Electrochemical studies of biological systems

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Electrochemical Studies of Biological Systems Donald T. Sawyer, EDITOR University of

California

A symposium sponsored by the Division of Analytical Chemistry at the 172nd Meeting of the American Chemical Society San Francisco, Calif., August 30, 1976

ACS SYMPOSIUM SERIES 38

AMERICAN

CHEMICAL

SOCIETY

WASHINGTON, D. C. 1977

In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

Library of Congress Œ

Data

Electrochemical studies of biological systems. (ACS symposium series; 38) Includes bibliographical references and index. ISSN 0097-6156 1. Electrochemical analysis—Congresses. 2. Biological chemistry—Congresses. I. Sawyer, Donald T . II. American Chemical Society. Division of Analytical Chemistry. III. Series: American Chemical Society. ACS symposium series; 38. QD115.E524 ISBN 0-8412-0361-X

Copyright ©

574.1'9283 ACSMC 8

76-30831 38 1-216

1977

American Chemical Society A l l Rights Reserved. N o part of this book may be reproduced or transmitted in any form or by any means—graphic, electronic, including photocopying, recording, taping, or information storage and retrieval systems—without written permission from the American Chemical Society. PRINTED IN T H E U N I T E D

STATES

OF

AMERICA


ACS Symposium Series Robert F. Gould, Editor

Advisory Board Donald G .

Crosby

Jeremiah P. Freeman E. Desmond Robert A .

Goddard Hofstader

J o h n L. Margrave N i n a I. M c C l e l l a n d J o h n B. Pfeiffer Joseph V . Rodricks A l a n C . Sartorelli Raymond B. Seymour Roy L. Aaron

Whistler Wold


FOREWORD The A C S S Y M P O S I U

a medium for publishin format of the SERIES parallels that of the continuing A D V A N C E S IN C H E M I S T R Y SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form.

As a further

means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book.

Papers published in the A C S S Y M P O S I U M

SERIES

are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.


PREFACE electrochemistry has enjoyed a renaissance

during the past decade

because of its use for chemical characterization. In particular, the subdisciplines of organic, inorganic, and biological chemistry have found electrochemical methods uniquely effective for determining the stoichiometrics, thermodynamics, and kinetics of electron transfer reactions. Although cyclic voltammetry and controlled potential electrolysis are by far the most used electrochemical techniques, a number of new methodologies that combine electrochemical and spectroscopic measurements have been developed in recen This symposium was trochemical methods for the characterization of biological systems needs to be brought to the attention of chemists and biochemists.

M u c h of

biology and biochemistry involves oxidation-reduction processes, atomtransfer reactions, and electron-transfer reactions.

Because the theory

and principles of electrochemistry are concerned with the same kinds of processes, as well as with the thermodynamics and kinetics of heterogeneous redox processes, substantial synergistic benefits can result from a coordinated, rational application of electrochemical principles

and

theories to the electron-transfer and oxidation-reduction chemistry of biology. T h e twelve papers of the symposium provide a representative cross section of the kinds of electrochemical methodologies that are used to study biological systems.

They also illustrate the kinds of biological

problems that are being studied by such methods.

Beyond cyclic volt-

ammetry and controlled potential coulometry, the use of optically transparent thin-layer electrodes

( O T T L E ) , rotating ring-disc enzyme elec-

trodes, mediator titrants, differential capacitance and phenomena,

and differential

pulse

polarography

are

electrocapillary discussed.

The

applications range from the analysis of N T A and E D T A in water samples to the characterization of the redox chemistry for several metalloproteins. Several chapters emphasize the development of improved electrochemical techniques and instrumentation for the study of biological systems. However, the major emphasis of the papers is the study of the redox properties

of model compounds

for biological systems.

The

specific

systems include vitamin B i , cytochrome c, cytochrome c oxidase, metal 2

porphyrins, nitrogenase, mitochondrial superoxide dismutase, purines and pyrimidines, and a model for a mammalian heart.

vii


The assistance of H . B. Mark, Jr., and G . S. Wilson, who chaired the two sessions of the symposium, is gratefully acknowledged.

M y sincere

thanks to John Miller, Chairman of the Analytical Chemistry Division, for his support and encouragement in the organization of the symposium and to Marian M a n n for her assistance with the correspondence

and

manuscript preparation. Department of Chemistry

DONALD

T.

SAWYER

University of California Riverside, Calif. 92502 November 18, 1976

viii


1 Spectroelectrochemical

Investigation

of

Vitamin

Β

12

and Related Cobalamins HARRY B. MARK, JR., and THOMAS M. KENYHERCZ Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221 PETER T. KISSINGER Purdue University, W. Lafayette, Ind. 47907

This paper discusses three aspects of the spectroelectro chemical study of Vitamin time resolved spectral study mins u n d e r various experimental conditions; (ii) the electro chemical behavior of the cob(III)alamins, a n d (iii) the electro chemical behavior of 5'-deoxyadenosylcobalamin. Several pre viously unknown features concerning the redox chemistry of these unusual but important complexes are reported. The

Autooxidation

of

B s Under Various Conditions 12

Recent spectroelectrochemical investigations of the oxida tion of cob(I)alamins to cob(III)alamins in various media has yielded the previously unobserved sequence of intermediates and steps involved in the mechanism of this biologically important reaction. This sequence of intermediates disagrees with pre viously speculated mechanisms . The time resolved visible-UV spectra for the reoxidation of the electroreduced Vitamin B type compounds: cyanocobalamin (B ), aquocobalamin (B ) and dicyanocobalamin (B -CN) are reported below. The autooxidation of the cob(I)alamins were carried out under both air and inert argon atmospheres, the electrochemical reoxidation was also studied under these conditions. Mechanisms consistent with the qualitative kinetic data obtained from time resolved spectra are presented. The r e c e n t l y d e v e l o p e d m e r c u r y c o a t e d n i c k e l m i n i g r i d s y s t e m was e m p l o y e d i n a t h i n l a y e r e l e c t r o l y s i s c e l l a s a n o p t i c a l l y t r a n s p a r e n t e l e c t r o d e , Hg-Ni 0TTLE. L L = U T h e Hg-Ni 0TTLE c e l l was m o u n t e d i n a c o m p u t e r i z e d H a r r i c k r a p i d s c a n n i n g d u a l t e a m spectrophotometer,ϋ w h i l e a l l experimental p r o c e d u r e s , i n t e r f a c e design, e l e c t r o c h e m i c a l i n s t r u m e n t a t i o n and computer programming i n c l u d i n g data a c q u i s i t i o n , p r o c e s s i n g a n d r e d u c t i o n have been described p r e v i o u s l y . ^ A l l s o l u t i o n s (except that noted i n F i g u r e 2 ) w e r e ImM i n c o b a l a m i n a n d 1.0M i n N a S 0 i * a s t h e s u p porting e l e c t r o l y t e (the solutions f o r the Bi -CN experiments 1

2

2-8

12

12

12a

12

2

2

1


2

ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS

w e r e a l s o 0.1M i n N a C N ) . T h e s o l u t i o n s w e r e i n t r o d u c e d i n t o t h e OTTLE c e l l w i t h c o b a l t i n t h e +3 v a l e n c e s t a t e . T h e s o l u t i o n s were t h e n p o t e n t i o s t a t e d a t -1.0 v o l t s v s . SCE u n t i l t h e s p e c t r a changed completely t o t h a t o f t h e Co(I) species » ( s e et h e f i r s t spectrum o f F i g u r e 1) and remained c o n s t a n t ( f o r a p p r o x i mately o n e - h a l f h o u r ) . The c o b ( I ) a l a m i n s o l u t i o n s were a l l o w e d to undergo a u t o o x i d a t i o n i n e i t h e r t h e presence o f a i r d i f f u s i n g i n t o t h e OTTLE c e l l o r u n d e r a n i n e r t a r g o n a t m o s p h e r e . T h e p o t e n t i o s t a t was d i s c o n n e c t e d d u r i n g t h e s e a u t o c a t a l y s i s e x p e r i m e n t s . D u r i n g e l e c t r o r e o x i d a t i o n s t e p - w i s e ( 5 0 mv i n c r e m e n t ) p o t e n t i o s t a t i c p o l a r i z a t i o n ( p o t e n t i o s t a t e d a t each p o t e n t i a l u n t i l s p e c t r a ceased changing) under an i n e r t argon atmosphere was e m p l o y e d . C u r v e A o f F i g u r e 2 shows t h e t y p i c a l t r a n s i e n t s p e c t r u m o f a p a r t i a l l y a i r reoxidized s o l u t i o n o fcob(I)alamin while curve Β i s t h e spectrum o f aquocobalamin The p e a k s o f i n t e r e s t t h e o x i d a t i o n s e q u e n c e o c c u r a t 4 1 0 nm a n d 4 7 5 nm. D u r i n g t h e large scale preparation o fB i t was o b s e r v e d t h a t t h e r e l a t i v e r a t i o o f t h e 4 1 0 a n d 4 7 5 nm p e a k s v a r i e d m a r k e d l y depending on t h e r a t e a t w h i c h o x y g e n was i n t r o d u c e d i n t o t h e c o b ( I ) a l a m i n s o l u t i o n . F o r example, curve A o f F i g u r e 2 i s t h e s p e c t r a ob t a i n e d on bubbling oxygen through a r a p i d l y s t i r r e d B solu t i o n ( t h e 4 1 0 nm p e a k i s much l a r g e r t h a n t h e 4 7 5 nm p e a k ) . How e v e r , t h e 4 7 5 nm p e a k s i n t h e c u r v e s shown i n F i g u r e 3 a r e l a r g e r t h a n t h e 4 1 0 nm p e a k s . Inthe l a t t e r case, a i r d i f f u s e s i n t o the B s o l u t i o n s l o w l y f r o m t h e e d g e s o f t h e OTTLE c e l l . Thus i t i s f e l t t h a t t h e 4 1 0 a n d 4 7 5 nm p e a k s r e p r e s e n t t w o d i f f e r e n t s p e c i e s even though p r e v i o u s workers have r e p o r t e d both peaks a s being c h a r a c t e r i s t i c o fthe s o - c a l l e d B ! The s p e c t r a o b t a i n e d f o l l o w i n g t h e e x h a u s t i v e r e d u c t i o n o f B i , B , a n d B - C N a t - 1 . 0 v o l t s v s . SCE w e r e i d e n t i c a l c o b (I)alamin species corresponding t othose p r e v i o u s l y designated B s . Though t h e e x a c t c o o r d i n a t i o n geometry f o r c o b ( I ) a l a m i n i s unknown, i t has been s u g g e s t e d t h a t t h e b e n z i m i d a z o l e i s i n a base-off c o n f i g u r a t i o n — w i t h water molecules occupying each of the axial positions. As B a n d B appear t o r e o x i d i z e a t comparable r a t e s and have s i m i l a r time r e s o l v e d s p e c t r a l c h a r a c t e r i s t i c s , a l l argu m e n t s made f o r B a r e e q u a l l y a p p l i c a b l e t o B i . A l s o , i t w a s found t h a t t h e time r e s o l v e d s p e c t r a l sequences and r a t e s o f peak c h a n g e s w e r e v i r t u a l l y t h e same i n t h e p r e s e n c e o f a i r o r a r g o n . The t i m e r e s o l v e d s p e c t r a f o r t h e a i r r e o x i d a t i o n o f a c o b ( I ) alamin s o l u t i o n obtained by the exhaustive reduction o f cyanoc o b ( I I I ) a l a m i n i s shown i n F i g u r e s 1 a n d 3. F i g u r e 1 s h o w s t h a t t h e c o b ( I ) a l a m i n , a s m o n i t o r e d b y t h e 3 8 5 nm p e a k , i s v i r t u a l l y r e o x i d i z e d c o m p l e t e l y t o a c o b ( I I ) a l a m i n i n t h e f i r s t 100 seconds. The p e a k w h i c h d e v e l o p s a t 4 7 5 nm c o r r e s p o n d s t o a c o b ( I I ) a l a m i n , — ( d e s i g n a t e d h e r e a s B ) g r o w s t o a maximum i n t h e f i r s t 400 s e c o n d s and t h e n s l o w l y d e c r e a s e s f i n a l l y v a n i s h i n g a t a b o u t i 2 a

1 2 s

1 2 S

i 2 r

2

1 2 a

i 2

i 2

i 2

i 2 a

i 2

2 a

i 2 r


Vitamin

MARK E T AL.

o.o Ll 280

I

1

320

360

1

B

12

1

and Related

I

400 440 480 WAVELENGTH . Ν M

Cobalamins

1

1

*-

520

560

600

ο·β

Figure 1. Time-resolved spectra for the reoxidation of cob(I)alamin to cob(II)ahmin in 1.0M lSla S0 at pH = 7.0 in 0-400 sec 2

300

350

400

ll

450 WAVELENGTH

500

550

600

650

, NM

Figure 2. Spectra of the air-reoxidized product formed from the electroreduction of the cyanocob(IU)alamin in 0.1M NaN0 . A, partially reoxidized cob(II)ahmin; B, totally reoxidized cob(IH)alamin, aquocob(III)alamin. 3


4


1

5 χ 10 * s e c o n d s . T h e c y a n o c o b ( I I I ) a l a m i n p e a k o f 3 6 0 nm w h i c h b e g i n s t o d e v e l o p a t 10 s e c o n d s i s c o m p l e t e l y r e c o n v e r t e d t o Bi2 b y 1 0 s e c o n d s ( a s s e e n i n F i g u r e 3 ) . T h e p r e v i o u s l y u n r e p o r t e d i n t e r m e d i a t e e x h i b i t s a 4 1 0 nm p e a k , w h i c h o c c u r s i n t h e same q u a l i t a t i v e t i m e p e r i o d o f t h e c h a r a c t e r i s t i c 4 7 5 nm c o b ( I I ) a l a m i n p e a k , a p p e a r s i n 2 0 0 s e c o n d s , r e a c h e s a maximum b y 5 χ 1 0 s e c o n d s a n d h a s d i s a p p e a r e d b y a b o u t 2 χ 10 s e c o n d s . As t h e r e a r e n o p e a k s i n t h e r e g i o n o f 3 7 0 nm w h i c h w o u l d i d e n t i f y e i t h e r Co(I) o r C o ( I I I ) s p e c i e s ^ during t h i s time i n t e r v a l , i t i s f e l t t h a t t h e p r o n o u n c e d 4 1 0 nm p e a k i n d i c a t e s a s e c o n d , d i f f e r e n t c o b ( I I ) a l a m i n i n t e r m e d i a t e which w i l l be i d e n t i f i e d as Bi2r- A s m e n t i o n e d p r e v i o u s l y , t h e t i m e r e s o l v e d s p e c t r a f o r t h e a i r r e o x i d a t i o n o f cob(I)alamin obtained by t h e exhaus t i v e r e d u c t i o n o f a q u o c o b ( I I I ) a l a m i n , i s q u a l i t a t i v e l y t h e same as r e p r e s e n t e d i n F i g u r e s 1 and 3 However t h e a i r r e o x i d a t i o n of cob(I)alamin obtaine cob(III)alamin (in th q u i t e d i f f e r e n t a s shown i n F i g u r e 4. F i r s t o f a l l , d i c y a n o c o b ( I I I ) alamin i s t o t a l l y regenerated i n l e s s than 400 seconds. The i n c r e a s e / d e c r e a s e i n t h e 2 9 0 nm b a n d , t h e r i s e a n d f a l l o f t h e 475 nm ( c o b ( I I ) a l a m i n ) p e a k , t h e f i n a l r i s e o f t h e 3 6 8 nm p e a k p l u s t h e t o t a l l a c k o f a 4 1 0 nm p e a k i n d i c a t e s t h a t t h e r e o x i d a t i o n o f c o b ( I ) a l a m i n i n t h e presence o f excess c y a n i d e goes through only a c o b ( I I ) a l a m i n , B type i n t e r m e d i a t e . Electrochemical r e o x i d a t i o n o f cob(I)alamin, obtained by the e l e c t r o r e d u c t i o n o f B12 u n d e r a n a r g o n a t m o s p h e r e , g o e s t h r o u g h b o t h t h e B i 2 r ( 4 7 5 nm) a n d B i ( 4 1 0 a n d 4 7 5 nm) i n t e r m e d i a t e s i n t h e p o t e n t i a l r e g i o n f r o m - 0 . 6 0 t o -0.01 v o l t s v s . SCE y i e l d i n g B12 a t +0.10 v o l t s v s . S C E . T h e c o n d i t i o n s o f t h e e l e c t r o c h e m i c a l r e o x i d a t i o n e x p e r i m e n t s i n d i c a t e t h a t t h e 4 1 0 nm p e a k i s not i n d i c a t i v e o f an oxygen adduct type o f cobalamin s p e c i e s . The t i m e r e s o l v e d a u t o o x i d a t i o n s p e c t r a o f a c o b ( I ) i n i m i d e — (no b e n z i m i d a z o l e m o i e t y o n t h e c o r r i n r i n g s y s t e m ) w a s a l s o examined. The a b s o r b a n c e - p o t e n t i a l r e d u c t i o n c h a r a c t e r i s t i c s o f the c y a n o a q u o c o b ( I I I ) i n i m i d e — s t a r t i n g m a t e r i a l and t h e r a t e o f autooxidation a r e p a r a l l e l t o those o f Vitamin B itself indi c a t i n g t h a t t h e l a c k o f t h e b e n z i m i d a z o l e moiety has n o t appre c i a b l y a l t e r e d t h e redox p r o p e r t i e s o f t h e c e n t r a l c o b a l t i o n . However, t h e time r e s o l v e d a u t o o x i d a t i o n s p e c t r a o f t h e c o b ( I ) i n i m i d e d o n o t e x h i b i t a 4 1 0 nm p e a k . As v a r i a t i o n o f p e a k s i n t h e 4 0 0 - 5 0 0 nm r e g i o n h a v e p r e v i o u s l y been a s s o c i a t e d w i t h changes i n t h e a x i a l l i g a n d s , — i t i s a t t r a c t i v e t o s p e c u l a t e a t t h e p o i n t t h a t t h e two c o b ( I I ) a l a m i n s represent c o n f i g u r a t i o n s where t h e benzimidazole i s e i t h e r c o o r d i n a t e d t o t h e C o ( I I ) , a base-on form, o r where t h e benzimi d a z o l e has been r e p l a c e d by a w a t e r i n t h e y - a x i a l p o s i t i o n , a b a s e - o f f f o r m . T h o u g h t h e p o s s i b i l i t y e x i s t s t h a t t h e r e i s some a l t e r a t i o n i n t h e c o r r i n r i n g s t r u c t u r e could a l s o account f o r the observed behavior, i t i s f e l t t h a t redox changes i n t h e r i n g would be i r r e v e r s i b l e . The f a c t t h a t t h e c o b i n i m i d e and k

5

3

h

1

2

r

2 r

i 2


Vitamin

MARK E T AL.

B

îg

and Related

Cobalamins

5.0

0.0

I

1 1 I I I I I I \L 2 . · 2 * > 3 2 O M 0 4 0 0 4 4 O 4 t 0 5 2 O M 0 t 0 0 WAVELENGTH. NM

Figure 3. Time-resolved spectra from the reoxidation of cob(H)ahmin to a cob(III)alamin in 1.0M Na SO at pH = 7.0 in 630-70,000 sec 2

0.0

I

I

280

I

I

320

360

I

1

1

400 440 4*0 WAVELENGTH . NM

Il

1

1

520

560

*- 0.6 600

Figure 4. Time-resolved spectra for the reoxidation of cob(I)alamin to dicyanocob(Ul)alamin in 1.0M Na SO^ and 0.1M NaCN at pH = 11.0 in 0-410 sec 2


6

ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS

d i c y a n o c o b a l a m i n e x p e r i m e n t s show n o 4 1 0 nm b a n d i s c o n s i s t e n t with the base-on/base-off suggestion. Furthermore, i f t h i s i s c o r r e c t , then t h e B l r would be t h e base-on c o n f i g u r a t i o n and B r would be t h e b a s e - o f f form. Q u a l i t a t i v e l y , t h e time resolved spectra i n d i c a t e that t h e reoxidation o f cob(I)alamin i n the presence o fstoichiometric o r l e s s a m o u n t s o f CN~ f o l l o w s t h e r e a c t i o n s c h e m e i l l u s t r a t e d below. As p r e v i o u s l y m e n t i o n e d , t h e p r e s e n c e o f e x c e s s c y a n i d e i o n s a f f e c t s t h e r e o x i d a t i o n scheme s u c h t h a t e i t h e r t h e B J species i s n o t formed o r t h a t t h e o x i d a t i o n o f t h e B species i s kineti c a l l y favored. I t has been p o s t u l a t e d by o t h e r s — t h a t c o b ( I ) alamin autooxidizes t o cob(II)alamin with t h e e v o l u t i o n o f hydro gen a n d t h a t c o b ( I I ) a l a m i n s d i s p r o p o r t i o n a t e i n t h e m e c h a n i s m t o form cob(I) and c o b ( I I I ) alamin s p e c i e s . Under present e x p e r i mental c o n d i t i o n s t h i alamin i s not d i r e c t l c o n c l u s i o n s o f B i r k e e t a l Λ who e s t i m a t e d t h a t t h e t h e r m o d y namic and k i n e t i c parameters f o r such a d i s p r o p o r t i o n a t i o n a r e very unfavorable. However, t h e time r e s o l v e d s p e c t r a l sequence observed does n o t r u l e o u t t h e p o s s i b i l i t y t h a t B undergoes disproportionation. I f the rate o fdisproportionation o f B i s very slow compared t o t h e r a t e o f o x i d a t i o n o f B i t h e same time r e s o l v e d s p e c t r a would be o b t a i n e d . I t i s interesting to n o t e t h a t we d o n o t f i n d a n y d i r e c t e v i d e n c e f o r H e v o l u t i o n w h i c h i s e a s i l y o b s e r v e d ( t r a p p e d s m a l l b u b b l e s ) i n t h e OTTLE c e l l f o r s y s t e m s w h e r e i t o c c u r s . We h a v e b e e n u n a b l e t o i d e n t i f y t h e o x i d i z i n g agent(s) thus f a r . F u r t h e r m o r e , i t has n o t been p o s s i b l e t o c a l c u l a t e m e a n i n g f u l k i n e t i c p a r a m e t e r s f r o m the time and p o t e n t i a l r e s o l v e d s p e c t r a a s no q u a n t i t a t i v e d i f f u s i o n model has been p o s t u l a t e d . T h e r e f o r e , c a l c u l a t i o n s o f d i f f u s i o n i n t h e OTTLE t y p e c e l l c a n n o t b e made. F u r t h e r m o r e , we c a n n o t t e l l i f B i oxidizes directly t o both B and B J r a t d i f f e r e n t r a t e s o r t h a t i f B J results simply from a r a p i d e q u i l i b r i u m with B as i l l u s t r a t e d by the d o t t e d a r r o w i n t h e p r o p o s e d m e c h a n i s m u ( C o b a l t +2 c o m p l e x o f t h i s t y p e a r e a l w a y s l a b i l e . — ) T h e same a r g u m e n t a p p l i e s t o t h e interpretation o fB and B ' o x i d i z i n g t o c o b ( I I I ) a l a m i n . However, i t does appear t h a t t h e r e o x i d a t i o n o f B t o dicyanoc o b ( I I I ) a l a m i n o c c u r s much m o r e r a p i d l y t h a n t h e r e o x i d a t i o n o f either B or B l t o B o r B . D i f f u s i o n s t u d i e s a r e now i n p r o g r e s s a s w e l l a s a s i m i l a r s t u d y w i t h m e t h y l a n d 5'-deoxyadenosylcob(III)alamin. Q u a n t i t a t i v e studies o f the chemical and e l e c t r o c h e m i c a l o x i d a t i o n k i n e t i c s a n d m e c h a n i s m s w i l l b e reported a t a f u t u r e date. 2

i 2

2 r

i 2 r

1

2

r

i 2 r

2 S

2

2 S

i 2 r

2

2 r

i 2 r

i 2 r

i 2 r

1

1

The

2

r

2 r

Electrochemical

1 2

Behavior

2

r

i 2 a

of Cob(III)alamins

The e l e c t r o c h e m i c a l b e h a v i o r o f v i t a m i n B (cyanocob(III)a l a m i n ) and r e l a t e d c o b a l a m i n compounds i n aqueous media i s o f i 2


1.

MARK E T A L .

Vitamin

B

12

and Related

7

Cobalamins

importance f o r e l u c i d a t i n g t h e biomechanistic r e a c t i o n sequences which i n v o l v e cobalamin species. There has been c o n s i d e r a b l e study o f the redox processes o f cobalamins using the convention al e l e c t r o a n a l y t i c a ltechniques o f p o l a r o q r a p h y , * " coulom e t r y , * * * and c y c l i c v o l t a m m e t r y , * _ and d i v e r s e working e l e c t r o d e m a t e r i a l s such a s m e r c u r y * ' ~ and p l a t i n u m . i * H o w e v e r , t h e i n t e r p r e t a t i o n o f t h e e l e c t r o c h e m i c a l d a t a t o unam b i g u o u s l y determine even t h e most fundamental parameters such a s t h e t h e r m o d y n a m i c r e d o x p o t e n t i a l s , t h e n u m b e r o f e l e c t r o n s (nvalues) i n v o l v e d i n t h e e l e c t r o n t r a n s f e r s t e p s , and t h e sequence o f s t e p s i n t h e mechanism has n o t been p o s s i b l e because o f numer ous c o m p l i c a t i n g c o n d i t i o n s . The c o m p l i c a t i o n s encompass s t r o n g a d s o r p t i o n o f both r e a c t a n t and product, i r r e v e r s i b i l i t y o f t h e r e d o x r e a c t i o n s , unusual medium e f f e c t s i n v o l v i n g t h e s o l v e n t system and t h e s u p p o r t i n g e l e c t r o l y t e and marked v a r i a t i o n o f electrode kinetics wit new t e c h n i q u e s e m p l o y i n t h i n l a y e r e l e c t r o l y s i s c e l l s — h a v e been d e v e l o p e d w h i c h have proved u s e f u l t o t h e study o f t h e b a s i c redox p r o p e r t i e s o f c y t o chrome c . - ^ This paper r e p o r t s t h e r e s u l t s obtained by using t h i n l a y e r m i n i g r i d electrode c e l l s t o study the electrochemical and s p e c t r o e l e c t r o c h e m i c a l b e h a v i o r o f c y a n o c o b a l a m i n ( B ) , aquocobalamin ( B i ) , and dicyanocobalamin (Bi -CN). 9

3

9

3 1

8

8

2 2

3 3

3 5

9

2 2

3 7

3 2

3

9

1 2

2 a

2

S p e c t r o e l e c t r o c h e m i s t r y o f t h e Cobalamin Systems. As changes i n t h e valence o f c o b a l t , t h e c e n t r a l metal i o n o f t h e cobalamins, a r e r e f l e c t e d by d i s t i n c t changes i n t h e v i s i b l e ab s o r p t i o n s p e c t r a , a c o u p l i n g o f e l e c t r o c h e m i c a l and s p e c t r o s c o p i c m e a s u r e m e n t s was p e r f o r m e d t o e l u c i d a t e t h e r e d o x b e h a v i o r o f t h e cobalamins. S p e c t r o e l e c t r o c h e m i c a l experiments were c a r r i e d o u t u s i n g t h e o p t i c a l l y t r a n s p a r e n t t h i n layer e l e c t r o c h e m i c a l c e l l s (OTTLE) i n t h e presence and a b s e n c e o f t h e e l e c t r o n t r a n s f e r mediator, 2 , 6 - d i c h l o r o p h e n o l i n d o p h e n o l T h e cobalamin-containi n g OTTLE c e l l s w e r e p o t e n t i o s t a t e d w h i l e t h e o p t i c a l a b s o r b a n c e o f a peak o f i n t e r e s t and c u r r e n t l e v e l s were monitored. When both t h e a b s o r b a n c e stopped changing and t h e c u r r e n t l e v e l s had f a l l e n t o e s s e n t i a l l y z e r o (

SCE

Figure 6. Potential-absorbance curves for the reduction (A) and oxidation (B) of 1.2 mM B monitored at 550 nm. 1.0 M Na SO^; 0.1 Μ NaN0 ; pH 7.0; Hg-Ni minigrid; cell thickness, 0.017 cm. 12

2

3



J

I

Ο

I

Ι

-0.

Figure 7. Potential-absorbance curves for the re duction (A) and oxidation (B) of 1.2 mM Β i2 momtored at 475 nm. 1.0 M Na SO>; 0.1 M NaNO ; pH 7.0; Hg-Ni minigrid; cell thickness, 0.017 cm. 2

Potential

s

Volts

vs

SCE

Figure 8. Potential-absorbance curves for the reduction (A) and oxidation (B) of 1.2 mM B CN monitored at 580 nm. 1.0 M Na S0 ; O.J M KCN; pH 10.4; Hg-Ni minigrid; cell thickness, 0.017 cm. 12

2

4


1.

MARK E T A L .

Vitamin

B

and Related

12

11

Cobalamins

p o t e n t i a l s f o r B12-CN r e d u c t i o n a n d r e o x i d a t i o n c u r v e s i n F i g u r e s 8 and 9 ) . Vitamin B was a l s o i n v e s t i g a t e d a t a H g - N i e l e c t r o d e i n a s i m i l a r set of experiments. C u r v e s A o f F i g u r e 1 0 ( t h e 530-nm p e a k ) a n d F i g u r e 11 ( t h e 475-nm p e a k ) show t h a t B undergoes an u n u s u a l t w o - s t e p p r o c e s s , a s i l l u s t r a t e d b y t h e b r e a k s a t a b o u t -0.06 and -0.65 V , b e f o r e c o m p l e t e c o n v e r s i o n t o a c o b ( I I ) a l a m i n s p e c i e s . C u r v e A o f F i g u r e s 1 0 a n d 11 shows t h a t t h e c o b ( I l ) a l a m i n i s then reduced to cob(I)alamin a s the p o t e n t i a l i n c r e a s e s f r o m -0.8 t o -1.0 V. On r e o x i d a t i o n , c u r v e Β o f F i g u r e 1 1 , t h e c o b ( I ) a l a m i n i s reversibly reoxidized to cob(II)alamin o v e r t h e same p o t e n t i a l r a n g e a s i n t h e negative scan. H o w e v e r , t h e Β c u r v e s i n b o t h F i g u r e s 1 0 and 11 i n d i c a t e t h a t t h e r e o x i dation which corresponds to a q u a n t i t a t i v e regeneration of B i a from the c o b ( I I ) a l a m i n s p e c i e s i s a s i n g l e step process which oddly occurs at a p o t e n t i a ( c a . -50 mV).u To c h e c k t h e u n i q u e s p e c t r o e l e c t r o c h e m i c a l p r o p e r t i e s o f B , other samples of B from d i f f e r e n t s o u r c e s and p r e p a r a t i o n s were examined, and, a l s o , the e l e c t r o c h e m i c a l p r e p a r a t i o n was r e c y c l e d a n u m b e r o f t i m e s . The s p e c t r o e l e c t r o c h e m i c a l b e havior at a p a r t i c u l a r wavelength f o r B from the v a r i o u s pre p a r a t i o n s gave s p e c t r o p o t e n t i o s t a t i c curves(OTTLEgrams) i d e n t i c a l with those presented herein. A l s o , s p e c t r o p o t e n t i o s t a t i c c y c l i n g of B gave r e p r o d u c i b l e s e t s of c u r v e s . I t i s i n t e r e s t i n g t h a t the a b s o r b a n c e - p o t e n t i a l waves f o r B i n t h e OTTLE e x p e r i m e n t s do n o t c o r r e s p o n d t o a n y p e a k s i n t h e c y c l i c v o l t a m m o g r a m o f B a t t h e same e l e c t r o d e . H o w e v e r , t h e t h r e e absorbance-poten t i a l "waves" f o r the r e d u c t i o n o f B i do c o r r e l a t e reasonably w e l l w i t h t h e t h r e e waves o b s e r v e d i n the p r e v i o u s l y r e p o r t e d polarography of B . > * . To understand the unusual two-step process i n the reduction of B t o a c o b ( I I ) a l a m i n s p e c i e s and to determine i f the e l e c t r o d e i t s e l f i s p l a y i n g a r o l e i n the e l e c t r o n t r a n s f e r k i n e t i c s , the mediator 2,6-dichlorophenolindop h e n o l was u s e d i n c o n j u n c t i o n w i t h t h e A u m i n i g r i d e l e c t r o d e . — The m e d i a t o r f u n c t i o n s a s t h e p r i m a r y e l e c t r o n t r a n s f e r a g e n t b e t w e e n t h e e l e c t r o d e and a r e d o x s y s t e m t h a t has v e r y s l o w heterogeneous e l e c t r o n t r a n s f e r r a t e s . Thus, the mediator a c c e l erates the o v e r a l l e l e c t r o c h e m i c a l r e a c t i o n of the system of i n t e r e s t . The c h o i c e o f t h i s m e d i a t o r was d e t e r m i n e d b y t h e po t e n t i a l r e g i o n o f i n t e r e s t i n t h i s c a s e (+0.2 t o -0.2 V v s . S C E ) . The A u m i n i g r i d e l e c t r o d e was u s e d t o e l i m i n a t e t h e p o s s i b i l i t y of o x i d a t i o n of the working e l e c t r o d e m a t e r i a l i n t h i s p o t e n t i a l r e g i o n and b e c a u s e t h e c y c l i c v o l t a m m o g r a m s o f B exhibited a m o r e w e l l - d e f i n e d wave a t a n i n t e r m e d i a t e p o t e n t i a l , a n d B appeared t o be l e s s s t r o n g l y adsorbed on the Au e l e c t r o d e . The a b s o r b a n c e c h a n g e s o f t h e 525-nm ( B i ) and 475-nm ( B ) b a n d s a s a f u n c t i o n o f t h e a p p l i e d p o t e n t i a l a r e shown i n F i g u r e s 1 2 a n d 1 3 r e s p e c t i v e l y . C u r v e A o f F i g u r e 1 2 s h o w s o n l y one " w a v e " w i t h a h a l f - a b s o r b a n c e p o t e n t i a l o f -0.15 V i n t h e +0.2 t o -0.6 V i 2 a

1

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a

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i 2 a

i 2 a

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9

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a

i 2 a

i 2 a

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Potential

Volts vs SCE

Figure 9. Potential-absorbance curves for the reduction (A) and oxidation (B) of 1.2 mM B CN monitored at 475 nm. 1.0 M Na S0 ; 0.1 M KCN; pH 10.4; Hg-Ni minigrid; cell thickness, 0.017 cm. 12

2

4

Figure 10. Potential-absorbance curves for the re duction (A) and oxidation (B) of 0.9 mM Β i2a monitored at 530 nm; 1.0 M Na SO,; 0.1 M NaNO ; pH 7.01; Hg-Ni minigrid; cell thickness, 0.017 cm. 2

s


1.

Vitamin

MARK E T A L .

B

12

and Related

13

Cobalamins

2,08

-0.2

-0.4

Potential

.

-0.6

-as

Volts vs SCE

-1.0

Figure 11. Potential-absorbance (A) and oxidation (B) of 0.9 mM B monitored at 475 nm. 1.0 M Na SO 0.1 M NaNO ; pH 7.0; Hg-Ni minigrid; cell thickness, 0.017 cm. 12a

2

i;

s

Figure 12. Potential-absorbance curves for the reduction (A) and oxi dation (B) of 0.9 mM B monitored at 525 nm. 1.0 M Na SO 0.1 M NaNO ; pH 7.0; Au minigrid and 2,6-dichlorophenolindophenol; cell thickness, 0.021 cm. 12a 2

0 Potential .

-0.2 Volts

vs

i;

s

-0.4 SCE

.13

Λ Ο

I

09

Figure 13. Potential-absorbance curves for the reduction (A) and oxi dation (B) of 0.9 mM B monitored at 475 nm. 1.0 M Na SO>; 0.1 M NaN0 ; pH 7.0; Au minigrid and 2,6-dichlorophenolindophenol; cell thickness, 0.021 cm. 12a 2

,07

3

0.2

0 Potential

-0.2 Volts

-0.4 vt

SCE

-0.6



14

p o t e n t i a l r e g i o n s c a n n e d . From c u r v e Β o f F i g u r e 12 i t c a n b e seen t h a t the produced B i s totally reoxidized t oB a with l i t t l e h y s t e r e s i s ( h a l f - a b s o r b a n c e p o t e n t i a l o f a b o u t -0.09 V f o r t h e r e o x i d a t i o n ) i n t h e p r o c e s s . T h e c h a n g e s i n t h e 475-nm a b s o r b a n c e p e a k ( F i g u r e 13) a g a i n i n d i c a t e o n l y one "wave" f o r t h e g e n e r a t i o n and s u b s e q u e n t r e o x i d a t i o n o f t h e B r w i t h h a l f - a b sorbance p o t e n t i a l s which correspond f a v o r a b l y t o those o f the B " w a v e s " i n F i g u r e 12. T h u s , t h e m e d i a t o r - A u e l e c t r o d e s y s tem r e f l e c t s a m o r e t y p i c a l r e d o x b e h a v i o r a s i t e l i m i n a t e s t h e unusual h y s t e r e s i s e f f e c t where the r e o x i d a t i o n o f B from B occurred a t p o t e n t i a l s negative t o the i n i t i a l reduction process ( s e e F i g u r e s 1 0 and 1 1 ) . H o w e v e r , a n e x a m i n a t i o n o f t h e m a g n i t u d e o f t h e a b s o r b a n c e c h a n g e o f b o t h t h e 5 2 5 - a n d 475-nm p e a k s s h o w s t h a t i t i s e x a c t l y t h e same a s t h a t f o r t h e f i r s t a b s o r bance waves f o r t h e Hg-Ni e l e c t r o d e — no m e d i a t o r system (see F i g u r e s 1 0 and 1 1 ) , i n d i c a t i n B i s only p a r t i a l l The t o t a l s p e c t r u m o f t h e s o l u t i o n p o t e n t i o s t a t e d a t -0.6 V a l s o i n d i c a t e s t h a t part o f the B ( a p p r o x i m a t e l y 35%) i s u n r e a c t e d . The same r e s u l t w a s a l s o o b t a i n e d f r o m t h e η - v a l u e s t u d i e s ( T a b l e I I I ) a t b o t h t h e H g - N i and A u m i n i g r i d e l e c t r o d e s . T h u s , t h e unusual two p o t e n t i a l processes necessary t o t o t a l l y reduce B appear t o be independent o f both working e l e c t r o d e m a t e r i a l a n d mediator p a r t i c i p a t i o n . Neither the spectra f o r B o r B -CN showed any s i g n i f i c a n t r e d u c t i o n e m p l o y i n g Au m i n i g r e d - m e d i a t o r s y s t e m . No s a t i s f a c t o r y m e d i a t o r w i t h t h e n e c e s s a r y o p t i c a l a n d p o t e n t i a l c h a r a c t e r i s t i c s t o e x p l o r e t h e -0.6 t o -1.0 V p o t e n t i a l a b s o r b a n c e b e h a v i o r a t a H g - N i m i n i g r i d e l e c t r o d e has b e e n f o u n d to date. The h a l f - a b s o r b a n c e p o t e n t i a l s f o r t h e c o b a l a m i n s p e c i e s i l l u s t r a t e d i n F i g u r e s 6 through 13 a r e p r e s e n t e d i n T a b l e I . i 2 r

1 2

1 2

1 2 a

1 2 a

1

2

r

1 2 a

i 2 a

i 2 a

i 2

i 2

η-Value Determination. C o n t r o l l e d p o t e n t i a l coulometry with a t h i n l a y e r m i n i g r i d e l e c t r o d e system^°ιM was used t o determine t h e number o f e l e c t r o n s ( η - v a l u e ) f o r v a r i o u s waves found i n t h e c y c l i c voltammograms o f each o f the c o b a l a m i n s . ^ A typical charge vs. time curve f o r B i s shown i n F i g u r e 14. I t w a s n e c e s s a r y t o e x t r a p o l a t e t h e f i n a l s l o p i n g p o r t i o n o f t h e Q-t curve back t o t = 0 t o c o r r e c t f o r edge e f f e c t s i n h e r e n t i n the t h i n l a y e r c e l l s y s t e m . ^ - The m e t h o d f o r c o r r e c t i o n and c a l c u l a t i o n o f η - v a l u e s f o r c h a r g i n g and r e s i d u a l c u r r e n t b y r e p e a t i n g t h e e x p e r i m e n t o n t h e s u p p o r t i n g e l e c t r o l y t e has b e e n d e scribed p r e v i o u s l y . T h e η-values, as well as the i n i t i a l and f i n a l v a l u e s o f t h e a p p l i e d p o t e n t i a l s t e p s , a r e shown i n T a b l e I I . F o r t h e t h r e e c o b ( I I I ) a l a m i n s y s t e m s u s i n g t h e Hg-Ni m i n i g r i d e l e c t r o d e , o n l y one r e d u c t i o n w a v e i s o b s e r v e d i n t h e -1.0 V v s . SCE p o t e n t i a l r e g i o n and t h e η - v a l u e o b t a i n e d i n e a c h c a s e f r o m t h e Q v s . t d a t a w a s e f f e c t i v e l y two ( 2 ) y i e l d i n g a c o b ( I ) a l a m i n p r o d u c t i n each c a s e w h i c h c o n f i r m s p o l a r o g r a p h i c and other p r e v i o u s l y reported r e s u l t s * * " As expected, the 1 2

2

8

9

2 2

3 7


Vitamin

MARK E T A L .

Table I .

B

12

and Related

Cobalamins

Half-Absorbance Potentials*

Monitor^ Working ed electrodes system Cobalamin Reduction Oxidation wavelength (OTTLE) species mV vs. SCE mV vs. SCE (nm) Hg-Ni

B, "

Hg-Ni

B i 2

Hg-Ni Hg-Ni

Bi2-CN*B, -C!N^

Hg-Ni

B,2a^

Hg-Ni

Au Au Au Au

+ + + +

mediator mediator mediator mediator

2

W

C

2

-625 (-875) -625 -875 -850 -825 -910 -60 -635

-180 (-880) -185 -875 -690 -689 -910

-634 -880

-176 -878

— —

— —

550 475 580 475 530

-188

B

B, " Bi -CN^ 2

C

2

2a

525 475

-93 -110

-155 -140

B, ^

a

The cobalamin concentration is 1 mM. It should be pointed out that no relationship between the half-absorbance potentials and the reversible potentials for these species exists at this time. Supporting electrolyte = 1.0 M Na2S0 . < Supporting electrob

4

\Λ \Λ \λ

\ 0J

300

Time

Figure 14. Charge-time curve for the application of a potential step from 0.000 to -0.970 to +0.100 V vs. SCE at a Hg-Ni OTTLE. (A) background, 1.0 M Na SO 0.1 M NaNOs. (B) B , 0.6 mM B , 1.0 M Na SO 0.1 M NaN0 . 2

lt

12

2

i>

h

3


16


background breakdown p o t e n t i a l s h i f t s p o s i t i v e on t h e Au m i n i g r i d e l e c t r o d e a n d o v e r l a p s t h e C o ( I I I ) - C o ( I ) wave o b s e r v e d o n t h e H g - N i e l e c t r o d e . On A u e l e c t r o d e s , B and B i - C N e x h i b i t s a s m a l l prewave a t -0.3 V a n d a broad i r r e v e r s i b l e a p p e a r i n g wave a t a b o u t - 0 . 8 V. T h e B e x h i b i t s a s i n g l e very broad p o o r l y d e f i n e d w a v e w i t h a p e a k p o t e n t i a l a t a b o u t - 0 . 5 V. P o t e n t i a l step experiments w i t h B -CN gave f r a c t i o n a l η-values r e g a r d l e s s of t h e magnitude o f t h e f i r s t a p p l i e d p o t e n t i a l , t h e meaning o f which c o u l d not be i n t e r p r e t e d from t h e e l e c t r o c h e m i c a l data. The η - v a l u e f o r B o n a p o t e n t i a l s t e p t o -0.6 V a l s o y i e l d e d a f r a c t i o n a l v a l u e o f a b o u t 0.65. I t h a s b e e n p r e v i o u s l y r e p o r t e d b y some w o r k e r s t h a t o n l y B can be c o u l o m e t r i c a l l y r e duced t o B ( c o b ( I I ) a l a m i n ) i n a o n e - e l e c t r o n s t e p a t a mer cury electrode a t intermediate p o t e n t i a l s ^ A t a Hg-Ni m i n i g r i d , t h e three c o b ( I I I ) a l a m i n s were c o u l o m e t r i c a l l y reduced t o c o b ( I ) a l a m i n a t -0.97 and t h e Q v s . t c u r v e a r e o x i d a t i o n η-value equal t o 2 found which i n d i c a t e s a v i r t u a l l y q u a n t i t a t i v e r e o x i d a t i o n t o B i . F r a c t i o n a l η-values ob tained f o r B and B i - C N d e r i v e d cob(I)alamins i n d i c a t e s t h a t o n l y p a r t o f these c o b ( I I I ) a l a m i n s a r e regeneraged even a t p o s i t i v e p o t e n t i a l s . However, these r e o x i d a t i o n η-values a r e d i f f i c u l t t o i n t e r p r e t as complicating e f f e c t s a r i s e from the i n t e r f e r i n g m e r c u r y ( I I ) c y a n i d e s p e c i e s w h i c h f o r m i n some c a s e s . A t t h e Au m i n i g r i d e l e c t r o d e s o n l y p a r t o f t h e c o b ( I I I ) a l a m i n s are reduced a s e x p l a i n e d above; however, i t appears from t h e r e o x i d a t i o n η-values t h a t t h e f r a c t i o n reduced i s q u a n t i t a t i v e l y regenerated a tp o s i t i v e p o t e n t i a l s . Thea b i l i t y o f the base-off cobalamin t o form complexes w i t h metal ions a l s o obscures t h e issue.~ F u r t h e r η - v a l u e i n f o r m a t i o n was o b t a i n e d b y f i x e d wave length o p t i c a l monitoring techniques coupled with c o n t r o l l e d p o t e n t i a l coulometry t odetermine η-values f o r appropriate redox processes i n v o l v i n g vitamin B . As mentioned p r e v i o u s l y B was c h o s e n f o r t h i s i n v e s t i g a t i o n a s e a r l i e r s t u d i e s h a d s u g gested that B underwent o n l y a s i n g l e two-electron r e d u c t i o n s t e p . * - . T h e m o n i t o r i n g w a v e l e n g t h o f 4 7 5 nm was c h o s e n a s t h i s peak i s i n d i c a t i v e o f t h e presence ( o r absence) o f a c o b ( I l ) a l a m i n . Monitoring t h i s wavelength, while c o u l o m e t r i c a l l y t h e number o f e l e c t r o n s t r a n s f e r r e d t o t h e c o b a l a m i n i n t h e p r o c e s s i s m e a s u r e d , y i e l d s t h e η - v a l u e f o r each s t e p o f t h e mech anism. Table I I I summarizes t h e r e s u l t s o ft h i s s p e c t r o e l e c t r o chemical study. I t i s e v i d e n t from t h e growth and decay o f t h e 475-nm p e a k t h a t a o n e - e l e c t r o n r e d u c t i o n d o e s o c c u r a t i n t e r mediate p o t e n t i a l s and t h a t t h i s s p e c i e s can undergo a f u r t h e r o n e - e l e c t r o n t r a n s f e r t o form c o b ( I ) a l a m i n . The η - v a l u e i n t h i s case cannot be determined d i r e c t l y because o f i n t e r f e r e n c e from background. This cob(I)alamin i s r e a d i l y r e o x i d i z e d t o a cob( I l ) a l a m i n ; η value equals one. E x i s t i n g experimental c o n d i t i o n s again d i d not allow f o r an accurate determination o f the n-value 1 2

1

2

2

a

1 2

X 2 a

1 2 a

x 2 r

2 6

3 1

2

i 2 a

2

i 2

i 2

9

22

33


i 2

1.

MARK E T A L .

Table I I .

Vitamin

B

12

and Related Cobalamins

17

n- • Value Results

Minigrid working electrode system

Potential step region mV vs. SCE

No. of electrons = η

Species

Reduction Coulometry 0 to -970 B -CN*>

Hg-Ni

1.90 2.00 1.96 0.013 0.126 1.37 0.65

12

B,2a*'

0 to -500 +300 to -400 + 100 to-1000 +300 to -600

Au

B,2 a

C

C

e

B -CN -* B -CN > 12

Û

12

B,2a*'

C

Hg-Ni Bi2-CN *

0.51 0.38 0.013 0.125 1.40 0.65

Bl2a

-500 toO -400 to +300 -1000 to+100 -600 to +300

Au

B,2 ' a

c

Bi2-CN '* fl

B -CN -

a b

X2

Bi2a ' f l

C

a

b

Supporting electrolyte = 1.0 M Na S0 . Supporting electrolyte = 0.1 M KCN. Supporting electrolyte = 0.1 M NaN0 . The cobalamin concentration is 1 mM. 2

4

c

3

Table I I I .

Spectropotential Step Λ-Values for B12

Working electrode system (OTTLE) Hg-Ni

a

Potential step V vs. SCE To From Rest -0.755 Rest -0.755 -1.000 -0.755

-0.755 0.200 -0.755 -1.000 -0.755 0.200

Monitored wavelength No. of electrons (nm) 475 475 475 475 475 475

0.98 a

0.99 0.93 1.04 a

Catalytic process, η > 2.


18


for

the r e o x i d a t i o n to a c o b ( I I I ) a l a m i n .

Conclusions. T h e r e s u l t s d e s c r i b e d a b o v e show t h a t , i n s p i t e o f the f a c t t h a t the e l e c t r o k i n e t i c data are vary compli c a t e d , u n u s u a l , and v i r t u a l l y i m p o s s i b l e t o i n t e r p r e t m e c h a n i s t i c a l l y , the o p t i c a l monitoring o f the s o l u t i o n composition using t h e OTTLE t e c h n i q u e g i v e s a g o o d p i c t u r e o f t h e n e t o r o v e r a l l redox r e a c t i o n s that take place. The f i r s t o b s e r v a t i o n o f s i g n i f i c a n c e i s t h a t a l l t h r e e c o b ( I I I ) a l a m i n s ( B , B12-CN, and B a ) u n d e r g o a q u a n t i t a t i v e o n e - e l e c t r o n r e d u c t i o n t o e i t h e r t h e same o r s i m i l a r c o b ( I I ) a l a m i n ( B i ) s p e c i e s a t i n t e r m e d i a t e p o t e n t i a l s i n t h e 0.0 t o -0.8 V range. Previous electrochemical s t u d i e s by other groups had claimed that only B c o u l d be reduced t o Β at intermediate p o t e n t i a l s . » ~ A s t h e p o l a r o g r a p h i c and c y c l i c v o l t a m m e t r i c s t u d i e s d i d not i n d i c a t range f o r B o r B -CN attempted c o u l o m e t r i c r e d u c t i o n s a t such p o t e n t i a l s . However, t h e OTTLE r e s u l t s c l e a r l y show t h a t t h e o n e - e l e c t r o n r e a c t i o n i s common t o a l l t h e s p e c i e s b u t t h a t i n t h e c a s e o f B a n d B - C N the k i n e t i c s o f the r e a c t i o n i s u n u s u a l l y slow even w i t h r e s p e c t t o t h e s l o w s c a n r a t e s e m p l o y e d i n p o l a r o g r a p h y and t h e c y c l i c voltammetry reported here. These one-electron processes f o r B a n d B - C N show u p o n l y d u r i n g p o i n t - b y - p o i n t p o t e n t i o s t a t i c OTTLE t e c h n i q u e s . The r e a s o n f o r t h e e x t r e m e l y s l o w k i n e t i c s o f t h i s o n e - e l e c t r o n r e a c t i o n has n o t b e e n e l u c i d a t e d a t t h i s t i m e . The e l e c t r o n t r a n s f e r r a t e i s f a s t e n o u g h f o r w a v e s t o b e ob served p o l a r o g r a p h i c a l l y or w i t h c y c l i c voltammetry only i n the Β c a s e . U n d e r t h e same c o n d i t i o n s t h e f u r t h e r r e d u c t i o n o f a l l the cobalamin systems from the Co(II) t o Co(I) o x i d a t i o n s t a t e was q u a n t i t a t i v e a n d " r e v e r s i b l e " . The a p p a r e n t h y s t e r e s i s i n v o l v i n g C o ( I I ) to C o ( I I I ) cobalamins i s not p r e s e n t l y w e l l u n d e r s t o o d b u t may r e s u l t f r o m c h e m i c a l r e a c t i o n s i n v o l v e d i n t h e mechanism. It i s i n t e r e s t i n g to note that B -CN i s t o t a l l y re-formed ( s h o w n i n c u r v e B, F i g u r e 9 ) w h i l e c y a n o c o b ( I ) a l a m i n d o e s n o t completely reoxidize to B . T h i s s u g g e s t s t h a t B and B i - C N may r e o x i d i z e b y s e p a r a t e p a t h w a y s . B e c a u s e o f t h e m a g n i t u d e o f the i r r e v e r s i b i l i t y o fthe B r e d o x c o u p l e and a l s o t h e f a c t that B i s n o t t o t a l l y r e - f o r m e d (some B i appears t o be a minor r e o x i d a t i o n product), i t i s thought that on electrochemical reoxidation that B i s t h e i n i t i a l p r o d u c t f o r m e d and t h a t B subsequently forms on a l i g a n d exchange r e a c t i o n i n v o l v i n g the cyanide i n s o l u t i o n ( i n i t i a l l y r e l e a s e d i n t o the s o l u t i o n phase during the reduction o f B t o B i r » a s shown b y t h e f a c t t h a t a -0.1 t o -0.8 V OTTLE e x p e r i m e n t ( c o b ( I I I ) a l a m i n cob(II)alamin) with vitamin B shows t h e same l a r g e i r r e v e r s i b i l i t y i n d i c a t i n g t h a t t h e CN" i s l o s t i n t h e f i r s t r e d u c t i o n s t e p ) . T h i s l i g a n d exchange r e a c t i o n o f B i w i t h C N " has b e e n shown t o b e v e r y f a s t . — However, the net r a t e i s slow because o f the d i l u t e i 2

1 2

2 r

i 2 a

6

2 6

i 2

1

2

Γ

3 1

i 2

1 2

1 2

1 2

1 2

a

1 2

i 2

i 2

2

i 2

1 2

2 a

X 2 a

i 2

i 2

2

i 2

2 a


1.

Vitamin

MARK E T AL.

B

12

and Related

19

Cobalamins

s o l u t i o n s employed. The c o m p l e t e r e g e n e r a t i o n o f B i s not pos s i b l e a s some CN' i s l o s t , p r o b a b l y t h r o u g h t h e f o r m a t i o n o f s t a b l e m e r c u r y ( I I ) cyanide complexes. I t was n o t e d t h a t t h e p e r cent recovery increased on a d d i t i o n of excess cyanide which i s c o n s i s t e n t w i t h t h i s i n t e r p r e t a t i o n . With r e s p e c t to B -CN, the f i n a l p r o d u c t i s formed d i r e c t l y upon r e o x i d a t i o n o r t h e f o l l o w up l i g a n d e x c h a n g e r e a c t i o n b e t w e e n t h e c o n c e n t r a t e d c y a n i d e s o l u t i o n a n d t h e B , f o r m e d b y t h e l o s s o f one e l e c t r o n f r o m B (with water molecules i n the a x i a l p o s i t i o n s ^ - ) , i s very fast. At t h i s time i t i s i m p o s s i b l e t o d i s t i n g u i s h between t h e s e two m e c h a n i s m s f o r t h e r e o x i d a t i o n o f B - C N . H o w e v e r , i t s h o u l d be n o t e d t h a t t h e B s p e c t r a ( a s i n d i c a t e d b y t h e 475-nm p e a k ) i n b o t h B and B - C N r e a c t i o n s a r e v i r t u a l l y i d e n t i c a l . P e r h a p s t h e m o s t u n u s u a l and d i f f i c u l t t o u n d e r s t a n d r e s u l t is the observation of f o r the reduction of B and t h e a p p a r e n t η - v a l u e d a t a i n d i c a t e t h a t B convertst o B ( a b o u t 65%) a t p o t e n t i a l s a r o u n d -0.05 V a t b o t h t h e H g - N i and Au e l e c t r o d e s w h i l e i t i s n e c e s s a r y t o r a i s e t h e p o t e n t i a l t o g r e a t e r t h a n -0.6 V w h e r e t h e s e c o n d w a v e c o r r e s p o n d i n g t o t h e r e d u c t i o n o f the r e m a i n i n g 35% o f the B i s o b s e r v e d . The m o s t obvious c o n c l u s i o n t h a t f i t s the data q u a l i t a t i v e l y i s t h a t the B e m p l o y e d i n t h e s e e x p e r i m e n t s was i m p u r e a n d c o n t a i n e d a b o u t 35% o f B itself (B was p r e p a r e d f r o m B ) . However, a s p o i n t e d o u t a b o v e , we f o u n d t h a t a l l b a t c h e s o f B gave the same r e s u l t s w h i c h a g a i n w o u l d n o t b e e x p e c t e d t o r e m a i n c o n s t a n t i f the various synthesis routes y i e l d e d only p a r t i a l conversion. A l s o t h e s p e c t r a l and p o l a r o g r a p h i c p r o p e r t i e s d o n o t s u g g e s t t h a t any a p p r e c i a b l e c o n c e n t r a t i o n o f B r e m a i n u n c o n v e r t e d and a l s o a r e i d e n t i c a l w i t h t h e s p e c t r a and p o l a r o g r a p h i c p r o p e r t i e s of vitamin B produced by the t o t a l l y d i f f e r e n t procedures. Furthermore, there i s considerable other i n d i r e c t evidence t h a t t h e r e i s no s i g n i f i c a n t u n c o n v e r t e d B i n the B samples. Note f i r s t o f a l l t h a t t h e r e i s no-0.6 V p o l a r o g r a p h i c wave f o r B t h a t c o r r e s p o n d s t o t h e wave f o r t h i s s e c o n d B species. ( I t i s i n t e r e s t i n g to note t h a t previous p o l a r o g r a p h i c s t u d i e s had r e f e r r e d t o t h e w a v e a t -0.6 V a s a n i m p u r i t y . ) > * * A l though the c y c l i c voltammogram f o r B does e x h i b i t an a n o d i c p e a k a t -0.28 V w h i c h c o u l d b e i n d i c a t i v e o f Hg o x i d a t i o n i n t h e p r e s e n c e o f a c o m p l e x i n g l i g a n d , t h i s w a v e i s a b o u t 5 0 mV p o s i t i v e t o t h e peak c o r r e s p o n d i n g t o m e r c u r y - c y a n i d e f o r m a t i o n i n the B c y c l i c voltammogram and t h e r e i s no c o r r e s p o n d i n g c a t h o d i c sweeps o f B i t s e l f . F i n a l l y high p r e s s u r e l i q u i d chroma t o g r a p h y ( u s i n g a m i x t u r e o f e i t h e r 80% i s o p r o p y l a l c o h o l and 20% w a t e r , o r 65% methanol and 35% w a t e r , a t 2000 p s i o n a n A m i n e x A - 4 c o l u m n , w i t h d e t e c t o r w a v e l e n g t h s e t a t λ 360 nm) o n Bi h a s e x h i b i t e d two c l o s e l y s p a c e d y e t d i s t i n c t p e a k s b o t h w i t h r e t e n t i o n times t h a t are d i f f e r e n t than B . Also, a thin l a y e r c h r o m a t o g r a p h i c c o m p a r i s o n o f B and B u s i n g a 65% 1 2

1 2

1 2 a

1 2 r

1 2

i 2 r

i 2

1 2

1

2

a

1 2 r

1

2

a

1 2 a

i 2

i 2 a

1 2

1

2

a

1 2

1

2

a

1 2

1

1 2

1

2

31

1

2

2

a

a

6

a

1 2

1 2

2 a

1 2

1 2

1

2

a


ELECTROCHEMICAL

20

STUDIES O F BIOLOGICAL

SYSTEMS

m e t h a n o l a n d 3 5 % w a t e r s o l v e n t s y s t e m has shown t h a t B and Bi2a have r e l a t i v e f r o n t s , t h o u g h no s e p a r a t i o n o f B a itself was o b s e r v e d . I t i s f e l t . t h a t r i n g s t r u c t u r e d i f f e r e n c e s would n o t a c c o u n t f o r t h e two u n i q u e B a s p e c i e s , a s t h e B a p r e p a r e d f r o m t h r e e t e c h n i q u e s ( p o t e n t i o s t a t i c , b i o l o g i c a l and c h e m i c a l ) would not g i v e i d e n t i c a l 65/35 r a t i o o f c o n c e n t r a t i o n s . Moreo v e r , i t i s h a r d t o u n d e r s t a n d how two d i f f e r e n t r i n g s , w h i c h w o u l d b e e x p e c t e d t o b e common t o a l l c o b a l a m i n s , e x h i b i t d r a s t i c reduction potential differences for B and n o t f o r B o r B CN. T h u s , i t i s a t t r a c t i v e t o s p e c u l a t e t h a t t h e two s p e c i e s r e present d i f f e r e n c e s i n a x i a l l i g a n d c o n f i g u r a t i o n . The simplest a n s w e r w o u l d b e t h a t one o f t h e B i species c o n t a i n s water molec u l e s i n t h e X and Y p o s i t i o n s ( t h e " b a s e - o f f " f o r m ) w h i l e t h e o t h e r i s i n t h e c o n f i g u r a t i o n w i t h one w a t e r i n t h e X p o s i t i o n and t h e 5 , 6 - d i m e t h y l b e n z i m i d a z o l e i n t h e Y p o s i t i o n ( t h e " b a s e on" f o r m ) . T h e s p e c t r o e l e c t r o c h e m i c a t h a t t h e two B specie T h u s i u s has shown t h a t t h e X p o s i t i o n o f B i s very l a b i l e ( r a t e c o n s t a n t s o f about 170-2300 M - s " ) . — However, no measurem e n t s h a v e b e e n made o n t h e Y p o s i t i o n b e n z i m i d a z o l e - H 0 e x c h a n g e r a t e s . — I t i s p o s s i b l e t h a t t h i s exchange c o u l d be very slow. The f a c t t h a t t h e d i a q u o c o b ( I I I ) i n a m i d e ( h a v i n g n o b e n z i m i d a z o l e a t t a c h e d t o t h e c o r r i n r i n g s i d e c h a i n ) has b e e n r e p o r t e d t o b e d i f f i c u l t t o r e d u c e ( t f w — -0.7 V ) — i s c o n s i s t e n t b u t n o t p r o o f of the "base-on"-"base-off" explanation. This f a c t suggests that t h e " b a s e - o n " a q u o c o b ( I I I ) a l a m i n f o r m has a c o n f i g u r a t i o n f a v o r a b l e t o r e d u c t i o n ( t h e -0.15 V w a v e ) and t h e " b a s e - o f f " f o r m which would c l o s e l y correspond t o a diaquocob(III)inamide c o n f i g u r a t i o n i s d i f f i c u l t t o r e d u c t (-0.6 V w a v e ) . - ^ H o w e v e r , r e c e n t s p e c t r o e l e c t r o c h e m i c a l s t u d i e s b y L e x a and S a v e a n t ^ - h a v e shown that a tplatinum grid electrodes B does not e x h i b i t t h i s two w a v e o n e - e l e c t r o n b e h a v i o r . O n l y one w a v e i s o b s e r v e d a t a b o u t 0.0 V. F u r t h e r m o r e , t h e y h a v e a l s o shown t h a t t h e d i a q u o c o b ( I l l ) i n a m i d e o x i d i z e s m e r c u r y m e t a l s p o n t a n e o u s l y and f o r m s t h e c o b ( I I ) i n a m i d e . Thus, t h e r e p o r t e d v a l u e s f o r t h i s compound * a r e i n c o r r e c t and r e a l l y c o r r e s p o n d t o t h e s u b s e q u e n t e l e c t r o chemical r e d u c t i o n o f the Cob(II)inamide. Thus, i t appears t h a t t h e m e r c u r y e l e c t r o d e i s i n some w a y t h e c a u s e o f t h i s two w a v e one-electron observation. I t i s o b v i o u s t h a t t h e r e a r e many u n a n s w e r e d q u e s t i o n s c o n c e r n i n g r a t e s o f the m i c r o s c o p i c precesses i n v o l v e d i n the redox c h e m i s t r y o f cobalamin complexes. However, the macroscopic r e sultant e f f e c t o felectrode potential i n s o l u t i o n composition i s now w e l l d e f i n e d . W i t h t h i s b a s i c o v e r a l l m e c h a n i s t i c i n f o r m a t i o n , a m o r e c o m p r e h a n s i v e s t u d y o f t h e e l e c t r o d e k i n e t i c s and time resolved s p e c t r a l s t u d i e s on p o t e n t i a l step experiments on t h e s e a n d o t h e r c o b a l a m i n s u n d e r v a r i a b l e c o n d i t i o n s o f pH, s u p p o r t i n g e l e c t r o l y t e , and e l e c t r o d e m a t e r i a l may e l u c i d a t e a l l t h e steps i n the o v e r a l l mechanism. i 2

i 2

i 2

X 2

1

2

a

i 2

1 2

2 a

i 2 a

i 2 a

1

2

2

1

1

2

a

1


7

1.

Vitamin

MARK E T A L .

B

and Related

12

Cobalamins

21

The E l e c t r o c h e m i c a l B e h a v i o r o f 5 ' - D e o x y a d e n o s y l - C o b a l a m i n enzyme B i T T

(Co-

The u n d e r s t a n d i n g o f t h e r e d o x p r o c e s s e s o f t h e c o b a l a m i n coenzyme, 5'-deoxyadenosylcobalamin (coenzyme B o r A d e n - B ) , is fundamental i n c o n s t r u c t i n g a v a l i d r e a c t i o n sequence f o r cob a l a m i n compounds i n n a t u r e . H i l l O f p a r t i c u l a r i m p o r t a n c e i s t h e o x i d a t i o n s t a t e o f c o b a l t i n A d e n - B and i t s i n f l u e n c e o n the r e a c t i v i t y of the cobalamin species. As a matter of notat i o n a l e a s e and t o m a i n t a i n c o n s i s t e n c y w i t h e x i s t i n g v i t a m i n B c o n c e p t s , t h e c o b a l a m i n c o e n z y m e has b e e n p r i m a r i l y i n t e r preted i n terms of a C o and a n a d e n s y l c a r b a n i o n . — R e d o x mesomers c o n s i s t i n g o f a C o and a n a d e n o s y l r a d i c a l o r a C o and a n a d e n o s y l c a r b o n i u m i o n h a v e a l s o b e e n f o r m a l l y c o n s i d e r e d . A l t h o u g h numerous p u b l i c a t i o n s have assumed the c o b a l t t o be i n t h e +3 o x i d a t i o n s t a t e , i s a v a i l a b l e . Some e v i d e n c t h e c o b a l t o f t h e c o b a l a m i n s h o u l d b e c o n s i d e r e d as a d i v a l e n t species.^-Z! S i m i l a r e l e c t r o c h e m i c a l and s p e c t r o e l e c t r o c h e m i c a l t e c h n i ques u t i l i z i n g a n amalgamated g o l d m i n i g r i d e l e c t r o d e i n a t h i n l a y e r c o n f i g u r a t i o n have been employed t o examine t h e r e d o x sequence o f 5'-deoxyadenosylcobalamin. This i n v e s t i g a t i o n suggests t h a t the cobalamin coenzyme undergoes a s i n g l e e l e c t r o n r e d u c t i o n to form v i t a m i n B ç , i n d i c a t i n g cleavage of the c o b a l t - c a r b o n b o n d . The r e o x i d a t i o n o f t h e r e d u c e d s y s t e m c o n t a i n i n g t h e B ^ o c c u r s v i a two c o n s e c u t i v e s i n g l e e l e c t r o n t r a n s f e r s r e s u l t i n g i n the q u a n t i t a t i v e formation of vitamin B , aquocobalamin. High performance l i q u i d chromatography confirmed t h a t 5'-deoxyadenos i n e i s the u l t i m a t e form of the cleaved 5'-deoxyadenosyl moiety. These r e s u l t s i n d i c a t e t h a t 5'-deoxyadenosyl-cobalamin i s reduced by a s i n g l e e l e c t r o n to form the c o b ( I ) a l a m i n , B , w h i c h i s t h e common r e d u c t i o n p r o d u c t o f a l l c o b a l a m i n s p e c i e s . — The f o r m a t i o n o f t h e c o b ( I ) a l a m i n , B , f r o m A d e n - B i s s i g n i f i c a n t as numerous p u b l i c a t i o n s ^ have a l l u d e d t o t h e f a c t t h a t due t o t h e h i g h n u c l e o p h i l i c i t y o f t h e c o b ( I ) a l a m i n s p e c i e s i t may b e t h e b i o l o g i c a l l y a c t i v e f o r m o f t h e c o e n z y m e . The r e s u l t reported herein i s the f i r s t electrochemical evidence f o r the f o r m a t i o n o f the c o b ( I ) a l a m i n from the cobalamin coenzyme. That B can be c o m p l e t e l y r e o x i d i z e d t o B , a c o b ( I I I ) a l a m i n , i s s i g n i f i c a n t i n t h a t e x i s t i n g c y c l i c biochemical mechanisms i n volve Aden-B a s the i n i t i a l l y r e a c t i v e s p e c i e s . ^ Furthermore, r i b o n u c l e o t i d e r e d u c t a s e has shown s p e c i f i c a c t i v i t y t o w a r d B s and A d e n - B i n t h e p r e s e n c e o f ô'-deoxyadenosine.^- -»- -!*1 2

1 2

1 2

1 2

3 +

2 +

1 +

i 2

1 2

1 2 a

i 2 S

1 2 S

1 2 S

1 2

X 2 a

1 2

1 2

3

7

1 2

Acknowledgments T h i s r e s e a r c h was s u p p o r t e d i n p a r t b y t h e N a t i o n a l S c i e n c e F o u n d a t i o n , NSF CHE76-04321 and t h e N a t i o n a l I n s t i t u t e s o f H e a l t h , GM-22713-01.


22

electrochemical studies of biological systems

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

14. 15.

16. 17. 18. 19. 20.

Pratt, J. M., "Inorganic Chemistry of Vitamin B ," Academic Press, New York, Ν. Y., 1972, pp. 15-17. Huennekens, F. M., in "Biological Oxidations," Thomas P. Singer, Ed., Interscience Publishers, New York, Ν. Y., pp. 482-502. Tackett, S. L . , Collat, J. W., and Abbot, J. C., Biochemis try, (1963), 2, 919. Collat, J. W. and Abbot, J. C., J. Amer. Chem. Soc., (1964), 86, 2308. Schrauzer, G. N., Deutsch, E. and Windgassen, R. J., J. Amer. Chem. Soc., (1968), 90, 2441. Yamada, R., Shimizu, S. and Fukui, S., Biochemistry, (1968), 2, (7), 1713. Rudiger, H., Eur Birke, R. L . , Brydon, , Boyle, , Chem., (1974), 52, 237. Kenyhercz, T. M. and Mark, Jr., Η. B., Anal. Lett., (1974), 7, 1. Heineman, W. R., Norris, B. J. and Goelz, J. Anal. Chem., (1975), 47, 79. Heineman, W. R., DeAngelis, T. P. and Goelz, J., Anal. Chem., (1975), 47, 1364 Kenyhercz, Τ. Μ., DeAngelis, T. P., Norris, B. J., Heineman, W. R. and Mark, Jr., H. B., J. Amer. Chem. Soc., (1975), 98, 2469. However, experiments were performed with the minigrid elec trode area increased to occupy the entire cell volume to determine if the edge effects from the diffusion of unreduced cob(III)alamins from solution not in immediate contact with the minigrid affected the time resolved spectra. No signifi cant difference was observed. Strojek, J. W., Gruver, G. and Kuwana, T., Anal. Chem., (1969), 41, 481. Mark, Jr., H. B., Wilson, R. M., Miller, T. L . , Atkinson, T. V., Yacynych, A. M., and Woods, H., "The On-Line Computer in New Problems in Spectroscopy: Applications to Rapid Scanning Spectroelectrochemical Experiments and Time Resolved Phosphorescence Studies" in "Information Chemistry; Computer Assisted Chemical Research Design," S. Fujiwara and H. B. Mark, Jr., Eds., University of Tokyo Press, Tokyo, Japan, 1975, pp. 3-28. Beaven, G. H. and Johnson, Ε. Α., Nature, (1955), 176, 1264. Ref. 1, p. 184. Ref. 1, p. 20 to 27. Ref. 1, p. 55 The cyanoaquocol(III)inimide was prepared by a previously described method; Ref. 1, p. 294. 12


1.MARKETAL. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.

23

Vitamin B and Related Cobalamins 12

Basolo, F. and Pearson, R. G., "Mechanisms of Inorganic Re actions," John Wiley and Sons, Inc., New York, Ν. Y . , pp. 141-144. Diehl, H., Sealock, R. R., and Morrison, J., Iowa State Coll. J. S c i . , (1950), 24, 433. Diehl, H., Morrison, J . I., and R. R. Sealock, Experientia, (1951), 7, 60. Diehl, J., and Morrison, J . I., Rec. Chem. Prog. (1972), 31, 15. Boos, R. N., Carr, J . E . , and Conn, J . B., Science, (1953), 117, 603. Jaselskis, B. and Diehl, H., J. Am. Chem. Soc., (1959), 81, 4345. Jaselskis, B. and Diehl, H . , J . Am. Chem. Soc., (1958), 80, 2147. Collat, J . W., an (1962), 4, 59. Tackett, S. L . , Ph.D. Thesis, Ohio State University, 1962. Kratochvil, B., and Diehl, H., Talanta, (1966), 13, 1013. Hogenkamp, H. P. C. and Holmes, S., Biochemistry (l970), 9, 1888. Lexa, D. and L'hoste, J . M., in "Biological Aspects of Elec trochemistry," G. Milazzo, P. E. Jones, and L. Rampazzo, Ed., Birkhauser Verlag, Stuttgart, 1971, pp. 395-404. Abd-el-Nabey, Β. Α., J . Electroanal. Chem., (1974), 53, 17. Das, P. K. et al., Biochim. Biophys. Acta., (1967), 141, 644. Tackett, S. L. and Ide, J . W., J . Electroanal. Chem. (l971 ), 30, 510. Swetik, P. G., and Brown, D. G., J. Electroanal. Chem., (1974), 51, 433. Kenyhercz, T. M. and Mark, J r . , Η. B., in preparation. Murray, R. W., Heineman, W. R., and O'Dom, G. W., Anal. Chem., (1967), 39, 1666. Provided by Dr. E. A. Deutsch, Department of Chemistry, University of Cincinnati. Beaven, G. H. and Johnson, Ε. Α., Nature (London), (1955), 176, 1264. It should be pointed out the reaction B = [H ] B (hydroxycob(III)alamin) has a pk of 7.8 and a more negative reduction potential than B : H. O. A. H i l l , "Inorganic Biochemistry," Vol. 2, G. Eichcon, Ed., Elsevier, New York, Ν. Y., 1973, Chapter 30. This proton equilibrium is un doubtedly fast and, thus, only the reduction of the B will be observed in these OTTLE experiments. McDuffie, B., Anderson, L. B., and Reilley, C. N., Anal. Chem., (1966), 38, 883. Cotton, F. A. and Wilkinson, G., "Advanced Inorganic Chemis try," 3d ed., Interscience, New York, Ν. Y., 1972, p. 519. Thusius, D. J . Am. Chem. Soc., (1971), 93, 2629. +

+

12a

12b

a

12a

12s

42. 43. 44.


24 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72.

ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYS Kenyhercz, T. M., Yacynych, Α. Μ., and Mark, J r . , H. B., Anal. Lett., (1976), 9, 203. Reference 6, p. 4346. Reference 11, p. 1889. Lexa, D. and Saveant, J . M., University of Paris, Private Communication, 1976. Babior, Β. M. in "Cobalamin: Biochemistry and Pathophysiol ogy," Β. M. Babior, Ed., John Wiley and Sons, New York, N.Y., 1975, p. 141. Pratt, J . M., "Inorganic Chemistry of Vitamin B12," Academic Press, London, 1972, p. 296. Mahler, H. R. and Cordes, Ε. Η., "Biological Chemistry," Harper and Row, Evanston, Ill., 1971, p. 427. H i l l , J . Α., Pratt, J . M., and Williams, R. J. P., J . Theor. Biol., (1962), 3, 423 Hogenkamp, H. P. C . Biochem. Biophys. H i l l , J . Α., Pratt, J . M. and Williams, R. J . P., J . Chem. Soc., (1964), 5149. Pratt, J . M., J . Chem. Soc., (1964), 5154. Huennekens, F. M. in "Biological Oxidations," T. P. Singer, Ed., John Wiley and Sons, New York, Ν. Y . , 1968, p. 483. Hogenkamp, H. P. C. and Holmes, S., Biochem., (1970), 9, 1889. Cotton, F. A. and Wilkinson, G., "Advanced Inorganic Chemis try," John Wiley and Sons, New York, Ν. Y. 1972, p. 888. Hughes, M. N., "The Inorganic Chemistry of Biological Pro cesses," John Wiley and Sons, New York, Ν. Y . , 1974, p. 187. Costa, C . , Puzeddu, A. and Reisenhofer, E . , Bioelectrochem. Bioenerg., (1974), 1, 29. H i l l , H. A. O. in "Inorganic Biochemistry," G. L. Eichhoren, Ed., Elsevier, New York, Ν. Y . , 1975, p. 1076. Babior, Β. M., Acc. Chem. Res., (1975), 8, 378. Abeles, R. H. and Dolphin, D., Acc. Chem. Res., (1976), 9, 114. Seki, H., Shida, T . , and Imamura, Μ., Biochem. Acta., (1974), 372, 106. H i l l , H. A. O., Pratt, J . M. and Williams, R. J . P., Disc. Farad. Soc., (1969), 16S. Kratochvil, B. and Diehl, H., Talanta, (1966), 13, 1013. Nowick, L. and Pawelkiewicz, Bull. Acad. Pol. Sci. C l . II., (1960), 17., 433. Johnson, A. W. and Shaw, N., Proc. Chem. Soc., (1960), 420. Bernhauser, K., Gaiser, P., Muller, O., Muller, E . , and Gunter, F . , Biochem. (1961), 333, 560. Johnson, A. W., Mervyn, L., Shaw, N., and Smith, E. L., J . Chem. Soc., (1963), 4146. White, Α., Handler, P., and Smith, E. L., "Principles of Biochemistry," McGraw H i l l , St. Louis, Mo., 1973, p. 1173. Schrauzer, G. N. and Sibert, J . W., J . Amer. Chem. Soc., (1970), 92, 1022, and references therein.


1.MARKETAL. 73. 74.

Vitamin B and Related Cobalamins 12

25

Hamilton, Α., Yamada, R., Blakley, R. L., Hogenkamp, H.P.C., Looney, F. D., and Winfield, M. E., Biochem. (1971), 10, 347. Tamao, Y. and Blakley, R. L., Biochem., (1973), 12, 24.


2 Bioelectrochemical Modelling of Cytochrome c CHARLES C. Y. TING and JOSEPH JORDAN 152 Davey Laboratory, Department of Chemistry, Pennsylvania State University, University Park, Penn. 16802 MAURICE GROSS Laboratoire d'Electrochimie et Chimie-Physique du Corps Solide, Université Louis Pasteur, BP 296 R/8, 67008 Strasbroug, France Numerous papers have in recent years been devoted to electrochemical studies of porphyrin cally, an investigation chrome c) is conspicuous by its absence. Heme c was first prepared from the naturally occurring protein in a classical piece of work by Theorell (1). Subsequently both heme c and its equatorial ligand (porphyrin c) became accessible by the synthetic route (2,3) outlined in Figure 1. The salient feature of porphyrin c is the bis-cysteinated substitution on the ring, which is unique in cytochrome c among hemoproteins (4). The corresponding substituents in hemoglobin and myoglobin are vinyl groups (5,6). In the present paper, we report some preliminary findings on the electrochemical behavior of porphyrin c and heme c. Experimental Materials. Porphyrin c and heme c were synthesized ad hoc using procedures referred to earlier in this write-up. Yields and elemental analyses are summarized in Table I. Table I Compound

Mol. % 1 Wt. Yield

Porphyrin c 805.0 40 Heme c 917.8 90

% Theory 0 6.08

Elemental Analysis Fe %S % Ν Actual Theory Actual Theory Actual 8.05 10.44 10.10 0.22 7.97 8.94 9.16 6.05 6.99 6.81

Authenticity was verified with the aid of the spectra illustrated in Figure 2 recorded with the aid of a Bausch and Lomb 505 Spectro photometer and quartz cells whose optical pathlength was 1 and 0.1 cm. P.C. Polarography. Current-voltage curves were recorded at a conventional (dropping mercury electrode (dme) which had 26


2.

TING E T A L .

Bioelectrochemical

the f o l l o w i n g c h a r a c t e r i s t i c s : c i r c u i t ) = 4.55 s e c o n d s .

Modelling

m

=

1.66

of Cytochrome

c

27

mg p e r s e c o n d ; t ( o p e n

C y c l i c V o l t a m m e t r y . K e m u l a ' s h a n g i n g chrop m e r c u r y e l e c t r o d e (hdme) s e r v e d a s i n d i c a t o r e l e c t r o d e . P o t e n t i a l s c a n r a t e s i n a r a n g e b e t w e e n 0.01 and 5 0 v o l t * s e c " w e r e u s e d . 1

Coulometry. Current-time i n t e g r a l s were determined a t appro p r i a t e c o n t r o l l e d p o t e n t i a l s , corresponding to well-defined polar o g r a p h i c d i f f u s i o n c u r r e n t d o m a i n s . The c a t h o d e was a m e r c u r y p o o l o f 2.60 s q . cm. Instrumentation, Solvents, Supporting E l e c t r o l y t e s , etc. A l l e x p e r i m e n t s w e r e c a r r i e d o u t a t 25°C. On s o l u b i l i t y c o n s i d e r a t i o n s DMF and w a t e r w e r e u s e d a s s o l v e n t s f o r heme c and p o r p h y r i n c r e s p e c t i v e l y . 0.1 M l y t e i n a l l experiments t h r o u g h o u t , u s i n g a s a t u r a t e d a q u e o u s £alomel r e f e r e n c e e l e c t r o d e (SCE) a n d a p l a t i n u m f o i l a u x i l i a r y c o u n t e r e l e c t r o d e . A l l e l e c t r o c h e m i c a l measurements were performed w i t h the a i d o f a m u l t i purpose instrument equipped w i t h advanced s o l i d s t a t e o p e r a t i o n a l a m p l i f i e r a n d f e e d b a c k c i r c u i t s , v i z , t h e M o d e l 170 E l e c t r o c h e m i c a l S y s t e m s u p p l i e d by £ r i n c e t o n A p p l i e d R e s e a r c h (PAR) C o r p o r a t i o n , P r i n c e t o n , N.J. O u t p u t s i g n a l s were a u t o m a t i c a l l y c o r r e c t e d f o r i R d r o p s a n d r e c o r d e d o n a b u i l t - i n X-Y pen r e c o r d e r a n d / o r w i t h t h e a i d o f a d i g i t a l o s c i l l o s c o p e ( M o d e l 1090 w i t h M o d e l 9 0 p l u g - i n u n i t , N i c o l e t Instrument Corporation, Madison, Wisconsin). The s c o p e was e q u i p p e d w i t h a mi n i - c o m p u t e r w h i c h had c a p a b i l i t i e s o f s t o r i n g d a t a i n a 4 0 9 6 χ 4 0 9 6 a r r a y memory a s f a s t as 5 y s e c p e r d a t a p o i n t . The c o u p l i n g o f t h e PAR i n s t r u m e n t w i t h t h e o s c i l l o s c o p e a l l o w e d measurement o f f a s t l i n e a r sweep voltammograms w i t h an a c c u r a c y o f 1 p e r c e n t . Whenever a p p r o p r i a t e , t o t a l c u r r e n t s were c o r r e c t e d f o r r e s i d ual c u r r e n t s to y i e l d the corresponding f a r a d a i c c u r r e n t s . Poten t i a l s are r e p o r t e d i n accordance w i t h the Stockholm Sign Conven t i o n o f t h e I n t e r n a t i o n a l U n i o n o f P u r e and A p p l i e d C h e m i s t r y ( 7 ) , i . e . , t h e more c a t h o d i c ( r e d u c i n g ) a p o t e n t i a l t h e more n e g a t i v e i t s assignment. R e s u l t s and

Discussion

Experimental 1.

f i n d i n g s are summaried below.

E l e c t r o a n a l y t i c a l Chemistry of Porphyrin c. D.C. p o l a r o g r a m s o f p o r p h y r i n c y i e l d e d two c a t h o d i c w a v e s w i t h w e l l - d e f i n e d l i m i t i n g c u r r e n t s whose c h a r a c t e r i s t i c s a r e l i s t e d i n Table I I .


28


Table I I Half-Wave Limiting Current Potential P o t e n t i a l Domain J idt (faradays-mole ) ( v o l t vs. SCE) (volt) - 1

-0.525 -0.730

-0.56 -0.88

2.02 ± 0.04 3.94 ± 0.02

t o -0.62 t o -1.0

Wave A n a l y s i s Slope (volt)

7

0.030 0.054

* S l o p e o f p l o t o f l o g [ ( i - i ) / i ] v e r s u s E. H

2. 3.

4.

Coulometry a t -0.615 v o l t v e r s u s SCE ( o n t h e f i r s t l i m i t i n g current plateau) substantiated a two-electron transfer i . e . two f a r a d a y s p e r m o l Coulometry a t -0.94 g current plateau) i n d i c a t e d t h e occurrence o f an o v e r a l l f o u r e l e c t r o n t r a n s f e r , i . e . , a t o t a l o f f o u r f a r a d a y s p e r mole o f porphyrin c. C y c l i c V o l t a m m e t r y a t t h e hdme y i e l d e d t h e f o l l o w i n g r e s u l t s . ( a ) A t r e l a t i v e l y f a s t p o t e n t i a l s c a n r a t e s (10

[i4]aneN [i5]aneN [i6]aneN Me [i4]aneN Me [l4]aneN 4

4

4

6

4

(volts)^

+3

+2

+.24 + .49 qr +.66 i + .27 +.38

4

2

2

-

+1

-2.3 i -2.1i -2.2 i -2.1 i

Abbreviations: q r , quasi-reversible; i , i r r e v e r s i b l e . F r o m Table IV the same effects are evident for iron complexes as for nickel complexes; i . e . , increasing ring size favors the lower state as does the presence of axial C H groups. Now, comparing these values (Table TV) with those in Table ΙΠ for unsaturated ligands, the following results are found (averaged to fit the several cases r e a s o n à ably well) : Et/2 = +140 m V for a pair of axial C H groups, +300 m V for an α-diimine group, and +50 mV for one isolated C=N. F r o m these values, it i s apparent that the enhanced sensitivity of the F e / F e couple to thejpresence of ligand unsaturation resides in the rather large effect of the α-diimine group. T h i s can be attributed to the special stability of the Fe -a-diimine r i n g structure (15). 3

3

2 +


3 +

3.

Potentials

BUSCH E T A L .

of Metal

Ion

Couples

in

41

Complexes

In view of the systematic structure-potential relationships observed for the N i / N i and F e / F e couples, it i s surprising that the C o / C o couple is insensitive to variations in the degree of ligand unsatura tion (16). A s i m i l a r observation (17) has been made on the complexes Co(MAC)(H 0) . T h i s is perhaps the result of the fact that the orbital receiving the added electron (d 2) is oriented on the ζ axis and relatively unaffected by the in-plane ligand field. F u r t h e r , since the unsaturated macrocyclic ligands are relatively inflexible, as compared to the satu rated tetraaza macrocyclic ligands discussed e a r l i e r , little rearrange ment is likely to occur upon reduction. The studies by Endicott et al (18) on the electrode potentials of the C o / C o couple in complexes containing a constant macrocyclic ligand and a variety of axial ligands supports this view. A strong dependence of the potential on the nature of the monodentate axial related to the ligand fiel is easily rationalized if the receptor orbital on C o is antibonding σ 2 +

3 +

2 +

3+

2 +

3 +

2

2

3

+

z

3 +

2 +

3 +

+ The C o ^ / C o couple is responsive to the extent and nature of the unsaturation of the ligands listed in Table HI. E / increases from - 2 . 3 V for [i4]aneN (7) to - 1 . 6 9 V for Me [14]4, l l - d i e n e N and -1.32 V for M e [ l 4 ] i , 4 , 8 , i l - t e t r a e n e N (Table ID). The effect of α-diimines is more extreme with E i / for the Me [14]i, 3-dieneN derivative being - 1 . 1 7 V and that for M e [ i 4 ] l , 3,8, iO-tetraeneN being -0.72 V . Since the v e r y negative E / for the saturated ligand system bears a large uncertainty, the magnitudes of these shifts are assessed by comparing values for the unsaturated ligand derivatives. O n that basis, Δ Ε ^ « +190 m V for each isolated C=N group and ~450 mV for each oediimine grouping. The greater sensitivity for the C o / C o couple as c o m pared to the isoelectronic N i / N i couple is attributed to the lower valence states involved in the former case. If back-bonding is a c o n s i deration it should be more important for lower states. Complexes With Anionic Tetraaza M a c r o c y c l i c Ligands. Ligands of general structures 3 and 4 have been utilized to characterize the oxidation-reduction behavior of complexes with macrocyclic anionic ligands. The N i complexes of ligands of structure 3 exhibit i r r e v e r sible oxidations at modest potentials (Ej/ - +.27 V for X = (CH ) and +.23 V for X = (CH ) and v e r y cathodic reductions (-2.30 V and -2.34 V, respectively (2)). These data show that the presence of charge on the ligand facilitates the oxidation process since the least positive values observed for this couple with neutral macrocyclic ligands is some 400 m V more positive. A l s o , reductions are made extremely unfavor able and the effect of ring size on E / has become v e r y s m a l l . The complexes of structure 4, having Ζ = Η and ring sizes of 14, 15, and 16 members, show i r r e v e r s i b l e oxidations at E / values of approximately ( d

z

2 K

1

t

4

2

6

6

4

4

2

2

4

4

t

4

2

2 +

3 +

1 +

2 +

2 +

2

2

2

3

x

2

t

2


2

42


-0.34 V , -0.39 V and - 0 . 2 9 V , respectively. Not only are these values extremely cathodic for the N i / N i couple, but greater uncertainties arise in their values because of their i r r e v e r s i b i l i t y . These uncertain ties and the proximity of their approximate E ^ values,suggest further that the oxidation reaction is not sensitive to ring size among these structures. T h i s insensitivity to ring size issiipported by data for Ni complexes of structure 4 having Ζ = - C ^ ç (Table V (2)). 2 +

3 +

2 +

H

The data of Table V has been obtained under Ihe same conditions as those for all the systems described above (19). A s w i l l be apparent, shortly, the derivatives of the ligands having stmcture 4 are more readily studied in a different solvent; however, the limited data available f r o m measurements in acetonitrile solutions is quite revealing. E a r l y Table V .

Half-Wave Potentials for N i

2 +

Complexes of Dianionic Ligands

of Structure 4, Having Differen Ag/AgN0

3

(0.1 M) Reference Electrode, 0.1 M n - B u 4 N B F

Supporting

4

Electrolyte. E A(volts) MÎT. .Mi T. 2+^ NiL-»NiL 1

Ring Size

N i L - N i L4T" *. "

Substituent

£

2

NiL -

NiL

15

H

-0.39 i

+1.77 i

-2.77 i

15

- C ^ ™

+0.26

+0.9

-2.53 i

14

~C^°

+0.25

+0.97 i

+0.24

+0.85 i

-2.38 q r

+0.27

+0.89 i

-2.39 qr

Fe(II)TPP-Py,

Δ

G

Δ Η

298°

Δ S

As

( a )

(

b

)

eu

Solvent

kcal/mole

DMSO

-3.6±0.2

-8.8±0.5

-17.411.2

-14.812.5

DMF

-7.3±0.2

-13.010.8

-19.212.0

-16.912.0

Butyronitrile

-8.9±0.1

-15.610.4

-22.511.8

-19.712.1

(a) (b)

kcal/mole

eu

1 Obtained from i n t e r c e p t o f l o g Κ vs_ Τ p l o t . Obtained from H e s s s law c a l c u l a t i o n i n v o l v i n g e l e c t r o n t r a n s f e r r e a c t i o n o f Fe(II)TPP i n the presence and absence of pyridine. 1

A t y p i c a l p l o t o f dE /dT f o r r e a c t i o n s 10 and 11 i s shown i n F i g u r e 4 and A s c a l c u l a t e d from these p l o t s i s l i s t e d i n Table V for several solvents. A l l entropy changes f o r Fe(II) complexa t i o n i n Table V are w i t h i n the range reported f o r other i r o n ( I I ) p o r p h y r i n s , but, as has been p o i n t e d out, As (as w e l l as AH) f o r b i n d i n g o f 2 p y r i d i n e molecules i s extremely s o l v e n t depenent.(13) x


KADISH E T A L .

Metalloporphyrins

in Aprotic

Solvents

[ C o l E D T P P · py]

_J 2.8

I 2.9

l 3.0

3.1

l/T Figure 3.

3.2

3.3 3

ο

X 10 ( κ

3.4

_ 1

3.5

)

Vant H off plot for pyridine binding by Co(II)TPP in DMSO, 0.1M TBAP

L 260

I

I

I

270

280

290

TEMPERATURE Figure 4.

L_ 300

< ° K)

Half wave potential as a function of temperature for Reactions 10 and 11



1/T Figure 5.

X

10

3

(°K" ) 1

Van t H off plot for pyridine binding by Fe(II)TPP in DMF, 0.1M TBAP


4. KADisH et al.

Metaîloporphyrins in Aprotic Solvents

63

In contrast to Co(II)TPP, AS for oxidation of Fe(II) to Fe(III) (see Table IV) is quite small in pyridine-DMF and pyridine-butyronitrile mixtures where both iron(II) and iron(III) form bis pyridine adducts. As can, in this case, be assigned entirely to the electron transfer step without contribution from changes in axial ligand coordination. The positive AS = 15.3 ± 3.2 eu in Py-DMSO implies a reduction in coordination on going from Fe(II) to Fe(III). Likewise, the small entropy changes in pyridine free solutions imply an identical coordination number of both the oxidized and the reduced forms of the complex. In order to corroborate the Hess s law determination for complexation with iron(II), as has been done for Co(II)TPP, we have calculated As directly from the temperature dependence of the measured stability constants. Calculations utilizing equation 2 gave aK= 2.2 χ 10 at 298° for FeTPP»Py in DMF and a similar Κ = 3.2 χ 10 varied and from the slop AH and As were calculated, and listed in Table V. No difference appears to exist between entropy changes calculated by the differ ent methods. 1

5

2

6

Acknowledgements. The support of Research Corporation is greatfully acknowledged. Literature Cited 1.

J.-H. Fuhrhop in "Structure and Bonding", Vol. 18, J. D. Dunitz, Ed., Springer-Verlag, New York, 1974. 2. Κ. M. Smith "Porphyrins and Metalloporphyrins", Elsevier Scientific Publishing Co., New York, Ν. Υ., 1975, chapter 14. 3. M. J. Carter, D. P. Rillema and F. Basolo, J. Amer. Chem. Soc., 96, 392 (1974). 4. H. C. Stynes and J. A. Ibers, J. Amer. Chem. Soc., 94, 1559 (1972). 5. F. A. Walker, J. Amer. Chem. Soc., 95, 1154 (1973). 6. D. V. Stynes, H. Stynes, J. A. Ibers and B. R. James, J. Amer. Chem. Soc., 95, 1142 (1973). 7. C. J. Weschler, D. C. Anderson and F. Basolo, J. Amer. Chem. Soc., 97, 6707 (1975). 8. C. J. Weschler, D. C. Anderson and F. Basolo, J. Amer. Chem. Soc., 96, 5599 (1974). 9. C. K. Chang and T. G. Traylor, Proc. Nat. Acad. Sci., U.S.A., 72, 1177 (1975). 10. C. J. Weschler, Β. M. Hoffman and F. Basolo, J. Amer. Chem. Soc., 97, 5278 (1975). 11. F. A. Walker, J. Amer. Chem. Soc., 95, 1150 (1973). 12. D. V. Stynes, H. C. Stynes, B. R. James and J. A. Ibers, J. Amer. Chem. Soc., 95, 1797 (1973).


64 ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

S. J. Cole, G. C. Curthoys and E. A. Magnusson, J. Amer. Chem. Soc., 92, 2991 (1970), ibid, 92, 2153 (1971). W. J. Moore, "Physical Chemistry", Prentice Hall Inc., Englewood Cliffs, N.J., 1972. I. M. Kolthoff and J.J. Lingane, "Polarography", Vol. 1, Interscience Publishers, New York, Ν. Y., 1952, chapter 12. R. H. Felton and H. Linschitz, J. Amer. Chem. Soc., 88, 1113 (1966). J. Fajer, D. C. Borg, A. Forman, D. Dolphin and R. H. Felton, J. Amer. Chem. Soc., 92, 3451 (1970). R. P. Van Duyne and C. N. Reilley, Anal. Chem., 44, 142 (1972). D. J. Quimby and F. R. Longo, J. Amer. Chem. Soc., 97, 5111 (1975). D. Dolphin, R. H. Felton, D. C. Borg and J. Fajer, J. Amer. Chem. Soc. K. A. Zachariass 22, 527 (1973). F. A. Walker, D. Beroiz, and Κ. M. Kadish, J . Amer. Chem. Soc., 98 3484 (1976). F. A. Walker, private communication. D. Lexa and J. M. Lhoste, Experimentia Suppl. 18, 395 (1971). D. Beroiz and Κ. M. Kadish and L. Bottomley, manuscript in preparation. L. A. Truxillo and D. G. Davis, Anal. Chem., 47, 2260 (1975). J. Manassen, Isr. J. Chem., 12, 1059 (1974). Κ. M. Kadish, M. M. Morrison, L. A. Constant, L. Dickens and D. G. Davis, J. Amer. Chem. Soc., in press.


5 Electrochemical Investigations of the Redox Properties of a N-Bridged Dimer, μ-Nitrido-bis[α,β,γ,δtetraphenylporphyriniron], in Nonaqueous Media Κ. M. KADISH* and J. S. CHENG Department of Chemistry, California State University, Fullerton, Calif. 92634 I. A. COHEN and D. SUMMERVILLE Brooklyn College of the City University of New York, Brooklyn, Ν. Y. 11210 Investigations of iron porphyrin redox properties in nonaque ous media have led, in several previously unreporte Iron(I) porphyrins have been characterized both chemically(2) and electrochemically(3,4) while electrochemical studies of porphyrin πcation radicals and dications have led to the proposed assign ment of quadrivalent heme iron(5,6). This latter oxidation state was generated by electrooxidation of either monomeric or dimeric iron(III) complexes of octaethyl or tetraphenylporphyrin(5,6). With the μ-οxο dimers, only one of the two iron atoms was oxidized to yield a mixed oxidation state Fe(III)-Fe(IV) dimer. Electroreduction of μ-οxο-bis-[tetraphenylporphinatoiron(III)], (FeTPP)O, in DMF has also been shown to yield a mixed Fe(III)-Fe(II) complex, which was characterized by e.s.r. at low temperature(4) before dissociation to [Fe(I)TPP]. Recently, synthesis of the first stable nonintegral or mixed oxidation state iron porphyrin dimer was reported by Summerville and Cohen(7). This is a nitrogen-bridged species, μ-nitrido-bis[α,β,γ,δ-tetraphenylporphinatoiron], written as (FeTPP)N, and similar to, but not isoelectronic with, (FeTPP)O. In neutral (FeTPP)O, the formal oxidation state on both irons is +3. In (FeTPP)N, however, the extra negative charge on the bridging atom Ν , when compared to Ο , leads to the average iron oxida tion state +3½. The neutral 17 valence electron nitrido complex is thus isoelectronic with the cationic species [(FeTPP)O] characterized by Felton(5,6) while the reduced nitrido complex, [(FeTPP)N] , is isoelectronic with the well characterized 18 valence electron (FeTPP) O.(4,9-12) Comparisons of the physical properties (7) and X-Ray struc ture (8) of the neutral (FeTPP)N and (FeTPP)O have recently been made. The most outstanding difference between these two systems is the extent of antiferromagnetic coupling between the iron atoms accross the bridge. Whereas (FeTPP)N is a completely coupled dimer, (FeTPP)O is only weakly magnetically coupled. Because of the relationship between spin coupling and bridge *Present address: Department of Chemistry, University of Houston, Houston, Texas 77004 65 2

2

2

2

2

3

2

+

2

2

2

2

2

2

2


66

ELECTROCHEMICAL

STUDIES OF BIOLOGICAL

SYSTEMS

mediated e l e c t r o n t r a n s f e r and i t s relevance to b i o l o g i c a l e l e c t r o n t r a n s p o r t i n cytochrome oxidase, we wish to compare a s e r i e s o f i s o e l e c t r o n i c hemin dimers u t i l i z i n g d i f f e r e n t b r i d g i n g groups. In t h i s paper, we r e p o r t the e l e c t r o n t r a n s f e r p r o p e r t i e s of μ - n i t r i d o - b i s [α,3,γ,ό-tetraphenylporphinatoiron] i n methylene chloride. Experimental Chemicals. A l l s o l v e n t s and chemicals were reagent grade and were used without f u r t h e r p u r i f i c a t i o n . The supporting e l e c t r o l y t e , tetrabutylammonium p e r c h l o r a t e (TBAP) was r e c r y s t a l l i z e d from absolute methanol and was d r i e d at reduced pressure over Ρι+Oio. (FeTPP) 2Ο was purchased from Strem Chemical Inc. (Danvers, Mass.) and was used as r e c e i v e d . (FeTPP)2N was synthe s i z e d from TPPFeN3 as describe Cohen (7) . E l e c t r o c h e m i c a l Measurements. A l l polarographic measurements were made on a PAR Model 174 Polarographic Analyzer u t i l i z i n g a three e l e c t r o d e system. The working e l e c t r o d e and counter e l e c trode were platinum and a commercial calomel e l e c t r o d e was u t i l i z ed as the reference e l e c t r o d e . This was separated from the bulk of the s o l u t i o n by a bridge f i l l e d with the same solvent and supporting e l e c t r o l y t e . Porphyrin concentrations were between 10 and 10 ** M. The o v e r a l l number of e l e c t r o n s (faradays per mole of i r o n monomer) was determined by c o n t r o l l e d p o t e n t i a l coulometry u t i l i z i n g a PAR Model 173 P o t e n t i o s t a t . E l e c t r o n i c i n t e g r a t i o n o f the current-time curve was achieved using a PAR Model 179 i n t e g r a t o r i n conjunction with the Model 173 P o t e n t i o s t a t . The Coulometric c e l l was s i m i l a r to t h a t used f o r c y c l i c voltammetry. A l a r g e c o i l e d platinum wire served as the anode and was separated from the cathodic compartment by means of a f r i t t e d d i s k . A platinum mesh e l e c t r o d e was used as the cathode and a saturated calomel e l e c t r o d e was the reference e l e c t r o d e . S t i r r i n g of the s o l u t i o n was achieved by means of a magnetic s t i r r i n g bar. Dearation of the s o l u t i o n was performed before commencing the experiment and a stream of high p u r i t y argon was passed above the s o l u t i o n throughout the experiment. A l l e x p e r i ments were c a r r i e d out i n a c o n t r o l l e d temperature room of 20±0.5° and a l l p o t e n t i a l s are reported vs_ the saturated calomel e l e c t r o d e (SCE). 3

O p t i c a l Spectroscopy. The e l e c t r o l y s i s of (FeTPP)2N was followed o p t i c a l l y using a Cary 15 Spectrophotometer. A s p e c i a l l y constructed quartz flow c e l l of path length 0.90 cm was used, which was attached t o the e l e c t r o l y s i s c e l l and c o u l d be removed f o r i n s e r t i o n i n t o the Cary 15. For the n e u t r a l s p e c i e s , quartz spectrophotometric c e l l s of path length 1.00 and 0.10 cm were also u t i l i z e d .


5.

KADisH E T A L .

N-BHdged

Dimer

in Nonaqueous

Media

67

Results C y c l i c Voltammetry and D i f f e r e n t i a l Pulse Polarography. The e l e c t r o c h e m i c a l r e d u c t i o n o f (FeTPP) N proceeds i n s e v e r a l d i s c r e t e steps without d e s t r o y i n g the porphyrin r i n g . In F i g u r e l a i s shown a c y c l i c voltammogram o f (FeTPP) N obtained i n CH C1 . A l s o shown i n t h i s f i g u r e are c y c l i c voltammograms o f (FeTPP) 0 and FeTPPCl i n the same s o l v e n t . F o r comparison, d i f f e r e n t i a l pulse polarograms are shown overlapping the c y c l i c voltammograms. The advantage o f d i f f e r e n t i a l pulse polarography i s t h a t peak c u r r e n t height may be a c c u r a t e l y measured f o r c l o s e l y overlapping r e a c t i o n s which cannot be analyzed by c y c l i c voltammetry. In a d d i t i o n , h a l f wave p o t e n t i a l s are r e a d i l y obtainable from the p o s i t i o n o f the peak ( E ~E a t small modulation amplitudes (13)). H a l f wave p o t e n t i a l s i n è H c ï were i d e n t i c a l by each method and are summarized i n Tabl 2

2

2

2

2

x

2

2

Table I H a l f Wave P o t e n t i a l s f o r E l e c t r o o x i d a t i o n - R e d u c t i o n o f Several S i m i l a r Porphyrins a t a Platinum E l e c t r o d e i n C H C l , 0.1M TBAP 2

2

Half Wave P o t e n t i a l ( v o l t s v s . SCE) (a) Reaction Compound

(4)

(3)

(2)

(1)

(5)

(FeTPP) N

1.76

1.51

1.15

0.15

-1.21

(FeTPP) 0

—

1.45

1.09

0.84

-1.17

—

1.63

1.42

1.14

-0.32

2

2

FeTPPCl

(a)

See F i g u r e 1 and t e x t f o r i d e n t i f i c a t i o n o f each peak

In order t o i n v e s t i g a t e each r e a c t i o n process o f (FeTPP) N s e p a r a t e l y and t o determine the existence o f any chemical react i o n s coupled t o the e l e c t r o n t r a n s f e r , c y c l i c voltammograms were taken over v a r i o u s sweep ranges. The p o t e n t i a l was i n i t i a l l y s e t at 0.4 V and scanned i n s u c c e s s i v e l y l a r g e r increments f i r s t up to -1.6 V i n a cathodic d i r e c t i o n and then up t o +1.9 V i n an anodic d i r e c t i o n . F o r e i t h e r s i n g l e o r m u l t i p l e scans between the range o f +1.9 V and -1.6 V, a d i f f u s i o n c o n t r o l l e d r e d u c t i o n and r e o x i d a t i o n was i n v a r i a b l y observed a t 0.15 V i n methylene c h l o r ide. T h i s i s l a b e l e d peak 1 i n F i g u r e l a and can be assigned t o 2



(a) ( F e T P P ) N

-

2

L

300

1.6 0

0.8 0 POTENTIAL

0.0 0 (VOLTS

μA

-0.8 0

VS S C E )

Figure 1. Cyclic voltammograms obtained at 100 mV'/sec on a platinum electrode ( ) and differential pulse polarograms at 2mV/sec, modulation amplitude 25mV/sec on a platinum electrode ( ) for (a) (FeTPP) N; (b) (FeTPP) 0; and (c) FeTPPCl in CH Cl 0.1U TBAP 2

z

2

2y


5.

KADISH E T A L .

the

N-Bridged

Dimer

in Nonaqueous

69

Media

transition: [TPPFe(III)-N-Fe(IV)TPP]° +e- J

[TPPFe(III)-N-Fe(III)TPP]"

T h i s same r e a c t i o n was observed a t a s i m i l a r p o t e n t i a l i n benzon i t r i l e (Table II) but i n p y r i d i n e t h i s r e d u c t i o n peak was s h i f t e d c a t h o d i c a l l y i n p o t e n t i a l t o -0.26 V. This was not due t o the r e d u c t i o n o f FeTPPCl*py2 which i s reduced a t 0.18 and -1.38 V i n neat p y r i d i n e (14). No f u r t h e r r e d u c t i o n was observed i n any range o f scans up t o cathodic p o t e n t i a l s o f a t l e a s t -1.1 V. Table I I H a l f Wave P o t e n t i a l s f o r Oxidation-Reduction s e v e r a l s o l v e n t s , 0.1M TBAP

o f (FeTPP)2N i n

H a l f Wav So1vent (a) Reaction (2)

(1)

(5)

1.15

0.15

-1.21

1.22

1.06

0.16

-1.17

(b)

(b)

(4)

(3)

1.76

1.51

Benzonitrile

(b)

Pyridine

(b)

CH C1 2

2

(a)

See F i g u r e l a f o r C H C 1

(b)

Beyond p o t e n t i a l range o f s o l v e n t

2

-0.26

-1.15

2

When the scan was extended t o -1.21 V a second r e d u c t i o n peak (5 o f F i g u r e l a ) was obtained. This i s a t almost an i d e n t i c a l p o t e n t i a l t o the f i r s t r e d u c t i o n o f (FeTPP) 0 a t -1.17 V i n CH2CI2 and the second r e d u c t i o n o f FeTPPCl a t -1.08 V. No f u r t h e r r e d u c t i o n was observed up t o the s o l v e n t l i m i t o f -1.7 V. Currents and h a l f wave p o t e n t i a l s f o r peaks 1,2 and 5 o f (FeTPP)2N are l i s t e d i n Table I I I f o r slow scan r a t e s . As seen from t h i s t a b l e the i n v a r i a n c e o f peak c u r r e n t with the square r o o t o f the scan r a t e , as w e l l as the constant h a l f wave p o t e n t i a l , indicates clearly a diffusion controlled electron transfer f o r each r e a c t i o n . Peak c u r r e n t s from d i f f e r e n t i a l p u l s e polarograms were constant f o r each r e a c t i o n o f (FeTPP)2N, and p o l a r o g r a p h i c wave a n a l y s i s gave a slope o f 63mV, i n d i c a t i n g again a s i n g l e e l e c t r o n t r a n s f e r step. 2



0.148

0.148

0.148

0.146

50

100

200

500

See f i g u r e l a .

0.148

20

(a)

0.147

(Volt)

10

(mV/sec)

Scan Rate

p,c

40.8

26.8

19.2

13.2

8.4

6.0

(μΑ)

i

(D

p,c'

1.82

1.90

1.92

1.88

1.88

1.90

2

mV

±

2

A* 1

]ΐΡί· sec

i

-1.216

-1.210

-1.210

-1.213

-1.211

-

(Volt)

2

E,

35.0

23.1

17.5

12.2

7.5

-

p,c (μΑ)

i

Reaction

-4 χ 10 M (FeTPP) Ν i n 0.1M TBAP i n Methylene C h l o r i d e .

2

1.57

1.63

1.75

1.73

1.68

-

2

mV

2

/v " p,c'

\iA*sec

i

C5)

p,a

24.4

-

-

17.6

12.4

8.4

-

(μΑ)

i

+1.144

+1.144

+1.147

+1.149

-

(Volt)

2

E,

(2)

-

1.73

1.76

1.75

1.88

-

mV

2

2

2

1 /v P,a ι )iA*sec

i

Scan Rate Dependence o f Half Wave P o t e n t i a l and Peak Current f o r Three E l e c t r o d e Reactions o f 8.14

Table I I I

5.

KADISH E T A L .

N-Bridged

Dimer

in Nonaqueous

71

Media

Controlled Potential E l e c t r o l y s i s . In order t o i d e n t i f y the products o f each e l e c t r o d e r e a c t i o n , the n e u t r a l species was both e l e c t r o r e d u c e d and e l e c t r o o x i d i z e d a t c o n t r o l l e d p o t e n t i a l and the number o f coulombs recorded by i n t e g r a t i o n o f the r e s u l t i n g current-time curve. The voltammogram before e l e c t r o l y s i s i s shown i n F i g u r e l a . The p o t e n t i a l was then s e t a t -0.6 V. T h i s i s on a p l a t e a u o f the f i r s t r e d u c t i o n wave but 600mV anodic o f the second r e d u c t i o n . C o n t r o l l e d p o t e n t i a l e l e c t r o l y s i s was complete a f t e r 10 minutes a t t h i s p o t e n t i a l and y i e l d e d an η = 0.50 electrons/monomeric u n i t . The p o t e n t i a l was then s e t t o 1.4 V and (FeTPP)2N was reduced a t a c o n t r o l l e d p o t e n t i a l . The 60mV s e p a r a t i o n o f the cathodic and anodic peak on the c y c l i c voltammogram (Figure 1) as w e l l as p o l a r o g r a p h i c wave a n a l y s i s i n d i c a t e a r e v e r s i b l e one e l e c t r o n t r a n s f e r r e a c t i o n a t =-1.21 V. However, c o n t r o l l e d p o t e n t i a l r e d u c t i o n d i d not y i e l d c u r r e n t time curves i n d i c a t i v e gave evidence o f a chemica t r a n s f e r step. C a l c u l a t i o n s o f η f o r t h i s step were not reproduc i b l e , suggesting t h a t the dimer was cleaved d u r i n g e l e c t r o r e d u c tion. E l e c t r o o x i d a t i o n s were performed a t +1.13, +1.65 and +1.90 V. In each case a w e l l d e f i n e d i n t e g r a t e d current-time curve was obtained, with a n=0.5, 0.99 and 1.35 electrons/monomeric u n i t as the s o l u t i o n c o l o r changed from brown t o green. Values o f η a t each p o t e n t i a l are summarized i n Table IV. Table IV C o n t r o l l e d P o t e n t i a l E l e c t r o l y s i s and Coulometry o f C H

2

C 1

(FeTPP)in

2

Potential(v)

Electrons Transfered Reaction

(b) -0.60 (c) 1.30 (d) 1.65 (e) 1.90

(a) (b) (c) (d) (e)

Values 750 mV 150 mV 140 mV 140 mV

ο

0.50 0.50 0.99 1.35

_

[Fe(IV)-N-Fe(III)]+e [Fe (III) -N-Fe ( H f ^ l ~_ [Fe(IV)-N-Fe(III)]Sj[Fe(IV) -N-Fe(III)].-+e , J [ Fe(IV)-N-Fe(III)] +2e" [Fe(IV)-N-Fe(III)], [Fe(IV)-N-Fe(III)] ^ [ F e ( I V ) - N - F e ( I I I ) ] +3e 0

given as electrons/monomeric u n i t more c a t h o d i c than Reaction CD more anodic than Reaction (2) more anodic than Reaction (3) more anodic than Reaction (4)


3

72

ELECTROCHEMICAL


SYSTEMS

O p t i c a l Spectra. Before e l e c t r o l y s i s the spectrum of (FeTPP) N c o n s i s t e d of a s p l i t Soret Band i n the UV region (λ = 408 and 385 nm) and two weaker bands i n the v i s i b l e r e g i o n (λ = 625 and 532 nm). A methylene c h l o r i d e s o l u t i o n of (FeTPP) N was s t a b l e i n a i r and showed no change i n s p e c t r a l p r o p e r t i e s f o r s e v e r a l days. Values of these molar a b s o r p t i v i t i e s are l i s t e d i n Table V, Group I. For comparison we h a v e a l s o l i s t e d λ and ε f o r the spectrum of i s o e l e c t r o n i c [(FeTPP) 0] obtained by Felton.(6) O p t i c a l s p e c t r a of the s i n g l y reduced species (Table 5, Group 2) show the f e a t u r e s of an u n s p l i t Soret band (λ = 396 nm) which i s of approximately the same molar a b s o r p t i v i t y as t h a t f o r the i s o e l e c t r o n i c (FeTPP) 0 but s h i f t e d toward the blue. There i s no s t r u c t u r e d absorption of [(FeTPP) N] i n the 500-700 nm region. T h i s species was s t a b l e i n the presence of 0 f o r over 24 hours and was unchanged from t h a t obtained under an i n s e r t argon atmosphere. Reoxidation i n a l s t a r t i n g spectrum and (FeTPP) 0 are d i s p l a y e d i n F i g u r e s 2 and 3. As p r e v i o u s l y mentioned, r e d u c t i o n at -1.4 V d i d not proceed i n a s i n g l e step and y i e l d e d , i n a l l cases, an ultimate monomeric product a f t e r s e v e r a l hours. Products of the o x i d i z e d species were a l s o unstable i n the time i n t e r v a l o f t h e experiment and the s p e c t r a resembled those reported f o r FeTPP .(€0 2

2

+

2

2

2

2

2

+

2

D i s c u s s i o n of

Results

Based on the data, the e l e c t r o o x i d a t i o n - r e d u c t i o n of (FeTPP) N can be accounted f o r by the f o l l o w i n g mechanism: 2

[TPPFe

IIl2

= 1.15

-N-Fei n * -

TPP]

\

V

2

[TPPFe

Ii:E

IV

-N-Fe TPP]

- If * e

e

1.51

[TPPFe

+

V

+e

^ III „ IV _+2 [TPPFe rPPFe -N-Fe TPP] r m T %

XT

11

m

-It [TPPFe

I I I

[TPPFe

= 0.15 -N-Fe

I I I

TPP]"

= -1.21 I I I

-N-Fe

I I I

V 1

V

TPP]"

2

f u r t h e r r e d u c t i o n products I I I

I V

-N-Fe TPP]

+ 3

The i n i t i a l r e d u c t i o n of (FeTPP) N i s q u i t e f a c i l e and y i e l d s i n i t i a l l y an i r o n ( I I I ) dimer i s o e l e c t r o n i c with (FeTPP) 0. Further r e d u c t i o n of the dimer occurs a t -1.21 V by a s i n g l e e l e c t r o n t r a n s f e r step and y i e l d s a product assigned not as a mixed F e ( I I I ) , Fe(II) dimer, but r a t h e r as a dimeric i r o n ( I I I ) anion r a d i c a l . 2

2



e

e

(a)

III

II

Group

region.

11.3

417

510 -4 ε x 10

1.34

10.0

0.71

4

ε x 1θ" 0.43

9.40 408

396

8.50

562

(a)

(a)

408

408 10.7

532 0.86

.-4 ε x 10 λ 613

-4 ε χ ίο

λ ε χ ίο" λ

No s p e c t r a l d e t a i l i n v i s i b l e

Fe(III)TPPCl

[Fe(III)-0-Fe(III)] '

[Fe(III)-N-Fe(III)]

[Fe(IV)-0-Fe(III)]

[Fe(IV)-N-Fe(III)]'

Compound

6.34

378

-

11.3

385

3.60

327

This work

Reference

Réf. 6

This work

This work

Réf. 6

Absorbance Maxima (nm) and Molar A b s o r p t i v i t i e s ( l m o l e cm ) f o r Several Iron Tetraphenylporphyrin Complexes i n Methylene C h l o r i d e . Values o f Molar A b s o r p t i v i t i e s Are C a l c u l a t e d Per Mole o f Iron.

Table V


12h

400

500

600

700

λ , nm Figure 2. Spectra of (FeTPP) N first reduction product in CH Cl , 0.1M TBAP. (FeTPP) N before electrolysis ( ); after controlled potential reduction at -0.60 V to yield [(FeTPP) N]~ ( ). The molar absorptivity is per monomeric iron. 2

2

2

2

t

12

10 \

(FeTPP) 0

/V \

2

- /A

8

// / / > / / / / / ι I

β-

4

-1

\

1

s

/

\\ 1

\\

(FeTPP) N" 2

1 \ \ \ \ \ \ \

1

\ \ \ \ \ \

\\ \\ \\ \\ \ χ

2~

\\

0

1

400

1

500

600

700

λ, ηm Figure 3. (FeTPP) 0 2

Spectra of [(FeTPP) N]~ ( ) and the isoelectronic ( ; in CH Cl , 0.1M TBAP. The molar absorptivity is per monomeric iron. 2

2

2


5.

N-Bridged

KADISH E T A L .

Dimer

in Nonaqueous

75

Media

O x i d a t i o n o f (FeTPP)2N occurs i n three w e l l d e f i n e d s i n g l e e l e c t r o n t r a n s f e r steps (see f i g u r e 1). We have chosen t o a s s i g n the r e a c t i o n as o c c u r i n g a t the porphyrin r i n g to y i e l d a c a t i o n r a d i c a l r a t h e r than a t the c e n t r a l metal t o y i e l d an F e ( I V ) , Fe(IV) dimer. The reasons f o r these assignments are based only on p o t e n t i a l s o f the redox r e a c t i o n s and w i l l be d i s c u s s e d i n the following sections. The s t a r t i n g m a t e r i a l (FeTPP)2N has been w e l l c h a r a c t e r i z e d from Môssbauer data and magnetic s u s c e p t i b i l i t y measurements. At room temperature the Môssbauer spectrum i s sharp and symmetrical and a t 80°K shows only a s l i g h t asymmetry. Magnetic s u s c e p t i b i l i t y measurements between 80 Κ and 300 Κ i n d i c a t e a simple paramag n e t i c s p e c i e s with μ = 2.04BM per (FeTPP)2N. Since the Môssbauer data presents a temperature independent doublet t h i s would i n d i c a t e t h a t e i t h e r the two i r o n atoms are i n the same o x i d a t i o n s t a t e (3i) an e x i s t s the occurance o centers ( Mo(IV)-Cys Mo(IV)-Cys + C H 2

(2)

Mo(IV)-Cys-C H 2

2

(3)

2

Mo(IV)-Cys-C H + 2H+->Mo(VI)-Cys + C H 2

2

2

(4)

4

Mo(VI)-Cys + 2e-(BH -) -> Mo(IV)-Cys

(5)

4

Despite the success of this model i n simulating many of the reactions of nitrogenase enzyme, relatively little is known about the oxidation-reduction chemistry of the binuclear Mo O4 center and the means by which an active catalyst is generated from this species. A major research objective i n our laboratory has been to characterize the electrochemical behavior of binuclear molybdenum complexes. We have recently reported detailed electrode reaction mechanism studies of the principal nitrogenase model compound, Na Mo O4(Cys) (4), and its EDTA analog, NaMoO(EDTA) (5), in aqueous borate, phosphate, and ammonia buffers. This work is now being extended to a series of cysteine and EDTA complexes containing μ-οχο-μ-sulfido (Mo O3S ) and 2+

2

2

2

2

2

2

4

2+

2

78


6.

SCHULTZ E T A L .

Reduction

of Nitrogenase

79

Substrates

2+

and d i - y - s u l f i d o (Mo20 S2 ) bridged Mo(V) cores (Figure 1) ( 6 ) . Not s u r p r i s i n g l y , we a l s o have found that e l e c t r o c h e m i c a l r e d u c t i o n of Na Mo20i (Cys) and other oxo- and s u l f i d o - b r i d g e d Mo(V) species i n the presence o f acetylene leads to c a t a l y t i c r e d u c t i o n of t h i s substrate (7). Aside from demonstrating that c a t a l y s i s can be i n i t i a t e d e l e c t r o c h e m i c a l l y , two o b j e c t i v e s i n t h i s work have been 1) to use information from e l e c t r o d e r e a c t i o n mechanism s t u d i e s to help determine the nature and o x i d a t i o n s t a t e of the a c t i v e c a t a l y s t , and 2) to use c o n t r o l of e l e c t r o c h e m i c a l v a r i a b l e s and s o l u t i o n c o n d i t i o n s to provide i n s i g h t to the mechanism of c a t a l y s i s . F o r the oxo- and s u l f i d o - b r i d g e d Mo(V) complexes features such as l i g a n d and b r i d g i n g atom s i g n i f i c a n t l y i n f l u e n c e the e l e c t r o c h e m i c a l and c a t a l y t i c p r o p e r t i e s of the b i n u c l e a r center. The r e s u l t s of these s t u d i e s provide a general framework f o r understanding the mode of production of a c t i v e c a t a l y s t s from b i n u c l e a which these species c a t a l y z substrates. 2

2

f

2

E l e c t r o c h e m i s t r y o f Oxo- and S u l f i d o - B r i d g e d Complexes of Mo(V) A l l of the oxo- and s u l f i d o - b r i d g e d Mo(V)-cysteine and EDTA complexes are reduced to b i n u c l e a r Mo(III) products i n a s i n g l e , d i f f u s i o n - c o n t r o l l e d step a t ca. -1.1 to -1.3 V vs. SCE i n 0.1 F_ Νβ2Βι θ7· Some experimental r e s u l t s are shown i n Figures 2 and 3 and Table I . C o n t r o l l e d p o t e n t i a l coulometry and compara t i v e voltammetric and chronoamperometric current measurements confirm that four e l e c t r o n s are t r a n s f e r r e d i n the r e d u c t i o n of each complex. The Mo(111)2 e l e c t r o d e products are r e o x i d i z e d to Mo(V) species at p o t e n t i a l s 200-500 mV p o s i t i v e of the i n i t i a l r e d u c t i o n peak. The q u a s i r e v e r s i b l e character of t h i s e l e c t r o n t r a n s f e r process i s dependent upon both the s t r u c t u r e of the complex and the composition of the b u f f e r i n g medium. Several important e f f e c t s of l i g a n d and bridge atom s u b s t i t u t i o n are apparent from the e l e c t r o c h e m i c a l data. Replacement of 0 by one or two b r i d g i n g S atoms g r e a t l y increases the r e v e r s i b i l i t y o f the Mo(V) /Mo(111)2 e l e c t r o n t r a n s f e r process, as i n d i c a t e d by the decrease i n ΔΕ . S u l f u r b r i d g i n g atoms a l s o i n f l u e n c e the s t a b i l i t y of t h i t i m e r i c Mo(III) e l e c t r o d e products. The reverse peak currents shown i n the c y c l i c voltam metric experiments i n F i g u r e 2 i n d i c a t e that the s u l f i d o - b r i d g e d M o ( I I I ) - c y s t e i n e products a r e l e s s s t a b l e than the di-μ-οχο analog, and that the r a t e o f decomposition increases i n the order: Mo 0 < Mo 0 S < Mo 0 S +. A l l of the oxo- and s u l f i d o - b r i d g e d Mo(III) -EDTA products a r e s t a b l e on the time s c a l e of c y c l i c voltammetry (Figure 3). The Μο 0ι+ (EDTA) *" complex can be c a r r i e d through a complete coulometric r e d u c t i o n and r e o x i d a t i o n c y c l e with i t s oxo-bridged s t r u c t u r e i n t a c t (5). However, changes i n absorption s p e c t r a f o l l o w i n g reduction of M02O2S2(EDTA) " i n d i c a t e that i t s r e d u c t i o n product undergoes a +

2

2

e

2 +

2

i +

2 +

2

3

2

2

2

2

2

2

2

2



80

Mo 0 X (cysteine) 2

2

2

2

.2-

Ο

Figure 1. Structures of the bi nuclear oxo- and sulfido-bridged molybdenum(V)-cysteine and EDTA complexes

Figure 2. Cyclic voltammetric curves for reduction of ImM oxo- and sulfido-bridged Mo(V)-cysteine complexes at a H g elec trode in 0.1F Na Bj,0 . Scan rate = 0.1 V/sec. Inset, 2c: scan rate = 20 V'/sec. 2

0

Mo 0 X (EDTA) 2

2

X=SorO

2

7

E,V vs SCE


In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977. 1 / 2

/Τ7

/v

C λ

k

l

2

3

2

2

2

1

(46.6)

100

7

0.9

0.004

(small)

(stable product) (stable product)

2

2

4

From references (4) , (5) and ( 6 ) . ^Data recorded at ν = 0. 1 V/s i n 0.1 F N a B 0 at a hanging Values i n parentheses recorded at Hg drop e l e c t r o d e (A = 0.022 cm ); p o t e n t i a l s i n V vs. SCE. ν = 20 V/s.

2

Na Mo 0 S (Cys)

2

(0.51)

(0.096)

-1.29(sh) (-1.39)

i+

2

49.2

0.296

0.079

-1.213

2

2

Na Mo 0 S(Cys)

2

2

2

57.0

0.522

0.056

-1.310

Na Mo 0 (Cys)

2

2

>0

57.9

0.170

0.055

-1.068

Na Mo 0 S (EDTA)

2

0

60.1

0

0.172

3

2

0.052

2

2

-1.079

2

2

Na Mo 0 S (EDTA)

c

A

Ρ , 1/2 1/2 „ (μΑ s /V mM) ( s " )

i

59.1

Ρ

0.382

ΔΕ

0.053

P

-1.248

E

pc" /2

N a M o 0 (EDTA)

E

(V)

pc (V)

Ε

Voltammetric Data f o r Reduction of Oxo- and S u l f i d o - B r i d g e d Mo(V) Complexes

(V)

Compound

Table I.

ELECTROCHEMICAL

82


SYSTEMS

slow d i s s o c i a t i o n r e a c t i o n . Some d i s s o c i a t i o n a l s o may occur f o l l o w i n g reduction of Mo 0 S(EDTA) ~. The dimeric Mo(III)-EDTA products are c l e a r l y more s t a b l e than the analogous c y s t e i n e compounds. We b e l i e v e t h i s greater s t a b i l i t y i s due p r i m a r i l y to the f a c t that EDTA bridges both Mo centers and thereby i n creases the i n t e g r i t y of the b i n u c l e a r u n i t . Another feature observed during e l e c t r o c h e m i c a l experiments i s that decomposition of the i n i t i a l Mo ( I I I ) - c y s t e i n e e l e c t r o d e products leads to species which c a t a l y z e H r e d u c t i o n at the mercury e l e c t r o d e . T h i s behavior i s p a r t i c u l a r l y pronounced f o r M o 0 S 2 ( C y s ) ~ , i n which case the voltammetric wave i s observed as a shoulder on the background discharge of H+. However, the c h a r a c t e r i s t i c M o ( V ) / M o ( I I I ) redox process f o r t h i s compound i s apparent at f a s t e r scan rates (Inset, F i g u r e 2c) where d i s s o c i a t i o n of the Mo (III)2 product i s l e s s extensive A mechanism f o r e l e c t r o c h e m i c a Mo(V) complexes i s show b u f f e r e f f e c t s has shown that the i n i t i a l step i n the r e a c t i o n i s a concerted 4-electron/4-proton t r a n s f e r i n which protonated b u f f e r species are involved i n the t r a n s i t i o n s t a t e of the e l e c t r o d e r e a c t i o n (4, .5)· h e protonated b u f f e r species probably i n t e r a c t with the terminal oxo groups of the Mo^^ "*" u n i t and f a c i l i t a t e coupled e l e c t r o n - p r o t o n t r a n s f e r to produce c o o r d i nated aquo groups i n the Mo (III) products. The b u f f e r species apparently r e p l a c e water molecules w i t h i n the Mo (III) coordina t i o n sphere soon a f t e r r e d u c t i o n of the Mo(V) dimer. For example, the s t a b l e oxo-bridged Mo (III) -EDTA products d i s p l a y v i s i b l e absorption bands which s h i f t with changes i n b u f f e r medium (5), and a b i n u c l e a r Mo(III) complex r e c e n t l y has been i s o l a t e d i n which acetate, EDTA, and oxo groups simultaneously bridge the two Mo atoms (8). S i m i l a r b r i d g i n g by borate or phosphate oxyanions (A) i s represented i n the e l e c t r o d e products i n F i g u r e 4. D i s s o c i a t i o n of the b i n u c l e a r Mo (III) products to c a t a l y t i c a l l y a c t i v e species i n v o l v e s a complicated s e r i e s of r e a c t i o n s . The process appears to be i n t r a m o l e c u l a r and to i n v o l v e a b u f f e r coordinated e l e c t r o d e product, s i n c e the r a t e of d i s s o c i a t i o n depends on b u f f e r type but not on pH, b u f f e r c o n c e n t r a t i o n , or a d d i t i o n of n u c l e o p h i l e s . For Μ ο 0 ( C y s ) " " i t has been deter mined that the rate-determining step i n v o l v e s cleavage of one of the μ-οχο bridge bonds to form a mono-oxo-bridged s p e c i e s . The l a t t e r species i s observed as a second anodic peak at slow voltam metric scan r a t e s , but vanishes a f t e r t o t a l e l e c t r o l y s i s . Further steps i n the sequence have not been f u l l y d e l i n e a t e d , but appear at l e a s t to i n v o l v e r e a c t i o n to a f u r t h e r Mo (III) dimer (bridged s o l e l y by oxo group or b u f f e r anion) i n e q u i l i b r i u m with Mo(III) monomer (which i s apparent from c a t a l y t i c s t u d i e s ) . The u l t i m a t e e l e c t r o d e r e a c t i o n products of Μ ο 0 ^ ( C y s ) ~ are n o n - e l e c t r o a c t i v e and have not been s u c c e s s f u l l y c h a r a c t e r i z e d to date. However, three bands (2 brown, 1 green) can be resolved by g e l column 2

2

3

2

+

2

2

2

2

2

T

2

2

2

2

4

2

2

2

2


2

6.

Reduction

SCHULTZ E T A L .

I -0.6

1

1

.

.

1

-0.8

I

of Nitrogenase

ι

I

-1.2

-1.0

-1.4

83

Substrates

Figure 3. Cyclic voltammetric curves for reduction of ImM oxo- and sulfido-bridged Mo(V)-EDTA complexes at a H g elec trode in 0.1F Na Bfi . Scan rate = 0.1 \ /sec. 2

7

7

E,VvsSCE

A" Ή

.Mo + 4HA + 4e" ^

? / 0 \ ? eq Mo^ Mo ^ : M o ^

Η

V ^ι

Mo ^ 0 ' ^Mo • 3/Γ

K

A"

Λ .0^ Mo;

Mo + 2HJ0

.Mo

T J Q * " Μσ H

Figure 4.

2°

OH

MO HO

further ^ Mo(m)dimer

2Mo(m)-cys

Proposed mechanism for electrochemical reduction of bi nuclear molybdenum(V) complexes


84

ELECTROCHEMICAL


SYSTEMS

chromatography f o l l o w i n g exhaustive e l e c t r o l y s i s of Mo20i (Cys)2 · A mixture of products i s c o n s i s t e n t with the r e a c t i o n scheme shown i n F i g u r e 4. The μ-οχο-μ-sulfido- and d i ^ - s u l f i d o - b r i d g e d complexes appear to undergo s i m i l a r d i s s o c i a t i o n r e a c t i o n s f o l l o w i n g reduction to the Mo (III) s t a t e . Table I shows estimated values of the d i s s o c i a t i o n r a t e constant, k}, f o r a l l compounds i n 0.1 F Na2Bi 0 . These estimates were made by c y c l i c voltammetry (9) or double p o t e n t i a l - s t e p chronocoulometry (10) assuming a pseudo f i r s t - o r d e r d i s s o c i a t i o n r e a c t i o n (EC mechanism) with k = /(l + ) £ k j . More d e t a i l e d s t u d i e s of the d i s s o c i a t i o n michanism Ire c u r r e n t l y underway i n our l a b o r a t o r y . As demonstrated i n the f o l l o w i n g s e c t i o n , t h i s d i s s o c i a t i o n r e a c t i o n plays a key r o l e i n the generation of c a t a l y t i c a l l y a c t i v e s p e c i e s . +

t

7

Qfe

e

E l e c t r o c a t a l y t i c Reductio Previous s t u d i e s of the chemical model system GL,3) have f a i l e d to answer a number of important questions regarding the nature of the a c t i v e c a t a l y s t and the mechanism of c a t a l y t i c substrate reduction. In attempting to answer the l a t t e r two p o i n t s we have decided to i n v e s t i g a t e i n d e t a i l the e l e c t r o c a t a l y t i c r e d u c t i o n of a s i n g l e s u b s t r a t e , acetylene, rather than to survey the behavior of a l l known substrates of the system. T h i s choice i s d i c t a t e d l a r g e l y by the f a c t that acetylene provides more e a s i l y assayed products and i s reduced more r a p i d l y i n the model system than the true b i o l o g i c a l sub s t r a t e , d i n i t r o g e n . While i n v e s t i g a t i o n s with N w i l l provide the u l t i m a t e b i o l o g i c a l relevance, our experiments with ΰ Η have provided s i g n i f i c a n t information regarding the o x i d a t i o n s t a t e and p r o p e r t i e s of the reduced Mo c a t a l y s t s . Our experiments a r e c a r r i e d out by c o n t r o l l e d p o t e n t i a l coulometry at a s t i r r e d Hg pool cathode i n a sealed c e l l equipped with gas sampling p o r t s (7). Vapor phase samples are withdrawn p e r i o d i c a l l y f o r gas chromatographic a n a l y s i s on Porapak N. Two procedures a r e used to study the e l e c t r o c a t a l y t i c reduction: (A) a s o l u t i o n of Mo20i (Cys)2 "" i s reduced d i r e c t l y under 1 atm of C2H2; (B) a s o l u t i o n of Mo20 (Cys)2 ~ i s prereduced to the Mo (III) s t a t e , the c e l l i s purged with 1 atm C H , and p o t e n t i a l i s r e a p p l i e d . The l a t t e r procedure i s more convenient because r a t e p l o t s are i n i t i a l l y l i n e a r and i n t e r s e c t the o r i g i n . In procedure A, 20-30 minutes pass before mass transport c o n t r o l l e d r e d u c t i o n of the complex i s complete and acetylene i s reduced a t a constant r a t e . T y p i c a l behavior of the e l e c t r o c h e m i c a l p a r t of the e x p e r i ment i s shown i n F i g u r e 5 using procedure Β f o r the r e d u c t i o n of C2H2 with Mo20i (Cys)2 ~ as c a t a l y s t . During c o n t r o l l e d p o t e n t i a l r e d u c t i o n of the complex at -1.40 V, current f a l l s to a r e l a t i v e l y high steady-state value (5-10 mA) due to r e d u c t i o n of hydrogen ion at the e l e c t r o d e s u r f a c e c a t a l y z e d by the u l t i m a t e e l e c t r o d e 2

2

2

+

2

I+

2

2

2

+


2

6.

scHULTZ E T AL.

Reduction

of Nitrogenase

85

Substrates

T n e

products of Mo20i+(Cys) · magnitude of the H reduction current i s p r o p o r t i o n a l to the square-root of i n i t i a l dimer con c e n t r a t i o n , thus i n d i c a t i n g c a t a l y s i s by a monomeric s p e c i e s . A f t e r the reduced complex i s purged with acetylene and p o t e n t i a l i s r e a p p l i e d , the current r i s e s to a much higher steady-state value than during the previous e l e c t r o l y s i s . The magnitude of t h i s current a l s o increases with negative p o t e n t i a l f o l l o w i n g an i n i t i a l reduction at -1.40 V. A p p l i c a t i o n of p o t e n t i a l to the reduced molybdenum s o l u t i o n under 1 atm C2H2 r e s u l t s i n the reduction of acetylene by an apparent f i r s t - o r d e r process ( i . e . , l o g [ C H ] = - k t + const.) and l i n e a r production of ethylene and ethane over a 2-3 hour p e r i o d . D i s t r i b u t i o n of these species during a t y p i c a l e x p e r i ment i s shown i n Figure 6. The C^îC^^ P °duct r a t i o i s about 4 or 5:1 and v a r i e s only s l i g h t l y with changes i n experimental c o n d i t i o n s . A considerabl concurrently with acetylen Figure 6 r e v e a l s that amounts of C^H^ and C H produced do not equal the quantity of C H reduced. An a d d i t i o n a l hydrocarbon product i s 1,3-butadiene (C^Hg), which i s produced at about 40% the l e v e l of ethylene ( i . e . , C H :C H :C Hg^5:2:l) . Butadiene a l s o has been discovered to be the major product of acetylene reduction i n the Μο 0^(Cys) "702^/6^"chemical model system (11), but i t i s not produced upon reduction of C H2 by the enzyme. With i n c l u s i o n of C^Hg a s u i t a b l e hydrocarbon balance i s obtained for the chemical model system (11), but the product balance f o r our e l e c t r o c a t a l y t i c system f a l l s as much as 60% short of the quantity of acetylene reduced. Thus, a d d i t i o n a l and as yet un detected products must be produced i n the e l e c t r o c h e m i c a l system. We d i d not discover the presence of butadiene or the discrepancy i n hydrocarbon balance e a r l y enough to take these f a c t o r s i n t o account i n a l l aspects of our i n v e s t i g a t i o n . Consequently, most r e s u l t s f o r the e l e c t r o c a t a l y t i c system are based e i t h e r on the r a t e of C H2 reduction or the r a t e of C ^ and C2H production. Our present r e s u l t s provide strong evidence that a monomeric Mo (III) complex i s the c a t a l y t i c a l l y a c t i v e species i n chemical models f o r nitrogenase based on molybdenum-sulfhydryl complexes. The e l e c t r o d e r e a c t i o n mechanism s t u d i e s of Μ θ 2 θ ( C y s ) 2 ~ and the analogous oxo- and s u l f i d o - b r i d g e d complexes with c y s t e i n e and EDTA e s t a b l i s h that these compounds are reduced d i r e c t l y to Mo (III) products with no evidence of the intermediate Mo(IV) o x i d a t i o n s t a t e . Involvement of Mo (III) i s e s t a b l i s h e d by the f a c t that C2H2 reduction occurs subsequent to the reduction of these complexes by two e l e c t r o n s per molybdenum. In a d d i t i o n , a p p l i c a t i o n of negative p o t e n t i a l to s o l u t i o n s containing equimolar Mo(III) (added as K M o C l ) and c y s t e i n e c a t a l y z e s acetylene r e d u c t i o n at approximately the same r a t e (see Table I I , procedure C). I t i s not l i k e l y that reduction proceeds beyond Mo(III) i n formation of the a c t i v e c a t a l y s t , because e l e c t r o c h e m i c a l l y r e duced s o l u t i o n s of M02O4(Cys)2 ~ or s o l u t i o n s of Mo(III) plus 2

2

2

r

2

2

6

2

2

I+

lf

6

2

2

2

2

2

2

2

g

4

3

2

6

2


86


120 100

CH 2

- 1.70V

2

-1.60 "

< Ε 60 Figure 5. Current-time curves ob served during electrocatalytic reduc tion of acetylene using procedure B. Experimental conditions: 1.70mM Na Mo 0 (Cys) 0.1F Ν α Β 0 , cell purged with 1 atm C H and potential reapplied as indicated.

ce

=> 40 ο —1.40V— 20

2

2

ll

2)

2

2

4

f Γ ^

-1.50 "

_____

7

50

2

100 TIME,min

100 TIME.min. Figure 6. Product-time and reactant-time behavior during electrocatalytic reduction of acetylene using procedure B. Experimental conditions: 1.70mM -1.40 V, 0.1F Na B 0 , Na Mo O (Cys) , 1 atm C H . 2

2

i

2

2

2

h

7

2


-140 -130 150

6.

Reduction

SCHULTZ E T A L .

Table I I .

a

Substrates

87

E l e c t r o c a t a l y t i c Reduction of Acetylene with Na Mo 0i (Cys)2 2

Proce dure

of Nitrogenase

2

+

Ε

C Hi . :

Rate

Cone.

2

4

app (μπιοί C Hi / min)

C H

(V)

(mM)

A A A

-1.40 -1.40 -1.40

1.00 1.00

9.6 9.2

0.204 0.264

4.8 4.9

Β Β Β Β

-1.40 -1.40 -1.40 -1.40

0.30 1.00 1.70 3.00

9.2 9.2 9.2 9.2

0.100 0.195 0.240 0.313

5.0 4.5 4.9 4.7

Β Β Β Β Β

-1.30 -1.40 -1.50 -1.60 -1.70

1.70 1.70 1.70 1.70 1.70

9.2 9.2 9.2 9.2 9.2

0.136 0.240 0.353 0.498 0.708

3.9 4.9 3.9 4.2 4.2

Β Β

-1.35 -1.27

1.00 1.00

8.3(P) 9.2(A)

0.139 0.106

2.4 2.9

C

-1.40

1.00

9.2

0.133

4.7

D

no Ε

1.00

9.2

0.003

-

2

+

2

6

P r o c e d u r e s : A. Complex reduced under 1 atm C H B. Complex prereduced at -1.40 V, c e l l purged with 1 atm C H , p o t e n t i a l r e a p p l i e d as i n d i c a t e d C. P o t e n t i a l a p p l i e d to 2mM K M o C l + 2mM c y s t e i n e under 1 atm C H D. Complex prereduced at -1.40 V, purged with 1 atm C H , and allowed to stand without p o t e n t i a l applied 2

2

2

3

2

2

2

6

2

2

b A l l s o l u t i o n s contain 0.1 F N a B 0 and A = ammonia (0.25 F) 2

i+

7

except Ρ = phosphate (0.5 F)


88

ELECTROCHEMICAL


SYSTEMS

c y s t e i n e e x h i b i t no voltammetric r e d u c t i o n peaks. A number of c o n t r o l experiments have been performed and demonstrate that Mo(III) plus an a d d i t i o n a l source of e l e c t r o n s (or Η ) i s r e q u i r e d f o r C2H2 r e d u c t i o n . For example, e l e c t r o l y s i s of s o l e l y C H2 at 1 atm i n borate b u f f e r at very negative p o t e n t i a l s , even i n the presence of t ^ - e v o l v i n g c a t a l y s t s such as c y s t e i n e , produces no reduced hydrocarbons. I f the Μθ2θ^(Cys)2 ~ complex i s reduced e l e c t r o c h e m i c a l l y , purged w i t h 1 atm C2H2, and allowed to stand, only minimal C R^ i s produced (Table I I , procedure D). When p o t e n t i a l i s r e a p p l i e d , c a t a l y s i s resumes at the normal r a t e . A sampling of data i s shown i n Table I I i l l u s t r a t i n g the e f f e c t s of various experimental c o n d i t i o n s and components on the e l e c t r o c a t a l y t i c r e d u c t i o n of acetylene. Notable f a c t o r s which i n c r e a s e the r a t e of C H.i+ production are decreasing pH, i n c r e a s ing concentration of complex S e v e r a l experiments ru that the r a t e of C U^ production i s decreased s l i g h t l y r e l a t i v e to borate b u f f e r . The e f f e c t of Μο 0ι (Cys) concentration on the rates of C E^ and C H production i s shown i n F i g u r e 7. These squareroot dependences on i n i t i a l concentration of complex demonstrate that the a c t i v e c a t a l y s t i s a monomeric species i n e q u i l i b r i u m w i t h a l a r g e r f r a c t i o n of dimeric m a t e r i a l . The same r e s u l t i s found i n the chemical model system regarding C E^ production (3,12) , but a l i n e a r dependence of C ]i^ formation on [Mo20i (Cys)2 ~] has not been noted p r e v i o u s l y . Production of a monomeric c a t a l y s t could be achieved through a sequence of chemical steps as o u t l i n e d i n Figure 4. Although we have not confirmed t h i s mechanism i n the d e t a i l depicted, s e q u e n t i a l d i s s o c i a t i o n of the i n i t i a l Mo ( I I I ) e l e c t r o d e product through one or more dimeric intermediates i s c o n s i s t e n t with the electrochem i c a l r e s u l t s , observation of s e v e r a l species i n product i s o l a t i o n attempts, and observation of an e q u i l i b r i u m c o n c e n t r a t i o n of monomeric c a t a l y s t . D i s s o c i a t i o n of Mo(III) atoms f o l l o w i n g r e d u c t i o n of the b i nuclear center i s c l e a r l y an important step i n the generation of an a c t i v e c a t a l y s t . For t h i s reason the e n t i r e s e r i e s of oxoand s u l f i d o - b r i d g e d Mo(V)-cysteine and EDTA complexes described e a r l i e r has been examined i n the acetylene r e d u c t i o n experiment. Results are shown i n Table I I I , and i n d i c a t e that s u l f u r b r i d g i n g atoms and l i g a n d play an important r o l e i n producing an a c t i v e c a t a l y s t and i n the r a t e and mechanism of C H2 r e d u c t i o n . The s u l f i d o - b r i d g e d c y s t e i n e complexes, Mo20 S(Cys)2 ~ and M02O2S2(Cys)2 ~, are reduced to Mo ( I I I ) products which d i s s o c i a t e r a p i d l y on the voltammetric time s c a l e . These products, however, provide only a marginal i n c r e a s e i n the r a t e of C H2 r e d u c t i o n and s i m i l a r G ^ H k ^ H g r a t i o s of 4 or 5:1. The di-μ-οχο EDTA complex, Μ ο 0 ^ ( E D T A ) * - , i s reduced to a s t a b l e b i n u c l e a r product which i s completely i n e f f e c t i v e i n acetylene r e d u c t i o n . 2

2

2

2

2

2

2

2

2

+

6

2

2

2

1

2

+

2

2

2

3

2

2

2

2


6.

SCHULTZ E T A L .

Table I I I .

Reduction

of Nitrogenase

E l e c t r o c a t a l y t i c Reduction of Acetylene with Various Oxo- and S u l f i d o - B r i d g e d Molybdenum(V) Complexes

Compound

Rate (ymol C Hi + C H /min)

C Hi : C Hg

0.302

3.9

0.323

4.0

0.324

4.8

2

+

Na Mo 0i (Cys)2 2

2

t

Na Mo 0 S(Cys) 2

2

3

2

Na Mo 0 S (Cys) 2

2

2

2

2

Na Mo 0 (EDTA) 2

2

89

Substrates

2

2

+

-

0.0

4

2

6

Na Mo 0 S(EDTA) 2

2

3

Na Mo 0 S (EDTA) 2

2

2

0.33

0.330

2

a) A l l s o l u t i o n s contain 1.70 mM complex i n 0.1 F Na Bi 0y, Ε -1.40 V, 1 atm C H , procedure Β (see footnote a, Table i f ? ? 2

2

+

2

2

Mo 0 S(EDTA) "", which shows some a c t i v i t y towards C H r e d u c t i o n , may d i s s o c i a t e s l i g h t l y a f t e r e l e c t r o c h e m i c a l reduction. The d i - y - s u l f i d o complex, M o 0 S ( E D T A ) ~ however, d i s s o c i a t e s slowly but q u i t e e v i d e n t l y f o l l o w i n g i t s e l e c t r o c h e m i c a l reduction. This reduced m a t e r i a l c a t a l y z e s production of C H and C H at a r a t e equal to the corresponding c y s t e i n e compound and y i e l d s an i n v e r t e d product r a t i o of 0 Η β : 0 Η ι = 3:1. F i g u r e 8 shows the s i g n i f i c a n t e f f e c t of e l e c t r o d e p o t e n t i a l on the e l e c t r o c a t a l y t i c process. The rates of 0 Η ^ and C Hg pro duction and acetylene r e d u c t i o n increase e x p o n e n t i a l l y with poten t i a l , as does the steady-state current f o l l o w i n g a p p l i c a t i o n of p o t e n t i a l to an acetylene-purged s o l u t i o n . One experimental ob s e r v a t i o n which p a r a l l e l s t h i s behavior i s the c a t a l y t i c evolu t i o n of H at the mercury e l e c t r o d e , which occurs concurrently with acetylene reduction and a l s o i n the presence of the Mo ( I I I ) e l e c t r o d e products alone. During a t y p i c a l experiment hydrogen e v o l u t i o n may account f o r 30-40% of the t o t a l coulombs passed, the remainder going to C H r e d u c t i o n . We b e l i e v e i t i s premature to dismiss hydrogen e v o l u t i o n as an experimental a r t i f a c t . Evolu t i o n of H i n the absence of substrates other than IT*" i s an im portant feature of the chemical model system (3,12) and of n i t r o genase enzyme (13). In the chemical model, f o r example: (a) NaBH^ i s a more e f f e c t i v e reductant than N a S 0 i and a l s o c a t a l yzes H e v o l u t i o n more s t r o n g l y (3), and (b) a d d i t i o n of c o c a t a l y s t s such as methyl v i o l o g e n (14) and F e ^ S ^ ( S R ) c l u s t e r s 2

3

2

2

2

2

2

2

9

2

2

2

l+

2

+

2

2

2

6

2

2

2

2

+

2


2

90


Figure /. Rates of ethylene and ethane production as a function of square root of Na Mo 0 (Cys) concentration. Other experimental as in Figure 6. 2

2

Jf

2

Figure 8. Effect of electrode potential on catalytic current, acetylene reduction rate, and ethylene and ethane production rates during electrocatalytic reduction of C H using procedure B. Experimental conditions as in Figure 5. 2

2

E.Vvs. SCE


SCHULTZ E T A L .

6.

Reduction

of Nitrogenase

91

Substrates

(12,15), which i n c r e a s e the C2H2 r e d u c t i o n r a t e , a l s o s t i m u l a t e the production of H . The e f f e c t of e l e c t r o d e p o t e n t i a l i n d i c a t e s that an important r e l a t i o n s h i p e x i s t s between H2 e v o l u t i o n and C H r e d u c t i o n i n the e l e c t r o c a t a l y t i c system. C l e a r l y , an e l e c trode a c t i v a t e d rather than a bulk s o l u t i o n process i s i n v o l v e d i n the rate-determining step of acetylene reduction. Four p o s s i b l e mechanisms which have been considered f o r the e l e c t r o c h e m i c a l l y c a t a l y z e d r e d u c t i o n of acetylene are l i s t e d i n Table IV. In each case formation of a Mo(III)-C2H2 adduct i s assumed to be the i n i t i a l step i n the mechanism, because the requirement of molybdenum i n d i c a t e s that some i n t e r a c t i o n between the reduced Mo species and acetylene must occur during c a t a l y s i s . I t i s not p o s s i b l e to confirm or r e j e c t any of these mechanisms with c e r t a i n t y . Mechanism D, however, i s c o n t r a d i c t e d l e a s t by present evidence. T h i s mechanism i s viewed as an e l e c t r o c a t a l y t i c hydrogénation i n whic c a t a l y s t i s to bind acetylen produced at the e l e c t r o d e surface. Hydrogen atoms are the product of hydrogen i o n r e d u c t i o n catalyzed by the Mo(III)-cysteine 2

2

2

Table IV.

P o s s i b l e Mechanisms f o r E l e c t r o c a t a l y t i c of Acetylene

Mo (III)

A.

2

2

Mo(V)

+ 2H

+

+ 2e~

+ 2H

+

+ 2e"

H-MoUlD-C^

+ H+

followed

2

+

Mo(V)

+ C^fy

£

Mo (III)

+

Mo (III)

+ C

+

H-MoUH)-^^ +

+

Mo (III)

+

H+

C^H,

E l e c t r o c a t a l y t i c Hydrogénation j.Mo(III)-Cys + 2H+ + 2e"

Ο Γ

2

Homogeneous Hydrogénation Mo ( I I I ) - C ^ +

D.

Mo(III)-C H

E l e c t r o c h e m i c a l Reduction of Adduct Mo ( I I D - C ^

C.

?

Homogeneous Redox Reaction Mo(III)-C H

B.

+ C ^

Reduction

+

Mo(III)-Cys +

Mo(III)-C H, + 2H+ + 2e"

->

Mo(III)-C H

Modll)-^!^

+

Mo(III) +

2

+ 2H-

2

2H+

2

2H-

C> \ 2


by:

92

ELECTROCHEMICAL


SYSTEMS

complex with or without C H bound to i t . This process i s much the same as the t r a d i t i o n a l e v o l u t i o n of H at mercury e l e c t r o d e s c a t a l y z e d by t r a n s i t i o n metal complexes with s u l f u r - c o n t a i n i n g ligands (16), and would e x h i b i t an exponential dependence on p o t e n t i a l . C a t a l y s i s presumably occurs when a M o ( I I I ) - c o o r d i nated s u l f u r atom (from e i t h e r c y s t e i n e l i g a n d or b r i d g i n g S group) i s protonated to form an -SH+ species and then reduced: 2

2

2

Mo(III)-S:

+ H+

Mo(III)-SH+ + e"

Mo(III)-SH+

(6)

+ Mo(III)-SH-

(7)

These species could r e a c t to form

H

2

2 Mo(III)-SH- -> 2 Mo(III)-S:

+ H

(8)

2

or, i n the presence of 2 Mo(III)-SH- + M o ( I I I ) - C H 2

+ 2 Mo(III)-S:

2

+ Mo(III) + 0 Η 2

4

(9)

The a l t e r n a t i v e mechanisms i n Table IV are c o n t r a d i c t e d by at l e a s t one piece of experimental evidence. In mechanisms A and Β the regeneration of c a t a l y s t and r e d u c t i o n of adduct are accomplished through d i f f u s i o n - l i m i t e d e l e c t r o c h e m i c a l processes. I t i s d i f f i c u l t to imagine how these e l e c t r o c h e m i c a l steps could be so i r r e v e r s i b l e as to d i s p l a y the observed p o t e n t i a l depen dence over a range of 400-500 mV. Mechanism A a l s o i s discounted by the f a c t that a d d i t i o n of C H to s o l u t i o n s of Mo (III) does not lead to s u b s t a n t i a l formation of 0 Η . Mechanism C i s s i m i l a r to known homogeneous hydrogénation r e a c t i o n s c a t a l y z e d by t r a n s i t i o n metal complexes (17). A s i m i l a r mechanism i n v o l v i n g a h y d r i d i c intermediate has been suggested f o r the chemical system (18). T h i s mechanism does not seem a t t r a c t i v e i n the e l e c t r o c a t a l y t i c system, because, as the quantity of H i n the c e l l i s constantly i n c r e a s i n g during e l e c t r o l y s i s , the r a t e of c a t a l y t i c acetylene r e d u c t i o n remains constant. Ethane and 1,3-butadiene are two a d d i t i o n a l products of e l e c t r o c a t a l y t i c acetylene r e d u c t i o n . Ethane i s produced i n constant p r o p o r t i o n to ethylene under a v a r i e t y of experimental c o n d i t i o n s , and ethylene i t s e l f i s not reduced i n the c a t a l y t i c system. Therefore, a separate b i n d i n g r e a c t i o n between Mo(III) and ethylene does not take p l a c e before r e d u c t i o n to C Hg. A l s o , i t i s not l i k e l y that a dimeric Mo (III) species i s r e s p o n s i b l e f o r r e d u c t i o n of C H to C H because ethane formation i s l i n e a r l y dependent on [ M o 0 i ( C y s ) ~ ] / (Figure 7) and the Mo(III) dimer produced by r e d u c t i o n of M o 0 ( E D T A ) i s completely i n a c t i v e i n acetylene r e d u c t i o n (Table I I I ) . We b e l i e v e i t i s more l i k e l y that about 20-25% of the time two a d d i t i o n a l hydrogen atoms are i n s e r t e d i n t o a bound acetylene molecule before d i s s o c i a t i o n from the Mo (III) c a t a l y s t takes p l a c e : 2

2

2

4

2

2

2

2

2

6

2

2

+

1

2

2

2-

2

t+


6.

SCHULTZ E T A L .

Reduction

Mo(III)-C H 2

2

of Nitrogenase

+ 2H. + Mo(III)-C Hi 2

(10)

+

Mo(III)-C H^ + 2H- + Mo(III) + C H 2

2

Formation of butadiene can occur i f C H M o ( I I I ) - C H adduct before r e d u c t i o n . 2

2

93

Substrates

2

(11)

6

i n t e r a c t s with the

2

-, CH

CH + HI . . . CH

Mo(III)^|| ^CH

OTT+ η — 8

'

> Mo (III) + H C=CH-CH=CH 2

2

(12)

Subsequent polymerization r e a c t i o n s or e l e c t r o c h e m i c a l r e d u c t i o n of butadiene could e x p l a i n the i n a b i l i t y to o b t a i n a t o t a l hydro carbon balance i n the e l e c t r o c h e m i c a l system. Reaction 12 could w e l l be favored a t the high C H p a r t i a l pressures used i n t h i s work. The e f f e c t of acetylen t r i b u t i o n has not been chemical c a t a l y t i c systems. Discussion Several years" study of these model systems has impressed upon us the complicated nature of molybdenum s o l u t i o n chemistry. S p e c i f i c and unique e f f e c t s of b r i d g i n g atom, l i g a n d , s o l u t i o n environment, and b u f f e r s a l t are evident i n the chemical, e l e c trochemical, and c a t a l y t i c p r o p e r t i e s of these compounds. Such complexity, incompletely understood, makes the e x t r a p o l a t i o n of r e s u l t s to an e q u a l l y complicated b i o l o g i c a l system somewhat tenuous. However, we b e l i e v e our e l e c t r o c h e m i c a l s t u d i e s have provided r e s u l t s which are d i r e c t l y u s e f u l i n understanding the behavior of chemical models f o r nitrogenase based on molybdenums u l f h y d r y l complexes. These r e s u l t s a l s o c o n t r i b u t e to the general knowledge of molybdenum chemistry which i s necessary i n i n t e r p r e t i n g the behavior of the enzyme. A number of s i g n i f i c a n t p o i n t s emanating from these e l e c t r o c h e m i c a l s t u d i e s are d i s c u s sed below. 1. Molybdenum Oxidation State. A l l b i n u c l e a r Mo(V) compounds we have examined undergo Mo(V) ->Mo(III) reduction under aqueous s o l u t i o n c o n d i t i o n s comparable t o those used i n s t u d i e s of the chemical model and the enzyme i t s e l f . There i s no evidence o f the intermediate Mo(IV) o x i d a t i o n s t a t e . I t seems l i k e l y , there f o r e , that Mo (III) and not Mo(IV) i s the o x i d a t i o n s t a t e of the a c t i v e c a t a l y s t i n the Mo-cysteine model system. Despite recent comments to the contrary (19), we a l s o b e l i e v e that Mo (III) i s a strong candidate f o r the o x i d a t i o n s t a t e of the reduced Mo center i n nitrogenase. Other evidence a l s o supports the p o s s i b l e impor tance of Mo (III) i n the molybdenum-containing reductases: the r e d u c t i o n of N and C H c a t a l y z e d by i n o r g a n i c Mo (III) species over a range of temperatures and pressures (20,21), c o n s t r u c t i o n 2

2

2

2

2


94

ELECTROCHEMICAL


SYSTEMS

of a s u c c e s s f u l model f o r n i t r a t e reduction based on the hexaaquomolybdenum(III) c a t i o n , M o ( H 2 0 ) (22), and t e n t a t i v e obser v a t i o n of Mo(III) epr s i g n a l s i n n i t r a t e reductase (23-25). Evidence f o r the molybdenum o x i d a t i o n s t a t e i n reduced n i t r o genase may be provided by x-ray absorption edge spectroscopy (26), but d e f i n i t i v e r e s u l t s are not yet a v a i l a b l e . 3+

6

2. Coupled Electron-Proton T r a n s f e r . Reduction of the b i nuclear complexes proceeds by coupled t r a n s f e r of four e l e c t r o n s and four protons i n a s i n g l e step. This observation i s s i g n i f i cant i n view of a recent proposal (27) that coupled e l e c t r o n proton t r a n s f e r i n m u l t i p l e s of two i s an important feature of molybdenum-containing enzymes. Furthermore, the unique b u f f e r e f f e c t s observed i n the e l e c t r o d e r e a c t i o n mechanism suggest that oxomolybdenum species may f a c i l i t a t e proton t r a n s f e r to or from substrates i n a h i g h l 3. D i s s o c i a t i o n of the B i n u c l e a r Center. D i s s o c i a t i o n of Mo(III) atoms f o l l o w i n g r e d u c t i o n of the b i n u c l e a r Mo(V) u n i t appears to be an e s s e n t i a l step i n the generation of an a c t i v e c a t a l y s t . Only those compounds which show evidence of d i s s o c i a t i o n f o l l o w i n g e l e c t r o c h e m i c a l reduction are e f f e c t i v e i n the c a t a l y t i c reduction of C H and H+. On the other hand, the dimeric Mo (III) r e d u c t i o n product of M02O4(EDTA) ~ does not c a t a l y z e r e d u c t i o n of C2H2, even though i t i s an extremely strong reducing agent (Ε°' = -1.06 V vs. SCE i n 0.1 F N a B i 0 ) . Also, i t i s apparent that d i s s o c i a t i o n of the b i n u c l e a r center occurs a f t e r , not p r i o r to Ç3), r e d u c t i o n to the Mo(III) s t a t e , and that s u l f u r b r i d g i n g atoms and ligands which do not bridge the two Mo centers i n c r e a s e t h i s tendency f o r d i s s o c i a t i o n . 2

2

2

2

+

7

4. S u l f u r B r i d g i n g Atoms. S u l f u r b r i d g i n g atoms i n c r e a s e the r e v e r s i b i l i t y of e l e c t r o n t r a n s f e r i n the b i n u c l e a r Mo(V) center and the ease of d i s s o c i a t i o n of b i n u c l e a r Mo(III) u n i t s . Both features enhance the c a t a l y t i c p r o p e r t i e s of the compounds we have studied. Thus, s u l f u r b r i d g i n g may be an important and d e s i r a b l e feature of Mo enzyme model chemistry. Several i n stances have been noted wherein s u l f i d o b r i d g i n g has imparted unusual s t a b i l i t y to b i n u c l e a r Mo(V) species (28,29), and i t has been suggested that such a feature may be undesirable i n terms of Mo enzyme model chemistry (29). In the Mo (III) o x i d a t i o n s t a t e , however, s u l f i d o b r i d g i n g enhances the r e a c t i v i t y of the b i nuclear u n i t and thus improves c a t a l y t i c a c t i v i t y . Presence of s u l f u r i n the Mo ( I I I ) c o o r d i n a t i o n sphere may i n c r e a s e the l a b i l i t y of t h i s o x i d a t i o n s t a t e and permit more f a c i l e b i n d i n g of substrates. S i m i l a r increases i n ease of s u b s t i t u t i o n promoted by t h i o l ligands have been noted r e c e n t l y i n chromium(III) chemistry (30-32).


95

Reduction of Nitrogenase Substrates

6. schultz et al.

5. Biological Function of Molybdenum. The primary function of the reduced molybdenum species may be simply to bind rather than transfer electrons to the substrate. In our electrochemical studies a number of mechanisms appear to be at least as probable as one involving a bulk solution Mo(V)/Mo(III) redox cycle. In the chemical model systems these alternative mechanisms could be translated to ones in which the chemical reductant [NaBH^, NaÔ^ or Fe^Si^SR)^-*] transfers electrons (or H ) to molybdenum-bound C H without need of reoxidizing the Mo(III) center. In nitrogenase, a similar process can be pictured in which the molybdenum center is first reduced to its substratebinding oxidation state. Once bound, the substrate is reduced by a flow of electrons or reactive hydrogen from a proximal site (presumably Fei+S^-type ferredoxin) and then released, leaving the molybdenum site in its reduced state. Transfer of two electrons and two protons or transfe ally equivalent mechanism or bridged by an atom such as sulfur which could facilitate both proton and electron transfer. 2

2

2

Acknowledgment This research has been supported by the National Science Foundation under Grant GP-38442X. We are particularly grateful to Drs. W. E. Newton, Ε. I. Stiefel, J. W. McDonald and J. L. Corbin of the Charles F. Kettering Research Laboratory, Yellow Springs, Ohio for many fruitful discussions and for disclosing to us their discovery of butadiene product in the chemical model system prior to publication. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9.

Schrauzer, G.N., Angew. Chem. Int. Ed., (1975) 14, 514, and references therein. Kay, A. and Mitchell, P. C. H., J. Chem. Soc. A, (1970), 2421. Schrauzer, G. N. and Doemeny, P. Α., J. Amer. Chem. Soc., (1971) 93, 1608. Ott, V. R. and Schultz, F. Α., J. Electroanal. Chem., (1975) 61, 81. Ott, V. R. and Schultz, F. Α., J. Electroanal. Chem., (1975) 59, 47. Ott, V. R., Swieter, D. S. and Schultz, F. Α., manuscript in preparation. Ledwith, D. A. and Schultz, F. Α., J. Amer. Chem. Soc., (1975) 97, 6591. Kneale, G. G., Geddes, A. J., Sasaki, Y., Shibahara, T. and Sykes, A. G., J. Chem. Soc. Chem. Commun., (1975), 356. Nicholson, R. S. and Shain, I., Anal. Chem., (1964) 36, 706.


96 10.

ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTE Ridgway, Τ. Η., Van Duyne, R. P. and Reilley, C. N., J . Electroanal. Chem., (1972) 34, 267, 283.

11.

Corbin, J. L . , Pariyadath, N. and Stiefel, Ε. I., J. Amer. Chem. Soc., in press. 12. Tano, K. and Schrauzer, G. N., J. Amer. Chem. Soc., (1975) 97, 5404. 13. Hardy, R. W. F., Burns, R. C. and Parshall, G. W., Advan. Chem. Series, (1971) 100, 219. 14. Ichikawa, M. and Meshitsuka, S., J . Amer. Chem. Soc., (1973) 95, 3411. 15. Schrauzer, G. N., Kiefer, G. W., Tano, K. and Doemeny, P. Α., J. Amer. Chem. Soc., (1974) 96, 641. 16. Mairanovskii, S. G., "Kinetic and Catalytic Waves in Polarography," Plenum Press, New York, 1968. 17. James, B. R., "Homogeneous Hydrogenation," Wiley, New York, 1973. 18. Khrushch, A. P., Shilov Amer. Chem. Soc., (1974) 96, 4987. 19. Wentworth, R. A. D., Coordin. Chem. Rev., (1976) 18, 1. 20. Denisov, N. T., Shuvalov, V. F., Shuvalova, Ν. I., Shilova, A. K. and Shilov, A. E . , Dokl. Akad. Nauk SSSR, (1970) 195, 879. 21. Shilov, Α. Ε., Denisov, N. T., Efimov, O. Ν., Shuvalov, V. F., Shuvalova, N. D. and Shilova, Α. Κ., Nature, (1971) 231, 460. 22. Ketchum, P. Α., Taylor, R. C. and Young, D. C., Nature (1976), 259, 202. 23. Forget, P. and DerVartanian, D. V., Biochim. Biophys. Acta, (1972) 256, 600. 24. DerVartanian, D. V. and Forget, P., Biochim. Biophys. Acta, (1975) 379, 74. 25. Orme-Johnson, W. Η., Jacob, G., Henzl, M. and Averill, Β. Α., ACS Centennial Meeting, New York, 1976, Abstr. INOR-137. 26. Cramer, S. P., Eccles, T. K., Kutzler, F. W., Hodgson, K. O. and Mortenson, L. E., J. Amer. Chem. Soc., (1976) 98, 1287. 27. Stiefel, Ε. I., Proc. Nat. Acad. Sci. U.S.A., (1973) 70, 988. 28. Spivack, B. and Dori, Z., J. Chem. Soc. Chem. Commun., (1973), 909. 29. Newton, W. E., Corbin, J. L., Bravard, D. C., Searles, J. E. and McDonald, J. W., Inorg. Chem., (1974) 13, 1100. 30. Weschler, C. J. and Deutsch, Ε., Inorg. Chem., (1973) 12, 2682. 31. Ramasami, T. and Sykes, A. G., Inorg. Chem., (1976) 15, 1010. 32. Asher, L. E. and Deutsch, Ε., Inorg. Chem., (1976) 15, 1531.


7 Manganese(II) and -(III) 8-Quinolinol Complexes. R e d o x M o d e l for M i t o c h o n d r i a l Superoxide Dismutase

JOHN K. HOWIE, MARK M. MORRISON, and DONALD T. SAWYER Department of Chemistry, University of California, Riverside, Calif. 92502

The discovery i n 1969 (1) that superoxide i o n O, i s a common r e s p i r a t o r organisms with i t s y super oxide dismutase (SOD) has revolutionized the i n t e r pretation of b i o l o g i c a l oxidation-reduction processes. A subsequent discovery was a manganese-containing version of superoxide dismutase which can be i s o l a t e d from b a c t e r i a l sources (2,3) and from mitochondria (4) as well as the o r i g i n a l l y discovered copper-zinc form from erythrocytes. L i t t l e i s known about manganese SOD. The c r y s t a l structure has not been determined and there i s still controversy as to whether the manganese SOD contains one or two manganese atoms per enzyme molecule (2,4) . The exact r o l e of the manganese atom(s) i n the enzyme, the o x i dation state(s) of the manganese atom(s), the degree of association of the two manganese atoms, i f two are indeed present, the type of ligands coordinated to the manganese atom(s), and the stereochemistry around the metal(s) are not known. Although little i s known about the structure and properties of manganese SOD, its c a t a l y t i c reactions with superoxide ion can be represented by (5) -

2

Such a mechanism requires that the manganese e x i s t i n three d i f f e r e n t oxidation states if the enzyme contains only one metal atom per molecule. However, 97


98

ELECTROCHEMICAL


SYSTEMS

only two formal oxidation states are required i f the enzyme contains two metal atoms. Because the manganese-containing enzymes catalyze oxidation-reduction reactions, electrochemical methods are p a r t i c u l a r l y a t t r a c t i v e f o r the study of the redox behavior of manganese complexes, both alone and i n the presence of substrate. The further objective i s to charact e r i z e the structures and coordination chemistry of these complexes i n s o l u t i o n by use of spectroscopic as w e l l as other p h y s i c a l c h a r a c t e r i z a t i o n techniques. The goal of the present research i s the i d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n of manganese complexes that mimic the enzyme i n reactions represented by Equations 1-4 and that can serve as models f o r manganese SOD. This pape ese(II) and - ( I I I model compounds. Experimental Measurements and Materials. C y c l i c voltammetric experiments were performed using a v e r s a t i l e i n s t r u ment constructed from P h i l b r i c k s o l i d - s t a t e operat i o n a l a m p l i f i e r s (6). The c o n t r o l l e d p o t e n t i a l e l e c t r o l y s i s experiments were performed using a Wenking Model 61RH potentiostat and i n t e g r a t i n g the current vs. time curve using a K&E Model 62005 compensating polar planimeter. The electrochemical c e l l employed i n a l l electrochemical experiments was described previously (7). A Beckman Model 39273 platinum i n l a y electrode was used as the working electrode f o r c y c l i c voltammetry and a platinum gauze electrode was employed as the working electrode i n the coulometric experiments. The reference electrode was composed of a Ag/AgCl electrode i n aqueous t e t r a methylammonium chloride s o l u t i o n (0.000 V vs. SCE) and a glass bridge tube which made contact with the bulk s o l u t i o n through a cracked glass-bead j u n c t i o n . The platinum f l a g a u x i l i a r y electrode was i s o l a t e d from the bulk s o l u t i o n by a f i n e porosity f r i t . Dimethyl sulfoxide (DMSO) ( J . T. Baker analyzed reagent grade) had a water content of 0.02 to 0.06% as s p e c i f i e d by the manufacturer. Pyridine (Burdick and Jackson) contained 0.009% water and a c e t o n i t r i l e (MC/B Spectroquality grade) contained a maximum of 0.02% water. The solvents were degassed with argon i n the electrochemical c e l l p r i o r to the addition of the compound to be studied. Tetraethylammonium perchlorate (TEAP) was used as the supporting e l e c t r o -


7.

Manganese(II)

HOWIE E T A L .

and -(HI)

8-Quinolinol

Complexes

99

l y t e i n a 50-to 100-fold excess over the concentration of the e l e c t r o a c t i v e species. S o l i d - s t a t e magnetic s u s c e p t i b i l i t y values were determined by the Guoy method and s o l u t i o n mag n e t i c s u s c e p t i b i l i t y values were determined by the nuclear magnetic resonance (nmr) method (8). Conductivity measurements were made with an Ind. Instr. Model RC16B conductivity bridge and a dip c e l l . Mole cular weight determinations were made c r y o s c o p i c a l l y i n DMSO s o l u t i o n using a Hewlett-Packard Model 2801A Quartz thermometer t o measure temperature changes. The apparatus was c a l i b r a t e d with b e n z i l and a l l s o l u tions were prepared from the same f r e s h l y opened b o t t l e of DMSO. The solutions were protected from contamination by water i n the a i r by an atmosphere of dry argon. 8-Quinolinol wa ous acetate tetrahydrate, Mn (OAc) ·4Η2θ, was ob tained from A l f a . Methanol was reagent grade and used without further p u r i f i c a t i o n . Argon was dried by passing i t through a column packed with Aquasorb (Mallinckrodt). 1.00 M HC10 i n water and 1.42 M tetraethylammonium hydroxide i n methanol (Eastman) were used i n the electrochemical experiments where hydrogen and hydroxide ions were employed. II

2

4

Preparation of the Complexes 1.

Bis£8-quinolinolato)manganese(II) dihydrate, Mn Q *2H20. The compound was prepared by the r e a c t i o n between 10 g (0.041 mole) Μη (0Αο)2· 4H20 and 11.8 g (0.082 mole) HQ i n 250 ml of deaerated 1:1 methanol/water. The yellow product was f i l t e r e d under argon, washed with deaerated water and methanol, and d r i e d i n vacuo at room temperature f o r 2 nr. Elemental a n a l y s i s : Calcd. for M n C N H 0 : Mn, 14.49; C, 57.00; N, 7.39; and H, 4.26. Found: Mn, 14.27; C, 56.63; N, 7.04; and H, 4.31. II

2

ΑΙ

18

2.

2

16

4

μ-0xo-bis(8-quinolinQlato-8-quinolinol)manganese(III) dimethanol, Mni 0Q (HQ) -2CH 0H. The compound was prepared by the r e a c t i o n of a i r with a saturated s o l u t i o n of Mn Qo*2H 0 i n 1:1 meth anol/ water. The product which formed as black c r y s t a l s was f i l t e r e d , washed with water and methanol, and dried i n vacuo at room temperature for 2 nr. Elemental a n a l y s i s : Calcd. f o r M n C N H O : Mn, 10.39; C, 63.64; N, 7.95; and H, 4.38. Found: Mn, 10.14; C, 63.70; N, 7.76; II

4

2

3

2

2

56

6

46

g


100


and H, 4.30. I][

3.

Bis(8-quinolinolato)magnesixim(II) , Mg Q , and b i s ( 8 - q u i n o l i n o l a t o ) z i n c ( I I ) , Zn Q - Both of the compounds were prepared by the procedure used to synthesize Mn Q -2H 0. 2

2

I]C

2

4.

2

Superoxide ion, 0 ". Superoxide ion was generated i n s i t u i n DMSO and pyridine solutions by cont r o l l e d p o t e n t i a l e l e c t r o l y s i s at -1.00 V at a gold f o i l electrode of oxygen-saturated s o l u t i o n s . The solutions were degassed with argon, prereduced at -1.00 V f o r 10 min, and then saturated with oxygen. The oxygen flow was continued throughout the e l e c t r o l y s i s . To avoid the formation of protons during e l e c t r o l y s i which migrat int the working electrod electrode compartmen t i o n of tetraethylammonium hydroxide i n water. The solutions were degassed with argon p r i o r to use. 2

Results D i s s o c i a t i o n and Magnetic S u s c e p t i b i l i t i e s of Manganese Complexes. Conductance measurements i n d i cate that MnilQ «2H 0 i n DMSO s o l u t i o n i s about 10% dissociated into a 1-to-l e l e c t r o l y t e and that s o l u tions of Mni 0Q (HQ) '2CH 0H are not dissociated into ionically-cénducting species. The molecular weight determinations indicate that MnQ *2H 0 i s about 80% dissociated into MnQ and H 0. The manganese(III) 8-quinolinol complex contains a high spin d manganese ion i n the s o l i d state (μ=5.0+0.1 B.M.), but i n DMSO s o l u t i o n the complex exhibits a decreased magnetic moment ( f X 4.56+ 0.10 B.M.) The magnetic moment i s close t o the spin only value i n a c e t o n i t r i l e s o l u t i o n ^ =4.8+0.2 B.M.) and i n pyridine s o l u t i o n ( μ = 4 . 9 1 + 0 . 0 7 B.M.). 2

2

i:i:

4

9

3

2

2

2

2

4

=

c o r r

οθΓΓ

Electrochemistry of Mn(II)- and Mn(III)-8-Quinoli n o l Complexes! C y c l i c voltammograms of MnilQ -2H 0 and Mn OQ (HQ) i n DMSO s o l u t i o n appear i n Figure 1. The two compounds share common redox products. Mn Q *2H 0 i s not reduced but i s oxidized at +0.16 and +0.75 V. The +0.16 V peak i s a r e v e r s i b l e one-electron per manganese oxidation based on peak currents; however, c o n t r o l l e d p o t e n t i a l coulometry at +0.25 V reveals that on the longer coulometric time scale («~-*20 min) the oxidation i s only a 0.5 e l e c t r o n process. I f an equivalent of hydroxide ions i s added 2

2

4

2

II

2

2


2

7.

HOWIE E T AL.

Manganese(II)

and -(III) 8-Quinolinol

Complexes

101

at t h i s point and the e l e c t r o l y s i s i s continued, another 0.25 e l e c t r o n per manganese atom i s trans ferred. Reversing the c y c l i c voltammetric scan d i r e c t i o n a f t e r the oxidation at +0.16 V reveals another catho dic peak at -0.31 V. This peak i s observed also with Mn* ^0Q4(BQ)2both complexes, c o n t r o l l e d poten t i a l coulometry at -0.50 V indicates that t h i s i s a one-electron (per manganese atom) reduction. For Mn^ *0Q (HQ)2> as seen by c y c l i c voltammetry, most of the reduction product formed at -0.50 V i s reoxidized at -0.16 V with a small a d d i t i o n a l amount being r e o x i dized a t +0.16 V. For Un Q ' 2° P y electrolyzed at +0.25 V, the s i t u a t i o n i s reversed with respect to the product y i e l d s at -0.16 and +0.16 V Addition of 1 equivalen manganese atoms) t a f t e r e l e c t r o l y s i s at -0.5 V y i e l d s c y c l i c voltammo grams i d e n t i c a l to those observed a f t e r the e l e c t r o l y s i s sequence described above f o r solutions of Mn. Q2. MniiQ has a second large anodic peak at +0.75 V which i s also present i n solutions of Mn *0Q (HQ>2· The oxidation i s i r r e v e r s i b l e i n DMSO and pyridine but q u a s i - r e v e r s i b l e i n a c e t o n i t r i l e . Again, t h i s appears to be a one-electron per manganese oxidation based on peak currents. Electrode f i l m i n g by the oxidation product precluded confirmation by c o n t r o l l e d p o t e n t i a l coulometry. When one equivalent of protons i s added to a solu t i o n of Mn Q , both anodic peak currents are reduced by one h a l f ana a new cathodic peak appears at -1.85 V which corresponds to the reduction of the hydroxy1 protons of free HQ (7). When another equivalent of protons i s added, the anodic peaks disappear e n t i r e l y and the only peak which remains i s the cathodic peak at -1.85 V. Addition of OH" ions to solutions of Mn**Q2 also decreases the o r i g i n a l anodic peak currents and a new anodic peak appears at +0^08 V which corresponds to the oxidation of free Q~ ions to dimeric Q2 Addition of one equivalent of protons per mole of manganese atoms to solutions of Mn|- 0Q (HQ) s l i g h t l y decreases the peak current f o r the reduction at -0.31 V and reveals a new small cathodic peak at +0.11 V. (The peak at +Q-H V i s a l s o observed as part of the r e v e r s i b l e MniiQ re-reduction following the oxidation at +0.16 V.) Addition of one equivalent of 0ΗΓ ions per mole of manganese to solutions of Mni 0Q (HQ) r e s u l t s i n a spontaneous chemical reduction, a decrease i n the o r i g i n a l cathodic peak at -0.31 V, F

o

r

1

4

I1

2E

9

r e v i o u s l

2

2

2

4

II

2

CI

4

2

2

i:[

4


2


102

and the formation of a new anodic peak at -0.16 V. Reactions of Mn(II)- and Mn(III)-8-Quinolinol Complexes with 0 ~ , 0 , and HgC^. Figure 2 i l l u s 2

2

t r a t e s c y c l i c voltammograms i n DMSO s o l u t i o n at a platinum electrode f o r a) 1.3 mM 0 ~ , b) 1 mM Mn Q -2H 0, and c) a mixture of 1.3 mM 0 " and 1 mM M n Q * 2 H 0 ten seconds a f t e r mixing. C l e a r l y a l l of the 0 " i s decomposed within the time i t takes to record the f i r s t c y c l i c voltammogram, and the complex remains i n i t s i n i t i a l oxidation state and i s not appreciably decomposed. The s o l u t i o n does not change c o l o r during the r e a c t i o n . In addition there are cathodic peaks at -0.83 V and -1.30 V, which c o r r e s pond to the reductio and an enhancement The r e l a t i v e y i e l d s of 0 and H 0 vary but are between 50 and 75% of the t h e o r e t i c a l values. A f t e r standing f o r 30 minutes the c y c l i c voltammograms look s i m i l a r to those obtained f o r solutions of pure i 0 Q ( H Q ) . At higher 0 Q ~ to Mn Qo*2H 0 concent r a t i o n r a t i o s there i s 50 to 80% immediate decompos i t i o n , but then the r a t e of decomposition decreases. A large anodic peak appears at +0.08 V which c o r r e s ponds to Q" oxidation. Mn Q3" (prepared i n s i t u by e l e c t r o l y s i s of Mn| OQ (HQ) solutions at -0.50 V) and M n 0 Q 4 ( H Q ) a l s o react with 0o" to form 0 and H2O0 but at a slower rate than Mn- Q »2H 0. In addition, Mn Q *2H 0 reacts slowly with both 0 and H 2 0 to y i e l d solutions which have c y c l i c voltammograms s i m i l a r to those of M n i 0 Q ( H Q ) . I t i s noteworthy that Mni 0Q (HQ) does not react with e i t h e r 0 or H 02 i n DMSO s o l u t i o n . When 0.39 mM 0 ~ and 1.25 mM Mn^Q^HgO are mixed i n p y r i d i n e tne r e s u l t s are q u a l i t a t i v e l y s i m i l a r t o those obtained i n DMSO s o l u t i o n . Hydrogen peroxide formation, however, i s more c l e a r l y v i s i b l e and the y i e l d s of 0« and H 0 are somewhat higher. At higher 0 "-to-MnÎÏQ *2H 0 concentration r a t i o s , although the decomposition of 0 ~ remains rapid, the complex i s destroyed as indicated by the absence of a l l peaks assignable to manganese species. C y c l i c voltammograms recorded a f t e r the r e a c t i o n show only 2

l:[

2

2

2

II

2

2

2

2

M n

2

2

11

I I

4

2

2

II

4

2

2

2

2

T

LI

2

2

i:i

2

2

2

2

II

4

2

Il:

4

2

9

2

2

2

2

2

2

2

0

2

and H o 0 . 2

Μη ^θ2·2Η2θ a l s o reacts slowly with H 0 i n pyridine to give solutions which a f t e r degassing with argon give c y c l i c voltammograms s i m i l a r to M n ^ 0 Q (HQ>2. However, Mn ^Q2»2H20 reacts very r a p i d l y with Ι

2

2

II

I


4

7.

HOWIE E T AL.

Manganese(II)


Complexes

103

0 i n pyridine to give a s o l u t i o n with c y c l i c voltam mograms devoid of Μη(II) or Mn(III) redox a c t i v i t y . This indicates the formation of an insoluble or e l e c trochemically i n a c t i v e species. The c y c l i c voltammetric experiments d e t a i l e d i n Figure 2 were also c a r r i e d out using M g ^ and Z n ^ i n place of Mn* Q *2H 0. In both cases the rate of decomposition f o r 0 "" i s as slow as i t i s f o r DMSO solutions without added metal complex (about 5-15% per hour). 2

1 1

1 1

I

2

2

2

Discussion and Conclusions Structure of the Complexes i n Solution. The conductance and molecular weight data indicate that MnllQg-HgO i n DMSO different equilibria y i e l d s about 80% M n Î Q and HgO, and a ligand transf e r or hydrolysis whicn y i e l d s about 10% of some charged species. The electrochemical data to be d i s cussed l a t e r support the formulation of the charged species as Mn Q and Mn Q . The manganese(III)-8-quinolinol complex also undergoes s t r u c t u r a l changes upon d i s s o l u t i o n into DMSO as i l l u s t r a t e d by i t s decrease i n magnetic moment. Because a l l known Mn(III)-DMSO complexes are high-spin (9), solvent e f f e c t s are u n l i k e l y to cause spin p a i r i n g . The decreased value of the magnetic moment implies some antiferromagnetic coupling of the type that would be expected f o r μ-οχο bridged species or d i ^ - h y d r o x o bridged species, but i s weaker than that observed f o r di-μ-οχο bridged species (10,11). The decrease i n magnetic moment i n the Mn(IlTT-8q u i n o l i n o l complex i s comparable to that observed f o r μ-οχο-bis(tetraphenylporphinato)dimanganese(III,III) where the s o l i d state magnetic moment at 295 °K i s reported as 4.12 B.M. (12). In contrast to the magnetic moment i n DMSO s o l u t i o n , the near t h e o r e t i c a l value f o r the mangane s e ( I I I ) - 8 - q u i n o l i n o l complex i n a c e t o n i t r i l e and p y r i dine solutions suggests monomeric structure i n aceto n i t r i l e and pyridine. The di-μ-οχο bridged manganese(IV) 1,10-phenant h r o l i n e complex, Mn 0 (1,10-phen)^(ClO^)^, has a s o l i d - s t a t e magnetic moment of 1.86 B.M. per manganese ion (10). This value corresponds to one unpaired electron per manganese ion rather than the expected three unpaired electrons. Unfortunately, the compound i s not soluble enough t o permit measurement of i t s s o l u t i o n magnetic moment. The one-electron I

2

II

+

II

3

v

2

2



104

Figure 2. Cyclic voltammograms in 0.1M TEAP-DMSO at a Ft electrode of (a) 1.32mU 0 (b) lOOmM Mn Q - 2H 0, and (c) a mixture of 1.32mU Of and l.OOmU Mn Q · 2H 0. Scan rate, 0.1 V s~*. 2y

n

2

2

11

2

2


7.

HOWIE

Manganese(II)

E T A L .


105

Complexes

IV

reduction product of t h i s dimer, MnJH" 0 (1,10-phen^~ ( C 1 0 ) , i s soluble, however, and has a magnetic moment of 1.56 B.M. per manganese ion i n a c e t o n i t r i l e s o l u t i o n (11). This value corresponds to an average of less than one unpaired electron per mangan ese instead of the expected 3.5. Similar r e s u l t s have been obtained f o r the di-μ-οχο manganese(III-IV) b i p y r i d y l complex Mn* ~ 0 ( b i p y ) ~ (ClO.)o* magnetic moment i n a c e t o n i t r i l e s o l u t i o n of 1.81 B.M. per manganese (11) i s again close to the spin-only value of one unpaired electron per manganese. These r e s u l t s imply strong anti-ferromagnetic coupling across the dioxo bridge and confirm the s t a b i l i t y of oxo-bridged species i n s o l u t i o n . The elemental analysis and s o l i d - s t a t e magnetic moment of the manganese(III)-8-quinolino imply that the comple chelate, Mn Q -jHgO-CHoOH, i n the s o l i d state and becomes a bridged dimer i n DMSO s o l u t i o n . There are several possible dimeric structures: a di-μ-οχο bridge, a μ-οχο bridge, or a d i ^ - h y d r o x o bridge. The dioxo bridged structure can be ruled out because of the lack of strong antiferromagnetic coupling. The d i ^ - h y d r o x y species, Mn£**(OH) Q (HQ) , and the μ-οχο species, Mn| OQ (HQ) , are a l t e r n a t i v e s . Magnetic behavior f o r these species cannot be predicted because w e l l characterized manganese(III) dimers with these types of bridging are unknown. The mono-oxo-bridged formulation i s a t t r a c t i v e , however, because the r e l a t e d i r o n ( I I I ) - 8 - q u i n o l i n o l complex, Fe Q *^H 0, becomes mono-oxo bridged i n DMSO s o l u t i o n (13). Although the structure of the complex i s s t i l l i n doubt, the mono-oxo bridged formulation i s reasonable and w i l l be used throughout the remaining discussion. 2

4

3

Ii:

IV

4

T

h

e

Ii:i

3

2

4

2

II

4

2

3

2

Redox Properties of Mn(II)- and Mn(III)-8QuinoTinol Complexes. A s e l f - c o n s i s t e n t redox mechanism f o r these compounds appears i n Table I. The Μ η ^ · 2 Η 0 complex i s not reducible but i s r e v e r s i b l y oxidized to M n Q at +0.16 V. Most of the oxidized species ends up as Mn^ OQ (HQ) . Mn Q therefore must undergo a h y d r o l y t i c reaction with r e s i d u a l water to form Mn Q (0H) and H . The IT ions thît are formed attack unoxidized M n ^ to form Mn -Q and HQ. This r e a c t i o n has been confirmed by the experiments i n which H ion i s added to solutions of mi Q . The l i b e r a t e d HQ reacts with Mn Q (0H) to form Bn^ÔQ^ (HQ) and H 0. Furthermore, c o n t r o l l e d ι:ι

2

2

IIi:

+

2

1

+

4

1IA

2

2

+

2

1 1

I3

+

iI

Ii:E

2

2

9

9



lJ

2

2

3

1 1

2

^

4

2

4

2

II

Mn^ 0Q (HQ)

4

3

2

II

AiA

Mn^ 0Q (HQ)

2

2

Q (OH) + 2HQ

+

+ H0

+ H

+

Q

JI

+ HQ

+

i:i

3

1 1

4

+ 20H"

2

II

3

2

2+

II

+ 2e~

» Mn Q " + Mn Q

4

^ = ± Mn 0Q (HQ)

V

2

* M n ^ O Q ^ H Q ^ + 1^0

+

2

3

Q (0H) + H

Mn Q

> Mn

b

0Q (HQ) + 2 e ~ ^ = ± 2Mn Q " + BÔ τττ , „ DMSO _ τττ 2 M n Q (solid) + H^0 ^ = = i M n J O Q ( H Q )

Mn

I]CI

Ii:

2Mn

Mn ^

2

III

Q

I I I

Mn

1

^ + HQ

3

d

2

+ 2H 0

Mn ^" " + e"

Mn

1 1

±A

2

Mn"Q' + Mn II _ > Mn Q

= ^

11

1

+

^

II

Mn Q

Mn ^

Mn Q " + H

ir

A1

2Mn -Q II Mn Q + Q

11

m 0%-2^0

+

+ Q

2

e

+ HgO

+0.70

-0.31

+0.11

+0.75

-0.16

+0.16

V vs. SCE Ε Ε pc pa

Manganese(II) and - ( I I I ) 8-Quinolinol Complexes*

Redox Reactions and Voltammetric Peak P o t e n t i a l s f o r

Table I


a platinum electrode

1

(scan rate, 0.1 V s"" ) i n 0.1 M TEAP/DMSO.

A c e t o n i t r i l e solution.

e

2

i n s i t u by the reduction of HQ.

9

Q~ was prepared electrochemically

I3[

d

AA

C y c l i c voltammetry indicates that 1 mM solutions of Mn Q -2H 0 i n DMSO contain about 10% Mn Qo , and conductance measurements indicate that such solutions d i s s o c i a t e about 10% into a 1-to-l e l e c t r o l y t e .

Cryoscopic molecular weight determination i n DMSO indicates compound i s 80% dissociated.

At

c

a


108

p o t e n t i a l coulometry of MnIlQ at +0.25 V indicates that 0.5 equivalent of electrons i s transferred per mole of complex instead of 1. This i s consistent with the o v e r a l l reaction 2

11

2 Mn Q

2

+ Mn Q + l e " ( 5 )

II

+JH 0

>iMn^ OQ (HQ)

2

4

I][

2

+

i:i

Mn* 0Q (HQ) i s reduced, i n the absence of acid, by a one electron-per-manganese i o n process at -0.31 V t o form MniiQ " and H 0. The electron stoichiometry has been confirmed by c o n t r o l l e d p o t e n t i a l coulometry at -0.50 V. The formulation of the reduced species as Mn^Qo" i s supported by experiments i n which Q~ i o n i s eleçtrochemically generated i n s i t u i n the presence of Mn Q . The f i r s t anodic peak s h i f t s from +0.16 to -0.16 V, the p o t e n t i a a f t e r reduction of H ions are present, Mn Q i s converted t o Mn *^ and HQ. The anodic peak corresponding to the reoxidat i o n of the product species s h i f t s back to +0.16 V and a cathodic peak appears at -1.85 V due t o HQ reduc tion. The Mn OQ (HQ) complex i s i r r e v e r s i b l y o x i dized at +0.75 V to form a manganese(IV) species which immediately oxidizes the solvent, the r e s i d u a l water present i n the solvent, or i t s own ligands, and forms a mixture of the two manganese(III) complexes again. F i n a l l y , Mnî fOQ (HQ) i s chemically reduced i n the presence of OH ions to give a species which i s oxidized at -0.16 V. This p o t e n t i a l corresponds to the oxidation of Mn Q "". The reducing agent i s probably Q~ which i s oxidized to Q . The Mn Q complex also i s highly susceptible to n u c l e o p h i l i c d i s placement of the Q" ligands by OH"ions. In addition, MnîQo reacts slowly with both 0 and H 0 and, based on the peak p o t e n t i a l s , the product species of the r e a c t i o n i s M n | 0 Q ( H Q ) « . The extra HQ ligands must of necessity come from Mn^Qg so the other product of the r e a c t i o n i s Mn Q . 4

2

3

2

i:[

2

+

I3:

2

4

11

2

I

i

4

2

i:t

3

2

2

2

2

2

II

4

lA

+

Reaction of the Complexes with 0 ~. 2

ese ( iTf-S^quIno^

The mangan-

and

MniiQ3, and the manganese(III)-8-quinolinol complex, Mn| 0Q (HQ) *2CH 0H, i n DMSO s o l u t i o n represent a system that undergoes oxidation-reduction chemistry which p a r a l l e l s much of that observed f o r mitochond r i a l superoxide dismutase. Mn Q accelerates the decomposition of 0 ~ to form nearly stoichiometric amounts of the correct products, 0 and a mixture of II

4

2

3

2

2

9


7.

Manganese(II)

H O W I E E T AL.


Complexes

109

H 0 and HO ". The increased peak current that appears t o be associated with the oxidation of Mn Q at +0.16 V probably i s due to the coincident oxida t i o n of the decomposition product, H0 " to 0 . When the scan i s reversed a f t e r the anodic peak at +0.16 V the reduction peak at -0.83 V i s enhanced. The c y c l i c voltammograms and the s o l u t i o n c o l o r i n d i c a t e that the complex i t s e l f i s i n i t s i n i t i a l oxidation state and i s not appreciably decomposed. The c a t a l y t i c properties of the system are com p l i c a t e d by the f a c t that the only source of protons for H O " and H 0 formation i s HÔ. The OH" ions thus generated attack the c a t a l y s t to form what appears to be an i n a c t i v e species. When the c a t a l y s t i s used at lower concentrations (0.5 mM or l e s s ) the decomposition of 0 quickly, presumably decompo s i t i o n of the c a t a l y s t , and also because the i n creasing b a s i c i t y of the medium decreases the proton a c t i v i t y , which i n turn i n h i b i t s peroxide formation. In addition, the 0« and H 0 which are formed react with M n ^ to form Mn^^QîHQ)^. Mn^ 0Q (HQ) and Mn Q ~ a l s o react with 0 ~ to give 0 and H 6 , although more slowly. Based on our observations, a reasonable mechanism f o r the Q 2 t a l y z e d disproportionation of 0 "" appears to be 2

2

ft

II

2

o

2

Λ

2

2

2

1 1

l:f

JÎ

4

2

2

2

M n I I

2

c a

2

II

Mn Q (H 0) 2

Mn

II]C

2

2

+ 0 ~

^Mn

2

III

Q ( 0 H ) ( H 0 ) + OH" 2

2

2

I I

Q (0 H)(H 0)+0 ^ 2 ^ M n Q ( H 0 ) 2

2

2

2

2

2

2

+ 0

2

(6)

+ H0 " (7) 2

with secondary reactions η

Μη 10 V c m ) DNA first aggregates and then undergoes a s t r u c t u r a l transition in which the a n g l e s o f the p u r i n e and p y r i m i d i n e bases w i t h r e s p e c t t o the h e l i x - a x i s . , are altered. In e l e c t r i c f i e l d s o f about 2x10 VcnT r i b o s o m a l RNA and p o l y n u c l e o t i d e s such as p o l y ( A ) . 2 p o l y ( U ) appear t o undergo a t r a n s i e n t o p e n i n g o f base p a i r s f o l l o w e d by o n l y p a r t i a l r e a s s o c i a t i o n o f the unfolded regions (3). Such s t r u c t u r a l e f f e c t s o f e l e c t r i c a l f i e l d s on n a t u r a l p o l y n u c l e o t i d e s have been i m p l i c a t e d i n the mechanisms o f nerve impulse t r a n s m i s s i o n and i n f o r m a t i o n s t o r a g e i n the c e n t r a l nervous system (2), perhaps as an i n i t i a l step i n the r e c o r d i n g o f b i o l o g i c a l memory ( 3 - 6 ) . 4

- 1

113


114


The p o t e n t i a l s t h a t e x i s t a t c e r t a i n b i o l o g i c a l membranes s u c h a s a c e l l membrane a r e t h o u g h t t o be of the order of 0.1V. In a b i o l o g i c a l f l u i d h a v i n g an i o n i c s t r e n g t h o f 0 , 1 t o 0 . 2 t h i s p o t e n t i a l w o u l d e x t e n d o v e r d i s t a n c e s o f 1 0 - 1 0 0 ^ (1^. Tljiis c o r r e s p o n d s t o e l e c t r i c f i e l d s o f 10 - 1 0 Vcm . Clearly, i f a b i o p o l y m e r s u c h as DNA o r RNA i s p r e s e n t i n c l o s e p r o x i m i t y t o s u c h a b i o s u r f a c e t h e n i t seems quite reasonable to suggest that macromolecular s t r u c t u r a l t r a n s i t i o n s might occur. In f a c t , i n l i v i n g o r g a n i s m s DNA, f o r e x a m p l e , i s p a r t i a l l y a s s o c i a t e d w i t h t h e n u c l e a r o r c y t o p l a s m i c membrane, o r w i t h the i n t e r f a c e o f the n u c l e o l u s (8-11). A theory has been advanced t h a t r e p l i c a t i o n c o u l d b e g i n a t the l e v e l of the n u c l e a r o r c e l l u l a r w a l l (10-12). Indeed, H i l l (1_) h a s and t h e i r v a r i a t i o n a c t as t h e t r i g g e r f o r d i v i s i o n o f g e n e t i c m a t e r i a l i n the c e l l p r i o r to s e l f - d u p l i c a t i o n . A very i n t e r e s t i n g aspect of e l e c t r i c a l a c t i v i t y a s s o c i a t e d w i t h b i o l o g i c a l processes i s the e x i s t e n c e o f the p o t e n t i a l o f i n j u r y at a trauma s i t e . A sign i f i c a n t o b s e r v a t i o n i s t h a t the i n j u r y p o t e n t i a l follows a d i f f e r e n t time course i n the h e a l i n g o f , for example, a limb amputation i n the case of a s p e c i e s w h i c h c a n r e g e n e r a t e t h e l i m b a s o p p o s e d t o one w h i c h e x h i b i t s o n l y s c a r f o r m a t i o n (13, 1 4 ) . I t has been found t h a t i m p l a n t a t i o n o f s m a l l e l e c t r o d e s at the i n j u r y s i t e i n a nonregenerating s p e c i e s (forci n g the i n j u r y p o t e n t i a l to approximate t h a t of a r e generating species) causes at l e a s t p a r t i a l limb r e g e n e r a t i o n even i n a complex s p e c i e s s u c h as t h e rat (15,16). T h i s i m p l i e s t h a t fundamental b i o l o g i cal processes ( u l t i m a t e l y a t t h e g e n e t i c l e v e l ) may be c o n t r o l l e d b y t h e n a t u r a l o r a r t i f i c i a l l y a p p l i e d e l e c t r i c a l environment at a t i s s u e r e p a i r s i t e . The application of small, l o c a l l y applied e l e c t r i c fields h a s b e e n u s e d f o r t h e s t i m u l a t i o n o f b o n e h e a l i n g as a v e r y p r a c t i c a l outcome o f s u c h s t u d i e s . Cope ( 1 7 - 2 4 ) h a s p r e s e n t e d some c o n v i n c i n g a r g u ments t h a t a c e l l s u r f a c e - b i o l o g i c a l f l u i d i n t e r f a c e may be r e g a r d e d as b e i n g v e r y s i m i l a r t o a l i q u i d s o l i d i n t e r f a c e which e x h i b i t s e l e c t r i c a l behavior a n a l o g o u s t o t h a t o c c u r r i n g a t an e l e c t r o d e - s o l u t i o n interface. An e l e c t r o d e - s o l u t i o n i n t e r f a c e i s generally c h a r a c t e r i z e d by a w e l l - d e f i n e d e l e c t r i c a l d o u b l e l a y e r b o u n d e d o n one s i d e b y t h e e l e c t r o d e surface and on t h e o t h e r s i d e by an i o n i c l a y e r a c r o s s w h i c h a r e l a t i v e l y high e l e c t r i c a l f i e l d develops (up t o


8.

KINOSHITA E T A L .

Interfacial

Behavior

of

Purines

115

ca. 10 V cm ) . This c o n s t i t u t e s the s o - c a l l e d i n n e r o r compact double l a y e r , the w i d t h o f which i s o n l y a few a t o m i c d i a m e t e r s . On t h e b o u n d a r y b e t w e e n the i n n e r and the d i f f u s e d o u b l e l a y e r s the f i e l d s t r e n g t h h a s o n l y a b o u t 1/10 o f i t s o r i g i n a l v a l u e , and i t t h e n d e c r e a s e s i n the d i f f u s e double l a y e r t o v i r t u a l l y zero. I n a medium o f i o n i c s t r e n g t h c a . 0. 1-0.2 ( t y p i c a l o f b i o l o g i c a l f l u i d s } the depth o f t h e d i f f u s e d o u b l e l a y e r i s a b o u t 100& ( 2 5 ) . An e x a c t l y s i m i l a r l y structured region exists at a c e l l membrane-biological f l u i d interface (26). A t p h y s i o l o g i c a l b u l k - p h a s e pH v a l u e o f 7.2 a l l mammalian c e l l s so f a r e x a m i n e d c a r r y a n e t n e g a t i v e charge at t h e i r s u r f a c e s . However, the s u r f a c e p o t e n t i a l of a c e l l i s not c o n s t a n t but can undergo some r a t h e r d r a m a t i i s o l a t e d f r o m t h e r e g e n e r a t i n g l i v e r s o f r a t s some days a f t e r p a r t i a l hepatectomy and c e l l s from n e o n a t e s have s i g n i f i c a n t l y h i g h e r e l e c t r o p h o r e t i c mob i l i t i e s t h a n l i v e r c e l l s from normal a d u l t s (21), 1. e . , c e l l p r o l i f e r a t i o n i s a s s o c i a t e d w i t h i n c r e a s e d net surface n e g a t i v i t y . S i m i l a r l y , the e l e c t r o p h o r e t i c m o b i l i t i e s o f c e r t a i n tumor c e l l s i n c r e a s e w i t h g r o w t h r a t e (28!) . A t the time o f m i t o s i s a v e r y s i g n i f i c a n t i n c r e a s e i n n e t s u r f a c e n e g a t i v i t y has been o b s e r v e d i n v a r i o u s t y p e s o f c u l t u r e d tumor c e l l s (29, 3 0 ) . I n d e e d , Ambrose e t a l . (31, 3 2 ) , have n o t e d a c o r r e l a t i o n between m a l i g n a n c y and i n c r e a s e d c e l l surface n e g a t i v i t y , although t h i s i s c e r t a i n l y not t h o u g h t t o be a u n i v e r s a l c o r r e l a t i o n (33). A s u b s t a n t i a l amount o f e v i d e n c e i s b e i n g d e v e l oped which i n d i c a t e s t h a t i n t e r a c t i o n s w i t h b i o l o g i c a l i n t e r f a c e s i s a p r e r e q u i s i t e f o r the m a n i f e s t a t i o n of the b i o l o g i c a l e f f e c t s of p o l y n u c l e o t i d e s i n mammalian c e l l s y s t e m s i n v i v o and i n v i t r o . Thus, F i e l d e t a l . (34) h a v e r e p o r t e d t h a t RNA d o u b l e s t r a n d s , and p a r t i c u l a r l y p o l y (I), poly(C) induce i n t e r f e r o n f o r m a t i o n i n mammalian c e l l s . Subsequently, S c h e l l (15) h a s shown t h a t p o l y (I) . p o l y (C) i s a d s o r b e d t o t h e o u t s i d e o f t h e c e l l f o l l o w e d by s t r a n d s e p a r a t i o n a n d u l t i m a t e l y by i n t e r f e r o n f o r m a t i o n . I t h a s f u r t h e r b e e n s u g g e s t e d (36) that othex b i o l o g i c a l e f f e c t s o f p o l y n u c l e o t i d e s , s u c h as adjuvant e f f e c t s and enzyme i n h i b i t i o n / a c t i v a t i o n r e q u i r e s i n t e r a c t i o n of the p o l y n u c l e o t i d e with the charged c e l l surface. T h u s , i n summary, t h e s u r f a c e s o f m a m m a l i a n c e l l s a n d o t h e r b i o l o g i c a l membranes c a r r y a n a p preciable electrical potential. The e l e c t r i c a l d o u b l e - l a y e r formed i n the immediate v i c i n i t y o f a


116

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STUDIES O F B I O L O G I C A L S Y S T E M S

charged membrane-biological f l u i d i n t e r f a c e i s essent i a l l y i d e n t i c a l t o t h a t f o r m e d a t an e l e c t r o d e surface. The p o t e n t i a l o f a c e l l membrane s u r f a c e is known t o a l t e r when p r o c e s s e s s u c h a s c e l l regenerat i o n o c c u r o r , o f t e n , when a c e l l b e c o m e s m a l i g n a n t and p a r t i c u l a r l y a t the time o f m i t o s i s . The p o t e n t i a l of i n j u r y at a s i t e o f trauma i s c l e a r l y i m p l i c a t e d i n c o n t r o l of the t i s s u e r e g e n e r a t i o n p r o c e s s . The e l e c t r i c a l f i e l d s i n t h e i m m e d i a t e v j c i n j t y o f a ^ c e l l s u r f a c e a r e p r o b a b l y v e r y l a r g e (10 - 1 0 Vcm ) although they extend over only very small d i s t a n c e s ( c a . 1 0 - 1 0 0 K) . Such i n t e n s e e l e c t r i c f i e l d s h a v e b e e n shown t o c a u s e s t r u c t u r a l t r a n s i t i o n s i n c e r t a i n n a t u r a l and b i o s y n t h e t i c p o l y n u c l e o t i d e s . In a d d i t i o n , the i n t e r a c t i o n (adsorption) of p o l y n u c l e o t i d e s at the charge a prerequisite for manifestatio e f f e c t s of the p o l y n u c l e o t i d e s . Because a charged c e l l s u r f a c e - b i o l o g i c a l fluid i n t e r f a c e i s s i m i l a r to a charged e l e c t r o d e e l e c t r o l y t e s o l u t i o n i n t e r f a c e , i t seems reasonable t h a t an u n d e r s t a n d i n g o f t h e i n t e r f a c i a l b e h a v i o r o f biomolecules at the l a t t e r i n t e r f a c e might r e v e a l s i g n i f i c a n t i n f o r m a t i o n regarding the i n t e r a c t i o n s of these molecules at b i o l o g i c a l interfaces. I t w o u l d seem t o be s e l f - e v i d e n t , h o w e v e r , t h a t i n t e r f a c i a l s t u d i e s o f n u c l e i c a c i d s and o t h e r p o l y n u c l e o t i d e s , and i n t e r p r e t a t i o n o f the d a t a so c o l l e c t e d , must r e l y on a f u n d a m e n t a l k n o w l e d g e o f t h e i n t e r f a c i a l b e h a v i o r o f the monomeric u n i t s , i.e., b a s e s , n u c l e o s i d e s and n u c l e o t i d e s . A number o f i n v e s t i g a t o r s have r e p o r t e d t h a t v a r i o u s monomeric p u r i n e and p y r i m i d i n e d e r i v a t i v e s are a d s o r b e d a t mercury e l e c t r o d e s (37-45). Such s t u d i e s , however, have g e n e r a l l y been v e r y q u a l i t a t i v e ; t h e y have r e v e a l e d v i r t u a l l y n o t h i n g about the s u r f a c e areas o c c u p i e d by t h e a d s o r b e d m o l e c u l e s and h e n c e t h e i r probable surface o r i e n t a t i o n s , the nature o f the a d s o r p t i o n i s o t h e r m s and t h e e f f e c t s o f p o t e n t i a l on the a d s o r p t i o n p r o c e s s e s , the i n t e r m o l e c u l a r i n t e r a c t i o n s between a d s o r b e d m o l e c u l e s and i n t e r a c t i o n s between the adsorbed m o l e c u l e s and the e l e c t r o d e surface. R e c e n t l y , N u r n b e r g e t a l . (46) h a v e r e p o r t e d o n t h e a d s o r p t i o n o f a d e n o s i n e and a d e n o s i n e m o n o n u c l e o t i d e s at a mercury e l e c t r o d e . Measurements o f the a m o u n t o f t h e a d e n i n e s p e c i e s a d s o r b e d w e r e accom-^ p u s h e d by use o f t h e t i m e i n t e g r a l o f t h e faradaic r e d u c t i o n peak o f t h e s e m o l e c u l e s a t a s t a t i o n a r y m e r c u r y d r o p e l e c t r o d e a t 5 ° C a n d a t pH 3 . 4 . This


8.

KiNosHiTA E T A L .

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117

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v o l t a m m e t r i c t e c h n i q u e , h o w e v e r , i s s u b j e c t t o many e x p e r i m e n t a l l i m i t a t i o n s and p r o b l e m s (£7). We h a v e b e g u n a s y s t e m a t i c i n v e s t i g a t i o n o f t h e i n t e r f a c i a l b e h a v i o r o f t h e p u r i n e and p y r i m i d i n e b a s e s , d e o x y n u c l e o s i d e s and d e o x y n u c l e o t i d e s found i n nucleic acids. In t h i s r e p o r t the i n t e r f a c i a l behav i o r o f v a r i o u s a d e n i n e d e r i v a t i v e s a t pH 9 a t m e r c u r y e l e c t r o d e s w i l l be d e s c r i b e d . Two m a j o r s u r f a c e elect r o c h e m i c a l t e c h n i q u e s were employed i n t h e s e studies — t h e c a p i l l a r y e l e c t r o m e t e r and d i f f e r e n t i a l capacitance measurements. Some p r e l i m i n a r y r e s u l t s u s i n g e l e c t r o e l l i p s o m e t r y w i l l a l s o be p r e s e n t e d . Experimental D i f f e r e n t i a l capacitanc o b t a i n e d by a p h a s e - s e l e c t i v e a . c . polarographic method. A Princeton Applied Research Corporation (PARC) M o d e l 174 P o l a r o g r a p h i c A n a l y z e r c o u p l e d w i t h a PARC M o d e l 1 7 4 / 5 0 AC P o l a r o g r a p h i c A n a l y z e r Interf a c e a n d a PARC M o d e l 121 L o c k - i n A m p l i f i e r / P h a s e D e t e c t o r were employed f o r d i f f e r e n t i a l capacitance measurements. A phase a n g l e o f 90° w i t h r e s p e c t t o t h e a p p l i e d a l t e r n a t i n g v o l t a g e was e m p l o y e d . At the pH v a l u e s e m p l o y e d i n t h i s s t u d y , a d e n i n e a n d i t s d e r i v a t i v e s are not e l e c t r o c h e m i c a l l y r e d u c i b l e . The dropping mercury e l e c t r o d e (DME) was s i l i c o n i z e d (39) a n d was e q u i p p e d w i t h a m e c h a n i c a l d r o p dislodger. A p o o l o f mercury i n s e r t e d a t the bottom o f a thermos t a t t e d 5ml c a p a c i t y c e l l s e r v e d a s t h e c o u n t e r e l e c trode. A saturated calomel reference electrode (SCE) was e m p l o y e d u s i n g a f i n e L u g g i n c a p i l l a r y p o s i t i o n e d c l o s e t o t h e t i p o f t h e DME. A . c . p o l a r o g r a m s were u s u a l l y o b t a i n e d a t a f r e q u e n c y o f 100Hz a n d w i t h a m o d u l a t i n g a m p l i t u d e o f lOmV p e a k - t o - p e a k . Thus, the c a p a c i t y r e s u l t s r e p o r t e d h e r e were a l l measured a t a f r e q u e n c y o f 100Hz a n d w e r e n o t e x t r a p o l a t e d to zero frequency. However, the c a p a c i t y v a l u e s were v i r t u a l l y i n d e p e n d e n t o f f r e q u e n c y b e t w e e n a b o u t 50 a n d 600 H z . A l l m e a s u r e m e n t s w e r e made w i t h o u t d a m p i n g o n t h e M o d e l 174 a n d u s i n g a c o n t r o l l e d d r o p t i m e of 2.00s. When t h e a . c . p o l a r o g r a m was r e c o r d e d o n a n X - Y r e c o r d e r ( H e w l e t t - P a c k a r d M o d e l 7001A) t h e d . c . p o t e n t i a l was s c a n n e d a t a sweep r a t e o f 0.005Vs" . H o w e v e r , i n some o f o u r l a t e r studies t h e a l t e r n a t i n g c u r r e n t was m e a s u r e d b y u s e o f a K e i t h l e y M o d e l 16 8 A u t o r a n g i n g D i g i t a l M u l t i m e t e r connected to the Y a x i s ( c u r r e n t ) o u t p u t o f t h e PARC Model 174. The c a p i l l a r y

electrometer

and

its

associated


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BIOLOGICAL

SYSTEMS

p r e s s u r e s y s t e m i s shown s c h e m a t i c a l l y i n F i g u r e 1. The P y r e x c a p i l l a r i e s h a d a d i a m e t e r , 1mm a b o v e t h e t i p , of 0.002-0.005mm. The c a p i l l a r i e s w e r e n o r m a l l y aged f o r s e v e r a l d a y s by s o a k i n g i n d e i o n i z e d w a t e r and s u b s e q u e n t l y s t o r e d w i t h the t i p immersed i n w a t e r . A B r i n k m a n n / W e n k i n g M o d e l L T 7 3 p o t e n t i o s t a t was u s e d . The l o c a t i o n o f t h e m e r c u r y c o l u m n i n t h e c a p i l l a r y was o b s e r v e d w i t h a G a e r t n e r 2 2 0 6 - A C a t h e t o m e t e r t h r o u g h a n o p t i c a l l y f l a t P y r e x window s e a l e d o n one side of the c e l l (Figure 1). The c e l l was w a t e r j a c k e t e d and m a i n t a i n e d a t a t e m p e r a t u r e o f 25±0.1°C. The p r e s s u r e a t t h e m e r c u r y - t e s t s o l u t i o n interface was v a r i e d b y a p p l y i n g p r e s s u r e t o t h e g a s a b o v e t h e m e r c u r y b y means o f a s y r i n g e a n d two m i c r o m e t e r burets. The c o a r s e a d j u s t u t i l i z e d a 20ml p l a s t i c s y r i n g e w h i l e f i n e p r e s s u r e a d j u s t m e n t was a c c o m p l i s h e d w i t h two G i l m o n M e n s o r C o r p o r a t i o n Q u a r t z M a n o m e t e r p r e s s u r e g a u g e was u s e d t o measure t h e gas pressure. T e s t s o l u t i o n s were d e a e r a t e d w i t h n i t r o g e n for a t l e a s t 15 m i n u t e s b e f o r e m e a s u r e m e n t s w e r e c a r r i e d out. A l l p o t e n t i a l measurements u t i l i z e d a s a t u r a t e d c a l o m e l r e f e r e n c e e l e c t r o d e at 25°C. D a t a p o i n t s w e r e t a k e n a t 50mV i n t e r v a l s from -0.2V to - 1 . 8 V . A d r o p o f m e r c u r y was e x p e l l e d f r o m the c a p i l l a r y before the e l e c t r o c a p i l l a r y curve at e a c h c o n c e n t r a t i o n was m e a s u r e d . The p r e s s u r e at e a c h a p p l i e d p o t e n t i a l was t h e n a d j u s t e d t o b r i n g the mercury to the reference p o i n t i n the c a p i l l a r y (1mm f r o m t h e t i p ) . The h e i g h t o f t h e m e r c u r y c o l umn was m e a s u r e d w i t h t h e G a e r t n e r c a t h e t o m e t e r t o a p r e c i s i o n o f 0.02mm b e f o r e a n d a f t e r m e a s u r e m e n t o f each e l e c t r o c a p i l l a r y curve. A c o r r e c t i o n was a p p l i e d t o the measured p r e s s u r e f o r t h i s h e i g h t and f o r the s m a l l back p r e s s u r e o f the s o l u t i o n . The b o r a t e b u f f e r pH 9 u t i l i z e d was c o n s t i t u t e d as f o l l o w s : 17.5g N a J 0 10H 0 , 6 7 . 7 g KC1 a n d 1 6 . 8 5 m l 1M HC& d i l u t e d t o I I w i t h d e i o n i z e d w a t e r . M c l l v a i n e b u f f e r pH 7 was c o n s t i t u t e d a s f o l l o w s : 5 8 . 9 g N a H P O . 1 2 H 0 , 3.7g c i t r i c a c i d . H 0 and 5.4g KC1 d i l u t e d t o 11 w i t h w a t e r . Both of these buffer s o l u t i o n s have an i o n i c s t r e n g t h o f 0 . 5 . Sample s o l u t i o n s w e r e p r e p a r e d by d i s s o l v i n g t h e a d e n i n e s p e c i e s i n the a p p r o p r i a t e volume o f buffer. A G a e r t n e r M o d e l L 1 1 9 E l l i p s o m e t e r was e m p l o y e d for e l l i p s o m e t r i c studies. A schematic diagram of t h e a p p a r a t u s u t i l i z e d i s shown i n F i g u r e 2 . The a n g l e o f i n c i d e n c e o f t h e l i g h t beam was 7 0 ° . Test s o l u t i o n s w e r e d e a e r a t e d f o r a b o u t 30 m i n u t e s a n d a n i t r o g e n a t m o s p h e r e was m a i n t a i n e d o v e r t h e s o l u t i o n 2

4

2


8.

Interfacial

KiNosHiTA E T A L .

Behavior

of

119

Purines

Potentiostat

Figure 1.

System for electrocapillary measurements

Potentiostat

Figure 2.

Electroellipsometry system


ELECTROCHEMICAL

120

STUDIES O F B I O L O G I C A L

SYSTEMS

when m e a s u r e m e n t s w e r e t a k e n . The c i r c u l a r m e r c u r y p o o l w o r k i n g e l e c t r o d e h a d an a r e a o f 5.07cm . The c u r v a t u r e o f t h e m e r c u r y s u r f a c e was r e d u c e d by p l a c i n g an amalgamated p l a t i n u m i n s e r t a r o u n d t h e p e r i p h e r y of the g o o l . A small c r o s s - s e c t i o n a l area l i g h t b e a m , 0.8mm , was u s e d t o m i n i m i z e t h e e f f e c t of c u r v a t u r e of the mercury e l e c t r o d e s u r f a c e . The p r o cedure used i n measuring t h i c k n e s s e s and changes i n t h i c k n e s s o f f i l m s w i t h t h e e l l i p s o m e t e r was t h e f o l lowing. F i r s t , with o n l y the background e l e c t r o l y t e (buffer) s o l u t i o n i n the c e l l , values o f the p o l a r i z e r a n g l e (P) p r o d u c i n g a minimum i n i n t e n s i t y o f t h e r e f l e c t e d beam w e r e d e t e r m i n e d a s a f u n c t i o n o f p o t e n t i a l i n the range - 0 . 2 t o - 1 . 6 V v s . SCE. (The a n a l y z e r a n g l e , A , was n o t v a r i e d d u r i n g t h e s e e x p e r i m e n t s s i n c e the optimum v a l u by t h e p r e s e n c e o f f i l m t h i c k and s i n c e t h e c h o i c e o f a n a l y z e r a n g l e i n t h e minimum r e g i o n d o e s n o t i n f l u e n c e t h e v a l u e o f Ρ l e a d i n g t o minimum i n t e n s i t y . ) Next, with a s o l u t i o n of a d s o r b a t e i n t h e c e l l , new v a l u e s o f Ρ p r o d u c i n g m i n i mum i n t e n s i t y w e r e d e t e r m i n e d . Values of f i l m t h i c k n e s s were c a l c u l a t e d from changes i n Ρ (at f i x e d A and f i x e d p o t e n t i a l ) by u s i n g a c o m p u t e r p r o g r a m d e v e l o p e d by M c C r a c k i n ( £ 3 ) . In p r a c t i c e , i t i s sometimes d i f f i c u l t t o r e p r o d u c e e x a c t l y t h e v a l u e s o f Ρ w h i c h p r o d u c e minimum i n t e n s i t y for a given adsorbate or background s o l u tion. However, the shapes o f the v a r i o u s Ρ v s . p o t e n t i a l c u r v e s are o r d i n a r i l y q u i t e r e p r o d u c i b l e , and i t i s p o s s i b l e t o use the e x t e n s i v e a d s o r p t i o n r e s u l t s a v a i l a b l e from the c a p a c i t a n c e and e l e c t r o c a p i l l a r y experiments to " n o r m a l i z e " the e l l i p s o m e t r i c data. Thus, simple v e r t i c a l displacement o f the o b s e r v e d Ρ v s . p o t e n t i a l c u r v e s u s u a l l y b r i n g s them i n t o c o i n c i d e n c e w i t h the background curve i n regions o f p o t e n t i a l w h e r e l i t t l e o r no a d s o r p t i o n o c c u r s . D i f f e r e n c e s between the d i s p l a c e d curves f o r the a d s o r b a t e s o l u t i o n s and t h e b a c k g r o u n d c u r v e can t h e n be a t t r i b u t e d d i r e c t l y t o t h e o p t i c a l e f f e c t o f t h e f i l m and i n t e r p r e t e d t o y i e l d f i l m t h i c k n e s s e s as a f u n c t i o n o f a d s o r b a t e c o n c e n t r a t i o n and p o t e n t i a l . Analysis

of

Capacitance

and E l e c t r o c a p i l l a r y

Data

U s i n g tê a . c . p o l a r o g r a p h i c method the c a p a c i t a n c e , pFcm , i s r e a d i l y d e t e r m i n e d from the o b s e r v e d a l t e r n a t i n g c u r r e n t , yA, f r o m t h e e q u a t i o n : C

=

I ΔΕΑ2πί


(1)

8.

KINOSHITA

Interfacial

E TAL.

Behavior

121

of Purines

w h e r e ΔΕ i s t h e a m p l i t u d e o f t h e a p p l i e d a l t e r n a t i n g v o l t a g e ( V ) , A i s t h e s u r f a c e a r e a o f t h e DME (cm ) at the time the c u r r e n t i s sampled, and f i s the frequency (Hz) o f a p p l i e d a l t e r n a t i n g v o l t a g e . This e q u a t i o n i s v a l i d when t h e r e s i s t a n c e o f t h e t e s t s o l u t i o n i s s m a l l and the frequency o f the a p p l i e d a l t e r n a t i n g v o l t a g e i s low (48). The g e n e r a l p r o c e d u r e i n v o l v e d i n p r o c e s s i n g capacitance data to obtain adsorption isotherms i n volved f i r s t , c a l c u l a t i o n of surface spreading pres s u r e v a l u e s , π, f o r t h e o r g a n i c compound a t t h e m e r c u r y - s o l u t i o n i n t e r f a c e by back i n t e g r a t i o n o f capacitance data. T h e n , f r o m v a l u e s o f π a t known p o t e n t i a l , E , and c o n c e n t r a t i o n s o r a c t i v i t i e s , t h e p a r a m e t e r s i n t h e F r u m k i n a d s o r p t i o n e q u a t i o n were c a l c u l a t e d by a n o n l i n e a The b a c k i n t e g r a t i o n m e t h o d (49) r e l i e s o n t h e assumption t h a t at s u f f i c i e n t l y negative p o t e n t i a l s , c a p a c i t a n c e v e r s u s p o t e n t i a l c u r v e s (C v s . E) f o r aqueous s o l u t i o n s o f an o r g a n i c a d s o r b a t e c o i n c i d e with the background C v s . Ε curve f o r the e l e c t r o l y t e alone. T h i s c o n d i t i o n was m e t f o r a l l t h e a d e n i n e systems d e s c r i b e d h e r e , i . e . , a t p o t e n t i a l s between - 1 . 6 V a n d - 1 . 8 V t h e C v s . Ε c u r v e s become c o i n c i d e n t a n d r e m a i n s o a t e v e n more n e g a t i v e potentials. I n t e g r a t i o n o f C vs_. Ε c u r v e s f o r t h e b a c k g r o u n d electrolyte solution (i.e., v s . E) g i v e s values of charge q , r e l a t i v e q * , the charge a t the mercurye l e c t r o l y t e solution interface at the s t a r t i n g poten t i a l E * ( t h i s was t y p i c a l l y - 1 . 8 V i n t h e s e studies). Thus, Ε - q * =/ CdE (2) . E* By m e a s u r e m e n t o f a n e l e c t r o c a p i l l a r y c u r v e o f i n t e r f a c i a l t e n s i o n v e r s u s p o t e n t i a l (γ v s . E) o n t h e background e l e c t r o l y t e s o l u t i o n the vallie o f the e l e c t r o c a p i l l a r y maximum p o t e n t i a l (ECM) may b e o b tained ( i . e . , Ε at γ max.). A t t h e ECM q = 0 , h e n c e the a b s o l u t e v a l u e o f q ^ a t any p o t e n t i a l E (q^(E)) i s q

w

w

q (E)

=

w

fq (E) w

-

q*]

-

[qÊCM)

-

q * ] (3) .

From t h e measured v a l u e s o f γ (Ε) d e t e r m i n e d from e l e c t r o c a p i l l a r y curves o f the background s o l u t i o n the v a l u e o f

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> - « _ ^ c o c o ^ r r r i r > w « > • • • υ ε CQ χ* ^ 1

g H

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Ε

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Ν M

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c

c

2

2 2

2-Amino-1,4naphthoquinone

Anthraquinone - 2 sulphonate

2 -Amino-4 -pteridone

s are i n p a r e n t h e s e s ,

2

b

m a x

450

X

(nm) a

l

1,500

e(cm~

O x i d i z e d Form

c

V

Q

u

e

s

o

f

1

M^ )

r e f . (51) gave Ε 1/2 l - 0 . 3 0 and - 0 . 90 v o l t w ith r e s p e c t to S C E for 2 -• a m i n o - 6 , 7 - d i h y d r o - 4 pteridone and 2 - a m i n o - 4 - p t e r i d o n e , respe c t r / e l y , i n \ DH 9 borate buffer.

(pH 7. 0 , i o n i c strength 0.15)

135

p

η AE (mV)

2-Amino-6,7-dihydro4-pteridone

Compound

^supporting e l e c trolyte u s e d i s phosphate buiEfer

^literature v a l u e

5 1

5 8

5 2

(-660)

(-225)

-133 (-137)

(-60)

a

E°'(mV,NHE) '

Reduced Form

Redox Compounds U s e f u l a s M e d i a t o r - t i t r a n t s

TABLE IV ( C O N T I N U E D )

9

C/3 KJ C/5

>

Ω

ο ο

W

ο

Μ

α

Η ci

> f

Μ

g

ο η

w

H—»

SZENTRIMAY E T A L .

Coulometric

_J

ι

Titration

of

I

Biocomponents

ι

L_

Figure 10. Plot of the applied potential (E ned) vs. the optically determined logarithm of concentration ratio of oxidized to reduced molybdenum octacyanide. Experimental conditions same as those in Figure 9. apv

Cytochrome c

Oxidase

Equivalents/Mole

Enzyme

Figure 11. Normalized plots of optical absorbance vs. charge for the ICT of cytochrome c and cytochrome c oxidase with molybdenum octacyanide and anthraquinone 2-sulfonic acid, (left) ICT of cytochrome c (22μΜ); (right) ICT of cytochrome c oxidase (12μΜ). M-T's of LOmM Mo(CN) ~ and 1.3mM anthra quinone 2-sulfonic acid. 13μΜ phenazine methosulfate added to the oxidase solution to insure equilibrium. 8

3


166

ELECTROCHEMICAL


SYSTEMS

and may c a u s e problems for o p t i c a l monitoring of b i o c o m p o n e n t s . The o x i d i z e d forms of these c o m p l e x e s may be q u a n t i t a t i v e l y generated at the t i n oxide Ο TE and M - T y s ^ M - T I C T s have been performed u s i n g b e n z y l v i o l o g e n as the other M - T w i t h s a t i s f a c tory r e s u l t s . H o w e v e r , o x i d a t i v e I C T ' s of r e d u c e d c y t o c w i t h these M - T ' s e x h i b i t e d d r a w n - o u t ΔΑ-q c u r v e s i n d i c a t i v e of o x i d a t i o n of more than the iron heme m o i e t y . It i s i n t e r e s t i n g to note that the p r e v i o u s l y d i s c u s s e d molybdenum c y a n i d e gave q u a n t i tative η = 1 titration of c y t o c w h i c h s u g g e s t s that o x i d a t i o n of other parts of c y t o c_ o c c u r s at p o t e n t i a l s above ca_. +800 mV. The s u b s t i t u t e d p - p h e n y l e n e d i a m i n e s have been p r e v i o u s l y e m p l o y e d as mediators and M a c k e y (4) demonstrated the q u a n t i t a tive e l e c t r o g e n e r a t i o n of the d i i m i n e i n the c a s e of t e t r a - m e t h y l p - p h e n y l e n e d i a m i n e ( T M P D ) . He a l s o obtained good r e s u l t s for the I C T of c y t o c_ o x i d a s e u s i n p h e n o l h a s been examined thoroughly i n the O T T L E c e l l and u s e d for potentiometry of c y t o c_ by Heineman (21). Although o n l y a few naphthaquinones appear i n the t a b l e , s e v e r a l other n a p h t h a q u i n o n e s , p a r t i c u l a r l y those s u b s t i t u t e d a p p r o p r i a t e l y for greater water s o l u b i l i t y , are b e i n g c h a r a c t e r i z e d . These naphthaquinones w i l l serve as M - T ' s i n the p o t e n t i a l range of +100 to +200 mV. Phenazine m e t h o - and e t h o - s u l f a t e are w e l l known mediators w h i c h have been w i d e l y u s e d i n potentiometric titrations of b i o c o m p o n e n t s . The pteridones h a v e been s u g g e s t e d for c o u p l i n g to N A D r e d u c t i o n by Kwee and Lund (33). W e hope that further work w i l l be forthcoming from their laboratory u t i l i z i n g these p t e r i d o n e s . 1

+

Acknowledgement The f i n a n c i a l support provided by NSF Grant MPS 73-04882 and N I H - P H S Grant N o . G M 19181 i s gratefully a c k n o w l e d g e d .

Literature Cited 1. Hawkridge, Fred and Kuwana, Theodore, Anal. Chem., (1973) 45, 1021. 2. Heineman, William and Kuwana, Theodore, Acc. Chem. Res., (1976) 9, 241. 3. Heineman, William and Kuwana, Theodore, Biochem. Biophys. Res. Commun., (1973) 50, 892. 4. Mackey, L.N., Kuwana, T., and Hartzell, C.R., FEBS Lett., (1973) 36, 326. 5. Rodkey, F.L. and Donovan, J.A. Jr., J. Biol. Chem., (1959) 234, 677.


9.

SZENTRIMAY ET AL.

Coulometric Titration of Biocomponents

167

6. Thevenot D. and Leduc, P., 3rd International Symposium on Bioelectrochemistry, Juelich (1975). 7. Ke, B. and Hawkridge, F.M., unpublished results. 8. Steckan, Eberhard and Kuwana, Theodore, Ber. Bunsenges. Phys. Chem., (1974) 78, 253. 9. Mackey, L.N. and Kuwana, Theodore, 3rd International Symposium on Bioelectrochemistry, Juelich (1975). 10. Szentrimay, Robert, (1976) Ph.D. Thesis, Ohio State University. 11. Hennig, Horst and Gürtler, Oswald, J. Organometal. Chem., (1968) 11, 307. 12. Mason, J.G. and Rosenblum, Myron, J. Am. Chem. Soc. (1960) 82, 4206. 13. Gubin, S.P. and Perevalova (1962) 143, 1351 14. Perevalova, E.G., Gubin, S.P., Smirnova, S.A. and Nesmeyanov, A.N., Dokl. Akad. Nauk. SSSR (1964) 155, 857. 15. Yu, C.A. and Yu, Linda, Biochem. Biophys. Res. Commun., (1976) 70, 1115. 16. Fujihira, Υ., Kuwana, T. and Hartzell, C.R., Biochem. Biophys. Res. Commun., (1974) 61, 488. 17. Yeh, P. and Kuwana, T., J. Electrochem. Soc., (1976) 123, 1334. 18. Penden, Α.Α., Leont'evskaya, P.K., L'vova, T.I. and Nikolskii, B.P., Dokl. Akad. Nauk. SSSR, (1969) 189, 115. 19. Strojek, J.W. and Kuwana, T., Electroanalytical Chemistry and Interfacial Electrochemistry, (1968) 16, 471. 20. Penden, Α.Α., Zakharevskii, M.S. and Leont'evskaya, P.K., Kinetika: Kataliz, (1966) 7, 1074. 21. Heineman, W.R., Norris, B.J. and Goelz, J.F., Anal. Chem., (1975) 47, 79. 22. Caughey, W.S., Wallace, W.J., Volpe, J.A. and Yoshikawa, S., in "The Enzymes" (P.D. Boyer ed.) Volume XIII Part C, p. 299, Academic Press, New York, 1976. 23. Schroedel, Nancy, (1976) Ph.D. Thesis, The Pennsylvania State University. 24. Reinhammar, Bengt R. M., Biochimica et Biophysica Acta, (1972) 275, 245. 25. Pecht, Israel, Israel Journal of Chemistry, (1974) 12, 351. 26. Mendez, Hernandez and Lucenta, F., An. Quim, (1968) 64, 71. 27. Mendz, Hernandez, J. Acta Salmanticensia, Cienc (19671968) 33, 41.


168


28. Cordova-Orellana, Lucena-Conde, F., Talanta (1971) 18 505. 29. Laitinen, H.A. and Conley, J.M., Anal. Chem., (1976) 48, 1224. 30. Kolthoff, I.M. and Tomsicek, Wm. J., J. Phys. Chem., (1936 40, 247. 31. Malik, W. andAli,S.I., Indian J. Chem., (1963) 1, 374. 32. Nickolls, P. and Chance, Β., in "Molecular Mechanisms of Oxygen Activation", (O. Hayaishi, ed.) p. 479 Academic Press, New York 1974. 33. Kwee, S. and Lund, Η., Bioelectrochemistry and Bioenerget i c s , (1975) 1, 137. 34. Schilt, Α.Α., Anal. Chem., (1963) 35, 1599. 35. Miller, R.R., Brandt Soc., (1955) 77, 3178 36. Dwyer, F.P. and McKenzie, H.A., J. Proc. Roy. Soc. N.S. Wales, (1947) 81, 93. 37. Fortune, W.B. and Mellon, M.G., Ind. Eng. Chem., Anal. Ed., (1938) 10, 60. 38. Harvey, A.E. and Manning, D.L., J. Am. Chem. Soc., (1952) 74, 4744. 39. Moss, M.L. and Mellon, M.G., Ind. Eng. Chem., Anal. Ed., (1942) 14, 862. 40. Schilt, Α.Α., "Analytical Applications of 1,10-Phenanthroline and Related Compounds", Pergamon Press, New York, (1969). 41. Dywer, F.P., Gibson, N.A. and Gyarfas, E.C., J. Proc. Roy. Soc. N.S. Wales, (1942) 84, 80. 42. Burstall, F.H., Dwyer, F.P. and Gyarfas, E.C., J. Chem. Soc., (1950), 953. 43. (a) Volke, J., Collect. Czechoslov. Chem. Commun., (1968) 33, 3044. (b) Volke, J. and Volkova, V., Collect. Czechoslov. Chem. Commun., (1969) 34, 2037. 44. Kolthoff, I.M. and Tomsicek, W.J., J. Phy. Chem., (1935) 39, 945. 45. Michaelis, L. and H i l l , E.S., J. Am. Chem. Soc., (1933) 55, 1481. 46. Albrecht, A.C. and Simpson, W.T., J. Am. Chem. Soc., (1955) 77, 4455. 47. Dutton, P.L., Wilson, D.F. and Lee, C.P., Biochem., (1970) 9, 5077. 48. Dickens, F. and McIlwain, H., Biochem. J., (1938) 32, 1615.


9. SZENTRIMAY ET AL.

Coulometric Titration of Biocomponents

169

49. Friedheim, E.A.H., Biochem. J., (1934) 28, 180. 50. Friedheim, Ε.A.H. and Michaelis, L., J. Biol. Chem. (1931) 91, 355. 51. Kwee, S. and Lund, H., Biochim. Biophys. Acta, (1973) 297, 285. 52. Fieser, L.F. and Fieser, M., J. Am. Chem. Soc., (1934) 56, 1565. 53. Conaut, J.B., Kahn, H.M., Fieser, L.F. and Kurtz, S.S., J. Am. Chem. Soc., (1922) 44, 1382. 54. Michaelis, L., Schubert, M.P. and Granick, S., J. Am. Chem. Soc., (1939) 61, 1981.


10 Rotating Ring Disk Enzyme Electrode for Biocatalysis Studies 1

RALPH A. KAMIN, FRANK R. SHU, and GEORGE S. WILSON Department of Chemistry, University of Arizona, Tucson, Ariz. 85721

In recent years there has been considerable interest i n catalytic surface reaction interest. This has bee development of immobilized enzyme technology (1,2) and electrochemical sensors based on electroactive product formation within an enzyme layer (3,4). In comparing the kinetic behavior of an immobilized enzyme with i t s soluble counterpart, it is necessary to establish that the overall reaction rate i s catalysis rather than mass transport limited. It has been shown, for example, that immobilized enzymes i n flowing streams give apparent Michaelis constants K ', that are flow rate dependent (5). Under conditions where the overall reaction i s limited by mass-transport supply of substrate to the catalytic surface, K ' i s larger than expected. One i s then tempted to conclude that the properties of the enzyme have been modified by immobilization. On the contrary, increasing flow (mass transport) rates may lead to a limiting value for K ' essentially identical to that of the soluble enzyme (6). The rotating disk electrode as described by Levich (7) appears to offer an experimentally facile means for varying the rate of substrate mass transport. The addition of a concentric ring (rotating ring disk electrode) (8) permits independent monitoring of the reaction at the disk surface. We have recently (9) derived the theory describing the response of the rotating disk enzyme electrode. In the present work we report further experimental studies in support of this theoretical model. The system selected for study i s the glucose/glucose oxidase reaction: M

M

M

Glucose + 0 2 o

l u c

s e

g ° ) oxidase

Gluconic Acid + H 0 22 o

o

(1)

The peroxide produced i s either monitored directly or coupled 1

Present address: Smith-Kline Instruments, 880 W. Maude Ave., Sunnyvale, CA 94086 170


10.

KAMIN

ET

Rotating

AL.

Ring

Disk Enzyme

171

Electrode

with the i n d i c a t o r r e a c t i o n : H 0 2

2

+ 2H

+

+ 2Γ

m

o

l

r

b

d

a

t

e

>

I

2

+ 2H 0 2

(2)

Experimental Instrumentation, The f our^ e l e c t r o d e p o t e n t i o s t a t used i n these s t u d i e s was s i m i l a r to that described by Shabrang and Bruckenstein (10). The r o t a t i n g d i s k e l e c t r o d e , Model DT-6, was purchased from the Pine Instrument Co., Grove C i t y , PA. The d i s k was a 0.5 cm deep c a v i t y with a radius of 0.382 cm ac cording to the manufacturer's s p e c i f i c a t i o n s . When f i l l e d with carbon paste the c a l c u l a t e d d i s k area was 0.46 cm^. The width of the platinum r i n g e l e c t r o d e was 0.024 cm and was separated from the d i s k by a 0.01 e f f i c i e n c y measured experimentall paste e l e c t r o d e (8) was 0.18 and was i n good agreement with experimental r e s u l t s . A platinum wire counter e l e c t r o d e and a Ag/AgCl reference e l e c t r o d e ( E = 0.200 v) were employed. A Pine Instruments Model PIR r o t a t o r was used to c o n t r o l e l e c trode r o t a t i o n speed. o f

Preparation of Glucose Oxidase E l e c t r o d e . The carbon paste was prepared i n the usual manner from 5 g of graphite powder #38 ( F i s h e r S c i e n t i f i c Co.) and 3 ml of Nujol except that 10 mg (except where otherwise s p e c i f i e d ) of n-octadecylamine ( t e c h n i c a l grade, A l d r i c h ) was a l s o added. The carbon paste was packed f i r m l y i n t o the d i s k c a v i t y of the DT-6 e l e c t r o d e which was then p o l i s h e d with a piece of weighing paper. A f t e r the r i n g and gap were c a r e f u l l y cleaned, the e l e c t r o d e was allowed to r o t a t e i n a 12.5% glutaraldehyde s o l u t i o n f o r 10 - 15 min. followed by a 1 minute washing with c o l d 0.2M phosphate b u f f e r pH 6.5. (Glutaraldehyde must be f r e s h l y p u r i f i e d and s t o r e d below 0°C as i t r e a d i l y polymerizes (11))· The r o t a t i n g e l e c t r o d e was dipped i n t o a bovine serum albumin s o l u t i o n (0.1 g/ml) (BSA F r a c t i o n V 96-99%, Sigma Co.). After 2 - 3 minutes the e l e c trode was washed f o r 1 minute i n c o l d phosphate b u f f e r . The e l e c t r o d e was then removed from the r o t a t o r and p o s i t i o n e d with the e l e c t r o d e surface f a c i n g up. A glucose oxidase s o l u t i o n prepared by d i s s o l v i n g 0.3 g of the enzyme (Glucose Oxidase E.C. 1.1.3.4 Sigma Type II 15,000 units/g) i n 1 ml. of 5% g l u t a r a l dehyde s o l u t i o n (buffered with phosphate at pH 6.5) was a p p l i e d to the d i s k s u r f a c e . A f t e r standing at room temperature f o r 5 min., the excess enzyme s o l u t i o n was discarded and the gap and r i n g were c a r e f u l l y cleaned. Rotating the e l e c t r o d e i n c o l d phosphate b u f f e r at 2500 rpm f o r 5 min. aids i n removing phys i c a l l y entrapped or weakly bonded enzyme. When not i n use the e l e c t r o d e was stored i n phosphate b u f f e r at 5°C.


172

ELECTROCHEMICAL

STUDIES O F

BIOLOGICAL

SYSTEMS

S o l u t i o n s and Reagents Unless otherwise mentioned, a l l chemicals used were reagent grade. The stock s o l u t i o n of 0.1 M glucose was allowed to mutar o t a t e at room temperature f o r at l e a s t 24 hr. before using. When the course of the r e a c t i o n was measured by f o l l o w i n g I2 formation (Reaction 2) a K I - b u f f e r c a t a l y s t described p r e v i o u s l y (12) was employed. Where d i r e c t monitoring of peroxide format i o n (Reaction 1) i s p o s s i b l e the glucose i s d i s s o l v e d i n a 0.05 M phosphate b u f f e r pH 6.5. Procedure. The enzyme e l e c t r o d e was allowed to r o t a t e f o r about 30 sec. i n the glucose s o l u t i o n at which time a p o t e n t i a l was a p p l i e d to the r e s p e c t i v e i n d i c a t i n g e l e c t r o d e . The i o d i n e formed i n Reaction 2 was monitored at the d i s k by applying a p o t e n t i a l of -0.2 V v mation (no i o d i d e present by h o l d i n g the p o t e n t i a l at -0.2 V followed by a step to 0.75 V at which p o i n t the current t r a n s i e n t was measured. The p o t e n t i a l was then returned to -0.2 V u n t i l the next measurement. Enzyme E l e c t r o d e T h e o r e t i c a l Model. The d e t a i l s of the d i g i t a l s i m u l a t i o n c a l c u l a t i o n s f o r t h i s e l e c t r o d e have been presented elsewhere (9). Our model assumes the existence of an enzyme l a y e r extending i n t o s o l u t i o n from the e l e c t r o d e surface (X=0). This uniformly d i s t r i b u t e d t h i n enzyme l a y e r i s assumed not to i n t e r f e r e with d i f f u s i o n of species to or from the e l e c t r o d e s u r f a c e . The enzyme l a y e r l i e s w i t h i n the minimum hydrodynamic l a y e r j u s t i f y i n g the assumption that s o l u t i o n flow i n the e l e c t r o d e v i c i n i t y i s a l s o unaffected by the immobilization process. Michaelis-Menten k i n e t i c theory i s assumed to describe the enzymatic r e a c t i o n . Figure 1 i l l u s t r a t e s the nature of the concentration gradi e n t s at the e l e c t r o d e surface f o r a p a r t i c u l a r set of condit i o n s . The steady s t a t e product (or coupled product) concentrat i o n gradient i s f i r s t simulated f o r the r o t a t i n g e l e c t r o d e at open c i r c u i t . Product concentration increases as substrate pene t r a t e s the enzyme l a y e r from the s o l u t i o n s i d e . I f a p o t e n t i a l i s a p p l i e d to the d i s k i n a region where the product i s e l e c t r o a c t i v e , i t s concentration at the e l e c t r o d e surface drops to zero. E v e n t u a l l y the steady-state c o n d i t i o n shown i n Figure 1 i s a t t a i n e d . I t w i l l be noted that the concentrations i n the outer p o r t i o n of the enzyme l a y e r are r e l a t i v e l y unaffected by the pot e n t i a l perturbation. The r a t e of product formation i s given by Michaelis-Menten theory d

[P] dt

k C /(K /[S]+l) 3

E

M


(3)

10.

Rotating

KAMiN E T A L .

Ring

Disk Enzyme

173

Electrode

where i s the rate constant f o r the i r r e v e r s i b l e conversion of the enzyme-substrate complex i n t o products and K^. the M i c h a e l i s constant. C^, i n t h i s case i s the a n a l y t i c a l concentration of a c t i v e enzyme i n the immobilized l a y e r and S the substrate con c e n t r a t i o n i n the enzyme l a y e r . In order to evaluate the r e l a t i v e e f f e c t s of c a t a l y s i s and convective mass transport a r e a c t i o n v e l o c i t y parameter, V, i s defined: = W k ^ I

v

(

4

)

The convection time constant, t ^ , has been derived p r e v i o u s l y by P r a t e r and Bard (13) and i s given by t

k

= (0.51)-

2 where ν i s the kinematic v i s c o s i t y (cm /sec) and ω the r o t a t i o n speed i n rad/sec. For a given enzyme e l e c t r o d e , V r e f l e c t s the amount of product formed i n a given time and i s dependent only on ω, to which i t i s i n v e r s e l y p r o p o r t i o n a l . For l a r g e values of V e.g. V > 10 the c a t a l y s i s rate i s extremely f a s t and the o v e r a l l r e a c t i o n becomes convection mass transport l i m i t e d . For V < 0.1 the enzymatic r e a c t i o n i s c a t a l y s i s r a t e l i m i t e d . Thus, by v a r y i n g the e l e c t r o d e r o t a t i o n speed, the f l u x of sub s t r a t e can be modulated to change the nature of the r a t e l i m i t i n g process. The r a t i o C/K^ where C i s the bulk substrate con c e n t r a t i o n a l s o serves to define the current response. We have a l s o shown (9) that an optimal r o t a t i o n speed f o r current mea surement w i l l r e s u l t from increased substrate mass transport on one hand and decreased product production due to short contact time with the c a t a l y t i c l a y e r on the other. The steady s t a t e current r e l a t i o n s h i p s are presented below: Case I - Mass Transport Limited Rate (V > 10) From s i m u l a t i o n i t can be shown (by analogy to a L i n e weave r-Burk p l o t (14)):

nFAdk C 3

1/2 E

k

2

" d ~

1.22

D t

- k_

C

t

3 E k C

(6)

where i i s the steady s t a t e current at the d i s k , d i s the en zyme l a y e r thickness; b i s a f u n c t i o n only of ω and D. A l l other parameters have the usual e l e c t r o c h e m i c a l s i g n i f i c a n c e . At low substrate concentrations the f i r s t term of Equation 6 i s much greater than b and the steady-state current becomes 0.65nFAD V 2 /

1 / 6

U)

1 / 2

C


(7)

174

ELECTROCHEMICAL

STUDIES O F

BIOLOGICAL

SYSTEMS

which i s p r a c t i c a l l y i d e n t i c a l to the L e v i c h equation (7) f o r a r o t a t i n g d i s k e l e c t r o d e as expected. Case I I - C a t a l y s i s Limited Rate (V lO^Qcm) were used, h i t t i n g the LVS with a metal object d i d produce measurable sinusoidal voltages. With e l e c t r o l y t e s of lower r e s i s t i v i t y , the e f f e c t was l e s s s i g n i f i c a n t . The Ueda e f f e c t does not account f o r the p u l s a t i l e p o t e n t i a l s measured i n the water bath, but i t d i d contribute a "sinusoidal noise" signal due to wave motions i n the bath. A factor that indicates that i t was indeed streaming p o t e n t i a l s that we measured was that the wave shape was dependent upon the rubber check valves. These valves were i n d i v i d u a l l y cast i n our laboratory by hand, using a s u r g i c a l latex. Each had a character of i t s own. As a r e s u l t , the opening and closing c h a r a c t e r i s t i c s of each


11.

FiNDL A N D KURTZ

Left Ventricle/Aorta

Figure

9.

Wave form parison

Simulator

com-


191

192

ELECTROCHEMICAL

STUDIES O F B I O L O G I C A L S Y S T E M S

differed. This resulted i n a noticeable variation i n s i g n a l waveform. I t i s much more r e a s o n a b l e t o assume t h e s e v a r i a t i o n s i n s i g n a l waveforms a r e due t o f l o w v a r i a t i o n s a n d t h u s s t r e a m i n g p o t e n t i a l s r a t h e r t h a n due t o a c o u s t i c e f f e c t s c a u s i n g Ueda potentials. As a f i n a l i n d i c a t i o n t h a t t h e p o t e n t i a l s m e a s u r e d were i n d e e d s t r e a m i n g p o t e n t i a l s , F i g u r e 9 i l l u s t r a t e s a c o m p a r i s o n b e t w e e n t h e wave s h a p e o f e l e c t r o d e s 4 and 5 and the flow o f blood i n t o the human a o r t a ( i ) . N o t e t h e c l o s e s i m i l a r i t y o f wave form. The p o t e n t i a l s m e a s u r e d u s i n g t h e L V S * s w i t h n o r m a l s a l i n e , were a n o r d e r o f m a g n i t u d e l o w e r than those t y p i c a l l y obtained i n v i v o , using mammals. I t was n o t o u r o b j e c t i v e t o d u p l i c a t e in vivo surface-electrolyt potential levels. However, i t i s f e l t t h a t such l e v e l s c a n be a t t a i n e d i n v i t r o , u s i n g b l o o d a s an e l e c t r o l y t e , c o l l a g e n l i n e d plumbing and p u l s a t i l e blood flow conditions as occur i n v i v o . I n summary, i t h a s b e e n shown t h a t p u l s a t i l e flow of s a l i n e e l e c t r o l y t e s generates electrok i n e t i c p o t e n t i a l s remarkably s i m i l a r to i n vivo EKG's. This fact, i n conjunction with p r i o r research^ 2), i n d i c a t e s that the present assumption t h a t EKG p o t e n t i a l s a r e d u e s o l e l y t o m u s c l e a c t i o n p o t e n t i a l s n e e d s t o be r e - e x a m i n e d . Acknowledgments The a s s i s t a n c e o f L i n d a Stormo a n d S i d n e y Golden o f our research s t a f f i n the conduct o f t e s t s and p r e p a r a t i o n o f t h i s paper i s g r a t e f u l l y acknowledged. Abstract Several left ventricle/aorta mechanical s i m u l a t o r s were f a b r i c a t e d t o e v a l u a t e t h e p o s sibility o f g e n e r a t i n g EKG like electrical signals by electrokinetic methodology. The s i m u l a t o r s produced p u l s e d t u r b u l e n t flows, s i m u l a t i n g mammalian h e a r t pumping c o n d i t i o n s . EKG like s i g n a l s were g e n e r a t e d by t h e m o t i o n o f t h e electrolyte through the simulators.


11. FINDL AND KURTZ Literature

Left Ventricle/Aorta Simulator

193

Cited

1.

I b e r a l l , Α., Cardon, S., Young, Ε., "On Pulsatile and Steady Arterial Flow, t h e GTS C o n t r i b u t i o n , " General T e c h n i c a l S e r v i c e s Inc., Upper Darby, Pa. (1973) LC 72-96894. 2. M i l l e r , J . R., Dent, R. F., Lab and Clin. Med., (1943), 28, 168. 3. Sawyer, P. Ν., Himmelfarb, Ε., L u s t r e n , I . , Z i s k i n d , Η., B i o p h y s . J . , (1966), 6, 641. 4. C i g n e t t e , Μ., "Streaming P o t e n t i a l s , Theory and Examples in Biological Systems," in P r o c . 1 I n t e r . Symp. Biol. A s p e c t s o f E l e c t r o c h e m . , S. M i l l a z o , P. E. Jones, L. Rampazzo eds., Birkhauser Verlag Basel (1971) 5. K u p f e r , Ε., J. 53, 16. 6. S r i n i v a s a n , S., Sawyer, P. Ν., J . Coll. I n t e r f a c . Sci., (1970), 32, 456. 7. Quinke, G., Ann. P h y s i k , (1859), 2, (107), 1. 8. Z o l l n e r , F., Ann. P h y s i k , (1873), 2, (148), 640. 9. Edlund, Ε., Ann. P h y s i k , (1875), 2, (156), 251. 10. Haga, Η., Ann. P h y s i k , (1877), 3, ( 2 ) , 326. 11. C l a r k , J . W., Ann. P h y s i k , (1877), 3, ( 2 ) , 335. 12. Helmholtz, H. L. F., Ann. P h y s i k , (1879), 3, (7), 337. 13. Dorn, E., Ann. P h y s i k , (1880), 3, ( 9 ) , 513. 14. Boumans, Α. Α., P h y s i c a , (1957), 23, 1038. 15. K u r t z , F., Findl, Ε., K u r t z , Α., Stormo, L., J . Coll. I n t e r f a c . S c i e n c e , ( i n p r e s s ) . 16. P r o c o p i u , S., Ann. P h y s i k , (1913), 37, 229. 17. Zucker, E. R., "A Critical Evaluation of Streaming P o t e n t i a l Measurements," Ph.D. T h e s i s , Columbia Univ., (1959). 18. Newberry, Α., T r a n s . E l e c t r o c h e m . Soc., (1934), 67, 25. 19. Ueda, T., e t al., J. E l e c t r o c h e m . Soc. Japan, (1951), 19, 142. 20. Yeager, Ε., Hovorka, F., J. A c o u s t i c a l Soc. America, (1953), 25, 445. 21. W i l l i a m s , Μ., Rev. Sci. Instr., (1948), 19, 640. 22. Packard, R. G., J . Chem. Phys., (1953), 21, 303. st


12 Differential Pulse Polarographic Analysis for Ethylenediaminetetraacetate ( E D T A ) and Nitrilotriacetate ( N T A ) i n Phytoplankton Media RICHARD J. STOLZBERG Harold Edgerton Research Laboratory of the New England Aquarium, Central Wharf, Boston, Mass. 02110 In t h e c o u r s e o f investigating t h e effects o f t r a c e metal s p e c i a t i o we have found it desirable tration of small q u a n t i t i e s o f strong organic l i g a n d s . These i n c l u d e artificial l i g a n d s such as e t h y l e n e d i a m i n e t e t r a a c e t a t e (EDTA), nitrilotriacetate (NTA), and t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e ( t r i s ) added by the e x p e r i m e n t e r and some l e s s w e l l - d e f i n e d e x t r a cellular m e t a l b i n d i n g o r g a n i c s (EMBO) added by t h e phytoplankton. Artificial l i g a n d s have traditionally been added by algal p h y s i o l o g i s t s because a wide variety o f a l g a e c a n be grown in media c o n t a i n i n g complexed t r a c e m e t a l s . In a d d i t i o n , precipitation of t h e medium is reduced, e n a b l i n g t h e e x p e r i m e n t e r t o p r e p a r e a more r e p r o d u c i b l e medium ( 1 ) . The h y p o t h e s i s has been made t h a t p l a n k t o n might a c t i v e l y produce EMBO f o r much t h e same r e a s o n - t o improve the medium f o r growth e i t h e r by d e t o x i f y i n g potentially t o x i c m e t a l s such as copper (2) o r by making i r o n a v a i l a b l e as a s o l u b l e c h e l a t e d s p e c i e s (3,4). Our work c u r r e n t l y i n v o l v e s correlating t h e c o n c e n t r a t i o n o f complexed and uncomplexed s p e c i e s o f copper w i t h p h y t o p l a n k t o n p r o d u c t i v i t y and w i t h t h e p r o d u c t i o n o f EMBO. W e l l d e f i n e d artificial media t h a t l e n d themselves t o c o n v e n i e n t c h e m i c a l manipu lation and which s u p p o r t a good growth o f a l g a e a r e used. Table I g i v e s the composition o f both the s y n t h e t i c seawater (SSW) used in t h e analytical development work and t h e artificial medium d e s i g nated Cu-IV. In media c o n t a i n i n g l e s s than 5 χ 10 M EDTA o r NTA, a c c u r a t e measurement o f specific l i g a n d concen t r a t i o n i s q u i t e important. Small v a r i a t i o n s i n l i g a n d c o n c e n t r a t i o n may i n c e r t a i n c a s e s produce -6

194


12.

EDTA

STOLZBERG

and NTA

in Phytoplankton

TABLE Composition

of

I

Synthetic

Component

195

Media

Seawater

and

Cu-IV

SSW

Cu-IV

NaCl

4.3

χ

ΙΟ"

1

M

4.3

χ

ΙΟ"

1

M

KC1

9.4

χ

1(Γ

3

M

9.4

χ

ΙΟ"

3

Μ

χ

ΙΟ"

3

Μ

7.5

χ

ΙΟ"

3

Μ

1.2

χ

10~

3

Μ

5

Μ

MgS0

4

2.

CaCl

2

9.5

NaN0

3

NaH P0

4

4.8

χ

10~

Na Si0

3

2.6

χ

ΙΟ"

4.4

χ

10~

to

5 χ

ΙΟ"

6

Μ

to

5 χ

1θ"

β

Μ

to

8 χ

10~

2

2

Boron EDTA

5 χ

ΙΟ"

ΝΤΑ

5 χ

ΙΟ""

Tris

8 χ

ΙΟ"

7

7

5

Μ

4

5

3

Μ

Μ

Μη

2.2

χ 1θ"

Ζη

2.2

χ

1θ""

7

Μ

5.4

χ

10~

8

Μ

3.7

χ

10~

9

Μ

Co

(inorganic)

Vitamin

Β

1 2


6

Μ

196

ELECTROCHEMICAL


SYSTEMS

i m p o r t a n t changes i n copper s p e c i a t i o n . Loss of l i g a n d due t o p h o t o d e g r a d a t i o n i s a d i s t i n c t p o s s i b i l i t y when c u l t u r i n g a l g a e , p a r t i c u l a r l y i n l o n g term experiments under h i g h l i g h t i n t e n s i t y . The s e n s i t i v i t y of ferric-EDTA to photodegradation has b e e n known f o r o v e r two d e c a d e s (5) . The s u s c e p t i b i l i t y o f b o t h f e r r i c - and c u p r i c - N T A t o p h o t o d e g r a d a t i o n has been documented r e c e n t l y {6,]_,S). Sorption of ligands can a l s o present experimental d i f f i c u l t i e s i n dense a l g a l c u l t u r e s . Changes i n m e t a l s p e c i a t i o n c a n be e x p e c t e d f r o m any o f t h e s e mechanisms t h a t m i g h t r e d u c e l i g a n d o r m e t a l c o n c e n trations. F i n a l l y , t h e p r o d u c t i o n o f EMBO b y t h e c e l l s s h o u l d be t a k e n i n t o c o n s i d e r a t i o n when calculating speciation. T h e q u a n t i t y o f EMBO p r o d u c e d by t h e c e l l s c o u l d b t o t a l complexing capacit EDTA o r NTA p r e s e n t . The a n a l y t i c a l methodology f o r s p e c i f i c and s e n s i t i v e d e t e r m i n a t i o n o f EDTA a n d NTA i n s a l i n e waters i s not w e l l developed i n s p i t e o f the wide r a n g e o f t e c h n i q u e s d e v e l o p e d f o r NTA i n f r e s h w a t e r and sewage s l u d g e (100 . The most w i d e l y used s p e c i f i c t e c h n i q u e s i n n o n - s a l i n e w a t e r a r e gas chromatography (11-13) and e l e c t r o c h e m i s t r y (14-18). The p r e s e n c e o f l a r g e q u a n t i t i e s o f d i s s o l v e d salts i n seawater c l e a r l y favors e l e c t r o c h e m i c a l techniques. E l e c t r o c h e m i c a l r e d u c t i o n o f t h e CdNTA c o m p l e x , f i r s t u s e d a n a l y t i c a l l y f o r t h e d e t e r m i n a t i o n o f NTA i n EDTA (14^) a n d t h e n l a t e r a d a p t e d f o r NTA d e t e r m i n a t i o n i n l a k e and r i v e r w a t e r s ( 1 6 , 1 7 ) , i s t h e system of c h o i c e . R e d u c t i o n o f NTA c o m p l e x e s o f l e a d , b i s m u t h , a n d i n d i u m (_15,1_8) h a s b e e n u s e d a n a l y t i c a l l y , b u t t h e CdNTA {19_,2C0 a n d C d E D T A ( 2 1 , ,22,23) e l e c t r o c h e m i s t r y h a s b e e n c h a r a c t e r i z e d i n d e t a i l and appears r e l a t i v e l y w e l l behaved. C l a s s i c a l DC p o l a r o g r a p h y h a s b e e n u s e d t o m e a s u r e 1 t o 10 ppm NTA i n l a k e w a t e r (16^) , b u t t h i s t e c h n i q u e c o u l d n o t be u s e d i n s e a w a t e r . The r e d u c t i o n c u r r e n t due t o t h e l a r g e q u a n t i t y o f cadmium a d d e d t o d i s p l a c e c a l c i u m f r o m t h e NTA w o u l d swamp t h e s m a l l c u r r e n t i n c r e m e n t s d u e t o CdNTA r e d u c t i o n . D i f f e r e n t i a l pulse polarography (DPP) i s more s e n s i t i v e t h a n DC p o l a r o g r a p h y , and i t c a n be u s e d t o measure s m a l l c u r r e n t s a t a p o t e n t i a l c a t h o d i c o f an e l e c t r o c h e m i c a l l y a c t i v e s p e c i e s p r e s e n t a t much g r e a t e r c o n c e n t r a t i o n .


12.

EDTA

STOLZBERG

Theoretical

and NTA

in Phytoplankton

197

Media

Considerations

T h e r e d u c t i o n o f t h e CdNTA c o m p l e x a t - 0 . 9 V (vs SCE) i s i r r e v e r s i b l e a n d d i f f u s i o n controlled (20). A t pH 8 t h e r e d u c t i o n o f t h e CdEDTA c o m p l e x a t - 1 . 2 t o - 1 . 3 V i s more i r r e v e r s i b l e t h a n t h a t for CdNTA. I t i s d i f f u s i o n c o n t r o l l e d o n l y when t h e c o n c e n t r a t i o n o f t h e s u p p o r t e l e c t r o l y t e i s h i g h (2_1) . S e p a r a t i o n o f t h e uncomplexed Cd (at - 0 . 6 V ) , the CdNTA, and t h e CdEDTA waves w i l l p r e s e n t no p r o b l e m s u s i n g DPP. I n s e a w a t e r , t h e CdEDTA c o m p l e x r e d u c t i o n c u r r e n t i s e x p e c t e d t o be d i f f u s i o n c o n t r o l l e d due to the presence of a high c o n c e n t r a t i o n of salts, and t h e s e n s i t i v i t y s h o u l d be s u f f i c i e n t f o r t h e determination of micromolar q u a n t i t i e s of ligand. The a d d i t i o n o f a l a r g a l a r g e f r a c t i o n o f b o t h l i g a n d s t o be p r e s e n t i n t h e b u l k o f t h e s o l u t i o n as t h e cadmium complex (see below). E q u a t i o n 1 d e s c r i b e s the g e n e r a l i z e d competition r e a c t i o n b e t w e e n c a d m i u m a n d c o m p e t i n g m e t a l (M) for EDTA o r NTA ( L ) . Charges have been o m i t t e d f o r clarity. C o n c e n t r a t i o n s t a b i l i t y c o n s t a n t s and m o l a r c o n c e n t r a t i o n s are used throughout. CdL + Μ ^

Cd + ML,

K„ = C

w

h

a

n

e

d

r

=

e

.

TmTTTlT

=

CdL

[ [ C

C

d

L

( l a )

(lb)

]

d]

(D

CdL

[L]

Using Ringbom's concept o f c o n d i t i o n a l s t a b i l i t y c o n s t a n t s a n d h i s n o t a t i o n (24^) , t h e c o m p e t i t i o n s t a b i l i t y c o n s t a n t , K , c a n be r e w r i t t e n as a conditional competition constant c

K'

=

'ML K '

K

c

d

[Cd'] [M

Electrochemical studies of biological systems

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Electrochemical studies of biological systems

NMR IN BIOLOGICAL SYSTEMS

Fundamentals of Electrochemical Corrosion

Fundamentals Of Electrochemical Deposition

Electrochemical Activation of Catalysis

Carbon-13 NMR Spectroscopy of Biological Systems

Dynamic Models and Control of Biological Systems

Automaton theory and modeling of biological systems

Transport Phenomena in Biological Systems

Information Processing and Biological Systems

Electrochemical Dictionary

Nanoscale technology in biological systems

Fundamentals of Electrochemical Deposition

Electrochemical activation of catalysis

Encyclopedia of Electrochemistry, Electrochemical Engineering

Encyclopedia of Electrochemistry, Electrochemical Engineering

Electrochemical Aspects of Ionic Liquids

Biological Magnetic Resonance: Distance Measurements in Biological Systems by EPR

Electrochemical Dictionary

Electrochemical Sensor Analysis

Electrochemical Aspects of Ionic Liquids

Electrochemical Toxicity Sensors

Electrochemical Process Simulation III

Molecular Association in Biological and Related Systems

Electrochemical Thermodynamics and Kinetics

Electrochemical Impedance Spectroscopy

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Molecular orbital calculations for biological systems

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