Electrochemical and Spectrochemical Studies of Biological Redox

7 The Heterogeneous Electron Transfer Properties of Cytochrome c Downloaded by NORTH CAROLINA STATE UNIV on November 11, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch007

E D M O N D F. BOWDEN and FRED M. HAWKRIDGE

1

Virginia Commonwealth University, Department of Chemistry, Richmond, VA 23284 HENRY N. BLOUNT

1

The University of Delaware, Brown Chemical Laboratory, Newark, D E 19711

The heterogeneous electron transfer kinetic parameters of horse heart cytochrome c were evaluated at pH 7.0. This work was directed at determining the formal heter ogeneous electron transfer rate constant, k°' , and the electrochemical transfer coefficient, α, at three different electrode surfaces: gold electrodes electrochemically modified with methyl viologen, fluoride-doped tin oxide optically transparent electrodes (OTEs), and tin-doped indium oxide OTEs. Kinetic parameters of cytochrome c were evaluated using samples in the totally oxidized and in the totally reduced forms. Kinetic effects arising from anion binding to cytochrome c were investigated for phosphate and chloride in the presence of the nonbind ing buffer tris(hydroxymethyl)aminomethane-cacodylic acid. The kinetic parameters were determined using single potential step chronoabsorptometry at all three electrodes and using rotating disk electrode voltam metry at the methyl viologen-modified gold disk elec trode. s,h

he t h e r m o d y n a m i c s a n d h o m o g e n e o u s e l e c t r o n transfer k i n e t i c s o f cytochrome c have been studied widely. Extensive reviews point to t h e i m p o r t a n t q u e s t i o n s t h a t r e m a i n u n a n s w e r e d r e g a r d i n g t h e m e c h a n i s m b y w h i c h electrons are transferred b y c y t o c h r o m e c i n m a m m a l i a n o x i d a t i v e p h o s p h o r y l a t i o n (1-8). T h e p a t h w a y b y w h i c h 1

To whom correspondence should be addressed. 0065-2393/82/0201-0159$06.00/0 © 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.

160

BIOLOGICAL R E D O X COMPONENTS

Downloaded by NORTH CAROLINA STATE UNIV on November 11, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch007

c y t o c h r o m e c accepts electrons from the m e m b r a n e - b o u n d cyto c h r o m e c reductase a n d then donates electrons to c y t o c h r o m e c oxi d a s e , w h i c h is a l s o m e m b r a n e b o u n d , r e m a i n s a p o i n t o f c o n t r o v e r s y . T h e i m p e t u s for s t u d y i n g the energetics a n d k i n e t i c s o f c y t o c h r o m e c e l e c t r o n transfer reactions d e r i v e s p r i m a r i l y from t h e n e e d t o u n d e r stand its e l e c t r o n transfer m e c h a n i s m ( s ) . S e v e r a l m e c h a n i s m s h a v e b e e n p r o p o s e d f o r c y t o c h r o m e c (J - 6 ) , all based o n indirect evidence. A nelectron hopping mechanism i n v o l v i n g transfer o f a n e l e c t r o n t h r o u g h v a r i o u s a r o m a t i c residues i n t h e p r o t e i n f a b r i c w a s p r o p o s e d (8). T h i s m e c h a n i s m w a s s u b s e q u e n t l y a b a n d o n e d b e c a u s e o f its f a i l u r e t o a c c o u n t for s t r u c t u r a l a n d e n e r g e t i c factors (9). T h e i n v o l v e m e n t o f a π - c a t i o n r a d i c a l i n t e r m e d i a t e w a s a l s o p r o p o s e d (JO) b u t w a s n o t e x p e r i m e n t a l l y v e r i f i e d . E l e c t r o n t u n n e l i n g w a s p r o p o s e d for b a c t e r i a l c y t o c h r o m e s (11-13) a n d t h i s m e c h a n i s m also m a y b e operative i n m a m m a l i a n c y t o c h r o m e c. P o s s i b l y the most w i d e l y a c c e p t e d m e c h a n i s m i n v o l v e s e l e c t r o n t r a n s f e r at t h e e x p o s e d h e m e e d g e o f c y t o c h r o m e c . T h i s m e c h a n i s m w a s first p r o p o s e d for c y t o c h r o m e c (14) a n d l a t e r f o r Rhodospirillum ruhrum c , a p h o t o s y n t h e t i c c y t o c h r o m e (15). T h i s o u t e r s p h e r e m e c h a n i s m w a s w i d e l y tested t h r o u g h use o f exogenous a n d endogenous redox reactants a n d t h r o u g h s t u d i e s o f t h e effect o f s o l u t i o n p H , i o n i c s t r e n g t h , a n d i o n b i n d i n g o n t h e h o m o g e n e o u s e l e c t r o n transfer k i n e t i c s o f c y t o c h r o m e c (J -5). S u p p o r t f o r t h e h e m e e d g e e l e c t r o n t r a n s f e r m e c h a n i s m w a s p r o v i d e d b y these h o m o g e n e o u s e l e c t r o n transfer k i n e t i c studies. H o w e v e r , t h e m e c h a n i s m b y w h i c h c y t o c h r o m e c transfers e l e c t r o n s p h y s i o l o g i c a l l y remains to b e established. 2

T h e d e t e r m i n a t i o n o f t h e e l e c t r o n transfer k i n e t i c s o f d i r e c t heterogeneous reactions b e t w e e n c y t o c h r o m e c a n d several electrode surfaces w a s t h e o b j e c t i v e o f this study. T h e reason for p u r s u i n g this t y p e o f m e a s u r e m e n t i s t h a t c y t o c h r o m e c p h y s i o l o g i c a l l y transfers e l e c t r o n s at m e m b r a n e i n t e r f a c e s . H e n c e , t h e p h y s i o l o g i c a l e l e c t r o n transfer reactions o f c y t o c h r o m e c m a y p r o c e e d v i a a m e c h a n i s m that contains e l e m e n t s o f a s i m p l e h e t e r o g e n e o u s e l e c t r o n transfer m o d e l . This work utilized n e w l y developed a n d previously reported elec trode surfaces a n d m e t h o d s .

Direct Electrochemical Studies of Cytochrome c C y t o c h r o m e c has been s t u d i e d extensively b y direct voltammetr i c m e t h o d s at m e r c u r y e l e c t r o d e s (16-24). S t r o n g a d s o r p t i o n o f c y t o c h r o m e c o n t h e m e r c u r y surface d u r i n g r e d u c t i o n has b e e n w i d e l y r e p o r t e d . T h e a d s o r b e d l a y e r h a s b e e n v a r i o u s l y d e s c r i b e d as f o r m i n g a flattened l a y e r w i t h p o r e s w h e r e r e d u c t i o n o f d i f f u s i n g c y t o c h r o m e c occurs (23), a l a y e r at w h i c h a self-exchange reaction occurs b e t w e e n the r e d u c e d adsorbed m o l e c u l e s a n d those diffusing to t h e e l e c t r o d e


7.

Electron Transfer Properties of Cytochrome c

BOWDEN E T AL.

161

(20), a n d an adsorbed layer o f denatured c y t o c h r o m e c (24). C y t o c h r o m e c a l s o h a s b e e n s t u d i e d d i r e c t l y at g o l d m i n i g r i d e l e c t r o d e s (25) , i n d i u m o x i d e thin-film o p t i c a l l y transparent electrodes

(OTEs)

( 2 6 ) , a n d at g o l d e l e c t r o d e s o n w h i c h 4 , 4 ' - b i p y r i d i n e w a s a d s o r b e d

(27-31). F o r m a l h e t e r o g e n e o u s e l e c t r o n t r a n s f e r k i n e t i c p a r a m e t e r s for t h e reduction of cytochrome c have been reported (24,29). Based on linear Downloaded by NORTH CAROLINA STATE UNIV on November 11, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch007

s w e e p v o l t a m m e t r y , the formal heterogeneous

e l e c t r o n transfer rate

c o n s t a n t (ks',h) a n d t h e e l e c t r o c h e m i c a l t r a n s f e r c o e f f i c i e n t (a) 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 at m e r c u r y w e r e e s t i m a t e d t o b e 1 0 " 10~

n

1 0

to

cm/s a n d ca. 0.5, r e s p e c t i v e l y (24). A t the 4 , 4 ' - b i p y r i d i n e / g o l d

e l e c t r o d e surface, ac i m p e d a n c e m e t h o d s w e r e u s e d to d e t e r m i n e that kt!h = 1-6 x 1 0 ~

2

c m / s , w i t h n o v a l u e g i v e n for t h e e l e c t r o c h e m i c a l

transfer coefficient (29).

Single Potential Step Chronoabsorptometry T h e m e t h o d o f single potential step chronoabsorptometry ( S P S / C A ) p e r m i t s the d e t e r m i n a t i o n o f heterogeneous e l e c t r o n transfer k i n e t i c p a r a m e t e r s for o p t i c a l l y a b s o r b i n g s p e c i e s at O T E s (32). T h e p r i n c i p a l a d v a n t a g e s o f t h i s m e t h o d c o m p a r e d to o t h e r e l e c t r o c h e m i c a l m e t h o d s a r e its i n s e n s i t i v i t y to c h a r g e c o n s u m i n g p r o c e s s e s o t h e r t h a n the reaction o f interest a n d the m o l e c u l a r specificity p r o v i d e d b y the optical p r o b e . A d e t a i l e d d e s c r i p t i o n o f the a p p l i c a t i o n o f this m e t h o d , w h i c h n e g l e c t s t h e effect o f t h e b a c k r e a c t i o n ( i r r e v e r s i b l e p r o c e s s e s ) , as w e l l as t h e m o r e r e c e n t a p p l i c a t i o n o f a m e t h o d t h a t a c c o u n t s for t h e b a c k r e a c t i o n ( q u a s i - r e v e r s i b l e p r o c e s s e s ) w a s p r e s e n t e d (33). T h e n e e d for t h e S P S / C A m e t h o d d i r e c t l y f o l l o w e d t h e r e p o r t s o f the e l e c t r o a c t i v i t y o f g o l d m i n i g r i d electrodes, w h i c h w e r e e l e c t r o c h e m i c a l l y m o d i f i e d w i t h m e t h y l v i o l o g e n , t o w a r d the d i r e c t r e d u c t i o n a n d o x i d a t i o n o f f e r r e d o x i n (34) a n d m y o g l o b i n (35). T h e a p p l i c a t i o n o f t h e S P S / C A m e t h o d to t h e d e t e r m i n a t i o n o f t h e h e t e r o g e n e o u s e l e c t r o n t r a n s f e r k i n e t i c p a r a m e t e r s w a s r e p o r t e d for t h e r e d u c t i o n o f m y o g l o b i n (36) a n d f e r r e d o x i n (37) at t h i s e l e c t r o d e s u r f a c e . R e c e n t w o r k e x t e n d e d t h e a p p l i c a t i o n o f S P S / C A to c y t o c h r o m e c , w h i c h w a s s t u d i e d at t h e m o d i f i e d g o l d m i n i g r i d s u r f a c e a n d at fluoride-doped t i n o x i d e a n d t i n - d o p e d i n d i u m o x i d e O T E s (38). T h e effects o f p H a n d i o n i c strength o n the h e t e r o g e n e o u s r e d u c t i v e e l e c t r o n transfer p a r a m e t e r s for m y o g l o b i n w e r e a l s o d e s c r i b e d (38). I n t h e p r e s e n t w o r k , S P S / C A w a s u s e d t o e v a l u a t e t h e effects o f i o n b i n d i n g to c y t o c h r o m e c o n h e t e r o g e n e o u s e l e c t r o n t r a n s f e r k i n e t i c p a r a m e t e r s at fluoride-doped t i n o x i d e O T E s . I n a d d i t i o n , i n i t i a l re s u l t s f r o m o x i d a t i v e S P S / C A m e a s u r e m e n t s for c y t o c h r o m e c w e r e o b t a i n e d at t h e m o d i f i e d g o l d m i n i g r i d s u r f a c e a n d at t h e fluorideIn Electrochemical and Spectrochemical Studies of Biological Redox Components; Kadish, K.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

162

BIOLOGICAL REDOX COMPONENTS

d o p e d t i n o x i d e O T E surface. T h e s e latter e x p e r i m e n t s w e r e per f o r m e d to d i r e c t l y m e a s u r e t h e r a t e c o n s t a n t s for t h e b a c k r e a c t i o n ( o x i d a t i o n ) to d e t e r m i n e t h e a g r e e m e n t o f t h e h e t e r o g e n e o u s e l e c t r o n transfer reactions o f c y t o c h r o m e c w i t h the s i m p l e e l e c t r o n transfer theory used i n k i n e t i c analyses.


Experimental Apparatus. T h e electrochemical a n d optical instrumentation was de s c r i b e d p r e v i o u s l y (36, 38). T h e spectroelectrochemical c e l l s were based on a p r e v i o u s l y reported design a n d h a d an optical pathlength o f ca. 1 m m (39). Rotating disk voltammetry was performed w i t h a P i n e Instrument C o m p a n y M o d e l A S R - 2 rotator. T h e g o l d m i n i g r i d electrodes were 200 lines per i n c h , 67% transmittant a n d 0.1 m i l n o m i n a l thickness from B u c k b e e - M e a r s C o . T h e g o l d rotating disk electrode, 7.5-mm diameter, was M o d e l D D 2 0 from P i n e Instrument C o . T i n - d o p e d i n d i u m oxide a n d fluoride-doped t i n oxide O T E s were ca. 20 ohms/square from P P G Industries. C h e m i c a l s . M e t h y l v i o l o g e n (Κ & Κ Laboratories) was r e c r y s t a l l i z e d three times from methanol. T h e phosphate buffer was prepared from T i t r i s o l , p H 7.0 ( E . M e r c k Co.) or from reagent grade salts. C a c o d y l i c a c i d a n d tris(hydroxymethyl)aminomethane, reagent grade, were obtained from S i g m a C h e m i c a l C o . T h e c a c o d y l i c a c i d was r e c r y s t a l l i z e d t w i c e from ethanol. A l l other chemicals were reagent grade and solutions were prepared i n glass d i s t i l l e d water. Procedures. G o l d electrodes were m o d i f i e d w i t h m e t h y l v i o l o g e n as p r e v i o u s l y d e s c r i b e d (36). T h e semiconductor O T E s were c l e a n e d b y succes s i v e l y subjecting them to 5 m i n o f ultrasonic agitation i n Alconox, ethanol, and d i s t i l l e d water (twice) after a p r e v i o u s l y d e s c r i b e d procedure (40). S P S / C A measurements were performed at 550 or 416 n m a n d Ac values of 21,100 (41) a n d 57,000 M ^ c m " (42), respectively, were used i n a l l calcula tions. T h e diffusion coefficient used i n a l l calculations was 1.1 x 1 0 " cm /s (28). T h i s value was experimentally verified ( ± 0 . 0 5 ) from the slope of plots of absorbance vs. t for 22 diffusion-controlled S P S / C A transients at a fluorided o p e d t i n oxide O T E . T h e formal potential for cytochrome c, w h i c h was used to determine overpotential step values, was 0.260 V vs. N H E (2). A l l experi ments were performed at 25 ± 2°C. 1

6

2

1/2

Reduction and Oxidation of Cytochrome c at Various Electrodes T a b l e I s u m m a r i z e s t h e e l e c t r o n t r a n s f e r k i n e t i c b e h a v i o r s e e n for h o r s e h e a r t c y t o c h r o m e c at t h e t h r e e e l e c t r o d e s u r f a c e s r e p o r t e d h e r e . T h e s e results w e r e a l l o b t a i n e d w i t h solutions c o n t a i n i n g 0.07 M p h o s p h a t e a n d 0 . 1 0 M N a C l , p H 7.0, u s i n g S P S / C A . R a t e p a r a m e t e r s o b t a i n e d f r o m p r e v i o u s r e d u c t i v e p o t e n t i a l s t e p e x p e r i m e n t s w i t h fer r i c y t o c h r o m e c ( E n t r i e s 1, 4 , 6) (38) a r e s h o w n i n F i g u r e 1. E n t r y 3 d a t a for c y t o c h r o m e c r e d u c t i o n at t i n o x i d e r e s u l t e d f r o m a r e c e n t e x p e r i m e n t t h a t d u p l i c a t e d t h e c o n d i t i o n s for E n t r y 4 . T h e c o n s i s t e n c y b e t w e e n t h e s e t w o sets o f d a t a o b t a i n e d 6 m o n t h s a p a r t is q u i t e g o o d .



77.3 77.3 101 103 103 43.0

1 2 3 4 5 6 -4.99(±0.12) -5.24(±0.05) -5.20(±0.08) -5.17(±0.05) -4.31(±0.07) -4.51(±0.05)

h

6

log k|,' , cm/s 0.24( 0.74( 0.28( 0.32( 0.95( 0.50(± 0.03) 0.02) 0.02) 0.01) o.oiy 0.04) /

d

c

6

° All solutions contained 0.07 M phosphate buffer, pH 7.0 and 0.1 M NaCl. Parentheses contain one standard deviation. Methyl viologen-modified gold minigrid electrode. Reductive SPS/CA performed on oxidized sample of cytochrome c. ' From Ref. 38. 'Oxidative SPS/CA performed on reduced sample of cytochrome c, value is from (1 - a). " Fluoride-doped tin oxide O T E . * Tin-doped indium oxide O T E .

[Cyt β ] , μ Μ «

2

3

2

2

2

9

M G M M G M Sn0 Sn0 Sn0 Ιη 0

Λ

C

Elec trode

6

6

reductive**' oxidative reductive reductive oxidative reductive

Potential steps

Heterogeneous Electron Transfer Kinetic Parameters for Cytochrome c at Various O T E s

Entry

Table I.


6

CO

3

ο

ri.

ο

3 "β

3

3

Ρ) Η

W Ο ΪΟ M Ζ


164


0.2

0.3

0.4

0.5

0.6

-η,ν Figure 1. Log k , vs. overpotential for the reduction of cytochrome c. Letter designation/electrode material/entry correspondence in Table I: a, tin-doped indium oxide, Entry 6; b, fluoride-doped tin oxide, Entry 5; c, methyl viologen-modified minigrid, Entry 1. f

h

A s n o t e d p r e v i o u s l y (38), t h e s a m e n e s s o f t h e r e d u c t i v e k i n e t i c r e s u l t s , w h e n c o m p a r e d to t h o s e o b t a i n e d at t h e 4 , 4 ' - b i p y r i d i n e / g o l d e l e c t r o d e s (29) a n d at m e r c u r y (24), a r g u e s for t h e e x i s t e n c e o f a s i m i l a r p r o t e i n / s o l u t i o n i n t e r f a c e at t h e s e t h r e e e l e c t r o d e s u r f a c e s . T h e p r e s e n c e o f a n a d s o r b e d p r o t e i n l a y e r at t h e s e e l e c t r o d e s u r f a c e s is a l i k e l y p o s s i b i l i t y b u t is n o t e s t a b l i s h e d . Oxidative S P S / C A experiments were performed i n a manner iden t i c a l to t h e r e d u c t i v e e x p e r i m e n t s e x c e p t t h a t p o s i t i v e o v e r p o t e n t i a l s t e p s w e r e a p p l i e d to t h e e l e c t r o d e s e x p o s e d to b u l k f e r r o c y t o c h r o m e c. A n a l y s i s o f a b s o r b a n c e - t i m e d a t a y i e l d s , for e a c h o v e r p o t e n t i a l , a


7.

BOWDEN E T A L .


165

kb h ( a s s u m i n g t h a t ZC/,Λ c o r r e s p o n d s t o r e d u c t i o n ) . A l i n e a r p l o t o f l o g t

kb,h

vs.

(1 -

a) f r o m t h e s l o p e . A c c o r d i n g t o B u t l e r - V o l m e r f o r m a l i s m (43),

overpotential

(η)

affords

kt'

h

from

the

intercept

and a

s i m p l e h e t e r o g e n e o u s e l e c t r o n transfer r e a c t i o n ( w i t h o n l y one p h y s i c a l p a t h w a y for b o t h o x i d a t i o n a n d r e d u c t i o n ) s h o u l d y i e l d t h e s a m e v a l u e o f k° [ , i n d e p e n d e n t o f w h e t h e r t h e m e a s u r e d r e a c t i o n is a r e d u c s

h

t i o n or a n o x i d a t i o n . F u r t h e r m o r e , t h e v a l u e s o f t h e t r a n s f e r c o e f f i c i e n t Downloaded by NORTH CAROLINA STATE UNIV on November 11, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch007

o b t a i n e d from the separate r e d u c t i v e a n d o x i d a t i v e e x p e r i m e n t s s h o u l d a g r e e . I n t h e e x p e r i m e n t s o f T a b l e I , t h e s e c r i t e r i a a r e n o t m e t for t h e f l u o r i d e - d o p e d t i n o x i d e O T E ( E n t r i e s 3 a n d 4 c o m p a r e d t o 5) or t h e m e t h y l v i o l o g e n - m o d i f i e d g o l d m i n i g r i d e l e c t r o d e E n t r i e s 1 a n d 2). F o r t h e f o r m e r e l e c t r o d e , t h e t r a n s f e r

(compare coefficients

d i f f e r b y c a . 0 . 6 5 , a n d t h e k° [ v a l u e s d i f f e r b y n e a r l y a n o r d e r o f m a g s

h

n i t u d e i n the oxidation a n d reduction experiments. S i m i l a r disparities a l s o e x i s t for t h e l a t t e r e l e c t r o d e , b u t to a l e s s e r d e g r e e . R e a s o n s for t h i s d i s c r e p a n c y m a y i n v o l v e i o n b i n d i n g to c y t o c h r o m e c, a p a t h w a y d e p e n d e n c e o n t h e r e a c t i o n d i r e c t i o n , a n d s e m i c o n d u c t o r s u r f a c e effects. W o r k is i n p r o g r e s s t o d e t e r m i n e t h e r e a s o n ( s ) for t h e s e d i s c r e p a n c i e s . T h e h e t e r o g e n e o u s e l e c t r o n t r a n s f e r k i n e t i c p a r a m e t e r s for t h e reduction

of cytochrome

c

were

also

investigated

at

a

methyl

v i o l o g e n - m o d i f i e d r o t a t i n g g o l d d i s k e l e c t r o d e ( R D E ) to c o m p a r e

the

r e s u l t s o f t h i s s t e a d y state t e c h n i q u e w i t h t h e r e s u l t s o b t a i n e d b y t h e S P S / C A t r a n s i e n t t e c h n i q u e . T h e g o l d R D E w a s first p o l i s h e d s u c c e s s i v e l y w i t h 1-, 0 . 3 - , a n d 0 . 1 - / x m a l u m i n a s l u r r i e s f o l l o w e d b y a n u l trasonic d i s t i l l e d water rinse. T h e g o l d R D E was then m o d i f i e d follow i n g t h e p r o c e d u r e d e s c r i b e d for g o l d m i n i g r i d s (36). S t a n d a r d R D E k i n e t i c a n a l y s i s (43) o f d a t a o b t a i n e d for a d e o x y g e n a t e d s o l u t i o n o f 1 6 6 μΜ c y t o c h r o m e c, 0 . 0 7 M p h o s p h a t e b u f f e r , a n d 0 . 1 0 M N a C l , p H 7.0, y i e l d e d v a l u e s for l o g k° [ o f - 5 . 2 4 ( ± 0 . 2 2 ) a n d a o f 0 . 2 1 ( ± 0 . 0 3 ) . s

h

T h e s e p r e l i m i n a r y r e s u l t s d e m o n s t r a t e t h a t R D E v o l t a m m e t r y at t h e m e t h y l v i o l o g e n - m o d i f i e d g o l d d i s k e l e c t r o d e c a n b e u t i l i z e d to m e a s u r e t h e h e t e r o g e n e o u s e l e c t r o n t r a n s f e r k i n e t i c s o f c y t o c h r o m e c. T h i s r e s u l t is i n a g r e e m e n t w i t h t h e r e s u l t s o f t h e S P S / C A t r a n s i e n t t e c h n i q u e (38).

Anion Effects on the Heterogeneous Electron Transfer Kinetics of Cytochrome c S p e c i f i c c a t i o n a n d a n i o n b i n d i n g to one or b o t h r e d o x forms o f c y t o c h r o m e c is a w e l l - e s t a b l i s h e d p h e n o m e n o n (44, 4 5 ) . C h a n g e s i n h o m o g e n e o u s e l e c t r o n t r a n s f e r rates b e t w e e n c y t o c h r o m e c a n d s o l u b l e r e d o x p a r t n e r s a l s o h a v e b e e n o b s e r v e d a n d a t t r i b u t e d to i o n b i n d i n g (2, 4 6 , 47). T h i s section describes e v i d e n c e w h i c h shows that


166



s p e c i f i c a n i o n effects, p r e s u m a b l y r e s u l t i n g f r o m b i n d i n g to c y t o c h r o m e c, c a n i n f l u e n c e h e t e r o g e n e o u s e l e c t r o n t r a n s f e r rates i n a measurable a n d r e p r o d u c i b l e fashion. F o r t h e s e e x p e r i m e n t s , c y t o c h r o m e c w a s d i s s o l v e d i n p H 7.0 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 ( 0 . 0 9 M ) / c a c o d y l i c a c i d ( 0 . 1 0 M) b u f f e r ( B u f f e r A ) o f c a l c u l a t e d i o n i c s t r e n g t h e q u a l to 0 . 0 8 M . T h i s b u f f e r s y s t e m is c o n s i d e r e d to b e n o n b i n d i n g w i t h r e s p e c t to c y t o c h r o m e c (46, 48). E v a l u a t i o n o f r e d u c t i v e e l e c t r o n t r a n s f e r k i n e t i c parameters was then performed at fluoride-doped tin oxide O T E s b o t h i n the presence a n d a b s e n c e o f a d d e d salts. F i r s t the results w i t h buffer alone w i l l b e presented i n some d e t a i l f o l l o w e d b y results o b t a i n e d i n the presence o f c h l o r i d e a n d phosphate. F i g u r e 2 s h o w s t y p i c a l a b s o r b a n c e - t i m e t r a n s i e n t s 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 i n B u f f e r A for a n u m b e r o f o v e r p o t e n t i a l s t e p s .

_l

0

I

I

6

12

l _

18

T i m e (s) Figure 2. Typical SPS/CA absorbance-time transients for the reduc tion of cytochrome c. Solution contained 97.6 μΜ cytochrome c, and Buffer A, pH 7.0 (ionic strength = 0.08 M) at a fluoride-doped tin oxide OTE from Table III, Entry 1. Trace Ioverpotential in mV/transient number in experimental sequence: a, —78, #10; b, —128, #6; c, —178, #20; d, -228, #14; e, -278, #18; f, -328, #4; g, -428, #1; h, -628, #5; and i, -728, #8.


7.


BOWDEN E T AL.

167

T h e r e p r o d u c i b i l i t y w a s v e r y g o o d a n d n o m e a s u r a b l e loss i n r e s p o n s e was seen d u r i n g the r a n d o m acquisition o f more than thirty S P S / C A transients. T a b l e I I s h o w s t h e k v a l u e s c a l c u l a t e d for transients a t h r o u g h g o f F i g u r e 2 . E x c e l l e n t fit t o t h e S P S / C A t h e o r y for i r r e v e r s i b l e e l e c t r o n transfer is i n d i c a t e d b y t h e s m a l l s t a n d a r d d e v i a t i o n s . T h i s a s s e r t i o n is f u r t h e r c o r r o b o r a t e d i n F i g u r e 3 , w h i c h p r e s e n t s t h e k i n e t i c w o r k i n g c u r v e a l o n g w i t h d a t a f r o m t r a n s i e n t s b , c , d , f, a n d g . O n l y t h e s e d a t a a r e s h o w n for c l a r i t y . F o r e a c h t r a n s i e n t , e x p e r i m e n t a l n o r m a l i z e d a b s o r b a n c e i s p l o t t e d for five o b s e r v a t i o n s (t = 6 , 9 , 1 2 , 1 5 , a n d 18 s) v s . l o g [(k t )/D ] u s i n g t h e a v e r a g e Rvalues f r o m T a b l e I I . I f e x p e r i m e n t fits t h e o r y , a l l five p o i n t s for e a c h t r a n s i e n t s h o u l d f a l l o n t h e w o r k i n g c u r v e a n d t h i s i s i n d e e d t h e c a s e . V a l u e s o b t a i n e d for t = 3 s were not i n c l u d e d i n a n y o f the k ^ a n d a determinations, because t h e i r fit t o t h e w o r k i n g c u r v e w a s n o t g o o d i n s o m e c a s e s . T h i s d e v i a t i o n is p r o b a b l y a r e s u l t o f t h e g r e a t e r r e l a t i v e e r r o r i n h e r e n t i n m e a s u r i n g t h e a b s o r b a n c e - t i m e r e s p o n s e at s h o r t t i m e s . .


fih

ll2

m

fth

0

F o r 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 at fluoride-doped t i n ox i d e O T E s i n p H 7.0 Buffer A , a v e r a g i n g the results i n E n t r i e s 1 a n d 2 o f T a b l e I I I y i e l d s logk° \ = - 4 . 7 5 a n d a = 0.32. E n t r i e s 3 a n d 4 o f the s a m e t a b l e i n d i c a t e t h a t t h e effect o f 10 m M p h o s p h a t e ( [ H 2 P O 4 ] / [ H P O l ] — 0.7) i n t h i s s a m e s y s t e m is t o d e c r e a s e l o g k° ' b y ca. 0 . 4 a n d a b y c a . 0.07. I n t h e e l e c t r o c h e m i c a l sense, c y t o c h r o m e c r e d u c t i o n at t i n o x i d e is m o r e i r r e v e r s i b l e i n t h e p r e s e n c e o f p h o s p h a t e . E x p e r i m e n t a l l y , t h i s fact is e v i d e n c e d b y a s i g n i f i c a n t r e d u c t i o n i n the m a g n i t u d e s o f the a b s o r b a n c e - t i m e transients s h o w n i n F i g u r e 2 u p o n a d d i t i o n o f p h o s p h a t e . T h a t t h i s o b s e r v e d d i f f e r e n c e is s i g n i f i c a n t is s h o w n b y t h e e r r o r l i m i t s a n d t h e e x p e r i m e n t a l r e p r o d u c i b i l i t y indicated i n Entries 1-4 of Table III. O n e experiment performed w i t h 10 m M N a C l a d d e d to t h e B u f f e r A s h o w e d a s l i g h t d e c r e a s e i n r e v e r s i b i l i t y as e v i d e n c e d b y a s m a l l e r t r a n s f e r c o e f f i c i e n t {see E n t r y 5 ) . H o w e v e r , c o m p a r e d w i t h t h e p h o s p h a t e effect, t h i s r e s u l t is n o t s t r i k i n g a n d repetitive experiments w i l l b e necessary to establish the v a l i d ity o f this difference. T h e k i n e t i c results presented i n T a b l e I I I are graphed i n F i g u r e 4. s

-

4

8 th

T h e results just p r e s e n t e d i n d i c a t e that specific i o n b i n d i n g c a n s i g n i f i c a n t l y i n f l u e n c e h e t e r o g e n e o u s e l e c t r o n t r a n s f e r rates o f c y t o c h r o m e c. U s i n g v a l u e s for i o n b i n d i n g constants p r e v i o u s l y r e p o r t e d (44), a 1 0 - m M concentration o f phosphate or c h l o r i d e is sufficient to b i n d e s s e n t i a l l y a l l o f t h e c y t o c h r o m e c m o l e c u l e s at 1 0 0 μΜ c o n c e n t r a t i o n . E v i d e n t l y , t w o a n i o n s b i n d t o e a c h c y t o c h r o m e c (49). T h e results presented i n T a b l e I I I a n d F i g u r e 4 support the v i e w that p h o s p h a t e a n d c h l o r i d e b i n d at d i f f e r e n t sites o n c y t o c h r o m e c (50) a n d suggest the i n v o l v e m e n t o f this m o l e c u l a r feature i n the r e d u c t i o n o f t h i s m e t a l l o p r o t e i n at t i n o x i d e O T E s .


168


Table II. Heterogeneous Electron Transfer Rate Constants for the Reduction of Cytochrome c at a Fluoride-Doped Tin Oxide O T E


η , mV - 78 -128 -178 -228 -278 -328 -428

k

f ) h

,

cm/s"

3.10(±0.20) 7.49(±0.07) 1.51(±0.02) 2.68(±0.03) 4.53(±0.07) 6.21(±0.02) 1.28(±0.05)

x x x x x x x

1(T ΙΟ" 1(T 1(T ΙΟ" ΙΟ ΙΟ

5

5

4

4

4

- 4

- 3

α

Rate constants are mean values of five observations taken at equal increments over the 6- to 18-s time domain. Parentheses contain one standard deviation. Note: Solution conditions are given in Figure 2.

1/2

1/2

Figure 3. Normalized absorbance vs. log [ ( k , t ) / D ] working curve with typical data for the reduction of cytochrome c at a fluoridedoped tin oxide OTE. Data shown correspond to appropriate transients in Figure 2 and were calculated using the k values from Table II. Key: Ο, η = -128 mV; Α, η = -178 mV; • , η = -228 mV; Φ, η = -328 mV; and Α, Ύ] = -428 mV. f

h

fh


7.

BOWDEN E T A L .

Electron

Transfer

Properties

of Cytochrome

c

169

Table III. Anion Effects on the Heterogeneous Electron Transfer Kinetic Parameters for Cytochrome c


Entry

1 2 3 4 5

log

k°/ , h

cm/s

-4.79(±0.05) -4.71(±0.01) -5.13(±0.05) -5.14(±0.02) -4.78(±0.03)

a

a

Electrolyte

0.30(±0.01) 0.34(±0.01) 0.24(±0.01) 0.26(±0.01) 0.28(±0.01)

Buffer Buffer Buffer Buffer Buffer

A A A + 10 m M p h o s p h a t e A + 10 m M p h o s p h a t e A + 10 m M N a C l

Note: SPS/CA at Sn0 OTEs, 96 to 98 μΜ cytochrome c, all solutions at pH 7.0, and all experiments are reductive. Parentheses contain one standard deviation. 2

α

I

0

1

0.1

I

I

0.2 0.3 -η,ν.

I

0.4

I

0.5

Figure 4. Anion effect on the SPS/CA reduction kinetics of cytochrome c at fluoride-doped tin oxide OTEs. Key: A, Buffer A, pH 7.0 (Entry 1 of Table III); • , Buffer A + 10 mM NaCl (Entry 5 of Table III); O, Buffer A + 10 mM phosphate (Entry 3 of Table III); andV, 0.07 M phosphate, 0.10 M NaCl, pH 7.0 (Entry 3 of Table I).



170


The results and conclusions just presented are the first reported evidence for specific ion effects on heterogeneous electron transfer kinetics for a biological redox molecule. Although the effect is thought to arise from binding to cytochrome c molecules, the anions may pos sibly be exerting an important effect on the oxide semiconductor sur face. Additional experiments, including variation of anion concentra tion, will be required to assess these potential causes. A final point that requires clarification concerns the phosphate effect. At p H 7.0, both H P O j and H P O l " are present at significant concentrations and it is not clear whether there is a difference in their ion binding behavior. Addi tional experiments performed over a p H range of ca. 6-8 should resolve this question. 2

Acknowledgments The support of the National Science Foundation (PCM79-12348), the National Institutes of Health (GM27208-02), and the University of Delaware Institute of Neuroscience (NIH Biomedical VII) is grate fully acknowledged. The assistance of Charlene D . Crawley and Eric E . Bancroft in some aspects of this work is gratefully acknowledged.

Literature Cited 1. Sutin, N. In "Bioinorganic Chemistry-II," Raymond, K. N., Ed.; ACS ADVANCES IN CHEMISTRY SERIES, No. 162, ACS: Washington, D.C.; 1977; pp. 156-172. 2. Cusanovich, M. A. In "Bioorganic Chemistry"; Ε. E . van Tamelen, Ed.; Academic: New York, 1978; Vol. 4, pp. 117-145. 3. Ferguson-Miller, S.; Brautigan, D. L.; Margoliash, E . In "The Porphy rins"; D. Dolphin, Ed.; Academic: New York, 1979; Vol. 7, pp. 149-240. 4. Timkovich, R., Ibid., pp. 241-294. 5. Wherland, S.; Gray, H. B. In "Biological Aspects of Inorganic Chemistry"; A. W. Addison et al., Eds.; Wiley: New York, 1977; pp. 289-368. 6. Williams, R. J. P.; Moore, G. R.; Wright, P. E., Ibid., pp. 369-401. 7. Walz, D. Biochim. Biophys. Acta 1979, 505, 279-353. 8. Dickerson, R. E.; Takano, T.; Kallai, Ο. B.; Samson, L. In "Structure and Function of Oxidation Reduction Enzymes"; A. A. Keison; A. Ehren berg, Eds.; Pergamon: Oxford, England, 1972; p. 69. 9. Dickerson, R. E.; Timkovich, R. In "The Enzymes"; P. D. Boyer, Ed.; Academic: New York, 1975; Vol. 11, Part A, pp. 397-547. 10. Dolphin, D.; Felton, R. H. Acc. Chem. Res. 1974, 7, 26. 11. DeVault, D.; Chance, B. Biophys. J. 1966, 6, 825. 12. Hopfield, J. J. Proc. Nat. Acad. Sci. U.S.A. 1974, 71, 3640. 13. Salemme, F. R. J. Mol. Biol. 1976, 102, 563. 14. Castro, C. E.; David, H. F. J. Am. Chem. Soc. 1969, 91, 5405. 15. Salemme, F. R.; Kraut, J.; Kamen, M. C. J. Biol. Chem. 1973, 248, 7701. 16. Griggio, L.; Pinamonti, S. Atti. Ist. Veneto Sci. Lett. Arti. 1965-1966, 124, 15. 17. Betso, S. R.; Klapper, M. H.; Anderson, L. B. J. Am. Chem. Soc. 1972, 94, 8197.



7. BOWDEN ET AL.


171

18. Scheller, F.; Jänchen, M.; Lampe, J.; Prümke, H. J.; Blanck, J.; Palecek, E . Biochim. Biophys. Acta 1975, 412, 157. 19. Scheller, F.; Prümke, H. J. Stud. Biophys. 1976, 60, 137. 20. Scheller, F. Bioelectrochem. Bioenerg. 1977, 4, 490. 21. Kuznetsov, Β. Α.; Mestechkina, N. M.; Shumakovich, G. P. Bioelec trochem. Bioenerg. 1977,4,1. 22. Haladjian, J.; Bianco, P.; Serre, P. A. J. Electroanal. Chem. 1980, 106, 397. 23. Kuznetsov, Β. Α.; Shumakovich, G. P.; Mestechkina, Ν. M. Bioelec trochem. Bioenerg. 1977, 4, 512. 24. Haladjian, J.; Bianco, P.; Serre, P. A. Bioelectrochem. Bioenerg. 1979, 6, 555. 25. Heineman, W. R.; Norris, B. J.; Goelz, J. F. Anal. Chem. 1975, 47, 79. 26. Yeh, P.; Kuwana, T. Chem. Lett. 1977, 1145. 27. Eddowes, M. J.; Hill, H. A. O. J. Chem. Soc., Chem. Commun. 1977, 771. 28. Eddowes, M. J.; Hill, H. A. O. J. Am. Chem. Soc. 1979, 101, 4461. 29. Eddowes, M. J.; Hill, H. A. O.; Uosaki, K. J. Am. Chem. Soc. 1979, 101, 7113. 30. Cass, A. E . C.; Eddowes, M. J.; Hill, H. A. O.; Uosaki, K.; Hammond, R. C.; Higgins, I. J.; Plotkin, E. Nature 1980, 285, 673. 31. Eddowes, M. J.; Hill, H. A. O.; Uosaki, K. Bioelectrochem. Bioenerg. 1980, 7, 527. 32. Albertson, D. E.; Blount, H. N.; Hawkridge, F. M. Anal. Chem. 1979, 51, 556. 33. Bancroft, E. E.; Blount, H. N.; Hawkridge, F. M., Anal. Chem. 1981, 53, 1862. 34. Landrum, H. L.; Salmon, R. T.; Hawkridge, F. M. J. Am. Chem.Soc.1977, 99, 3154. 35. Stargardt, J. F.; Hawkridge, F. M.; Landrum, H. L. Anal. Chem. 1978, 50, 930. 36. Bowden, E. F.; Hawkridge, F. M.; Blount, H. N. Bioelectrochem. Bioenerg. 1980, 7, 447. 37. Crawley, C. D.; Hawkridge, F. M. Biochem. Biophys. Res. Commun. 1981, 99, 516. 38. Bowden, E . F.; Wang, M.; Hawkridge, F. M. J. Electrochem. Soc. 1980, 127, 131C. 39. Shu, F. R.; Wilson, G. S. Anal. Chem. 1976, 48, 1676. 40. Armstrong, N. R.; Lin, A. W. C.; Fujihira, M.; Kuwana, T. Anal. Chem. 1976, 48, 741. 41. Van Gelder, B. F.; Slater, E. C. Biochim. Biophys. Acta 1962, 58, 593. 42. Van Buuren, K. J. H.; Van Gelder, B. F.; Wilting, J.; Braams, R. Biochim. Biophys. Acta 1974, 333, 421. 43. Bard, A. J.; Faulkner, L. R. "Electrochemical Methods"; Wiley: New York, 1980. 44. Nicholls, P. Biochim. Biophys. Acta 1974, 346, 261. 45. Osheroff, N.; Koppenol, W. H.; Margoliash, E . In "Frontiers of Biological Energetics"; P. L. Dutton; J. S. Leigh; A. Scarpa, Eds.; Academic: New York, 1978; Vol. 1, p. 439. 46. Cusanovich, Μ. Α.; Miller, W. G. Bioelectrochem. Bioenerg. 1974, 1, 448. 47. Cummins, D.; Gray, H.B.J.Am. Chem. Soc. 1977, 99, 5158. 48. Barlow, G. H.; Margoliash, E. J. Biol. Chem. 1966, 241, 1473. 49. Margalit, R.; Schejter, A. Eur. J. Biochem. 1973, 32, 500. 50. Osheroff, N.; Borden, D.; Koppenol, W. H.; Margoliash, E. J. Biol. Chem. 1980, 255, 1689. RECEIVED for review June 2, 1981. ACCEPTED November 4, 1981.


Electrochemical and Spectrochemical Studies of Biological Redox

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