Electrochemical and Spectrochemical Studies of Biological Redox

8 Binding as a Prerequisite for Rapid Electron Transfer Reactions of Metalloproteins M. J. EDDOWES and H. A. O. HILL

Downloaded by YORK UNIV on November 11, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch008

Inorganic Chemistry Laboratory, South Parks Road, Oxford, OX1 3QR, England

The electrochemistry of cytochrome c at a surface-modified gold electrode is described. Cyclic voltammetry and ac impedance studies show that the protein undergoes a rapid quasi-reversible electron transfer reaction, with k° = 1.5 x 10 m/s. Rotating disk electrode studies show that the protein binds to the electrode prior to electron transfer. The electrochemistry of cytochrome c, cytochrome c , and ferredoxin at the mercury electrode, on which these proteins adsorb, is also discussed. A general mechanism, involving binding of the protein to the electrode prior to electron transfer, and hence, analogous to that observed in the physiological redox reactions of cytochrome c, is proposed to account for the observed electron transfer reactions of metalloproteins at electrodes. Such binding is, most likely, a prerequisite for rapid electron transfer in the reactions of metalloproteins. -4

s

3

' I *he e l e c t r o n t r a n s f e r r e a c t i o n s o f c y t o c h r o m e c , a l o w - m o l e c u l a r ·*• w e i g h t , s o l u b l e , h e m e p r o t e i n c o m p o n e n t o f t h e m i t o c h o n d r i a l res piratory c h a i n , were the subject o f intense b i o c h e m i c a l a n d p h y s i c o c h e m i c a l studies to d e t e r m i n e h o w t h e p r o t e i n achieves t h e o b s e r v e d e a s e o f e l e c t r o n transfer a n d s t i l l m a i n t a i n s t h e d e g r e e o f s p e c i f i c i t y a n d c o n t r o l n e c e s s a r y for i t s e f f i c i e n t f u n c t i o n . K i n e t i c s t u d ies ( 2 - 5 ) i n d i c a t e d t h a t c y t o c h r o m e c f o r m s a c o m p l e x w i t h i t s p h y s i o l o g i c a l r e d o x p a r t n e r s p r i o r t o e l e c t r o n transfer, a n d v a r i o u s e q u i l i b r i u m s t u d i e s (6-10) c o n f i r m e d t h e e x i s t e n c e o f s u c h p r o t e i n protein complexes. T h e lysine residues o f cytochrome c were s h o w n (11-14) b y c h e m i c a l m o d i f i c a t i o n a n d o t h e r s t u d i e s t o b e i m p o r t a n t t o i t s p h y s i o l o g i c a l r e d o x r e a c t i o n s , c o n s i s t e n t w i t h (15) t h e i r d i s t r i 0065-2393/82/0201-0173$07.25/0 © 1982 A m e r i c a n C h e m i c a l Society In Electrochemical and Spectrochemical Studies of Biological Redox Components; Kadish, K.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

174

BIOLOGICAL REDOX COMPONENTS

b u t i o n on the surface o f the p r o t e i n . D e t a i l e d a n d specific c h e m i c a l m o d i f i c a t i o n s t u d i e s (16-25) h a v e s i n c e d e f i n e d t h e b i n d i n g d o m a i n on c y t o c h r o m e c ( w h i c h interacts w i t h its p h y s i o l o g i c a l redox partners, c y t o c h r o m e c reductase, c y t o c h r o m e c peroxidase, a n d cytochrome c oxidase) to b e a r o u n d t h e exposed h e m e edge o f c y t o c h r o m e c a n d h a v e c o n f i r m e d the i m p o r t a n c e o f p a r t i c u l a r l y s i n e


residues i n this interaction. T h e e l e c t r o n transfer reactions b e t w e e n s m a l l m o l e c u l e r e d o x re agents a n d c y t o c h r o m e c also w e r e investigated, a n d b i n d i n g b e t w e e n the reactants w a s o b s e r v e d . F o r e x a m p l e , the r e d o x reaction b e t w e e n c y t o c h r o m e c a n d the f e r r o - f e r r i c y a n i d e c o u p l e was s t u d i e d (26-29) u s i n g a variety o f t e c h n i q u e s , a n d apparently the reaction proceeds v i a a t i g h t l y b o u n d precursor c o m p l e x , the formation o f w h i c h precedes the e l e c t r o n transfer e v e n t itself. A s i m i l a r r e a c t i o n s e q u e n c e also was i n d i c a t e d b y k i n e t i c studies o f the redox reactions b e t w e e n s m a l l m o l e c u l e r e d o x c o u p l e s a n d o t h e r m e t a l l o p r o t e i n s , s u c h as t h e b l u e c o p p e r p r o t e i n s (30-33) a n d t h e f e r r e d o x i n s (34-36). Conventional electrochemical methods, w h i c h might b e consid e r e d t h e m o s t d i r e c t m e t h o d for t h e s t u d y o f r e d o x s p e c i e s , e n j o y e d l i m i t e d success i n their a p p l i c a t i o n to the study o f c y t o c h r o m e c a n d r e d o x proteins i n general. D e s p i t e the facility o f e l e c t r o n transfer be tween p h y s i o l o g i c a l redox-partner proteins, rapid, direct electron transfer b e t w e e n e l e c t r o d e s a n d m e t a l l o p r o t e i n s i n s o l u t i o n has b e e n r e p o r t e d i n f r e q u e n t l y . W h e r e r a p i d e l e c t r o n transfer is o b s e r v e d , a d sorption o f the p r o t e i n at the electrode surface g e n e r a l l y occurs. F o r e x a m p l e , at the m e r c u r y e l e c t r o d e , w e l l - d e f i n e d p o l a r o g r a p h i c waves w e r e o b s e r v e d for t h e r e d u c t i o n o f c y t o c h r o m e c (37-41), c y t o c h r o m e c (42, 43), a n d f e r r e d o x i n (44); i n e a c h c a s e a d s o r p t i o n effects w e r e c l e a r l y i n d i c a t e d . I n a d d i t i o n , w e s h o w e d (45-49) t h a t a t a s u r f a c e modified gold electrode, where well-defined voltammetric waves are o b s e r v e d for t h e r e d u c t i o n a n d o x i d a t i o n o f c y t o c h r o m e c , r a p i d a n d r e v e r s i b l e b i n d i n g o f t h e p r o t e i n to t h e e l e c t r o d e o c c u r s . T h i s b i n d i n g , w h i c h is a n essential feature o f the e l e c t r o d e process, m a y b e analo g o u s to t h a t b e t w e e n c y t o c h r o m e c a n d its p h y s i o l o g i c a l r e d o x p a r t n e r s i n t h a t i t a p p e a r s t o d e p e n d (47) o n t h e l y s i n e r e s i d u e s o f t h e c y t o c h r o m e c. 3

A s u i t a b l e b i n d i n g i n f r a c t i o n w o u l d a p p a r e n t l y b e o f major i m portance i n d e t e r m i n i n g the k i n e t i c s o f the e l e c t r o n transfer reactions o f redox proteins. W e here r e v i e w the current e x p e r i m e n t a l data con c e r n i n g t h e e l e c t r o n t r a n s f e r r e a c t i o n s o f m e t a l l o p r o t e i n s at e l e c t r o d e s . I n particular w e consider the i n v o l v e m e n t o f adsorption p h e n o m e n a i n s u c h reactions a n d their relation to c o m p l e x formation i n other redox reactions o f m e t a l l o p r o t e i n s . T h e r o l e o f b i n d i n g i n the rate enhance ment, c o n t r o l , a n d specificity o f p h y s i o l o g i c a l r e d o x processes is also discussed.

In Electrochemical and Spectrochemical Studies of Biological Redox Components; Kadish, K.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

8.

EDDOWES AND H I L L

Reactions

of

175

Metalloproteins

Electrochemistry of Cytochrome c at a Modified Gold Electrode C y c l i c Voltammetry Studies.

A s r e p o r t e d (46), t h e d c a n d a c c y c

l i c v o l t a m m o g r a m s o f h o r s e h e a r t c y t o c h r o m e c at a g o l d e l e c t r o d e i n t h e p r e s e n c e o f 4 , 4 ' - b i p y r i d y l ( F i g u r e 1) s h o w t h a t a q u a s i - r e v e r s i b l e , o n e - e l e c t r o n p r o c e s s o c c u r s , w i t h a h a l f - w a v e p o t e n t i a l , £1/2 = + 0 . 2 5 V v s . ( N H E ) . T h i s e l e c t r o d e p r o c e s s is a t t r i b u t a b l e to t h e r e d u c t i o n a n d oxidation o f the h e m e iron prosthetic group o f c y t o c h r o m e c. C o n trolled potential reduction a n d oxidation, followed

spectrophotome-

trically, confirm that the e l e c t r o d e process i n v o l v e s the h e m e

iron.

T h e e l e c t r o d e r e a c t i o n a l s o w a s s t u d i e d ( 4 7 , 48) u s i n g a c i m p e d a n c e Downloaded by YORK UNIV on November 11, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch008

methods,

which

s h o w that the reaction

is r a p i d w i t h

a

standard

4

e l e c t r o c h e m i c a l r a t e c o n s t a n t , kg = 1.5 x 1 0 ~ m / s . C

-0.2V

-E->

W.2V

Figure 1. Cyclic voltammetry (dc) of horse heart ferricytochrome c (5 mglmL) in 0.1 M NaCl0 , 0.02 M phosphate buffer at pH 7, in the presence of ΙΟ^ M 4,4'-bipyr idyl in the potential range +0.20 to -0.20 V vs. SCE, at sweep rates a, 20 mVIs; b, 50 mV/s; and c, 100 mV/s. The scan rate independent separation of forward and reverse peaks of 60 mV indicates that, under these conditions, the reaction is essentially diffusion controlled. (Reproduced from Ref. 46. Copyright 1979, Amer ican Chemical Society.) 4

2



176

,


T h e r e a c t i o n is n o t o b s e r v e d i n t h e a b s e n c e o f 4 , 4 - b i p y r i d y l , b u t 4 , 4 ' - b i p y r i d y l a l o n e is n o t e l e c t r o a c t i v e i n t h e p o t e n t i a l r e g i o n w h e r e the v o l t a m m e t r i c w a v e s are o b s e r v e d a n d therefore cannot b e a c t i n g as a c o n v e n t i o n a l m e d i a t o r i n t h i s c a s e . F u r t h e r m o r e , t h e o b s e r v e d v o l t a m m e t r y indicates that the e l e c t r o d e process i n v o l v e s d i r e c t e l e c tron transfer b e t w e e n t h e e l e c t r o d e a n d t h e p r o t e i n . 4 , 4 ' - B i p y r i d y l a p p e a r s t o a c t (46, 4 8 , 5 0 ) b y a d s o r b i n g o n t h e g o l d e l e c t r o d e s u r f a c e , t h e r e b y m o d i f y i n g i t , a n d t h u s p r o v i d i n g a s u i t a b l e i n t e r f a c e at w h i c h the electrode reaction o f cytochrome c may take place. T h e e l e c t r o d e r e a c t i o n o f c y t o c h r o m e c at t h e m o d i f i e d g o l d e l e c trode shows (47) some s t r i k i n g similarities to its reaction w i t h its p h y s i o l o g i c a l r e d o x p a r t n e r s i n t h a t b o t h a r e i n h i b i t e d (11-14) b y c h e m i c a l modification o f the c y t o c h r o m e c l y s i n e residues a n d b y the c o m p e t i t i v e i n h i b i t o r , p o l y - L - l y s i n e ( F i g u r e 2). T h e s e results suggest the p o s s i b i l i t y o f a n interaction b e t w e e n c y t o c h r o m e c a n d t h e m o d i fied e l e c t r o d e s u r f a c e a n a l o g o u s t o t h a t b e t w e e n c y t o c h r o m e c a n d its p h y s i o l o g i c a l p a r t n e r s a n d , as s u c h , i n d i c a t e t h a t c y t o c h r o m e c b i n d s t o t h e e l e c t r o d e p r i o r t o e l e c t r o n transfer. R o t a t i n g D i s k E l e c t r o d e Studies. B y analogy w i t h its p h y s i o l o g i c a l redox reactions, a s i m i l a r m u l t i - s t e p m e c h a n i s m is o u t l i n e d i n S c h e m e I for t h e e l e c t r o d e r e a c t i o n o f c y t o c h r o m e c i n w h i c h i t b i n d s to t h e e l e c t r o d e s u r f a c e b e f o r e t h e e l e c t r o n t r a n s f e r e v e n t i t s e l f o c c u r s . T h i s m e c h a n i s m w a s i n v e s t i g a t e d (49) u s i n g t h e r o t a t i n g d i s k t e c h nique. A »

Ao

transport i n solution

k

D

Ao

A ds a

adsorption a n d desorption of reactant

Unties Γ

Α ^ β . Λ ads τ—— #ads + #

electron

Bads *

desorption a n d adsorption of product

ΓιΜ-e

Bo

transfer

* a d s

BQ

Κ

kg ^ Boo k

transport i n solution

D

Scheme I S c h e m e I r e p r e s e n t s t h e r e a c t i o n A^B + e w h e r e A is f e r r o c y t o c h r o m e c , Β is f e r r i c y t o c h r o m e c , a n d T lmo\ m ~ is t h e n u m b e r o f a d s o r p t i o n sites p e r u n i t a r e a o n t h e m o d i f i e d e l e c t r o d e . T h e v a r i o u s rate c o n s t a n t s d e s c r i b e t h e rates o f t h e f o l l o w i n g p r o c e s s e s : t h e r a t e c o n s t a n t kolva s is t h e m a s s t r a n s p o r t rate c o n s t a n t f o r a r o t a t i n g d i s k electrode, d e s c r i b i n g the transport o f material from the b u l k o f the s o l u t i o n t o t h e e l e c t r o d e , a n d is g i v e n b y t h e L e v i c h e q u a t i o n (51 ) 2

L

- 1


8.

EDDOWES A N D H I L L

Reactions m

of m

111

Metalloproteins 112

k = 1.55 D v- W D

1 2

= BW '

(1)

w h e r e W is t h e r o t a t i o n s p e e d i n H e r t z . T h e r a t e c o n s t a n t fc ds d e s c r i b e s a

t h e rates o f a d s o r p t i o n o f b o t h t h e r e d u c e d a n d o x i d i z e d f o r m s o f c y t o c h r o m e c a n d has t h e u s u a l d i m e n s 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 a t e c o n s t a n t . T h e r a t e c o n s t a n t s fc and / c _ a r e t h e p o t e n t i a l - d e p e n d e n t r a t e e

e


c o n s t a n t s for t h e f o r w a r d a n d b a c k w a r d e l e c t r o n t r a n s f e r r e a c t i o n s b e -

E ( V vs. S C E ) Figure 2. Cyclic voltammograms (dc) of horse heart ferricytochrome c (5 mglmL) in 0.1 M NaCl0 , 0.02 M phosphate buffer at pH 7 in the presence of 1,2-bis(4-pyridyl)ethylene in the potential range +0.2 V to -0.2 V vs. SCE with poly-h-lysine at a, 0; b, 1; and c, 1.5 mglmL. The dc potential scan rate was 100 mVls. The increasing irreversibility with increasing poly-h-lysine concentration illustrates its inhibitory effect due to blockage of adsorption sites on the electrode surface. (Reproduced from Ref. 47. Copyright 1979, American Chemical Society.) 4


178


t w e e n a d s o r b e d s p e c i e s a n d t h e e l e c t r o d e i n u n i t s o f r e c i p r o c a l sec onds, a n d are given b y k = k° ex e

e

[ ^ r ( E - E ° ) ]

V

(2a)

for t h e a n o d i c p r o c e s s a n d

fc_ = k° exp [ e

(E- E°)]

e

(2b)

for t h e c a t h o d i c p r o c e s s , w h e r e k° is t h e s t a n d a r d first-order e l e c t r o n t r a n s f e r rate c o n s t a n t b e t w e e n a d s o r b e d s p e c i e s a n d t h e e l e c t r o d e at the standard e l e c t r o d e p o t e n t i a l o f the system, E ° . T h e coefficients a a n d β a r e t h e n o r m a l a n o d i c a n d c a t h o d i c c h a r g e transfer c o e f f i c i e n t s . T h e rate c o n s t a n t k d e s c r i b e s t h e rates o f d e s o r p t i o n o f t h e r e d u c e d a n d o x i d i z e d f o r m s o f c y t o c h r o m e c a n d is a l s o i n u n i t s o f r e c i p r o c a l seconds.


e

ues

E q u a t i o n s that d e s c r i b e the d e p e n d e n c e o f current o n p o t e n t i a l a n d r o t a t i o n s p e e d f o r t h e p r o p o s e d r e a c t i o n s c h e m e at t h e r o t a t i n g disk electrode are d e r i v e d b y consideration o f the f o l l o w i n g equations for t h e flux, j i n m o i s p e r s q u a r e m e t e r p e r s e c o n d , o f A t o Β : j = k (dao — a ) D

= = = =

(transport o f reactant)

0

\

&ads(l ~ θ - 0 )a - T kuesQa ( n e t a d s o r p t i o n o f r e a c t a n t ) I r k6 - Y k- 6 ( e l e c t r o n transfer) > rk e - fcadsU - θ ~ B )b ( n e t d e s o r p t i o n o f p r o d u c t ) I k (b — boo) (transport o f p r o d u c t ) J a

L

e

L

des

D

a

b

L

e

0

L

b

b

α

b

(3)

0

0

w h e r e θ a n d B a r e t h e f r a c t i o n o f sites o c c u p i e d b y A a n d B, r e s p e c tively. Elimination of θ , B a n d b hy e q u a t i n g t h e s e flux e q u a t i o n s gives the general result: α

b

α

l

j~ katek Y {k a«> ues

L

e

by

Q

l

- k- boo) = k ~ k k esYL(k e

D

ads

d

e

+ k-e)

ads#» + k à$boo)(k + k- + &des) a

e

e

W h e n t h e e l e c t r o d e i s s u f f i c i e n t l y p o s i t i v e , k w i l l b e so l a r g e a n d kso c o r r e s p o n d i n g l y s m a l l t h a t t e r m s c o n t a i n i n g k w i l l d o m i n a t e a n d the l i m i t i n g current, i w i l l b e o b s e r v e d . T h e l i m i t i n g current is d e s c r i b e d , for oxidation, b y E q u a t i o n 5, the appropriate form o f t h e K o u t e c k y - L e v i c h e q u a t i o n (52) for S c h e m e I: e

e

e

Ly

i - = ( F A a „ ) - ί τ τ+ i T -+ f t

L *D

Κ ads

1

f

- + n

ZAdes

1

H

(5)

XAdes J l

T h i s e x p r e s s i o n , t o g e t h e r w i t h E q u a t i o n 1, p r e d i c t s t h a t p l o t s o f h~ a g a i n s t W ~ s h o u l d b e l i n e a r w i t h a g r a d i e n t , r e p r e s e n t i n g t h e trans port term, proportional to the r e c i p r o c a l reactant concentration, α » " . 1 / 2

1


8.

Reactions of Metalloproteins

EDDOWES A N D H I L L

179

T h e i n t e r c e p t , / , c o r r e s p o n d i n g to t h e rate o f t h e s u r f a c e s t e p i n t h e e l e c t r o d e r e a c t i o n , is g i v e n , for o x i d a t i o n , b y ί = (FA)"

1

L-J—

+

+ ττγ—

1

( ) 6

I n this expression the terms i n the square b r a c k e t represent the ratelimiting

processes

at t h e

electrode

surface.

The

first

and

second

terms d e s c r i b e the adsorption a n d desorption o f reactant a n d p r o d u c t r e s p e c t i v e l y , a n d the

third term

describes

the

competition of

the

r e a c t a n t , A , a n d t h e p r o d u c t , B , for a d s o r p t i o n sites, t h a t i s , p r o d u c t


i n h i b i t i o n . E q u a t i o n 6 p r e d i c t s that, i n the a b s e n c e o f p r o d u c t i n b u l k s o l u t i o n (fooo = 0 ) , ί s h o u l d v a r y l i n e a r l y w i t h t h e r e c i p r o c a l o f t h e 1

concentration o f the reactant (α»" ) a n d that the c o r r e s p o n d i n g plots s h o u l d h a v e a s l o p e p r o p o r t i o n a l to

_1

fc ds a

and an intercept

propor

1

t i o n a l to fcdes" - F u r t h e r m o r e , i t p r e d i c t s t h a t t h e r e a c t i o n s h o u l d b e i n h i b i t e d b y a d d i t i o n o f the p r o d u c t to the b u l k s o l u t i o n , s u c h that, for a c o n s t a n t v a l u e o f r e a c t a n t c o n c e n t r a t i o n , a ,

I varies linearly

œ

w i t h product concentration, An

b. œ

e q u a t i o n d e s c r i b i n g the c u r r e n t - v o l t a g e

curve may be

ob

t a i n e d f r o m t h e g e n e r a l e x p r e s s i o n , E q u a t i o n 4 ; for o x i d a t i o n i n t h e case w h e r e

p r o d u c t is a b s e n t f r o m t h e b u l k s o l u t i o n (boo = 0) i t is

given by

fe)exp[-«$

Electrochemical and Spectrochemical Studies of Biological Redox

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