredoxin was first discovered in 1964 by Peter Reichard et al. in Sweden as a co-enzyme of proton-donor from NADPH to ribonucleotide reductase. 26 Later it has been studied intensively by Arne Holmgren et al. Thioredoxin is a small protein having oxidoreductase activity via its redox-active disulfide/dithiol site within the conserved active sequence, -Cys-Gly-Pro-Cys-.14 Reduced thioredoxin can reduce protein disulfide bonds and oxidized thioredoxin is reduced by NADPH and thioredoxin reductase cascade (Fig. 1). Thioredoxin appears to be present in essentially all living cells including prokaryotes as well as plant.14 It is considered as a more primitive redox regulating molecules than GSH, because it exists in the life lacking GSH. We identified an active cytokine-like principle named adult T-cell leukemia (ATL)-derived factor (ADF) from HTLV-I positive cell line ATL-2. After purification of ADF and cloning of the cDNA, ADF was found to be a human homologue of thioredoxin.27 Several cytokine-like factors proved to be identical or closely related to thioredoxin, indicating that thioredoxin has multiple functions in extracellular as well as intracellular, environment.20 We will mention about them later in the "Extracellular function of thioredoxin". Thioredoxin reductase has a selenium-containing active center in the C-terminals and there exist several isoforms of thioredoxin reductase. 28 In the past years, new members of thioredoxin-related molecules in the mammalian system have been identified. They share the similar active sites: -Cys-X-Y-Cys- and they are called thioredoxin superfamily. Table 1 summarizes the members of human thioredoxin superfamily. Glutaredoxin (GRX) was discovered as another proton-donor for ribonucleotide reductase in the Escherichia coli lacking thioredoxin 14 GRX

Table 1. TRX superfamily.

Thioredoxin Thioredoxin 2 TRX related protein (TRP32) Glutaredoxin (GRX) Nucleoredoxin Protein disulfide isomerase (PDI) Ca binding protein-1 (CaBPl) Ca binding protein-2 (ERp72) Phospholipase C E{

kDa

Localization

Active Site Sequence

12 12 32 12 48 55 49 72 61

Cytosol Mitochondria Cytosol Cytosol Nucleus Endoplasmic reticulum Endoplasmic reticulum Endoplasmic reticulum Endoplasmic reticulum

-Cys-Gly-Pro-Cys-Cys-Gly-Pro-Cys-Cys-Gly-Pro-Cys-Cys-Gly-Tyr-Cys-Cys-Gly-Pro-Cys-[Cys-Gly-His-Cys] -[Cys-Gly-His-Cys] -[Cys-Gly-His-Cys] -[Cys-Gly-His-Cys] '

2232-

^ i3 |L o HTO* ^ B" g55 |. §• 3' S' 3" #

a* a* 3"

3" . M™?

CNJMS^T)

* PEBP2 PEBP2 / P53 I //-►GeneExpressioi T » / • differentiation ' • growth

Fig. 2. Intracellular a n d extracellular activities of thioredoxin.

known as thioltransferase, has GSH-disulfide oxidoreductase activity with redox-active site, -Cys-Pro-Tyr-Cys-.29 GRX reduces low molecular weight disulfides and proteins in concert with NADPH and GSH reductase. There is accumulating evidence that GRX as well as thioredoxin plays an important role in redox regulation of signal transduction. Grx regulates the activation of transcription factors such as nuclear factor I,30 OxyR31 and PEBP-2.32 We detected differential expressions of GRX and thioredoxin in the differentiation of macrophage 33 and mouse embryos. It is also reported that GRX is detected within the HIV-1 virus and regulates the activity of glutathionylated HIV-1 protease. 34 Mammalian thioredoxin 2 (Trx2) has high homology with thioredoxin and has an active site Cys-Gly-Pro-Cys with thiol-reducing activity.35 It has mitochondrial insertion signal and is specifically localized in mito chondria. Recent studies have shown that mitchondorial thioredoxin reductase, Trx2 and mitchondorial peroxiredoxin III compose one of the mitochondorial antioxidant system as well as manganese-superoxide dismutase (Mn SOD) and mitchondorial GSH/GPx system.36,37 To studys the function of Trx2, we cloned the chicken Trx2 cDNA and generated conditional Trx2 deficient cells expressing a tetracycline-repressible Trx2

The Role of Thioredoxin in Regulatory Cellular Functions 121

Table 2. Peroxiredoxin family in human, (modified from the Refs. 38 and 85).

Prxl : PrxII : Prx III : Prx IV : PrxV : Prx VI :

Name

Length in Amino Acids

Localization

PAG, NKEFA TSA, NKEFB AOP-1 AOE372, TRANK AOEB166 ORF6

199 198 256 271 214 224

Cystosol and nucleus Cytosol Mitochondria Cytosol / secreted Mitochondoria / microsome Cytosol

transgene, using a DT 40 cell line.86 The growth of Trx2 deficient cells was significantly retarded and most of Trx2 deficient cells fell into apoptosis. And intracellular ROS levels increased in Trx2 deficient cells. Thioredoxin 2 deficient cells were more sensitive to exogenous hydrogen peroxide and GSH depletion. Moreover, cytochrome c was released into the cyto plasm and caspase-9 was activated in Trx2 deficient cells. These results indicate that Trx2 not only regulates the generation of ROS through Trx2/ peroxiredoxin system in mitchondoria but also plays a crucial role in the mitchondorial apoptotic signal pathway. However, the biological func tions of these members have not yet been fully clarified. Peroxiredoxins are considered to be members of a new family for intracellular hydrogen peroxidase 38 (Table 2). Six members of peroxire doxin family have been identified in human, all of which utilizes thiore doxin as the electron donor except peroxiredoxin VI. The features and functions of peroxiredoxin family were well described in a recent review elsewhere. 39 Thus, the thioredoxin system is composed of several related molecules forming a network of recognition and interaction through its active site cysteine residues.

4. Thioredoxin Knock Out and Transgenic Mice To analyze the biological functions of thioredoxin, we developed thiore doxin knock out mice. Taketo et al. characterized the mouse genome, which contain one active thioredoxin gene on chromosome 1 and one processed pseudogene on chromosome 4.40 The thioredoxin gene extends over 12 kb and contains five exons separated by four introns. 41 To develop thioredoxin knock out mice, a part of the mouse thioredoxin gene including


Fig. 3. Thioredoxin overexpressing transgenic mice (modified from Ref. 8) (a) Design of transgene of thioredoxin. (b) Expression of thioredoxin in various tissues of transgenic mice.

the translation start codon was deleted by homologous recombination in embryonic stem (ES) cells. Heterozygotes are viable, fertile and appear normal. Thioredoxin hetero-knock out mice are now available and under investigation for stress sensitivity. In contrast, homozygous mutants die shortly after implantation at the egg cylinder formation stage.42 One pos sible explanation for the early lethality of thioredoxin homo-knock out embryos is impaired DNA replication after maternal thioredoxin is lost in the embryo. Interestingly, Ref-1 deficient mice also die shortly after implantation at day 5.43 Since Ref-1 and thioredoxin operate coordinately in the redox-sensitive activation of transcription factor such as AP-1 or in the DNA repair/replication, both Ref-1 and thioredoxin may be essential for early embryonic development. Then, we have developed thioredoxin overexpressing transgenic mice (Trx-Tg mice) where human thioredoxin is overexpressed in C57/BL6 strain mice systemically by using beta-actin promoter. 8 Human thiore doxin cDNA was inserted between the beta-actin promoter and the betaactin terminator and used to generate the transgenic mice [Fig. 3(a)]. The apprearance and behavior of Trx-Tg mice are normal. Thioredoxin-Tg mice contain several fold larger amounts of h u m a n thioredoxin protein in the most organs compared with endogenous mouse thiore doxin protein level [Fig. 3(a), 4].

The Role of Thioredoxin in Regulatory Cellular Functions 123

Fig. 4. Expression of human thioredoxin (hTrx) in the brain of transgenic mice. Immunohistochemical study of thioredoxin in Tg mice hTrx was observed in cortex (A, C) and hippocampus (B, D) of Tg mice but not of WT mice (E, F). A hTrx signal was shown in only Tg mice, (modified from Ref. 8.)

5. Characteristics of Trx-Tg Mice 5.1 Resistance Against Focal Cerebral Ischemic Injury8 Focal cerebral ischemia was induced by the occlusion of the middle cerebral artery using the intraluminal filament technique in mature male mice under general anesthesia. Twenty four hours later, the animal was sacrificed and the brain section was analyzed. The infarcted areas and volume in Trx-Tg mice were significantly smaller than in wild type C57BL/6 mice (Fig. 5). Since oxidative modification of proteins is accompanied by the gener ation of protein carbonyl derivatives, the protein carbonyl contents of the soluble fraction of crude brain cortical extract preparations were analyzed at 24 hrs after ischemia. The protein carbonyl contents in Trx-Tg mice were significantly less than in wild type mice.

5.2 Resistance Against Excitotoxic Hippocampal Injury9 Thioredoxin-Tg mice also showed a resistance against kainic acid-induced excitotoxicity, in which the oxidative stress is involved. Mice were injected intraperitoneally with 20 m g / k g kainic acid. The mice were observed for seizure incidence for 1 hr after the injection. Although seven of ten Trx-Tg

124


(mm3) 120 100 "

n=each9, *p► ►

protein-S-SR protein-S' protein-S* protein-SOH

+ + + +

RS" ROO" RH/HOH H20

protective reaction after (2) or (3) leading to S-thiolation: glutathione-SH ► [protein-S-S*" - glutathione] >~ protein-S-S-glutathione + 02'+ H+ (5) protective reaction after (4) leading to S-thiolation: glutathione-SH > - protein-S-S-glutathione + H 2 0

protein-SOH

+

protein-S'

oxidative reaction after (2) (3) or (4) leading to damage: . (0 2 ) ► protein-SOOH/protein S 0 3 H

+

(1) (2) (3) (4)

(6)

(7)

However, thiol disulfide exchange is a rather slow reaction that requires high GSSG concentrations that seem unlikely in intact cells.4 In addition, experiments such as those with neutrophils and hepatocytes cited above have shown that protein S-thiolation can occur without significant increases in GSSG. Thus, a glutathione-dependent trapping mechanism in which oxi dized protein sulfhydryls are generated as either a thiyl radical or sulfenic acid [Reactions (2) - (4)] may be a primary mechanism for formation of S-thiolated proteins. These activated protein intermediates react with the pool of cellular glutathione to produce a mixed disulfide adduct of protein and glutathione [Reactions (5) and (6)]. Such a mechanism depends on a substantial supply of reduced glutathione to effectively trap partially oxidized protein cysteines (thiyl radical or sulfenic acid forms). This pro posed mechanism also leads to the hypothesis that in the absence of suffi cient glutathione, partially oxidized forms of protein cysteine may react with oxygen or other oxidants to produce extensively oxidized species such as sulfinic and sulfonic acids [Reaction (7)]. The effectiveness of glutathione in this role may be compromised by some oxidative event that depletes the glutathione pool significantly. This suggestion accounts for the importance of a large cellular pool of glutathione and provides a model for protein sulfhydryl damage that is related to the concentration of cellular glu tathione. In general, researchers have assumed that GSH is more reactive towards oxidants than proteins, while in fact, the opposite seems to be true.12 The model for protein S-thiolation has been extensively studied with pure proteins.12"14 Superoxide, H 2 0 2 , and peroxynitrite 15 have been shown

146


to directly cause S-thiolation in protein model systems and in cells. Secondary reactions of these molecules result in formation of various radicals (peroxyl, alkyl, thiyl and others), some of which can be shown to cause protein S-thiolation in protein model systems (unpublished results).

2.2.2.

S-nitrosylation

The protein oxidative effects of nitrogen-based reactive species are not as well understood, but analogies to S-thiolation suggest that the molecular mechanisms may have much in common. Attempts to understand both forms of protein modification draw heavily on the chemistry of low molecular weight thiols. This rationale assumes that the complex chemistry of protein thiols with all the potential modifications that result from the protein environment, and that of low molecular weight thiols, is similar. This model has not always provided explanations for observations that include the fact that nitroso glutathione may S-thiolate some cysteines while S-nitrosylating others, 1617 the fact that some cysteines do not form S-glutathiolated species as a consequence of charge interactions, 18 and the fact that protein S-nitrosylation apparently occurs in the presence of a very large excess of glutathione in intact cells.19 First, the mechanism of formation of protein-NO adducts may involve several fundamentally different mechanisms reminiscent of those proposed for protein S-thiolation (see reactions below). If cellular responses to nitrosative stress are similar to responses for oxidative stress, protein S-nitrosothiols will occur to an extent greater than or equal to S-nitrosylation of the glutathione pool. It has been suggested that transnitrosation of proteins from low molecular weight S-nitrosothiols is a feasible mecha nism for protein S-nitrosylation [Reaction (8)]. This reaction is quite remi niscent of thiol/disulfide exchange and probably is significant only when high concentrations of S-nitrosoglutathione are manifested in cells. Transnitrosation could therefore be significant in causing S-nitrosylation of intracellular proteins if scavenging of NO occurs to a significant extent in the extracellular space. Data indicate that low levels of S-nitrosothiols occur in vivo (approximately 10 uM in plasma, 23 less than 100 pmol/mg of cellular protein in NIH-3T3 cells19). However, in order to cause significant modification of S-nitrosothiols in cells, external concen trations of S-nitrosothiols such as S-nitrosocysteine or S-nitroso glutathione must be in millimolar concentrations or greater.

Monothiol Modification in Redox Regulation + + + + + + NA + Protein-SH Protein-S-N"-Or][ +

Protein-SH or Protein-SH Protein-SH Protein-S* 4NO"

(Protein-SH Protein-SH

+ +

RSNO -M RSNO < ONOO" NO"

°2

► ► >. >-

►

Protein-SH NO-

—^~ ^=*~

o2

—►

NO" RSNO

+

electron

Protein-SNO Protein-SSR Protein-S" Protein-SNO 2N203 HNO : Protein-S-N"-OH Protein-SNO

+ +

+ + +

RSH NO"

Protein-SNO

o- 2 -

147 (8)

+

H* (9) (Ref 20) (10) (Ref 21) (11) (Ref 22)

- ► Protein-SNO + reduced acceptor) acceptor + NO". -► Protein-SSR

Thus, in order for S-nitrosylation to occur in vivo, S-nitrosothiol concen trations must be at least 100-fold greater than at resting levels. Thus, transnitrosylation for the formation of S-nitrosylated proteins and thioldisulfide exchange for the formation of S-thiolated proteins may suffer from the same limitation, i.e. the concentration of available low molecular weight reactants seems too low to account for significant protein sulfhydryl modification. However, there are two important differences between S-nitrosylation and S-thiolation systems. First, GSSG may S-thiolate sites relatively slowly because of its net negative charge (-2) and steric interference by the bulk of a glutathione molecule regardless which sulfur is attacked by the thiolate nucleophile. Thus, direct oxidation of proteins [Reactions (2) - (6)] should have a greater contribution to S-thiolation than would thiol-disulfide exchange. However, GSNO has a less negative charge (-1) without the bulk of the glutathione moity to interfere with a nucleophilic attack on the nitrogen of the N O [Reaction (8)]. The orientation of the S-nitrosothiol during the nucleophilic attack (i.e. attack on the nitrogen of the N O or the sulfur atom of the glutathione) may be influenced by the charge characteristics of the site in question. 1617 Positively charged residues in close proximity to the thiolate may be able to orient the glutathione mol ecule in some cases so that the nucleophilic attack is on the sulfur atom and an S-glutathiolated protein is produced [Reaction (8)] as is the case for creatine kinase. Alternatively, GSH may associate with some protein sites and an S-glutathiolated end product may result via an S-nitrosylated protein intermediate. Thus charge and steric factors may account for the reported occurrence of both S-thiolation and S-nitrosylation of proteins in cells treated with exogenous S-nitrosocysteine18 and may ultimately deter mine the relative contribution of transnitrosylation versus radical medi ated mechanisms for S-nitrosylation of protein thiols. Secondly, GSSG is normally not able to reach very high concentrations in cells because of the efficacy of the glutathione disulfide reductase


enzymatic system and because any disulfides produced in the extracellular space do not readily cross the cellular membrane. In contrast, S-nitrosothiols are able to cross cellular membranes, apparently by transport processes that have not been completely elucidated.19,24 Thus, cells exhibit selectivity for uptake of low molecular weight S-nitrosothiols and S-nitrosocysteine is more readily taken up than S-nitrosoglutathione.19,25 The uptake of low molecular weight S-nitrosothiols may be cell-type specific. Protein disulfide isomerase (PDI), which exhibits differential expression with cell type, has also been implicating in transporting NO into cells via trans-nitrosation.24 The inability of cells to take up S-nitrosothiols may be a mechanism by which the nitrosative effects are blunted in the cells proximal to immune cell activation. To date, no quantitative measure of the nitrosative stress that results from the oxidative burst has been made, although relative lev els have been assessed in cell culture conditions.26 Measurements of intracellular S-nitrosothiols have not been made in cells proximal to immune cell activation. Thus, even if S-nitrosothiols are scavenged in the extracellular space, there is the potential for their entering nearby cells. It is possible that concentrations of GSNO do not have to be as high for S-nitrosylation and S-thiolation to occur as GSSG concentrations would need to be for signifi cant S-thiolation to occur. However, transnitrosation is most likely to be a mechanism for redistribution of S-nitrosothiols among protein thiols and for denitrosation of protein S-nitrosothiols. A second mechanism similar to that proposed for S-thiolation may require formation of either a reactive protein intermediate [thiyl radical— Reactions (2), (3) or (9)] or some reactive low molecular weight species other than S-nitrosothiol [Reaction (9) and (10)]. Because nitrosative stress occurs in an environment of oxidative stress during immune cell activation, and may itself cause oxidative events [Reaction (11)], mechanisms of this type may ultimately play a very large role in modification of protein thiols.

2.2.3. Interaction of S-nitrosylation Irreversible Oxidation

and S-thiolation

or

The potential for interchange between the S-nitrosylated state and the disulfide state of the cysteine is significant. S-thiolated proteins and low molecular weight disulfides form rapidly inside cells that are treated with extracellular S-nitrosothiols.19 Disulfide formation may be a physiologi cally important step in the degradation of S-nitrosothiols, since disulfides are a measured end product of nitrosothiol degradation 27 and free thiols seem to be requisite in S-nitrosothiol degradation in vivo.28 A recent report

Monothiol Modification in Redox Regulation

149

has suggested that glutathione disulfide S-oxide, also a degradation pro-duct of GSNO, is a much more effective thiolating agent than GSSG,29 but the study does not investigate the formation of a mixed proteinglutathione S-oxide, which may form just as readily in mixtures of proteins, GSH and GSNO. Reaction (8) is a simplification of what has proven to be a complex reaction path, 30 but in vivo studies bear out the overall stoichiometry remarkably well. 19 In purified protein systems, GSNO S-nitrosylates all four available cysteines on H-ras with only minimal S-glutathiolation,18 while it has recently been shown that S-nitrosothiols can selectively S-glutathiolate one cysteine on creatine kinase without formation of the protein S-nitrosothiol.17 It has been suggested that S-nitrosothiols are converted to irreversible oxidation products of thiols although scant physical evidence exists of these reactions. This lack of evidence may, however, be due to the lack of methods for measuring these modifications easily (see Sec. 2 of this review). The oxidized product of Reaction (11) can react with N O to form the protein sulfenic acid and nitrous oxide:27 Protein-SN'OH

+

NO

►

Protein-SOH

+

N20.

(12)

Since protein sulfenic acids may be intermediate to protein S-thiolation, combining rapidly with intracellular GSH [Reaction (6)], this reaction could be responsible for protein denitrosylation in vivo. Additionally, oxidative events apparently convert S-nitrosylated cysteine to sulfinic or sulfonic acid,30,31 although the reaction path for this is not well understood. Protein-SNO

+

electron acceptor

► Protein-Sox

(x = 2,3).

(13)

Studies have shown complex reaction pathways in which NO and a reduced thiol can be oxidized to an S-nitrosothiol. Because NO can cross cellular membranes, it remains uncertain whether NO generated extracellularly reacts with thiols in the extracellular space or whether S-nitrosothiols form largely inside the cell. If S-nitrosothiol formation occurs mainly in the extracellular space, then one would expect that much of the S-nitrosothiol would not be able to enter cells, particularly the protein S-nitrosothiols such serum albumen, reportedly the most abundant S-nitrosothiol in plasma. 32 Cells also exhibit selectivity for uptake of low molecular weight S-nitrosothiols. Data indicate that S-nitrosocysteine is more readily taken up than S-nitrosoglutathione. 1925 Because amino acid transporters may be involved in this transport, the uptake of low mole cular weight S-nitrosothiols may be cell-type specific. Protein disulfide isomerase (PDI), which exhibits differential expression with cell type, has

150


also been implicating in transporting N O into cells via transnitrosation. 24 The inability of cells to take up S-nitrosothiols may be a mechanism by which the nitrosative effects are blunted in the cells proximal to immune cell activation. To date, no quantitative measure of the nitrosative stress that results from the oxidative burst has been made, although relative levels have been assessed in cell culture conditions 33 and measurements of intracellular S-nitrosothiols have not been made in cells proximal to immune cell activation. While there are many studies showing S-nitrosylation of proteins in vitro, few studies show S-nitrosylation of specific proteins isolated from cellular systems. The cardiac calcium release channel was found to be S-nitrosylated on one thiol per subunit (each subunit contains 21 cysteine residues) in isolated canine hearts.34 Methionine adenosyltransferase was significantly S-nitrosylated in rat hepatocytes treated with S-nitrosoglutathione monoethyl ester, resulting in enzyme inhibition. 35 Caspase-3, which has a reactive cysteine residue required for activity, was immunoprecipitated from three different human B- and T-cell lines and found to be S-nitrosylated constitutively 36 Fas stimulation decreased caspase-3 S-nitrosylation. Most recently, H-ras found to be S-nitrosylated in immunoprecipitates from NIH-3T3 cells treated with S-nitrosocysteine.18 Since the hypothetical control of metabolic and signaling pathways depends upon modification of thiol groups, these studies represent a start to research that will undoubtedly increase significantly in the future.

1.3 Molecular Mechanism of Dethiolation and Denitrosylation Since both of these mild oxidative events are reversible in intact cells, the reversal reactions may also contribute significantly to the importance of each modification. Both glutaredoxin and thioredoxin proteins are poten tially involved as reductants in dethiolation. 37-39 Other chapters in this book provide details on the cellular roles of these proteins, but it should be emphasized that S-glutathiolated proteins are probably uniquely sensitive to glutaredoxin. This aspect of glutaredoxin's action was recently highlighted when the structure of S-glutathiolated carbonic anhydrase III became available. Subsequent modeling studies with both glutaredoxin and thioredoxin showed that glutaredoxin could form a productive com plex with the S-glutathiolated protein without significant protein-protein interactions between the glutaredoxin and carbonic anhydrase III. The figure shows the complex that can be formed between these two proteins


151

Fig. 1. A molecular model of the complex between S-glutathiolated carbonic anhydrase III and glutaredoxin. This model was developed from the published struc tures of carbonic anhydrase III (1FLJ) and glutaredoxin (1GRX). The bond angles for the three sulfur intermediate between these two molecules were optimized.

in which the carbonic anhydrase Ill-bound glutathione acts as a docking site for the glutaredoxin molecule. A productive 3-sulfur complex (left figure) is easily demonstrated with this model. On the other hand, thioredoxin could not be modeled into a complex without considerable overlap between the two proteins. It seems likely that the action of thioredoxin would have less specificity and require higher concentrations since it did not easily form a reductive complex. Experiments in two laboratories have supported this probability. Denitrosation of proteins has not been studied as thoroughly and relevant information must again be obtained from experiments with low molecular weight S-nitrosothiols. Recent experiments in intact cells showed that denitrosation of intracellular proteins occurs at nearly the same rate as dethiolation of glutathiolated proteins. 19 It is interesting that the removal of high molecular weight thiols, but not low molecular weight thiols, significantly diminished the ability of plasma to degrade GSNO.32 Similar studies have demonstrated the critical role of thiols in nitrosothiol degradation as well as a minor role for divalent cations.28 To date evidence is inconclusive with resect to an enzymatic system for

152


Table 1. Methods used to study protein cysteine oxidative modification Method

Detection Limit Advantages

Disadvantages

Gel Electrofocusing

Micrograms of protein

Techniques for method require some practice. Issues unresolved for detecting proteins by immunoblot.

Capture/Release

Micrograms of protein (pmol of adduct)

Radioactive Detection


MAL-PEG

Nanograms of protein

MAL-Biocytin

Nanograms of protein

Readily Quantitated Adduct can be identified. Multiple Samples per determination. A single method can be used for S-nitrostylation, S-thiolation, and irreversible oxidation. Adduct can be identified. Quantitation possible. Individual assays available for S-nitrosylation and S-thiolation. Identification of modified protein.

Very sensitive. Quantitaion is possible. Multiple samples per determination. Differentiates between reversible and irreversible oxidation. Very sensitive. Multiple samples per determination. Can differentiate between reversible and irreversible oxidation.

Extensive sample preparation protocol. No method for irreversible oxidation. Assay is specific. Protein synthesis must be inhibited. Adduct identity not possible. Not method for S-nitrosylation or irreversible oxidation. Cannot differ entiate between possible reductionsensitive adducts.

Quantitation is questionable. cannot differentiate between possible reductionsensitive adducts.


153

Table 1. Continued Method

Detection Limit Advantages

Sulfinic / Sulfonic Acid Analysis


Protein Activity/Function

Below nanograms of protein

Disadvantages

Unambiguous Extensive sample identification of preparation. protein modification. Multiple samples increase labor. Very sensitive Cannot identify methods. protein Activity correlates modification. with cellular Each protein function. requires specific methodology.

denitrosylation of protein or low molecular weight S-nitrosothiols, but degradation of S-nitrosothiols may be linked mechanistically to the reduc tion of protein and low molecular weight thiols (see above and Ref. 40).

1.4 Effects of Oxidative Modification on Protein Function The importance of post-translational covalent modification has a long history that places it in a central position with regard to cellular regulation. Most often the concepts that evolved to understand allosteric regulation have been incorporated into our understanding of the mechanisms for these protein effects. Thus, effects of protein phosphorylation can clearly be understood in terms of a two or more state protein model in which modification either favors some protein conformation or where it occurs only on a particular conformation of the affected protein. The structural elucidation of the glycogen phosphorylase model demonstrated that phosphorylation may cause very specific protein conformation changes that result in dramatic changes in protein function. To date, there is no similar model or concept to explain the importance of oxidative modifi cation in metabolic regulation. It seems clear that exposed protein sulfhydryls can be modified almost randomly, lacking the specificity that seems inherent in the phosphorylation system. In addition oxidative events such as S-glutathiolation, S-nitrosylation, or irreversible oxidation have generally been shown to have similar effects on the modified protein. Each of these modifications produces distinct chemistry where the addition product is negatively charged (S-glutathiolation and irre versible oxidation), or even uncharged (S-nitrosylation). The specific

154


Table 2. Model for oxidation of H-ras Cysteine Modified 118 181,184 186

Type of Modification

Effect of Modification

S-nitrosylation S-nitrosylation, S-glutathiolation S-nitrosylation, S-glutathiolation

Activation of GTP turnover Inhibition of palmitoylation, Inactivation Inhibition of farnesylation, Inactivation

protein changes generated by these modifications have been documented only for carbonic anhydrase III. In that case the S-glutathiolation seems to have no effect on the protein, and the attached glutathione molecules do not perturb the protein structure in the least. The generation of structural information on other proteins is one of the fundamental needs that must be undertaken in the future in order to support a regulatory role for protein sulfhydryl modification.

2. Analytical Methods for Protein Oxidation The single most important limitation to understanding the biology of protein cysteine oxidative modification is the availability of sensitive methods for each specific protein modification. The following is a brief overview of methods which have been used successfully to date, empha sizing sensitivity, selectivity, and applicability to experiments with intact cells and tissues.

2.1 Protein Separation by Charge 2.1.1 Gel Isoelectric Focusing The most versatile method for the study of oxidation of protein cysteines is gel isoelectric focusing (IEF). Proteins differing by as little as one charge can be separated by IEF, providing both quantitative and qualitative information about protein oxidation.12'13,30 Detection of IEF-separated pro teins is similar to SDS-PAGE (i.e. Coomassie blue staining), and analysis of 1 (Xg or less are routine. Coomassie blue stained band densities are proportional to protein content over approximately 5-fold range, and multiple dilutions of samples are beneficial. Qualitative information can


155

include the number of sites modified, which sites are modified, and the type of oxidative modification, i.e. S-glutathiolation, irreversible oxidation, or S-nitrosylation. While IEF is most readily implemented on proteins with only one or two reactive cysteines, when used in combination with point mutants, proteins with as many as four sites can be studied effec tively. The method depends on appropriate chemical modification of protein samples including reducing agents such as dithiothreitol (DTT) to identify irreversible modifications such as sulfinic or sulfonic acids, and alkylating agents such as iodoacetamide (uncharged) and iodoacetic acid (single negative charge). Thus, uncharged adducts (S-nitroso or S-cysteine groups) can be detected by alkylation with a charged agent, and charged adducts (S-glutathionyl groups) require an uncharged agent. Multiple samples can be separated on a single IEF gel, routinely up to twenty samples can be applied per gel. Native gel IEF is commonly used, but urea-based denaturing IEF gels have been of some use as well. Native IEF has been useful for some cytosolic proteins, however IEF has pro duced mixed results when separations of membrane-associated proteins or protein sub-domain constructs were attempted. An advantage which may seem at first to be a disadvantage is that proteins which appear pure by other methods such as SDS-PAGE separate as multiple bands with IEF. Even when other methods will be used to assay protein oxidation, IEF is an useful tool for determining the purity of the protein substrate. IEF has been used to study a single protein in cellular extracts in combi nation with Western blotting techniques. The sensitivity for Western blotting appears to be much less than for SDS-page Western blotting. The decreased sensitivity results from unidentified interference with antibody binding to the transferred protein. Theoretically, this methodology will detect picogram quantitities of proteins in complex mixtures, making IEF-Western blotting potentially very powerful if the technical issues are resolved.

2.1.2 Capture/Release - Measurement of Adducts from Isolated Proteins

Released

Glutathione may be released from protein with DTT and the glutathione may be measured by various methods such as HPLC, mass spectrometry, scintillation counting (35S-glutathione), or a combination of these. There are several attractive features shared by these methods regardless of the ultimate means of detection. Among the advantages is the identifica tion of the adduct by either molecular weight, comigration with standard

156


compounds in HPLC separations, or by incorporation of radioactive label. Quantitation is achieved by normalizing the results of the assay for protein content, which is trivial for pure proteins, but does introduce significant error into the assay. When measuring modification of proteins from cellular extracts, protein content may be normalized against known amounts of pure protein separated by SDS-PAGE and detected by Western blotting. In both purified and mixed protein systems, the protein must be separated from any low molecular weight thiols in the reaction mix ture. This may be accomplished by dialysis, precipitation with trichloroacetic acid (TCA), or by immunoprecipitation (IP). Great care must be taken to ensure that all non-covalently bound thiols are removed from the protein (proteins may have to be dialyzed up to five days to remove non-covalently bound thiols in cellular extracts) before release of covalently bound thiols with DTT. Detection limits for HPLC methodology vary, but, typically, picomoles of GSH can be detected. If cells are radioactively labeled with 35S methionine/cysteine mixture, the maxi mum specific activity which can be expected is ~250 cpm/pmol placing the lower limit of detection at the picomole level. Thus, if one expects 1-10% (mole/mole) modification of the protein, ~1 ug of protein is required to make an accurate determination of S-glutathiolated protein. While mass spectrometry is not quantitative, approximately 1 picomole of compound is needed per assay, making detection limits similar to HPLC and radiolabeling methodologies. S-nitrosylation can be detected by releasing the adduct from the protein. If Hg 2+ is added to the S-nitrosothiol under acidic conditions, nitrous acid (nitrite at higher pH) is formed. Nitrite is readily quantified by a variety of assays including the Greiss reagent assay for which a con servative detection limit is on the order of 100 picomoles.41 Fluorometric assay systems such as the diamino-naphthalene (DAN) assay reliably detect as little as 10 picomoles of nitrite. 42 Measurement of photolysisreleased NO using chemiluminescence provide quantitation of as little as 1 picomole of NO, 36 placing the sensitivity for this methodology around the same level as that for S-glutathiolation. Again, great care must be taken to ensure that proteins are separated from any low molecular weight contaminants which may interfere with the assay. These methodologies may also be applied to protein mixtures (e.g. total cytosolic proteins) and in this case sensitivity is not an issue. Because a wide variety of proteins may be immunoprecipitated from cells routinely, these methodologies are suited for most proteins.


2.1.3 Radioactive

157

Methods

Cells can be incubated with 35S methionine/cysteine to label the cellular glutathione pools and proteins from these cells may be separated by SDS-PAGE and the dried gel may be exposed to autoradiography film.10 This method is advantageous in that it provides a direct link between the radiolabeled adduct and the protein in question and so may be used in conjunc tion with the release and detect methodology described above to help confirm the results. A disadvantage of the technique is that cycloheximide must be used in order to prevent incorporation of 35S-cysteine into proteins. Incubation with the labeling medium must be short to minimize alterations in cellular metabolism during labeling. This short incubation time prevents complete incorporation of label into the cellular glutathione pool, resulting a cellular glutathione specific activity which is about 1/10 that of labeling cysteine. For rapid turnover proteins, the need for cycloheximide may be critical. As a general rule, to be able to detect S-thiolation of a given protein, there should be ~1 ug of protein per lane on an SDS-page. Thus, this tech nique is about as sensitive as the DTT-release methodologies above. Although glutathione accounts for >90% of all protein bound thiols, the iden tity of the protein adduct cannot be ascertained directly. Theoretically, it should be possible to quantify the amount of adduct if one is able to normalize for amount of protein as described above, provide a radioactive standard for use with the gel, and determine the specific activity of the glutathione pool. However, in practice, this methodology remains qualitative.

2.1.4 Maleimide-Derivatized

Polyethylene

Glycol

(Mal-PEG)

Detection of oxidatively modified cysteines on proteins. Cellular proteins with reactive cysteines are substrates for reaction by with maleimidederivatized polyethylene glycol (Mal-PEG).43 The resulting protein adducts have an increased size that is easily detected by SDS-PAGE. In conjunction with Western blotting, the detection limits for this method are considerable better than those already described. For each reactive sulfhydryl that reacts with Mal-PEG, the apparent molecular weight of the protein increases by the size of the PEG adduct. Thus, multiple sites produce several bands of increased size. When a specific reactive cysteine is oxidized, the reaction with Mal-PEG is blocked. If all reactive cysteines are blocked, Mal-peg has no affect on the molecular size of the protein.


This method can be used to detect either reversible oxidative/ nitrosative modification of a protein (modifications that are readily reversed by addition of dithiothreitol such as S-thiolation, S-nitrosylation, or protein disulfide formation), or irreversible modification of a protein (the oxidative modification is not affected by the addition of di-thiothreitol). This technique provides two distinct advantages over other cellular protein methods. First, the sensitivity is equivalent to that of SDSPAGE/Western blotting, i.e. protein amounts can be easily detected in the low nanogram range. Thus this technique is about 1000 times more sensi tive than any of the other techniques. Second, the number of adducts per mole of protein can be quantified using densitometry. Thus, no external standards are needed. Using DTT to remove all reducible sulfhydryl modifications, one is left with irreversibly oxidized sulfhydryls that will not react with Mal-PEG. The primary disadvantage of this technique is that there is no way to determine the identity of any oxidative adduct. While it is useful to determine the exact nature of modification in cells, the ability to monitor the kinetics of modification of the protein in concert with activation and inactivation in situ makes this technique extremely valuable for future research.

2.1.5 Maleimide-derivatized Biocytin (MAL-biocytin) Fluorescent Detection of Oxidatively Modified Cysteines on Proteins The use of biocytin-conjugated maleimide (MAL-Biocytin) was first introduced some years ago,44 this technology has recently been adapted for use in determining surface exposed loops in membrane proteins. 45 Like the MAL-PEG assay, the sensitivity is essentially the same as SDSPAGE/Western blot. Modification can be quantified as a percentage of total thiols for each protein, but determining the stoichiometry of reac tive thiols is not trivial. This technology could also be used to measure total protein thiols in an ELISA-type of assay using an automated plate reader.

2.2.6 Irreversible

Oxidation

Most recently, our laboratory has developed a robust method for deter mination of both sulfinic and sulfonic acid in purified proteins. 46 The


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method requires protection of reduced sulfhydryls and subsequent acid hydrolysis of the protein and separation by reverse phase HPLC. Treatment of replicate samples with sodium hypochlorite before hydrolysis oxidizes any protein sulfinic acid to a sulfonic acid. The method utilizes other amino acids in the protein to normalize the extent of modification for any protein with a known amino acid sequence. The detection limit for this assay is in the low picomolar range, typically requiring between 5 and 10 |J.g of protein. Thus, the sensitivity is within an order of magnitude of the detection of S-nitrosylated or S-glutathiolated species.

2.1.7 Protein Activity

and Binding

Assay

Because thiols are critical for the activity of some proteins, protein activity may be used to indirectly measure oxidative damage to these proteins. However, kinetic analysis of reversible modification of sites on these proteins is complicated, since stopping oxidation/reduction of reactive sites requires either addition of reducing agents or alkylating agents. With the careful use of controls it should be possible to measure recovery of activity in previously alkylated samples upon reduction with DTT of reversibly oxidized proteins and loss of activity due to irreversible oxida tion by comparison of DTT treated samples compared with activities of untreated samples. However, great care must be taken to insure that reac tions at secondary sites have no effect on protein activity. Obviously, correlation of oxidative events in cells with protein activity is of great value, but characterization of modification by some of the other method should be carried out as well.

3. Oxidative Modification of Reactive Cysteines in Selected Proteins We will divide the following discussion into abundant proteins and those of less abundance. In general the data available for abundant proteins provides a basis for understanding potential changes in less abundant proteins, but it seems clear that the most important regulatory effects of oxidative modification may occur on the less abundant proteins involved in signal-transduction and gene regulation.

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3.1 Abundant Proteins Much of the basic information about protein sulfhydryl oxidation has been derived from proteins that are found at relatively high concentration in vivoF^ For some proteins, the only data about oxidative modification has been obtained by assessment of either enzyme or binding activity. These types of experiments have relied heavily on establishing that the oxidative modification of interest affected the purified protein in a manner consistent with activity or binding changes that occur in intact cells. On the other hand, several studies have examined specific molecular changes in pro teins associated with oxidative or nitrosative stress of intact cells. The combination of these two types of experiments, i.e. assessment of mole cular modification of a specific protein in vivo correlated with changes in the activity of that protein, have not often been achieved.

3.1.1 Glyceraldehyde 3-P Dehydrogenase GAPDH has a reactive cysteine that is directly involved in the catalytic mechanism of the protein since it forms a covalent intermediate with the substrate during catalysis. Any oxidative modification of this cysteine produces a completely inactive enzyme. Since it is an abundant protein in many cells, it has been the subject of many reports describing oxidative modification during both oxidative and nitrosative stress. In cultured monocytes, the extent of S-glutathiolation correlated with the initiation of the oxidative burst, while dethiolation correlated with the cessation of the oxidative burst. 11 S-glutathiolation of the protein has also been observed in endothelial cells,50"52 and it was suggested that the protein might be oxidized to the S-glutathiolated form even in NO-treated cells. The propensity of this protein to form S-glutathiolated species may be related to the acidic nature of the reactive cysteine. In experiments with the yeast, two different isoforms of GAPDH were studied. Surprisingly, only one of these isoforms was regulated by S-thiolation during oxidative stress.53 S-ADP-ribosylation of the protein was reported in NO-treated cells.54,55

3.2.2 Carbonic anhydrase III CAIII is one of many isoforms of carbonic anhydrase and it was discovered that it contained two reactive cysteines several years ago.9 Interestingly, no


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function has yet been determined for these reactive cysteines. Since the protein is expressed at high levels in some cell types, it has been relatively easy to study the oxidative modification of the protein. Recently the struc ture of S-glutathiolated form of this protein was published. 56 The reactive cysteines183186 both reside in areas of negative surface charge density. One cysteine183 is less reactive and has at two conformations, one of which is clearly more buried. The protein has been reported to be S-glutathiolated 9 and possibly even irreversibly oxidized in both cultured cells and whole animals. Recent evidence suggests that its expression is related to oxida tive stress.57iS8 Molecular mechanisms of both S-glutathiolation and dethiolation of CAIII have been studied extensively. It has been suggested that the protein is S-glutathiolated by direct oxidation 1213 and that glutaredoxin is a very efficient catalyst of the dethiolation reaction.14 The kinetics of S-glutathiolation and dethiolation in vivo correlated with the amount and duration of added oxidants. S-glutathiolated carbonic anhydrase has been found in aged rats suggesting that the oxidation state of protein cysteines is altered with aging.59

3.1.3 Creatine Kinase The cytoplasmic form of CK has one reactive cysteine per subunit and although the cysteine is not a part of the catalytic mechanism, it is clearly important for enzyme activity.5 The protein is very abundant in a number of muscle cells and it is available from commercial sources at high purity. It has been used for a number of model studies in which S-thiolation, S-nitrosylation, dethiolation, and irreversible oxidation have been explored. Oxidative modification of the reactive cysteine completely inhibits enzyme activity. It is one of the most acidic protein cysteines and is prob ably completely ionized at neutral pH. Recent publication of the structure of this protein 60 has made it possible to understand oxidation experiments at the molecular level. The reactive cysteine clearly resides directly between an area of surface positive charge density and an area of surface negative charge. The cysteine is important for substrate binding in the active site. Early experiments showed that the protein was inhibited by S-thiolation, and that oxidative inactivation of the enzyme in cardiac cells could be explained by this mechanism. 13 Recently, it has been demon strated that the acidic cysteine in this protein reacts in a unique manner

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with S-nitroso glutathione, forming S-glutathiolated creatine kinase in preference to the S-nitrosylated protein.61 This property may be related to the surface charge properties of creatine kinase, changing the nucleophilic character of the thiolate anion necessary for reaction with appropriate molecules. This protein is easily S-glutathiolated by several reactive oxygen species if a pool of reduced glutathione is present.13'62 When the glu tathione concentration is inadequate, such reactions produce irreversibly oxidized forms of the protein that recently were identified as the sulfinic acid and sulfonic acid species.46 Surprisingly, the sulfinic acid form of the protein was present in abundance. Recent experiments indicate that cellular proteins may contain little sulfonic acid but significant amounts of the sulfinic acid.

3.1.4 Glycogen

Phosphorylase

Glycogen metabolism utilizes several enzymes that contain reactive sulfhydryls, i.e. glycogen synthase, glycogen phosphorylase, protein phosphatases and kinases. Enzymes involved in glycogen metabolism were some of the first in which oxidative mechanisms were thought to represent important regulatory mechanisms. 63 ' 64 Phosphoryase b (dephosphorylated) has been used as a model protein for study of protein sulfhydryl oxidation because commercially available protein of high purity is suitable for definitive studies on the mechanism of both S-thiolation and dethiolation.13 It has two reactive cysteines per subunit and oxidative modification by either S-thiolation or S-nitrosylation of these cysteines does not cause any apparent activity change, although the protein may have less affinity for the glycogen particle in the oxidized state (unpublished observations). The protein is easily studied by gel electrofocusing and by other molecular techniques. 30 It has been used as a substrate protein to study the enzymology of protein dethiolation. 37

3.1.5 Glutathione

S-Transferase

mGST (micorsomal glutathione S-transferase) is a rather unusual form of this enzyme that is closely associated with a number of membranes. It has an unusual trimeric structure and it is the only protein known to be acti vated by formation of an S-glutathiolated form. 65 Although other


163

enzymes including glucose 6-P dehydrogenase, 66 and APS reductase from plants 67 are activated under conditions that might lead to S-glutathiolation, mGST remains the only protein in which activation by S-glutathiolation has been demonstrated by molecular techniques.

3.1.6 Actin Actin is a cytoskeletal protein that has a single reactive cysteine. It has been suggested that S-glutathiolation,10 S-nitrosylation,68 and S-ADPribosylation69 modifications can alter the biological function of the protein. Since actin is found as both a soluble pool of protomers and a polymer ized filament in cells, it has been suggested that oxidative modification of the cysteine may have regulatory effects on the polymerization/ depolymerization process.70 Actin's role in neutrophil function has been of particular interest since these cells produce copious amounts of superoxide anion, nitric oxide, and hypochlorite on stimulation. It has also been suggested that oxidation of actin monomers may lead to the generation of covalently linked dimers or even higher oligomers. 71

3.2.7 Hemoglobin Hemoglobins from several eukaryotes including man, are known to have at least two reactive cysteines per tetramer. 72 The protein can be either S-glutathiolated or S-nitrosylated in vitro, and this reaction has been implicated in both transport of protein-bound NO, 73 and in oxidative regulation of red blood cell function during oxidative stress.74 It has been shown that hemoglobin is probably attack by oxidative mechanisms that result in formation of thiyl radicals that may be trapped by added spin traps. 75 Thus, the abundance of hemoglobin in red blood cells makes it a target for several different kinds of oxidative modification.

3.2 Less Abundant Proteins These proteins are generally present at low concentrations in cells and consequently, less is known about molecular events that lead to their oxidative modification in cells.

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3.2.1 Protein

phosphatases

The effect of oxidative and nitrosative stress on protein phosphorylation may occur by oxidative modification of either protein kinases, protein phos phatases, or the phosphoprotein substrate for this modification.76 Evidence for a direct role of oxidative stress as regulators of both protein kinases and protein phosphatases has been published. 77 Recently, it was reported that protein kinase C-a was easily S-thiolated and inactivated, and subsequent experiments with NIH3T3 cells confirmed that it could be S-thiolated in vivo.™ Additionally, S-glutathiolated protein tyrosine phosphatase IB was demonstrated in A431 cells after hydrogen peroxide treatment.79

3.2.2 NF-kappaB and c-Jun/AP-1 Much research effort has been designed to understand the "redox" regu lation of the NF-kappaB and c-Jun transcription factors. Recent reviews have treated the subject extensively,48,49 pointing to the very strong possi bility that redox regulatory effects are complex. In an important molecular study with c-Jun protein, it was recently reported that S-glutathiolation may be an important aspect of this process. 80 When an NO-generating agent was incubated with the protein in the presence of glutathione, c-Jun became S-glutathiolated. It was suggested that this modification inhibited DNA binding activity of the protein.

3.2.3 p53 The tumor supressor, p53, plays a major role in the transcription ("reading") of DNA, in cell growth and proliferation, and in a number of metabolic processes. Because p53 suppresses abnormal cell proliferation (it acts like an "emergency brake" in the cell cycle), it may represent an important mechanism for protection against cancer. It also appears to be involved in programmed cell death, or apoptosis. When a mutation in the p53 gene results in the substitution of one amino acid for another, p53 loses its abil ity to block abnormal cell growth. Indeed, some mutations produce a p53 molecule that actually stimulates cell division and promotes cancer. Almost 50% of human cancers contain a p53 mutation — including cancers of the breast, cervix, colon, lung, liver, prostate, bladder, and skin — and these cancers are more aggressive, more apt to metastasize, and more often fatal. p53 is a potent transcription factor and once activated, it


165

represses transcription of one set of genes (several of which are involved in stimulating cell growth) while stimulating expression of other genes involved in cell cycle control. It is a phosphoprotein of about 390 amino acids which can be sub divided into four domains: a highly charged acidic region of about 75 to 80 residues, a hydrophobic proline-rich domain (position 80 to 150), a central region (from 150 to about 300), and a highly basic C-terminal region. The sequence of p53 is well conserved in vertebrate species, but there have been no proteins homologous to p53 identified in lower eukaryotic organ isms. p53 is phosphorylated at many sites by casein kinases I and II, JNK1, cdk's, DNA-PK, and these sites reside in the N- and C-terminal domains of the protein. Recently, it has become clear that oxidative mechanisms can also regulate p53 function. The central region of the protein, whose struc ture is shown in the accompanying figure is responsible for DNA-binding activity. It is the only domain of p53 that contains cysteine residues and it does not contain phosphorylation sites. Mutations observed in human tumors and malignancies almost always map to this region of the protein. Apparently, three of the cysteines 176,238, and 242 are essential for a zinc binding site as labeled in the figure. Cys277/182 are clearly the most surface exposed cysteines that are located in the DNA/protein interface. Although mutation of these residues to serine had no affect on biological activities of p53, adducts to these two cysteines would probably impede proper DNA binding. Cysteine 277 is a highly conserved cysteine in p53 as divergent as human and squid, and it is also found in p53-like proteins p51/63 and p73. Mutational studies have shown that cys/ser conversion of cysl76, 238, and 242 affects DNA binding, and transactivation and transformation suppression activity of p53. Similar changes to cys 124, 135,141, and 275 affect both transactivation and transformation suppres sion activities. Recent work on the effects of PDTC (pyrrolidine dithiocarbamate) on cellular p53, has suggested that oxidative modification of p53 can indeed be responsible for altered expression of p53-related gene products. Oxidation of p53 was detected by a specific protocol that depends on a mobility shift of p53 when modified by a sulfhydryl reactive form of polyethylene glycol.81 Further experiments with this method have shown that PDTC produces 25% oxidation of p53 suggesting that at least one sulfhydryl on p53 was sensitive to oxidation. Oxidation correlated with a decrease in the activation of p53 downstream effector genes and altered subcellular localization of the protein. 82 The oxidative modification was reversible and additional studies showed that Ref-1 and thioredoxin were

166


Fig. 2. Molecular models of p53 interaction with a DNA as depicted in the file 1TUP. effective reductants of the oxidized protein. 83 The site of oxidation has not yet been clarified.

3.2.4 H-ras H-ras (p21hras, Ha-ras) is a low molecular weight (~19 kDa protein) G-protein which is critical to activation of several signal transduction pathways, including the extracellular signal-regulated kinases (Erk-1 and Erk-2). These pathways are activated when several different cell types are exposed to reactive oxygen or reactive nitrogen species.84,85 In most stud ies, H-ras was an essential component for the activation of Erk-1/2 by ROS or RNS. H-ras has two types of lipid modifications that are directly bound to reactive cysteines on the protein.86,87 These modifications were thought to be the only mechanisms for covalent regulation of the function of this protein. Recently we reported that H-ras is S-thiolated in NIH-3T3 cells which are exposed to diamide and both S-glutathiolated and S-nitrosylated in cells exposed to S-nitrosocysteine.18 H-ras has four potentially reactive cysteine residues (118,181,184, and 186), the latter three of which reside near the C-terminal prenylation site of the protein. Published structures of H-ras lack information about the three C-terminal cysteines, presumably because this part of the protein has much freedom of movement.88,89 Both X-ray crystal and NMR structures


167

show that Cysll8 is surface exposed near the beginning of a critical loop for binding the guanine nucleotide di- or tri-phosphate. It resides in a region of surface negative charge density. Oncogenic forms of H-ras have mutations that result in a loss of the ability of the protein to hydrolyze GTP, thus leaving the protein in a continuously active, GTP-containing state. It has been suggested that S-nitrosylation of Cysll8 can activate H-ras by increasing the turnover of the guanine nucleotide by an unknown mechanism which presumably involves changing the conformation of this loop and affecting the bound guanine nucleotide. S-glutathiolation of this site could not be demonstrated, 18 and it is suggested that either steric restraints around Cysll8 or, more likely, charge repulsion prevents the addition of negatively charged glutathione molecules. Potentially, activa tion of H-ras in cells by low levels of H 2 0 2 could occur by S-thiolation with an uncharged thiol like cysteine or by oxidation to a sulfenic, sulfinic or sulfonic acid. The remaining three reactive cysteine residues at the C-terminal of the protein must be lipidated for H-ras to function properly in cells. Cysl86 is farnesylated while Cysl81 and Cysl84 are palmitoylated. Farnesylation is a prerequisite for palmitoylation, and both farnesylation and palmitoylation are presumed to be essential for activation of H-ras, since mutating any of the three C-terminal cysteines interferes with membrane localization and transformation of cells by oncogenic forms of the protein. Because oxidative modification of any of these three cysteines might incur the same loss of function as a Cys mutation by blocking lipid modification reactions, oxidation of these residues is likely to inactivate H-ras. Farnesylation of Cysl86 is an irreversible modification, so oxidation of this residue will only occur on newly synthesized H-ras. Approximately 10% of Cysl86 is available for oxidation in NIH-3T3 cells, since 90% or more of H-ras is farnesylated in these cells. Thus, in cases of acute oxidative insult, oxidative modification of Cysl86 would only occur on the fraction of the cellular H-ras that was not yet farnesylated. Chronic oxidative stress has the potential to modify a larger fraction of Cysl86 and could affect H-ras activity by trapping newly synthesized H-ras in the cytosol. Cysl81 and 184 are normally palmitoylated in cells, and may be more important targets for oxidation. Palmitoylation is a transient modification, with significant rates of turnover of palmitate during the life of the protein. It is known that the palmitates of H-ras turn over more rapidly when cells are incubated with S-nitrosocysteine.90 The mechanism of this increased turnover is speculative at present, but it is tempting to suggest that modification of Cysl81/184 might be involved. Because other signal

168


transduction proteins such as the trimeric G-proteins are normally palmitoylated, oxidative events may also affect other signal transduction systems for which H-ras may be a model. All three C-terminal cysteines of H-ras react with S-nitrosoglutathione, generating S-nitrosylated forms of these cysteines. Two of the cysteines are also readily S-glutathiolated. N O adducts have a small size and are neutral while glutathione adducts are considerably more bulky and have a negative charge. Evidence suggests that glutathione is more likely to form stable adducts with neutral or positively sites. The positively charged residues in close proximity to the C-terminus cysteines of H-ras are thought to be important for palmitoylation of a protein. Palmitoylated cysteines may thus represent a subset of cysteine residues which are susceptible to S-glutathiolation. Because surface exposure is a strong determinant for oxidative modification, the localization of palmitoylated and farnesylated cysteines at the C-termini of proteins, which are often unstructured and solvent exposed, makes competition between lipidation and oxidation a likelihood in cells. The following table summarizes the potential modification of specific H-ras cysteines. Minimal nitrosative events may modify any of the four cysteines. If only a small amount of H-ras is activated by S-nitrosylation of Cysll8, it may be sufficient to activate the ERK-1/2 and other pathways. Inactivation of a small fraction of H-ras by blocking of lipidation would have little or no effect on the pathway, since a greater fraction of the protein remains unmodified. At higher levels of ROS or RNS, oxidation of H-ras becomes extensive enough to act as an effective competitor for lipidation. Such events would drastically reduce the participation of H-ras in signal transduction. Thus, H-ras in oxidant-treated cells should become resistant to activation by extracellular ligands such as TNF. A recent study has shown just such an inactivation when cells are exposed to high levels of S-nitrosocysteine,90 although the exact mechanism of inactivation of the pathway was not elucidated. Further studies in this system should explore the interaction between lipidation and oxidation of each cysteine as well as the membrane localization of H-ras and the over all activation state of pathways in which H-ras is a participant.

4. Perspective — Questions in Need of Answers The fundamental principles for the oxidative modification of the large pool of exposed and reactive protein cysteines in intact cellular proteins


169

are largely untested, but enough progress has been made to suggest the following overall concepts. Protein oxidation probably results from any number of different oxidizing molecules that abstract an electron from various protein locations. Subsequently, the electronic complexity of the protein structure produces electron deficient sulfur atoms at exposed cysteines. These become the reactive sites most likely to be modified by further chemical events. The protein sulfhydryls may be oxidized by a variety of mechanisms including reversible oxidative addition of a cellular metabolic product such as glutathione or nitric oxide. These adducts result in S-glutathiolated or S-nitrosylated proteins. In some cases ADP-ribosylation may be included as a reversible modification. S-thiolation and S-nitrosylation appear to be in direct competition with irreversible oxidation that results from incomplete protection. The attack of readily available oxygen or derived molecules further oxidizes protein cysteine to sulfinic acids and possibly even sulfonic acids. Protein sulfhydryls that are susceptible to these oxidative mechanisms may have several roles in the affected protein. They may be simply antioxidants, residing on surface sites that have no biological role other than reversible oxidation (carbonic anhydrase III). They may be involved directly in catalysis, or in binding substrate to an enzyme (GAPDH, creatine kinase, caspases). They may be necessary for attachment of lipids or other important protein modifications (H-ras). They may be integral to binding sites that are important in transcriptional regulation (p53, Jun-1, NF-kappaB). They are part of internal structures such as iron-sulfur centers or zinc binding sites (aconitase, p53, alcohol dehydrogenase). The functional differences between an S-glutathiolated, S-nitrosylated, or irreversibly oxidized protein cysteine may be very subtle. It is not clear that such differences exist. The different forms of reversible protein sulfhydryl modification are the direct result of different protein surface chemistries, or simple abundance of a particular protein adduct. Although it is important to study these phenomena, the interpretation of the experimental observations will undoubtedly change considerably as we learn more about the metabolic principles that affect these processes. Important progress in this aspect of metabolism will only come with improved methods for detecting protein modifications of interest in unique biological model systems. Methods for detecting protein S-nitrosylation are considerable less effective at present than those for detecting either S-glutathiolation or irreversible oxidation. The use of gel electrofocusing for study of the S-nitrosylation of specific proteins would considerably

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improve this problem. Thus, it is important to consider this and other innovative methods for improving experimentation on S-nitrosylation. Either Mal-PEG of some other reagent of a similar nature seems to pro vide a general method for at least detecting the extent of protein sulfhydryl oxidation for low abundance proteins in model systems. Used in conjunction with studies on purified proteins, this method could pro vide the first quantitative information on protein sulfhydryl modification of signal-transduction and transcription factor proteins during oxidative or nitrosative stress. Importantly, this same method (Mal-PEG) may also provide valuable information about irreversible damage to specific pro teins under these same conditions. Some of the most interesting aspects of protein sulfhydryl modifica tion for future study include: (1) Assessing each form of protein modifi cation in a model system responsive to several different types of oxidative or nitrosative stress. (2) Assessing the extent and ramifications of irreversible damage to protein sulfhydryls. Indeed, it will be interesting to determine whether protein sulfinic and sulfonic acids are "irreversible" in the biological sense. (3) By direct studies of a single protein one could assess the effectiveness of S-glutathiolation, S-nitrosylation, and irreversible oxida tion as modifiers of a specific biological function. (4) It will be important to determine whether proteins containing cysteines of different functional properties are actually modified by either S-glutathiolation or S-nitrosylation in vivo. There is still much to be done to understand the important role of protein sulfhydryls in the normal progression of oxidative and nitrosative stress.

Acknowledgment "H.S. is a Fellow of the National Foundation for Cancer Research (NFCR), Bethesda, MD. Support by Deutsche Forschungsgemeinschaft (SFB 575/B4) is gratefully acknowledged."

References 1. 2. 3. 4.

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Chapter 8 Radical Scavenging by Thiols: Biological Significance and Implications for Redox Signaling and Antioxidant Defense Christine C Winterbourn Free Radical Research Group, Department of Pathology, Christchurch School of Medicine and Health Sciences, PO Box 4345 Christchurch, New Zealand [email protected]

1. Summary The ability of thiols to undergo reversible oxidation and reduction enables them to contribute to many cell functions. Thiol groups are involved in the activity of numerous enzymes and have a major role in the antioxidant defenses of the cell. Additionally, there are a large number of cell proteins containing reduced thiol groups that are not known to parti cipate in enzyme catalysis. It is becoming clear that many of these are bind ing or regulatory proteins, the function of which can be modified by oxidation of the sulfhydryl group. Thus, sulfhydryl oxidation can affect cell function not only through inactivating thiol enzymes, but also by altering the binding characteristics of molecules involved in signaling pathways.1"4 Cells undergo a number of responses when exposed to oxidative stress. 5 Thiols are considered to be prime targets for oxidation. On the one hand, the redox state of the cell (which reflects the relative concentrations of oxidized and reduced thiols) influences the sensitivity of the cell to reactive oxidants. On the other, the behavior of different thiols dictates what that response will be. Cells encounter a range of oxidants that differ considerably in their chemical properties. These include peroxides and hypochlorous acid which undergo predominantly two electron reactions with thiols, and free radical species which prefer one electron pathways. Although frequently grouped together as reactive oxygen species, or ROS, it is important to recognize that this is a generic term rather than an entity 175


Table 1. Potential radical sources in the cell. Mitochondrial respiration Metal catalysed oxidations Peroxidase-mediated oxidations Redox cycling or autoxidation of xenobiotics UV and y-radiation Oxidoreductases e.g. NADPH oxidases, xanthine oxidase, lipoxygenase, nitric oxide synthase Lipid peroxidation

and reactive oxidants vary considerably in reactivity and selectivity. They are likely to undergo quite different reactions in the cell and it should not be assumed that they will all have the same influences on regulatory pathways. The importance of GSH plus glutathione peroxidase as an antioxidant defense system against peroxides is well recognized and understood. 5 The antioxidant role of the thioredoxin peroxidases (peroxiredoxin) systems, which are linked to thioredoxin/thioredoxin reductase, has more recently become apparent.3,6,7 These enzymatic systems act through nonradical mechanisms and are ultimately dependent on NADPH for reducing equivalents. Both may also be involved in redox regulation. Activity of the GSH/glutathione peroxidase system, by altering the GSH : GSSG ratio, has the ability to influence the overall redox state of the cell. However, as discussed elsewhere in this book, there is increasing evidence that more specific redox changes may be more critical. The thioredoxin peroxidase system, by causing selective oxidation of thiore doxin and the thiol proteins that it controls, could provide such specificity. How cells control free radicals is less well characterized, at least for mammals. Cells are continually exposed to free radicals from a variety of sources (Table 1). Superoxide dismutase is ubiquitously present to remove superoxide radicals enzymatically, but it is generally considered that other radicals are scavenged chemically by low molecular weight antioxidants such as glutathione, ascorbic acid, a-tocopherol, and dietary components such as the carotenoids and polyphenolics. 5,8 Vitamin E is important in the lipid phase where it is a good scavenger of peroxyl radicals and inhibitor of lipid peroxidation. GSH and ascorbate are aqueous antioxidants. Scavenging by vitamin E generates the tocopheroxyl radical, which must be recycled if vitamin E is to retain its antioxidant capacity. This can occur through reaction with ascorbate and

Radical Scavenging by Thiols 177

Table 2. Examples of oxidation of GSH to its thiyl radical. Compound or Class of Compound

System

Ref.

Tyrosine Phenols Sugars DNA bases Nitrogen dioxide and peroxynitrite Aromatic amines Phenothiazines Ethanol (hydroxyethyl radical)

Peroxidase Peroxidase, radiolysis Radiolysis Radiolysis Direct reaction Peroxidase, autoxidation Peroxidase Thermal decomposition

28,57 26, 30, 57-59 60 61 62 26, 30, 58, 63 58 64

possibly glutathione, 9 resulting in radical transfer from the lipid to the aqueous phase. Ultimately, therefore, protection against lipid peroxyl rad icals requires effective aqueous phase scavenging systems. This article con siders the radical scavenging properties of GSH and ascorbate and their roles in the antioxidant defenses of the cell. It also considers thiol proteins as potential radical scavengers and whether radical reactions could be involved in regulating redox-sensitive cell functions.

2. Radical Scavenging by GSH GSH reacts with a wide range of radical species. These include hydroxyl, phenoxyl, alkoxyl, arylamino, peroxyl, semiquinone and carbon centred radicals 1011 as exemplified in Table 2. Some of the parent compounds that give rise to these radicals occur physiologically, others are drugs or envi ronmental chemicals. Some, such as the flavonoids, are themselves radical scavengers and of interest for their potential health benefits as antioxidants. It is possible that an abilty to channel radicals to physiologi cal antioxidants such as GSH may be an important factor in this regard. GSH is typically present inside cells at millimolar concentrations. It is theoretically possible, therefore, for it to scavenge a large proportion of the radicals generated within a cell. For this to be the case, GSH must react sufficiently rapidly with the radicals it encounters to outcompete other potential targets. Furthermore, if it is to provide antioxidant protection, then products of the scavenging reaction must be benign. A characteristic of radical scavenging reactions is that they generate another radical, in this case the thiyl radical, GS". As described in more detail elsewhere,12"14

178


there are features of thiyl radical chemistry that are critical for GSH and other thiols to act as effective scavengers and antioxidants. Scavenging by GSH is reversible (Reaction (1), where R" is a geneiic radical), and in many cases, the equilibrium lies far to the left (e.g. for acetaminophen K = 3 x 10"4). R"

+

GSH

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