Electrochemical and Spectrochemical Studies of Biological Redox

9 Electrochemical Studies of Cytochrome c of Desulfovibrio vulgaris 3

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K A T S U M I NIKI—Yokohama National University, Department of Electrochemistry, Hodogaya-ku, Yokohama 240, Japan T A T S U H I K O YAGI—Shizuoka University, Department of Chemistry, Shizuoka 422, Japan H I R O O INOKUCHI—Institute for Molecular Science, Okazaki 444, Japan

The isolation, purification, biological function, and structure (including tertiary structure) of cytochrome c from Desulfovibrio vulgaris are briefly discussed. The electrochemical reaction rate of the adsorbed cytochrome c film on a mercury electrode is very rapid and it becomes electrochemically inactive once the film is reduced. However, the inactive film does not hinder the electrode reaction of cytochrome c from the bulk of the solution. Potentiometric, polarographic, and cyclic voltammetric results reveal that the electrode reaction is reversible and diffusion controlled. The formal potential of the ferricytochrome/ferrocytochrome couple is -0.528 V (-0.287 V vs. NHE). The electrode reaction rate constant is 0.7 cm/s. From the simulation of the deferential pulse polarographic data, the best fit values of the individual standard potentials were obtained as -0.467, -0.519, -0.539, and -0.580 V. Cytochrome c , adsorbed on a mercury electrode, catalyzes the reduction of dioxygen to water, and the highest catalytic activity is attained when one-half of the electrode surface is covered by cytochrome c . 3

3

3

3

3

/ C y t o c h r o m e c is a n e l e c t r o n c a r r i e r p r o t e i n p r e s e n t i n t h e s t r i c t l y 3

anaerobic dissimilatory sulfate-reducing bacteria, Different

kinds

of cytochrome C 3were

discovered

Desulfovibrio. from

different

0065-2393/82/0201-0199$06.00/0 © 1982 A m e r i c a n C h e m i c a l Society

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

200

BIOLOGICAL R E D O X COMPONENTS

s p e c i e s o f s u l f a t e - r e d u c i n g b a c t e r i a i n g e n u s Desulfovibrio (1,2), a n d now cytochrome c reducing

bacteria

3

is a m a r k e r p r o t e i n i n t a x o n o m y o f t h e s u l f a t e distinguishing

Desulfovibrio

from

Desulfoto-

maculum (3). C y t o c h r o m e c i s d e s c r i b e d as " l o w p o t e n t i a l l o w s p i n 3

cytochrome w i t h some thioether b i n d i n g side c h a i n o f heme

as i n

m i t o c h o n d r i a l c y t o c h r o m e c . . . I t e x i s t s i n m u l t i h e m e f o r m as a monomer

. .

(4).

Cytochrome

c

isolated

3

from

Desulfovibrio

vulgans M i y a z a k i is s i m i l a r t o m a m m a l i a n c y t o c h r o m e c i n m o l e c u l a r weight a n d i n absorption spectrum. H o w e v e r , it contains four hemes i n a single p o l y p e p t i d e c h a i n a n d has a very negative redox potential.


T h i s c h a p t e r is c o n c e r n e d w i t h t h e 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 a s p e c t s o f c y t o c h r o m e c o f Desulfovibrio 3

vulgaris M i y a z a k i a n d i t s

unusual electrochemical behavior.

Biochemical Aspects Isolation and Purification of Cytochrome c . 3

C y t o c h r o m e c is a 3

major c y t o c h r o m e c o m p o n e n t present i n the s o l u b l e fraction o f the c e l l - f r e e e x t r a c t o f Desulfovibrio (5, 6). I t i s a b a s i c p r o t e i n w i t h a n i s o e l e c t r i c p o i n t n e a r o r o v e r 1 0 ( 1 , 2 , 7 - 1 0 ) , e x c e p t for t h a t f r o m D . gigas w h o s e i s o e l e c t r i c p o i n t i s 4 . 5 (11). P a s s a g e o f t h e s o l u b l e f r a c t i o n o f D . vulgaris t h r o u g h a c o l u m n o f A m b e r l i t e C G - 5 0 ( N H - f o r m ) a n d s u b s e q u e n t e l u t i o n b y 0.1 M N H f r o m t h e c o l u m n separate cyto c h r o m e c a n d some basic proteins from most o f t h e other proteinaceous substances. Purification b y gel-filtration chromatography w i t h a S e p h a d e x G - 5 0 (fine) c o l u m n f o l l o w e d b y r e c h r o m a t o g r a p h y w i t h a n Amberlite C G - 5 0 ( N H ) yields a pure cytochrome c preparation w i t h a p u r i t y i n d e x o f 3 . 0 , w h i c h i s d e f i n e d as t h e r a t i o o f t h e a b s o r b a n c e at 5 5 2 m m ( a l p h a p e a k ) i n t h e f e r r o - f o r m t o t h a t at 2 8 0 m m i n t h e f e r r i f o r m ( 2 , 8). +

4

3

3

+

4

3

Spectral Properties. T h e a b s o r p t i o n s p e c t r a o f c y t o c h r o m e c a r e s i m i l a r to c-type c y t o c h r o m e s . T h e s p e c t r u m o f the ferri-form c a n b e r e c o r d e d from t h e v i s i b l e to t h e U V r e g i o n . H o w e v e r , that o f the ferroform i n the U V region cannot b e r e a d i l y recorded because o f the i n tense absorption o f N a S 0 u s e d i n r o u t i n e m e t h o d s to r e d u c e cyto chromes. T h e w h o l e s p e c t r u m o f the ferro-form c a n b e taken w h e n c y t o c h r o m e c is r e d u c e d b y t h e c a t a l y t i c a c t i o n o f a t r a c e o f p u r i f i e d h y d r o g e n a s e ( E C 1.12.2.1) u n d e r t h e h y d r o g e n a t m o s p h e r e ( 2 ) . T h e s p e c t r a l c h a r a c t e r i s t i c s o f c y t o c h r o m e c e x t r a c t e d f r o m D. vulgaris a r e s h o w n i n Table I. 3

2

2

4

3

3

O n e spectral characteristic o f c y t o c h r o m e c that w a s o v e r l o o k e d is t h e p o s i t i o n i n g o f i s o s b e s t i c p o i n t s o n b o t h s i d e s o f t h e γ - p e a k o f t h e f e r r o - f o r m , t h a t i s , 4 1 2 a n d 4 3 2 n m (2, 12) ( T a b l e I ) . T h e d i s t a n c e b e t w e e n these t w o isosbestic p o i n t s is o n l y 2 0 n m i n t h e case o f c y t o 3


9.

Table I.

201

Cytochrome C3 o f D e s u l f o v i b r i o vulgaris

NIKI E T A L .

S p e c t r a l D a t a for C y t o c h r o m e c f r o m Desulfovibrio vulgaris 3

Peak positions i n the ferri-form (nm): 530, 4 1 0 ( γ ) , a n d 350(δ) P e a k p o s i t i o n s i n t h e f e r r o - f o r m ( n m ) : 5 5 2 ( a ) , 524(0), 4 1 9 ( γ ) , 3 2 3 ( δ ) , a n d 280(protein) Isosbestic points (nm): 560, 542, 532, 508, 432, 412, 343, a n d 254 M i l l i m o l a r a b s o r p t i v i t y at 5 5 2 n m i n t h e f e r r o - f o r m : 110 m M " · c m A b s o r b a n c e ratios: • ferro-a/ferri-280 = 3.0 • ferro-0/ferro-a = 0 . 5 2 • ferro-y/ferro-a = 6.3 • ferri-y/ferro-a = 4.0 • f e r r o - ô / f e r r o - a = 1.05 • ferri-ô/ferro-a =0.77


1

- 1

c h r o m e c , whereas t y p i c a l l y i n those o f other c-type cytochromes hav i n g one h e m e , the corresponding isosbestic points are 406 a n d 431 n m , w i t h t h e distance b e t w e e n t h e m b e i n g 2 5 n m (the γ - p e a k o f t h e e u c a r y o t i c c y t o c h r o m e c is b l u e - s h i f t e d b y 6 n m f r o m t h a t o f Desul fovibrio c y t o c h r o m e s , a n d so a r e t h e i s o s b e s t i c p o i n t s ) . W e o b s e r v e d r e p e a t e d l y t h a t w h e n c y t o c h r o m e c l o s t i t s b i o l o g i c a l f u n c t i o n as a n e l e c t r o n c a r r i e r f o r Desulfovibrio h y d r o g e n a s e b y a n y m e t h o d , t h e isosbestic p o i n t shifted from 4 3 2 to 4 3 7 n m . T h e n a r r o w i n g o f the γ - p e a k o f c y t o c h r o m e c m i g h t b e a reflection o f its intramolecular h e m e - h e m e interaction i n its tetraheme structure. 3

3

3

B i o c h e m i c a l F u n c t i o n . S i n c e its d i s c o v e r y i n strict anaerobes, c y t o c h r o m e c has b e e n a s s u m e d to b e a n e l e c t r o n m e d i a t o r i n t h e b a c t e r i a l e l e c t r o n transfer s y s t e m . C y t o c h r o m e c e n h a n c e d t h e rate o f h y d r o g e n u p t a k e b y t h e c e l l - f r e e e x t r a c t o f D . vulgaris H i l d e n b o r o u g h i n the presence o f thiosulfate or tetrathionate, b u t not i n the presence o f s u l f i t e (1 ). T h e a d d i t i o n o f c y t o c h r o m e c r e s u l t e d ( 7 ) i n a r e c o v e r y o f the f o r m i c hydrogenase a c t i v i t y o f the cell-free extract from w h i c h cytochrome c was r e m o v e d b y passing through an A m b e r l i t e C G 50 ( N H ) c o l u m n (13). T h i s c y t o c h r o m e was i n v o l v e d i n the reduction o f h y d r o x y l a m i n e a n d e l e m e n t a r y s u l f u r ( 1 3 ) , s u l f i t e (14), a n d a d e n o s i n e p h o s p h o s u l f a t e (15) i n a h y d r o g e n a t m o s p h e r e . T h e s e o b s e r v a t i o n s w e r e i n a c c o r d w i t h the i d e a that the bacterial hydrogenase system m i g h t b e d i r e c t l y l i n k e d t o c y t o c h r o m e c , w h i c h possesses t h e s a m e e l e c t r o n c a r r i e r f u n c t i o n as f e r r e d o x i n i n t h e c l o s t r i d i a l h y d r o g e n a s e ( E C 1.18.3.1) s y s t e m (16). D i r e c t p r o o f t h a t Desulfovibrio h y d r o g e n a s e r e d u c e s c y t o c h r o m e c w a s r e p o r t e d (17,18). H y d r o g e n is p r o d u c e d b y p a r t i a l l y p u r i f i e d Desulfovibrio h y d r o g e n a s e f r o m a n e l e c tron donor, N a S 0 , o n l y i n the presence o f c y t o c h r o m e c , a n d not i n t h e p r e s e n c e o f f e r r e d o x i n . C y t o c h r o m e c is a d i r e c t e l e c t r o n c a r r i e r for t h e h y d r o g e n a s e p r e p a r a t i o n f r o m b a c t e r i a i n g e n u s Desulfovibrio, 3

3

3

3

+

4

3

3

2

2

4

3

3


202

BIOLOGICAL REDOX COMPONENTS

t h a t i s : t h e h y d r o g e n a s e s f r o m D . vulgaris M i y a z a k i (17-21), D. vul garis H i l d e n b o r o u g h , (22), a n d D . gigas (23). T h e p r o d u c t i o n o f h y d r o g e n from formate (formic h y d r o g e n l y a s e r e a c t i o n ) is c a t a l y z e d b y t h e e n z y m e s y s t e m o f h y d r o g e n a s e a n d for mate dehydrogenase [formate:ferricytochrome c-553 oxidoreductase (24)] i n t h e p r e s e n c e o f t w o e l e c t r o n c a r r i e r p r o t e i n s , c y t o c h r o m e c a n d c y t o c h r o m e c - 5 5 3 . T h e s u l f i t e r e d u c t i o n is c a t a l y z e d b y a n e n z y m e system o f hydrogenase a n d sulfite reductase i n t h e presence o f t w o e l e c t r o n c a r r i e r s , c y t o c h r o m e c a n d flavodoxin ( 2 5 ) . H y d r o g e n p r o duction from pyruvate b y the phosphoroclastic e n z y m e system re q u i r e s t h e c o o p e r a t i o n o f c y t o c h r o m e c a n d f e r r e d o x i n for D. vulgaris (26), a n d c y t o c h r o m e c a n d flavodoxin for D. gigas (27). T h e o x i d a t i o n o f l a c t a t e i s c a t a l y z e d b y a n e n z y m e , D - l a c t a t e d e h y d r o g e n a s e (Dl a c t a t e : f e r r i c y t o c h r o m e c - 5 5 3 o x i d o r e d u c t a s e ) (28), a n d t h e r e d u c t i o n o f a d e n y l y l s u l f a t e is r e p o r t e d t o b e l i n k e d t o c y t o c h r o m e c (15). T h e s e results i n d i c a t e that c y t o c h r o m e c p l a y s a n i m p o r t a n t role i n t h e elec t r o n transfer s y s t e m . I t is n o t p r o v e d w h e t h e r t h e N A D ( P ) / N A D ( P ) H s y s t e m p a r t i c i p a t e s i n t h e e l e c t r o n t r a n s f e r s y s t e m i n D . vulgaris.

3

3


3

3

3

3

+

Structure. T h e p r i m a r y structures o f several c y t o c h r o m e c m o l e c u l e s p u r i f i e d f r o m d i f f e r e n t s p e c i e s o f Desulfovibrio w e r e e l u c i d a t e d . T h e s e i n c l u d e t h e c y t o c h r o m e s f r o m D. vulgaris M i y a z a k i ( 2 9 ) , D. vulgaris H i l d e n b o r o u g h (30, 31), D. gigas (32), D. desulfuricans (33), D. desulfuricans N o r w a y (34), a n d D. salexigens (35). T h e s e s e q u e n c e s a r e g i v e n i n T a b l e I I . I t is s h o w n t h a t c y t o c h r o m e c M i y a z a k i c o n t a i n s 107 a m i n o a c i d r e s i d u e s a n d its m o l e c u l a r w e i g h t is 13,995. T h e s e sequences d i s p l a y rather poor h o m o l o g y . A l t h o u g h there are e i g h t c y s t e i n e r e s i d u e s i n t h e s e s e q u e n c e s i n c o n f o r m i t y t o t h e i r t e t r a h e m e s t r u c t u r e s , t h e h e m e - a t t a c h i n g sites a r e n o t i d e n t i c a l . T h e c y t o c h r o m e s f r o m b o t h D . vulgaris a n d D. gigas c o n t a i n t w o C y s - x - y C y s - H i s sequences a n d t w o Cys-a-b-c-d-Cys-His sequences, whereas the other c y t o c h r o m e s c o n t a i n three C y s - x - y - C y s - H i s sequences a n d only o n e Cys-a-b-c-d-Cys-His sequence. B e c a u s e these hemea t t a c h i n g sites m u s t h a v e d i f f e r e n t c o n f o r m a t i o n s , t h e c y t o c h r o m e s f r o m D. vulgaris a n d D. desulfuricans n a t u r a l l y d o n o t s h a r e a c o m m o n p r e c i p i t a t i o n a n t i g e n i c d e t e r m i n a n t as j u d g e d b y a n i m m u n o d i f f u s i o n test (36). 3

3

C y t o c h r o m e c f r o m D . gigas i s a n a c i d i c p r o t e i n w i t h p i = 4 . 5 , w h e r e a s m o s t o t h e r c y t o c h r o m e s c a r e b a s i c p r o t e i n s w i t h p i o v e r 10. T w o k i n d s o f proteins h a v i n g c o m p l e t e l y different i o n i c properties have the i d e n t i c a l b i o l o g i c a l function i n different b u t c l o s e l y related microorganisms. T h e tertiary structure o f c y t o c h r o m e c from b o t h D . desulfuricans N o r w a y (34) a n d D . vulgaris M i y a z a k i (37) w a s e l u c i d a t e d . T h e c r y s t a l s t r u c t u r e o f D . vulgaris M i y a z a k i b e l o n g s t o t h e o r t h o r h o m b i c system (space g r o u p Ρ 2χ2ι2ι), w i t h the c e l l u n i t o f a = 3

3

3



c c c c c c

G G S D G G

V V I I V V

C c c c c c

N S I s N (B

H H H H H H

L V K A K K

D D D E E S)

M M L L T T

E E D L

T V K K

K K K K K A F R S K K (K

A S D T S A

A A A K

G G G K

K K K K K G K

D D D D

K K K A A A

C C C c C C

H H H H H H Ρ Ρ Ζ Χ Κ Κ

V V Ρ Χ W W N N G A D D

G G B D G G

L L L Ρ Ρ Ρ

T T T T T T

G G G A K K

M M V F F F

K K K G A K

T T E Ρ T T

D D D T E S

K K A Q R K

K K G K Ρ K

K K K G K K C C C C C C

Q Q N A A A A G T T Q S

T T T T A A

A A D S S E

G G G G G G

Ρ Ρ L L Q Κ T Ρ Ρ

K S E S D I

s E Ρ T Ρ Ρ

K

S T N K N K K

s s

G T K F K S N T K F K S K G G A K Ρ T

N Y Q D Y R Y A A V K A I Q E V K

K A G K

C K G s K C H C K K s K C H C K G s A C H C G K C H C T E C H C G D C H

H H H H H H

E E 0 G G A

D E D F I A G K Ρ K G D

Y H A H V Y K V Y K L V Ε S A M A Y S A Y

H H H H H H

K K K E κ K K D L K K K K K Ρ Τ G M K K G L K K A Τ G

D D B Q T K

A D A Α A D A Α D D K Ε D S Α

G G Β V Κ τ G Y G Y S W D I S F K) F

K K K E D D

A D G L K Μ A D G L K Μ A D G A K I Ε G M K Α Α G A K V Α G A K Μ

S A κ A κ S V Ν s A N A Κ D D G K S T Ρ

V V V V M L

Ρ Ρ Ρ Ρ Ρ Ρ

0

Sequence

c

b

"Species are as follows: 1, D. vulgaris M i y a z a k i ; 2, D. vulgaris Hildenborough; 3, D. gigas; 4, D. desulfuricans Norway; 5, D. desul furicans; and 6, D. salexigens. A m i n o acids are as follows: A , alanine; B , aspartic acid or asparagine; C , cysteine; D , aspartic acid; E , glutamic acid; F, phenylalanine; G , glycine; H , histidine; I, isoleucine; K , lysine; L , leucine; M , methionine; N , asparagine; P, proline; Q , glutamine; R, arginine; S, serine; T, threonine; V , valine; W, tryptophan; X , unknown; Y, tyrosine; and Z , glutamic acid or glutamine. Sequences i n parentheses are estimated.

1 2 3 4 5 6

C

D D N D A V

Η Η Η Η Η Η

H H H H H H

C C C c c c

T T T K G G

1 2 3 4 5 6

S S S T K K

H H H H H H

C C C C C C

N N N Ρ S s

V V V Ρ A D

V V V V V V

1 2 3 4 5 6

F F F F F F

A A V A A A

A Ρ K A Ρ K V D A D A Ρ G D D Y V I S V D A Ρ A D M V I K V L K V D A Ρ G D M

Amino Acid

3

Comparison of the Amino Acid Sequences of Cytochromes c from Species of Desulfovibrio

1 2 3 4 5 6

0

Species

Table II.


204

BIOLOGICAL R E D O X COMPONENTS

52.8, b = 6 8 . 1 , a n d c = 34.9 À. T h e shape o f the c y t o c h r o m e C m o l e c u l e is a n e l l i p s o i d w i t h o v e r a l l d i m e n s i o n s o f 3 3 x 3 4 x 3 9 A . T h e i r o n - t o - i r o n d i s t a n c e s r a n g e d f r o m 1 1 . 3 to 18.1 A , a n d t h e a n g l e s b e t w e e n p o r p h y r i n r i n g s are s h o w n i n T a b l e I I I . E a c h h e m e is l i n k e d to t w o c y s t e i n e r e s i d u e s t h r o u g h t h i o e t h e r b o n d s as for c y t o c h r o m e c. T h e 3q

fifth a n d s i x t h l i g a n d s o f e a c h i r o n a t o m a r e n i t r o g e n o f i m i d a z o l e i n t h e h i s t i d y l r e s i d u e . E a c h h e m e is e x p o s e d to a d i f f e r e n t e n v i r o n m e n t a n d , c o n s e q u e n t l y , is e x p e c t e d to h a v e a d i f f e r e n t r e d o x p o t e n t i a l .


Electrochemical Aspects Adsorption of Cytochrome c on M e r c u r y Electrode. 3

T h e adsorp

t i o n b e h a v i o r o f c y t o c h r o m e c at t h e e l e c t r o d e s u r f a c e is i m p o r t a n t i n 3

the e l u c i d a t i o n o f not o n l y e l e c t r o c h e m i c a l r e d o x reactions b u t also b i o l o g i c a l f u n c t i o n s o f c y t o c h r o m e c as a n e l e c t r o n c a r r i e r i n t h e res 3

piratory chain. T h e differential c a p a c i t a n c e - t i m e curves o f the d r o p p i n g m e r c u r y e l e c t r o d e i n c y t o c h r o m e c solutions r e v e a l e d that the adsorption o f the 3

h e m e p r o t e i n o n t h e m e r c u r y e l e c t r o d e is i r r e v e r s i b l e a n d

diffusion

c o n t r o l l e d . T h a t is, the a m o u n t o f the a d s o r b e d h e m e p r o t e i n c a n b e evaluated b y u s i n g Koryta's equation (38): Γ = 0.736

112

C (D t) p

p

3

w h e r e C is t h e c o n c e n t r a t i o n o f p r o t e i n ( m o l / c m ) , D is t h e d i f f u s i o n c o e f f i c i e n t o f p r o t e i n ( c m / s ) , a n d t is t h e t i m e e l a p s e d f r o m t h e b i r t h o f t h e m e r c u r y d r o p (s). T h e s u r f a c e c o v e r a g e d e f i n e d as θ = I 7 r was e v a l u a t e d from the concentration d e p e n d e n c e o f the differential c a p a c i t a n c e at t h e d r o p p i n g m e r c u r y e l e c t r o d e , w h e r e Γ is t h e s u r f a c e concentration o f cytochrome c and r the m a x i m u m surface concen tration (39). T h e m a x i m u m surface concentration, r , was calculated f r o m t h e c o n c e n t r a t i o n at θ = 1 b y u s i n g K o r y t a ' s e q u a t i o n . T h e m a x i m u m c o n c e n t r a t i o n o b t a i n e d w a s 0 . 9 2 x 1 0 ~ m o l / c m at - 0 . 9 0 V [ a l l e l e c t r o d e p o t e n t i a l s i n t h i s c h a p t e r a r e r e f e r r e d to a s a t u r a t e d c a l o m e l e l e c t r o d e ( S C E ) at 2 5 ° C ] , w h i c h a g r e e s w i t h t h e r e s u l t o b t a i n e d b y t h e p o t e n t i a l s t e p c h r o n o c o u l o m e t r i c m e t h o d (40). A c c o r d i n g l y , t h e a r e a p

p

2

m a x

3

m a x

m a x

n

2

T a b l e I I I . H e m e - H e m e D i s t a n c e s ( u p p e r right i n A n g s t r o m u n i t s ) a n d H e m e - H e m e A n g l e s ( l o w e r left i n degrees) (37)

Heme 1 2 3 4

1

2

3

4

16.3

18.1

12.8


9.

205

Cytochrome C 3 of D e s u l f o v i b r i o vulgaris

NIKI E T A L .

2

o c c u p i e d b y t h e a d s o r b e d c y t o c h r o m e c m o l e c u l e is 1 8 0 0 Â , w h i c h is 3

1.35 t i m e s g r e a t e r t h a n t h e p r o j e c t e d a r e a o f t h e c y t o c h r o m e c

3

mole

c u l e i n t h e c r y s t a l . T h e e x t e n t o f t h e d e f o r m a t i o n (or u n f o l d i n g ) o f t h e cytochrome c

3

m o l e c u l e at t h e m e r c u r y e l e c t r o d e is c o n s i d e r e d t o b e

s m a l l . O n the other h a n d , the s p r e a d i n g o f the c y t o c h r o m e c m o l e c u l e at t h e e l e c t r o d e s u r f a c e is 1.8 t i m e s g r e a t e r , t h a t o f t h e

ribonuclease

m o l e c u l e is 2 . 2 - 2 . 9 t i m e s , a n d t h a t o f t h e l y s o z y m e m o l e c u l e is 1 . 3 2 . 0 t i m e s g r e a t e r (41 ).

Electrochemical Properties of Adsorbed Cytochrome c .

A fresh

3

mercury drop (hanging m e r c u r y drop electrode, H M D E ) was e q u i l i 5

4

b r a t e d for a b o u t 1 m i n i n c y t o c h r o m e c s o l u t i o n ( 1 0 ~ ~ 1 0 ~ M ) . A f t e r Downloaded by CORNELL UNIV on May 18, 2017 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch009

3

the m e r c u r y d r o p was r i n s e d t h o r o u g h l y b y p h o s p h a t e buffer s o l u t i o n , the H M D E was transferred i n t o the d e a e r a t e d 0.03 M p h o s p h a t e buffer solution w i t h o u t c y t o c h r o m e c a n d then c y c l i c voltammograms o f the 3

adsorbed cytochrome c

3

w e r e r e c o r d e d . T h e r e d u c t i o n p e a k o f fer

r i c y t o c h r o m e c w a s o b s e r v e d o n l y o n t h e first s c a n at - 0 . 5 V as s h o w n 3

i n F i g u r e 1 (40).

N o reoxidation peak o f ferrocytochrome c

3

was de

t e c t e d o n the reverse scan, a n d no peak was o b s e r v e d on further r e p e t i tion o f the scanning. T h e electrochem ically inactive adsorbed layer o f c y t o c h r o m e c w a s s t a b l e a n d w a s n o t r e o x i d i z a b l e b y e x p o s u r e to t h e 3

oxygen atmosphere. T h e same electrochemical behavior was reported as w a s for t h e c y c l i c v o l t a m m e t r y o f t h e a d s o r b e d m o n o l a y e r o f c y t o c h r o m e c on the m e r c u r y electrode

(42).

T h e potential step chronocoulometric technique

(43) w a s

em

p l o y e d to i n v e s t i g a t e t h e r e d u c t i o n o f t h e a d s o r b e d c y t o c h r o m e c t h e m e r c u r y e l e c t r o d e (40).

on

3

After the c o m p l e t e saturation o f the d r o p

p i n g m e r c u r y e l e c t r o d e b y f e r r i c y t o c h r o m e c , the e l e c t r o d e potential 3

w a s s t e p p e d f r o m - 0 . 3 to - 0 . 7 V . T h e n u m b e r o f e l e c t r o n s i n v o l v e d i n t h e r e d u c t i o n o f t h e a d s o r b e d f e r r i c y t o c h r o m e c w a s f o u r a n d its r e a c 3

tion was almost instantaneous. T h e reduction o f ferricytochrome c

3

in

t h e b u l k o f t h e s o l u t i o n w a s c o n f i r m e d to b e d i f f u s i o n c o n t r o l l e d . O n the other h a n d , the reoxidation o f the adsorbed ferrocytochrome

c , 3

w h i c h w a s r e d u c e d e l e c t r o c h e m i c a l l y at t h e e l e c t r o d e , w a s n o t o b served. T h e c h e m i c a l l y generated ferrocytochrome c

3

was also e l e c -

t r o c h e m i c a l l y i n a c t i v e w h e n it was adsorbed on the m e r c u r y electrode. T h e a d s o r b e d f e r r i c y t o c h r o m e film w a s s t a b l e a n d n o a p p r e c i a b l e d e s o r p t i o n w a s o b s e r v e d after 3 0 m i n i n t h e p h o s p h a t e b u f f e r s o l u t i o n . T h e a d s o r b e d f e r r o c y t o c h r o m e c w a s c o n f i r m e d to b e s t a b l e b y m o n i 3

t o r i n g the differential capacitance o f the e l e c t r o d e t h r o u g h o u t the re d u c t i o n a n d o x i d a t i o n processes o f the a d s o r b e d c y t o c h r o m e c

3

film.

T h e electron exchange b e t w e e n the electrode a n d c y t o c h r o m e c

3

i n t h e b u l k o f t h e s o l u t i o n ( d i s c u s s e d l a t e r i n t h i s c h a p t e r ) is n o t r e s t r i c t e d b y t h e m o n o l a y e r o f c y t o c h r o m e c at t h e e l e c t r o d e s u r f a c e . I n 3

a d d i t i o n , the a m o u n t o f the a d s o r b e d c y t o c h r o m e o n the m e r c u r y e l e c -


206



-0.10

0.051 0

,

,

,

I -1.0

-0.5 E l e c t r o d e p o t e n t i a l (V)

Figure 1. Cyclic voltammogram of the adsorbed cytochrome c (monolayer) on hanging mercury drop electrode in 0.03 M phosphate buffer solution at pH 7.0. Scan rate, 100 mV/s; A = 0.0143 cm (40).

3

2

2

t r o d e s u r f a c e is so s m a l l (1 m i c r o c o u l o m b / c m ) a n d t h e e l e c t r o c h e m i c a l r e a c t i o n rate c o n s t a n t is so l a r g e t h a t t h e e x p e r i m e n t a l errors c a u s e d b y the a d s o r b e d species i n c y c l i c v o l t a m m e t r y a n d p u l s e p o l a r o g r a p h y are i n s i g n i f i c a n t (40). Potentiometric Studies. The potentiometric measurements were c a r r i e d o u t i n 0 . 0 3 M p h o s p h a t e b u f f e r s o l u t i o n at p H 7.0 w i t h a s l o w d r o p p i n g m e r c u r y e l e c t r o d e ( d r o p t i m e ~ 3 0 s) as a n i n d i c a t o r e l e c t r o d e . T h e r a t i o o f t h e f e r r i - f o r m to t h e f e r r o - f o r m is v a r i e d b y t h e e n z y m a t i c r e d u c t i o n o f the ferri-form w i t h a c o n t r o l l e d a m o u n t o f h y d r o g e n i n the presence o f a s m a l l a m o u n t o f hydrogenase ( h y d r o g e n : f e r r i c y t o c h r o m e c o x i d o r e d u c t a s e , E C 1.12.2.1.). T h e p l o t o f l o g (C /CR) vs. electrode potential produces a straight l i n e w i t h a Nernst s l o p e o f 9 3 m V as s h o w n i n F i g u r e 2 . T h e a p p a r e n t n u m b e r o f e l e c trons i n v o l v e d i n t h e o v e r a l l e l e c t r o d e r e a c t i o n 3

0

ferricytochrome c

3

+ 4e ^

ferrocytochrome c

3

is c a l c u l a t e d t o b e 0.64. T h e a p p a r e n t f o r m a l p o t e n t i a l , w h i c h is t h e


9.

Cytochrome

NIKI E T A L .

207

C 3 of D e s u l f o v i b r i o vulgaris

m e a n r e d o x p o t e n t i a l o f t h e f o u r i n d i v i d u a l h e m e s i n t h e m o l e c u l e , is - 0 . 5 2 8 ± 0 . 0 0 1 V ( - 0 . 2 8 7 V v s . N H E ) (44). l i n e a r p l o t at t h e C /C 0

T h e d e v i a t i o n from the

r a t i o less t h a n 0.5 p r e d i c t s a m u l t i s t e p r e d u c

R

tion of cytochrome c . 3

Pulse Polarographic Studies. well-defined

Cytochrome c

3

produces a single

r e d u c t i o n w a v e w i t h a s m a l l p r e c e d i n g w a v e at

d r o p p i n g m e r c u r y e l e c t r o d e , as s h o w n i n F i g u r e 3 . T h e

the

half-wave

p o t e n t i a l is - 0 . 5 2 7 V , w h i c h is e q u a l to t h e a p p a r e n t f o r m a l p o t e n t i a l i n p o t e n t i o m e t r y , a n d t h e l o g a r i t h m i c p l o t is a s t r a i g h t l i n e w i t h a n inverse slope o f 0.085 m V , from w h i c h the n u m b e r o f electrons i n


v o l v e d i n t h e e l e c t r o d e r e a c t i o n is e s t i m a t e d to b e 0 . 7 0

(44).

T h e polarogram obtained b y a scan-reversal pulse polarography d e p e n d s m a r k e d l y on the r e v e r s i b i l i t y o f the e l e c t r o d e reaction In the c y t o c h r o m e c

3

(45).

s o l u t i o n the ratio o f the l i m i t i n g currents

for

c a t h o d i c a n d a n o d i c scans is u n i t y a n d t h e h a l f - w a v e p o t e n t i a l o n t h e c a t h o d i c s c a n is e q u a l to t h a t o n t h e r e v e r s e s c a n . T h e s e r e s u l t s p r o v i d e s t r o n g e v i d e n c e for t h e r e v e r s i b l e e l e c t r o d e chrome c

3

reaction of cyto

on the m e r c u r y electrode. O u r galvanostatic d o u b l e p u l s e

m e a s u r e m e n t s r e v e a l e d t h a t t h e a p p a r e n t e l e c t r o d e r e a c t i o n rate c o n stant o f the f e r r i c y t o c h r o m e c / f e r r o c y t o c h r o m e c 3

T h e diffusion coefficients o f f e r r i c y t o c h r o m e c c

3

c a l c u l a t e d from the

pulse

3

3

s y s t e m is 0.7 c m / s .

and

polarographic data

ferrocytochrome were

0.94 x 1 0 ~

10

U υ

ο 1.0

0.1

-0.4

-0.5

-0.6

E l e c t r o d e potential (V) Figure 2. Equilibrium potentials chrome c in 0.03 M phosphate 3

of the ferricytochrome/ferrocyto buffer solution at pH 7.0 (44).


6


208


0

-0.2

-0.4

-0.6

-0.8

-1.0

E l e c t r o d e p o t e n t i a l (V) Figure 3. Normal pulse polarograms of the reduction of chrome c and of the base solution (0.03 M phosphate Cytochrome c concentration, 1.24 x 10 ~ M ; m = 2.82 sampling time, 50 ms ( 44).

ferricytobuffer). mgldrop;

3

4

3

2

c m / s . T h i s v a l u e is i n a g r e e m e n t w i t h t h a t o f h o r s e h e a r t f e r r i c y t o c h r o m e c d e t e r m i n e d b y a h y d r o d y n a m i c t e c h n i q u e (46). Cyclic Voltammetric Studies. T h e cyclic voltammogram o f c y t o c h r o m e c is d i s t o r t e d a n d b r o a d e r t h a n e x p e c t e d f r o m t h e f o u r e l e c t r o n r e a c t i o n (47). T h e s a m e is t r u e i n c - t y p e c y t o c h r o m e s f r o m D. vulgaris H i l d e n b o r o u g h a n d D . desulfuricans N o r w a y (48). T h e p e a k p o t e n t i a l s o f t h e s e c y t o c h r o m e s a r e i n d e p e n d e n t o f b o t h t h e s c a n rate a n d the concentration o f c y t o c h r o m e c w i t h i n the investigated range. T h e p e a k c u r r e n t varies l i n e a r l y w i t h t h e square root o f the scan rate a n d t h e r a t i o o f t h e c a t h o d i c t o a n o d i c p e a k c u r r e n t is a l m o s t u n i t y . 3

3

3

T h e c a t h o d i c p e a k c u r r e n t is o n l y 1/2J5 o f t h e p e a k c u r r e n t e x p e c t e d from a reversible four-electron process. T h e cathodic a n d anodic peak potentials are - 0 . 5 5 a n d - 0 . 4 6 V , respectively. T h e n u m b e r o f e l e c t r o n s i n v o l v e d i n t h e e l e c t r o d e p r o c e s s is c a l c u l a t e d t o b e 0.61 f r o m t h e p e a k - t o - p e a k s e p a r a t i o n (47). I n t h e c a s e o f c y t o c h r o m e c f r o m D . vulgaris H i l d e n b o r o u g h t h e c a t h o d i c a n d a n o d i c p e a k p o t e n t i a l s a r e - 0 . 6 2 a n d - 0 . 5 1 V , r e s p e c t i v e l y (48). O n t h e o t h e r h a n d , c y t o c h r o m e c f r o m D. desulfuricans N o r w a y s h o w s t w o c a t h o d i c p e a k s at - 0 . 4 5 a n d - 0 . 6 3 V a n d t h e a n o d i c p e a k at - 0 . 5 4 V (48). T h e c a t h o d i c p e a k at - 0 . 2 V o b s e r v e d i n b o t h cases is p r o b a b l y d u e t o t h e reduction o f a c o n t a m i n a t e d o x y g e n . T h e electrode process o f cyto c h r o m e c f r o m D . vulgaris H i l d e n b o r o u g h at —0.56 V i s l i k e l y to c o r r e s p o n d to the redox reaction o f f e r r i c y t o c h r o m e c / f e r r o c y t o 3

3

3

3


9.

Cytochrome

NIKI E T A L .

209

C 3 of D e s u l f o v i b r i o vulgaris

c h r o m e C3 o f M i y a z a k i because o f the s i m i l a r i t y i n the p r i m a r y struc t u r e b e t w e e n c y t o c h r o m e c f r o m D . vulgaris M i y a z a k i a n d t h a t f r o m 3

D . vulgaris H i l d e n b o r o u g h ( t h e d e g r e e o f h o m o l o g y b e t w e e n

these

c y t o c h r o m e s c is 8 7 % ) as s h o w n i n T a b l e I I . H o w e v e r , c y t o c h r o m e 3

c

3

f r o m D . desulfuricans N o r w a y h a s t h e l e a s t h o m o l o g y t o o u r c y t o

chrome c

3

(the d e g r e e o f h o m o l o g y is o n l y 2 8 % ) , a n d t h e different

electrode process o f c y t o c h r o m e c can b e expected. 3

Differential

Pulse Polarographic Study.

T h e differential pulse

p o l a r o g r a m o f f e r r i c y t o c h r o m e c i s w e l l - d e f i n e d , as s h o w n i n F i g u r e 3

4. T h e p o l a r o g r a m is s l i g h t l y d i s t o r t e d f r o m t h e s y m m e t r y w i t h t h e


peak potential o f - 0 . 5 2 2 V , from w h i c h the half-wave potential o f a r e v e r s i b l e s y s t e m i n n o r m a l p u l s e p o l a r o g r a p h y is c a l c u l a t e d to b e - 0 . 5 2 7 V . H o w e v e r , t h e p e a k h e i g h t is s m a l l e r t h a n t h a t e x p e c t e d f r o m a r e v e r s i b l e f o u r - e l e c t r o n r e a c t i o n a n d t h e h a l f - p e a k w i d t h is also b r o a d e r t h a n t h a t e x p e c t e d . T h e h a l f - w a v e p o t e n t i a l s o f D. vulgaris Hildenborough cytochrome c

3

are estimated

to b e - 0 . 4 9 V ( i l l -

defined) a n d - 0 . 5 8 V (main wave) from the differential pulse polarog r a p h y , a n d t h o s e o f D . desulfuricans N o r w a y a r e e s t i m a t e d i n t h e s a m e w a y t o b e - 0 . 4 0 V ( w e l l - d e f i n e d ) a n d - 0 . 5 8 V ( m a i n w a v e ) (49).

-0.3

-0.4

-0.5

-0.6

-0.7

E l e c t r o d e p o t e n t i a l (V) Figure 4. Differential pulse polarogram for the reduction of ferricytochrome c (1.24 x 10~ M) in 0.03 M phosphate buffer solution (—) and best fit differential pulse polarography simulation (O) (57). m = 2.99 mgldrop; sampling time, 70 ms; modulation amplitude, 10 mV. 4

3


210


Elucidation o f the Redox Potential o f H e m e s i n Cytochrome c . T h e r e d o x states o f c y t o c h r o m e c f r o m D. vulgaris H i î d e n b o r o u g h w e r e s t u d i e d e x t e n s i v e l y b y E S R (50-52), M o s s b a u e r s p e c t r o s c o p y (53), N M R (54, 55), a n d E S R c o u p l e d w i t h p o t e n t i o m e t r y (56). I n t h e c o u r s e o f t h e r e o x i d a t i o n o f f e r r o c y t o c h r o m e c w i t h o x y g e n , a s t a b l e i n t e r m e d i a t e at a h a l f - r e o x i d i z e d state w a s o b s e r v e d i n t h e E S R m e a s u r e m e n t s (50, 51 ). T h e r e d o x c y c l i n g b y c h e m i c a l r e d u c tion a n d reoxidation w i t h o x y g e n r e v e a l e d that the i n d i v i d u a l hemes w e r e r e o x i d i z e d at d i f f e r e n t rates i n d i c a t i n g d i s s i m i l a r h e m e s at 8.5 Κ (52). M c D o n a l d e t a l . (54) r e p o r t e d t h a t t h r e e d i f f e r e n t o x i d a t i o n states appear to d e v e l o p i n the reoxidation o f ferrocytochrome c ; I, o n e f e r r i - h e m e a n d t h r e e f e r r o - h e m e s ; I I , t w o f e r r i - h e m e s a n d t w o ferrohemes; a n d III, fully o x i d i z e d hemes. O n the other h a n d , D o b s o n et al. 3

3

3


3

(55) o b s e r v e d t h a t t h e r e d u c t i o n o f f e r r i c y t o c h r o m e c w i t h d i t h i o n i t e proceeds i n t w o t w o - e l e c t r o n steps. 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 was p r e d i c t e d to p r o c e e d i n four one-electron steps w i t h u n e q u a l l y s p a c e d standard potentials (the spacings are -26, - 9 , a n d - 5 m V ) 3

3

(56) . T h e f o r m a l p o t e n t i a l i s e s t i m a t e d to b e -0.56 V . O u r i n v e s t i g a t i o n s h o w e d t h a t c y t o c h r o m e c f r o m D . vulgaris M i y a z a k i contains four h e m e groups a n d the v o l t a m m e t r i c data i n d i cate that these four r e d o x centers are c h a r a c t e r i z e d b y four separate but closely spaced redox potentials, w i t h each center e x h i b i t i n g revers i b l e e l e c t r o n transfer o n a m e r c u r y e l e c t r o d e . W e d e v e l o p e d theoreti c a l r e s p o n s e s for t h e r e d u c t i o n o f m o l e c u l e s c o n t a i n i n g f o u r s i m i l a r b u t n o n e q u i v a l e n t r e d o x sites f o r c y c l i c v o l t a m m o g r a m a n d d i f f e r e n t i a l p u l s e p o l a r o g r a m (57). 3

Definition o f the four i n d i v i d u a l standard potentials is g i v e n b y t h e f o l l o w i n g s c h e m e w h e r e C is t h e f u l l y o x i d i z e d a n d C t h e f u l l y reduced cytochrome: 0

Co

4

C\ * E\

C *

C *

2

E\

C

3

E%

4

E\

a n d b y the expression: Ε = Ei -

(RT/F)ln(Q/C

i + 1

)

T h e i n d i v i d u a l standard potentials are m a c r o s c o p i c rather than m i c r o scopic parameters. T h u s , C does not signify reduction o f a particular h e m e g r o u p b u t is s i m p l y t h e e q u i l i b r i u m d i s t r i b u t i o n o f a l l m o l e c u l e s t h a t h a v e o n e r e d u c e d site a n d t h r e e o x i d i z e d sites, n a m e l y , a c o l l e c tion o f the species R i 0 0 0 , 0 ^ 0 0 , 0 ! 0 R 0 , a n d 0 ! 0 0 R . T h e theoretical v o l t a m m e t r i c responses w e r e generated b y d i g i t a l s i m u l a t i o n w i t h t e c h n i q u e s d e v e l o p e d p r e v i o u s l y (58). t

2

3

4

2

3

4

2

3

4

2


3

4

9.

NIKI E T A L .

211

Cytochrome C 3 of D e s u l f o v i b r i o vulgaris

Cyclic Voltammetry (CV).

F o r the C V simulations, semi-infinite,

l i n e a r d i f f u s i o n to a p l a n e e l e c t r o d e w a s e m p l o y e d . A n u m b e r o f s i m u l a t i o n s for e q u a l l y s p a c e d E ° v a l u e s w e r e p e r f o r m e d , a n d t h e v a l u e s o f the difference b e t w e e n a n o d i c a n d cathodic peak potentials, Δ Ε , a n d Ρ

t h e c a t h o d i c p e a k c u r r e n t f u n c t i o n , λ/π

χ (at)

are g i v e n i n F i g u r e s 5

a n d 6 as f u n c t i o n s o f t h e s p a c i n g b e t w e e n t h e s t a n d a r d Δ Ε ° = E°

M

112

(Fv/RT)

~ El

T h e c u r r e n t f u n c t i o n is d e f i n e d ( 5 9 ) b y

potentials, m

i/FAD C*

w h e r e i is t h e c u r r e n t , A is t h e e l e c t r o d e a r e a , D is t h e

d i f f u s i o n c o e f f i c i e n t o f the r e a c t a n t , C * is t h e b u l k c o n c e n t r a t i o n o f t h e r e a c t a n t , ν is t h e

scan rate, a n d the other s y m b o l s have


n o r m a l m e a n i n g . P o s i t i v e values o f the

their

s p a c i n g c o r r e s p o n d to

r e a c t i o n t h a t b e c o m e s e a s i e r as r e d u c t i o n p r o c e e d s a n d i n t h e

the limit

o f v e r y p o s i t i v e s p a c i n g , Δ Ε a p p r o a c h e s 14.2 m V , w h i c h c o r r e s p o n d s Ρ

to t h e r e v e r s i b l e f o u r - e l e c t r o n r e a c t i o n . S i m i l a r l y , t h e c a t h o d i c p e a k current function approaches the theoretical v a l u e o f 3.57 (59). F r o m a c o m p a r i s o n o f t h e p r e s e n t r e s u l t s w i t h t h e v a l u e s i n F i g u r e s 5 a n d 6, t h e s p a c i n g o f t h e s t a n d a r d p o t e n t i a l s is e s t i m a t e d to b e

-35

mV

p r o v i d e d that the s t a n d a r d p o t e n t i a l s are e q u a l l y s p a c e d . Differential

Pulse Polarography (DPP).

T h e simulations

were

b a s e d o n s e m i - i n f i n i t e l i n e a r d i f f u s i o n to t h e e x p a n d i n g p l a n e m o d e l for t h e d r o p p i n g m e r c u r y e l e c t r o d e ( 5 8 ) . T h e b e s t fit s i m u l a t i o n w i t h the e x p e r i m e n t a l results was o b t a i n e d from the analysis o f D P P data

300

200 a.

Electrochemical and Spectrochemical Studies of Biological Redox

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