Electrode Reactions of Protein Prosthetic Groups - American Chemical

10 Electrode Reactions of Protein Prosthetic Groups F. SCHELLER and G. STRNAD

Downloaded by UNIV OF ARIZONA on November 11, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch010

Central Institute of Molecular Biology, Academy of Science of the German Democratic Republic, 1115 Berlin-Buch, German Democratic Republic

The exchange rate of the adsorbed molecules and the degree of structural changes determine the character of the electrode process. Additionally, the ability of the first adsorption layer to transfer electrons to molecules of the bulk phase, that is, the rate of the intermolecular electron transfer between the layers, is responsible for deviations from a reversible polylayer electrode process: decrease of limiting current, overvoltage, and suppression of reoxidation. The electrode process seems to be more similar to the mechanism in biological electron transfer chains than to the random collision processes following an outer or inner sphere mechanism.

/

I ' h e fast o x i d a t i o n o f r e d u c i n g o r g a n i c s u b s t a n c e s l i k e N A D H , g l u c o s e , u r i c a c i d , a n d d i f f e r e n t d r u g s is a n i m p o r t a n t p r e c o n d i t i o n for the function o f l i v i n g systems. T h e s e redox reactions are c a t a l y z e d b o t h b y s i n g l e e n z y m e s a n d b y e l e c t r o n transfer c h a i n s ( J ) . O n t h e o t h e r h a n d , at m e t a l e l e c t r o d e s t h e s e o x i d a t i o n p r o c e s s e s r e q u i r e a h i g h anodic overvoltage a n d m a y b e m a s k e d b y the background dis charge. T h u s , the c o m b i n a t i o n o f e n z y m a t i c substrate oxidation w i t h the transfer o f the r e d u c i n g e q u i v a l e n t s to t h e e l e c t r o d e p r o m i s e s a n i m p r o v e m e n t i n t h e rate a n d s p e c i f i c i t y o f t h e e l e c t r o d e p r o c e s s e s t h a t a r e a p p l i e d i n b i o - f u e l c e l l s , e n z y m e r e a c t o r s , a n d sensors. T h e k e y p r o b l e m i n the use o f enzymes i n accelerating electrode p r o c e s s e s is t h e o p t i m i z a t i o n o f t h e e l e c t r o n t r a n s f e r b e t w e e n t h e e n z y m e a c t i v e site a n d the e l e c t r o d e . I n p r i n c i p l e , t w o different a p p r o a c h e s m a y b e o f f e r e d ( F i g u r e 1): 1. L o w m o l e c u l a r r e d o x s y s t e m s c a l l e d m e d i a t o r s a r e s u i t e d to transfer electrons b e t w e e n the e n z y m e a n d the electrode. This principle frequently was used w i t h glu0065-2393/82/0201-0219$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.


220

BIOLOGICAL REDOX COMPONENTS

Figure

1.

Principles

of the electron site to the

transport from the enzyme electrode.

active

cose oxidation c a t a l y z e d b y glucose oxidase i n e n z y m e e l e c t r o d e s ( 2 , 3 ) a n d f u e l c e l l s ( 3 , 4). 2. T h e f e a s i b i l i t y o f d i r e c t e l e c t r o n t r a n s f e r f r o m t h e e n z y m e a c t i v e site t o t h e e l e c t r o d e a l s o w a s e s t a b l i s h e d w i t h s e v e r a l p r o t e i n s (5a-5c). 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 is a t t r a c t i n g i n c r e a s i n g a t t e n t i o n i n b i o t e c h n o l ogy (6). I n t h i s c h a p t e r t h e p r e s e n t state o f p r o t e i n e l e c t r o c h e m i s t r y w i l l be i l l u s t r a t e d u s i n g data from the literature a n d results o b t a i n e d w i t h t h e flavoprotein g l u c o s e o x i d a s e . T h e m e c h a n i s m o f t h e e l e c t r o n t r a n s fer b e t w e e n p r o t e i n s a n d e l e c t r o d e s w i l l b e d i s c u s s e d o n t h e b a s i s o f correlations b e t w e e n the c o n d u c t i v i t y o f p r o t e i n films a n d the charac ter o f the r e s p e c t i v e e l e c t r o d e process.

Results and Models of the Electrode Process A l t h o u g h t h e p r o t e i n - f r e e p r o s t h e t i c g r o u p s , for e x a m p l e , f l a v i n (7) a n d h e m i n ( 8 ) , s h o w a d i f f u s i o n - c o n t r o l l e d e l e c t r o n e x c h a n g e w i t h the e l e c t r o d e , the h i g h m o l e c u l a r w e i g h t a n d the surface a c t i v i t y o f the proteins m a y cause drastic changes i n their electrochemical behavior. W i t h b i o p o l y m e r s , s t r u c t u r a l factors m u s t a l s o b e t a k e n i n t o a c c o u n t ,

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

10.

221

Protein Prosthetic Groups

S C H E L L E R A N D STRNAD

s u c h as t h e a c c e s s i b i l i t y o f t h e r e d u c i b l e g r o u p s a n d s t r u c t u r a l c h a n g e s on adsorption. W i t h p r o t e i n s the e l e c t r o d e - a c t i v e part a n d the a d s o r b i n g tails are u n i t e d i n o n e m o l e c u l e , so t h e rate o f a d s o r p t i o n e x c h a n g e d i r e c t l y d e t e r m i n e s the n a t u r e o f the e l e c t r o d e

process.

Rapid Adsorption Exchange A r a p i d e x c h a n g e o f the a d s o r b e d m o l e c u l e s b y particles i n the Downloaded by UNIV OF ARIZONA on November 11, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch010

b u l k o f the s o l u t i o n was f o u n d o n l y w i t h several p e p t i d e s (9). T h e l i m i t i n g current increased w i t h the concentration o f the solution, w i t h out r e a c h i n g a saturation v a l u e .

Irreversible Adsorption of First Layer A s demonstrated b y adsorption studies w i t h radioactively l a b e l e d proteins (20) a n d the " f i l m transfer m e t h o d " ( I I ) , the m o l e c u l e s a d s o r b e d i m m e d i a t e l y at t h e e l e c t r o d e e x c h a n g e v e r y s l o w l y . A t h i g h e r c o n c e n t r a t i o n s a d d i t i o n a l p r o t e i n m o l e c u l e s a c c u m u l a t e d at t h e e l e c trode b y the formation o f p o l y l a y e r s (12). T h e e l e c t r o d e p r o c e s s is a h e t e r o g e n e o u s r e a c t i o n w h e r e t h e r a t e is i n c r e a s e d i n o n e d i r e c t i o n a n d d e c r e a s e d i n t h e o p p o s i t e d i r e c t i o n b y t h e a p p l i e d e l e c t r i c field. T h e e l e c t r i c field at t h e e l e c t r o d e / e l e c t r o l y t e i n t e r p h a s e e x t e n d s o v e r a d i s t a n c e o f 5 - 8 A at t h e i o n i c strength used. P r o v i d e d that the p r o t e i n adsorption does not d r a s t i c a l l y c h a n g e the p o t e n t i a l d i s t r i b u t i o n , the e l e c t r o d e reaction c a n b e a c c e l e r a t e d b y t h e a p p l i e d e l e c t r o d e p o t e n t i a l o n l y u p to t h e first l a y e r . Therefore a polylayer electrode t h r o u g h the adsorption layer.

process

requires

electron

transfer

T h e i r r e v e r s i b l e p r o t e i n a d s o r p t i o n at e l e c t r o d e s is e q u i v a l e n t t o a c h e m i c a l modification o f the electrode surface. Therefore, the elec t r o d e processes m a y e x h i b i t a character a n a l o g o u s to c h e m i c a l l y m o d ified electrodes possessing a p o l y m e r layer that contains r e v e r s i b l e r e d o x c e n t e r s ( 1 3 , 14). T h e c o n d u c t i v i t y o f t h i s l a y e r is b a s e d o n t h e e l e c t r o n transfer b e t w e e n r e d o x centers, w h i c h w o u l d i n v o l v e the j u m p i n g o f e l e c t r o n s f r o m o n e g r o u p to a n o t h e r ( " h o p m e c h a n i s m " ) . W i t h proteins, m e t a l c o m p l e x e s are the o v e r w h e l m i n g majority o f s u c h e l e c t r o n transfer c e n t e r s ; flavins, q u i n o n e , a n d t h i o l g r o u p s are a l s o s u i t e d for e l e c t r o n transfer. T h e s e c e n t e r s p r o v i d e a p a t h o f 1 0 - 1 5 A t h r o u g h the p r o t e i n fabric. F i g u r e 2 demonstrates that a p a r a l l e l i s m e x i s t s b e t w e e n t h e r e s i s t i v i t y o f a n h y d r o u s p r o t e i n films ( 1 5 ) a n d t h e deviations from the r e v e r s i b l e p o l y l a y e r e l e c t r o d e process i n d i c a t e d b y the m o n o l a y e r r e d u c t i o n , d e c r e a s e o f l i m i t i n g current, i i , shift o f the half-wave potential E , or i n h i b i t i o n o f the r e o x i d a t i o n . i m

m


222



reversible polylayer Process

•

* 5

irreversible polylayer process

•

*

*

1

• 10

1

1

monolayer process

1

' 15

1

' lg resistivity 1

1

Figure 2. Schematic representation of the relation between the resis tivity of anhydrous protein films ana the character of the electrode reaction.

Reduction of the First Adsorption Layer T h e resistivity, p , o f films f o r m e d b y the s i m p l e proteins l y s o z y m e or t r y p s i n is s o h i g h t h a t n o e l e c t r i c c u r r e n t is o b s e r v e d ( ρ > 1 0 Ω · c m ) (15). F o r l y s o z y m e a n d a n u m b e r o f proteins c o n t a i n i n g d i s u l f i d e b r i d g e s (16-20) o r F e S c e n t e r s (21 - 2 5 ) , t h e e l e c t r o d e p r o c e s s is r e s t r i c t e d t o t h e first a d s o r p t i o n l a y e r . 1 4

T h e l i m i t i n g c u r r e n t l e v e l s o f f at t h e c o m p l e t e s a t u r a t i o n o f t h e e l e c t r o d e s u r f a c e . T h i s p h e n o m e n o n h o l d s for t h e p o l a r o g r a p h i c a l l y r e v e r s i b l e r e d u c t i o n o f F e S centers i n a d r e n o d o x i n ( F i g u r e 3). T h i s re sult demonstrates, i n agreement w i t h the h i g h resistivity o f the protein film, t h e i n h i b i t i o n o f t h e e l e c t r o n t r a n s f e r i n t o t h e p o l y l a y e r . T h e flavoproteins c h o l e s t e r o l o x i d a s e (Schizophyllum commune) a n d c h o l i n e o x i d a s e (Cylindrocarpon didymum) e x h i b i t s i m i l a r e l e c t r o c h e m i c a l c h a r a c t e r i s t i c s . A n d o e t a l . (26) f o u n d a n o x i d a t i o n a n d a r e d u c t i o n d c w a v e at t h e h a n g i n g d r o p m e r c u r y e l e c t r o d e ( H D M E ) , w h i c h w a s attributed to the c o v a l e n t l y b o u n d F A D o f the e n z y m e adsorbed o n the e l e c t r o d e surface.


10.

S C H E L L E R AND STRNAD

Protein

Prosthetic

223

Groups

T h e macroscale electrolysis [in a potential region where o n l y one m o n o l a y e r is r e d u c e d 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 ( D M E ) or H D M E ] l e a d s to t h e r e d u c t i o n o f t h e p r o t e i n s w i t h F e S c e n t e r s or d i s u l f i d e b r i d g e s , r e s p e c t i v e l y (16,22) i n t h e b u l k s o l u t i o n . T h i s r e s u l t shows that d u r i n g the electrolysis the p r o t e i n m o l e c u l e s i n the

first

a d s o r p t i o n l a y e r are e x c h a n g e d w i t h those i n the b u l k o f the s o l u t i o n . T h i s p r o c e s s is o b v i o u s l y t o o s l o w to b e d e t e c t e d d u r i n g t h e l i f e t i m e o f the reaction b y the D M E . F o l l o w i n g this m e c h a n i s m , the e n z y m a t i c i n a c t i v i t y o f t h e r e d u c t i o n p r o d u c t s o f f e r r e d o x i n (11) a n d a d r e n o d o x i n Downloaded by UNIV OF ARIZONA on November 11, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch010

(27) m a y b e e x p l a i n e d b y the d i r e c t i n t e r a c t i o n o f the r e a c t i n g proteins w i t h the

electrode

surface, thus l e a d i n g to i r r e v e r s i b l e

structural

changes. A l s o , proteins w i t h o u t a n y p r o n o u n c e d w a v e i n the

polarograms

m a y b e i r r e v e r s i b l y r e d u c e d i n t h e first a d s o r p t i o n l a y e r . C o n t r a r y t o our earlier interpretations

(28)

o f the d c p o l a r o g r a m s o f s o l u b i l i z e d

fractions o f l i v e r c y t o c h r o m e P - 4 5 0 , the c h r o m a t o g r a p h i c a l l y p u r i f i e d P-450 L M

2

does not e x h i b i t any polarographic w a v e . T h e

polaro

g r a p h i c w a v e at t h e h a l f - w a v e p o t e n t i a l o f - 5 8 0 m V i n d i c a t e s t h e r e d u c t i o n o f c y t o c h r o m e b , a constituent o f the s o l u b i l i z e d fractions. 5

0.04

002

5

1 0

1 5

c

[fM(]

Figure 3. Concentration dependence of the peak current in the differ ential pulse polarograms with adrenodoxin (O) and trypsin (Φ). Solu tions contain 0.1 M phosphate buffer, pH 7 (background with adreno doxin), 0.1 Μ Na B 0 , and 0.1 M KCl, pH 9.2 (background with trypsin). At 1 mV/s where t = 2 s, and d = 25 mV. 4

4

7


224



N e v e r t h e l e s s , i n t h e m a c r o s c a l e e l e c t r o l y s i s at t h e p o t e n t i a l o f - 8 0 0 m V at t h e m e r c u r y p o o l e l e c t r o d e , w e o b t a i n e d a p r o d u c t c o n t a i n i n g native ferrous c y t o c h r o m e P-450. W i t h a c a r b o n cathode, t h e i n a c t i v e but not completely destroyed reduction product, cytochrome P-420, w a s o b t a i n e d ( 2 9 a , 29b). F o r c y t o c h r o m e P - 4 5 0 , w a s c a l c u l a t e d a n e x c h a n g e r a t e p e r m o n o l a y e r o f —100 s ( 3 0 ) , b o t h f r o m t h e p r e p a r a t i v e electrolysis a n d the substrate conversion b y the adsorbed protein. T h e s e c o n d a d s o r p t i o n l a y e r is i n v o l v e d i n t h e c a t h o d i c r e d u c t i o n o f i n s u l i n (16,31 ). T h i s e l e c t r o n t r a n s f e r t o t h e s e c o n d l a y e r r e q u i r e s a h i g h e r e l e c t r i c e n e r g y t h a n t h e r e d u c t i o n o f t h e first l a y e r . W i t h t r y p sin, i n analogy w i t h the d c polarographic l i m i t i n g current (16), the p e a k h e i g h t i n t h e 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 s ( F i g u r e 3) s h o w s n o s a t u r a t i o n at h i g h b u l k c o n c e n t r a t i o n s . A n e x p l a n a t i o n o f t h i s r e s u l t m a y b e a fast e x c h a n g e o f t h e s e c o n d a d s o r p t i o n l a y e r . B i a n c o a n d H a l a d j i a n ( 2 1 ) f o u n d w i t h f e r r e d o x i n (Desulfovibrio gigas), a b o v e t h e s a t u r a t i o n o f t h e e l e c t r o d e , a f u r t h e r i n c r e a s e o f t h e current, whereas the peak potential varies o n l y slightly. P r o b a b l y , the reduction process extends to several layers.

Reduction of Polylayers W i t h t h e h e m e p r o t e i n s c y t o c h r o m e c (32, 33), c y t o c h r o m e c (34, 35), c y t o c h r o m e c ( 3 6 ) , c y t o c h r o m e b (29a), m e t h e m o g l o b i n ( 3 7 ) , a n d m e t m y o g l o b i n ( 3 8 ) , a n d t h e flavoprotein g l u c o s e o x i d a s e ( 3 9 ) , t h e c u r rent increases l i n e a r l y w i t h concentration above the c o m p l e t i o n o f the first a d s o r p t i o n l a y e r . T h e e l e c t r o n t r a n s f e r is o b v i o u s l y n o t r e s t r i c t e d to t h e first l a y e r . 3

7

5

Irreversible Polylayer Reduction T h e r o l e o f t h e p r o s t h e t i c g r o u p for t h e e l e c t r i c r e s i s t i v i t y is d e m o n s t r a t e d b y t h e fact t h a t t h e a p o p r o t e i n i t s e l f is a n i n s u l a t o r ( ρ > 1 0 Ω · c m ) a n d t h e r e s i s t i v i t y o f m o n o h e m o p r o t e i n s is a b o u t 1 0 - 1 0 Ω · c m (15). A s mentioned, the structure o f the proteins drastically influences the electrode process. 1 4

9

n

A t t h e D M E , i n t a c t g l u c o s e o x i d a s e f r o m Aspergillus niger a n d f r o m Pénicillium notatum e x h i b i t s o n l y a v e r y s m a l l p e a k i n t h e 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 s . O n t h e o t h e r h a n d , w e e s t a b l i s h e d a fast electrode reaction w i t h glucose oxidase samples submitted to a l i m i t e d proteolysis d u r i n g purification procedures. I n this process a partial d e g r a d a t i o n o f the g l u c o s e oxidase takes p l a c e a n d leads to a decrease o f t h e m o l e c u l a r w e i g h t . T h e s e f r a g m e n t s possess a h i g h s p e c i f i c a c t i v i t y , as w a s p r o v e d b y t h e o x y g e n c o n s u m p t i o n m e t h o d . I n t h e d i f f e r e n -


10.

S C H E L L E R AND STRNAD

Protein

Prosthetic

225

Groups

t i a l p u l s e p o l a r o g r a m s this g l u c o s e oxidase s h o w e d a w e l l - s h a p e d p e a k ( 3 9 ) , t h e s u m m i t p o t e n t i a l o f w h i c h c o r r e s p o n d e d v e r y w e l l to t h e r e d o x p o t e n t i a l E°' o f - 3 0 5 m V v s . S C E (40).

Free

flavin

adenine

dinucleotide ( F A D ) showed an ~ 1 0 0 - m V more cathodic peak.

The

p e a k (39), the s u m m i t p o t e n t i a l o f w h i c h c o r r e s p o n d e d v e r y w e l l to the t h e p e a k p o t e n t i a l is i n d e p e n d e n t o f t h e p r o t e i n c o n c e n t r a t i o n . T h e e n z y m a t i c activity o f the a d s o r b e d g l u c o s e oxidase fragments was established b y the

finding

that the r e d u c t i o n o f the F A D groups b y

g l u c o s e b r i n g s a b o u t a d e c r e a s e o f t h e c a t h o d i c c u r r e n t (the s u m o f t h e Downloaded by UNIV OF ARIZONA on November 11, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch010

b a c k g r o u n d a n d the glucose oxidase r e d u c t i o n current) b y about t w i c e the v a l u e o f the step h e i g h t o f the o x i d i z e d g l u c o s e o x i d a s e . T h i s result demonstrates

that the d e e p e r c u r v e o f F i g u r e 4 reflects the

oxidation o f the p r o t e i n F A D H

2

anodic

g r o u p s . T h i s o x i d a t i o n c u r r e n t is i n

c r e a s e d u p t o a factor o f t w o w i t h r i s i n g g l u c o s e c o n c e n t r a t i o n . T h e e n z y m a t i c b a s i s o f t h i s effect w a s d e m o n s t r a t e d b y t h e i n e f f e c t i v i t y o f d e n a t u r e d e n z y m e . T h e i n c r e a s e i n c u r r e n t is b a s e d o n t h e r é g é n é r a -

i(M)

-300

-400

-500

E(mV) Figure 4. Normal pulse polarograms of glucose oxidase. Solution contains 0.1 M phosphate buffer, ρ H 7. At 2 mV/s where t = 2 s. Curve 1 = 7 μ Μ glucose oxidase and Curve 2=7 μΜ glucose oxidase + 10 μΜ glucose.


226

BIOLOGICAL R E D O X COMPONENTS


tion o f the r e d u c e d glucose oxidase b y the substrate glucose. T h e Brutto reaction represents the a n o d i c oxidation o f glucose. I n the reac t i o n c y c l e g l u c o s e o x i d a s e acts as a r e a l b i o c a t a l y s t a c c e l e r a t i n g t h e o x i d a t i o n o f its s u b s t r a t e . T h e p r o t e i n structure also d e p e n d s o n the conditions o f adsorption (41 ). T h e e l e c t r o d e r e a c t i o n o f a d e n a t u r e d c y t o c h r o m e c l a y e r f o r m e d b y a d s o r p t i o n at l o w b u l k c o n c e n t r a t i o n is i r r e v e r s i b l e . O n l y i n t h e first s c a n is t h e r e d u c t i o n o f t h e f e r r i - f o r m i n d i c a t e d b y a p e a k i n t h e c y c l i c v o l t a m m o g r a m s , a l t h o u g h t h e r e o x i d a t i o n w a s n o t o b s e r v e d (33). I n accordance w i t h this result, the absence o f a faradaic p e a k i n the ad s o r p t i o n r e g i o n o f t h e a c p o l a r o g r a m s w a s e s t a b l i s h e d (42). T h e r e d u c t i o n o f t h e p o l y l a y e r f o r m e d at h i g h e r c o n c e n t r a t i o n s (33) r e q u i r e d a l o w e r o v e r v o l t a g e t h a n t h a t o f t h e m o n o l a y e r o f d e n a t u r e d c y t o c h r o m e c m o l e c u l e s . B e c a u s e o f t h e s m a l l c o n t a c t a r e a at h i g h s u r f a c e c o n c e n t r a t i o n , n a t i v e m o l e c u l e s c o e x i s t i n t h e first a d s o r p t i o n layer. M o s t l i k e l y , these m o l e c u l e s are r e d u c e d m o r e e a s i l y a n d m a y transfer electrons i n t o the next layers. T h e p o l y l a y e r r e d u c t i o n p r o c e s s w i t h c y t o c h r o m e c, c y t o c h r o m e b j , m e t m y o g l o b i n , a n d m e t h e m o g l o b i n has the f o l l o w i n g characteris t i c f e a t u r e s at m e r c u r y e l e c t r o d e s : 1. T h e c a t h o d i c r e d u c t i o n g i v e s a c u r r e n t s i g n a l i n p o l a r o g r a m s or c y c l i c v o l t a m m o g r a m s , b u t t h e a n o d i c o x i d a t i o n cannot be d e m o n s t r a t e d . 2 . W i t h m o n o h e m o p r o t e i n s , t h e l i m i t i n g c u r r e n t is s m a l l e r t h a n t h a t c a l c u l a t e d b y t h e I l k o v i c h e q u a t i o n for t h e diffusion-controlled electrode process. 3. B o t h p H a n d i o n i c s t r e n g t h i n f l u e n c e t h e c u r r e n t . 4. T h e a d d i t i o n o f the r e s p e c t i v e r e d u c e d p r o t e i n does not influence the current. A n excess o f s e v e r a l i n e r t g l o b u l a r p r o t e i n s , for e x a m p l e , b o v i n e s e r u m a l b u m i n w i t h c y t o c h r o m e c, d o e s n o t d e c r e a s e t h e l i m i t i n g c u r r e n t (12). I n contrast, the a d d i t i o n o f p o l y - L - l y s i n e i n h i b i t s the e l e c t r o d e p r o c e s s . T h e r e f o r e , t h e s t r u c t u r e o f t h e first a d s o r p t i o n l a y e r s e e m s to d e t e r m i n e t h e r a t e o f t h e e l e c t r o d e process. 5. T h e v a l u e o f i i d e p e n d s l i n e a r l y o n t h e s q u a r e r o o t o f t h e m e r c u r y c o l u m n h e i g h t , as is p r e d i c t e d for a d i f f u s i o n - c o n t r o l l e d e l e c t r o d e p r o c e s s ( 3 2 ) . T h i s state m e n t is u n d e r l i n e d b y t h e finding t h a t t h e p e a k c u r r e n t o f the cathodic r e d u c t i o n o f c y t o c h r o m e c d e p e n d s l i n e a r l y o n t h e s q u a r e r o o t o f t h e s c a n rate at t h e H D M E ( 3 3 ) . I n c o n t r a s t , at t h e a m a l g a m a t e d g o l d e l e c t r o d e t h e p e a k c u r rent g r o w s l i n e a r l y w i t h i n c r e a s i n g scan rate, i n d i c a t i n g t h a t t h e f e r r i c y t o c h r o m e c is r e d u c e d i n t h e a d s o r b e d state (43). i m


10.



227

6. T h e h a l f - w a v e p o t e n t i a l is m o r e n e g a t i v e t h a n t h e f o r m a l redox potential. T h i s overvoltage cannot be e x p l a i n e d on the basis o f the h e t e r o g e n e o u s rate constant k c a l c u l a t e d b y the M a r c u s theory u s i n g the rate constant k o f the e l e c t r o n s e l f - e x c h a n g e r e a c t i o n (8, 44, 45). el

n

7. T h e v a l u e o f E becomes more negative w i t h increasing p r o t e i n concentration a n d it d e p e n d s on p H a n d i o n i c strength. m

A t e l e c t r o d e s these proteins b e h a v e s i m i l a r l y to l o w m o l e c u l a r Downloaded by UNIV OF ARIZONA on November 11, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch010

w e i g h t r e d o x s y s t e m s (e.g., h e a v y m e t a l i o n s ) o n a d d i t i o n o f s u r f a c e active substances, i n that they s h o w a decrease o f l i m i t i n g current a n d shift o f the w a v e p o s i t i o n . In

accordance

with

the

inhibition

effects

found

with

a low-

m o l e c u l a r w e i g h t r e d o x s y s t e m (46), t h e f o l l o w i n g a p p r o a c h e s m a y b e p r o p o s e d to e x p l a i n t h e r e s u l t s ( F i g u r e 5): T h e decrease o f the active e l e c t r o d e surface b y the i r r e v e r s i b l y a d s o r b e d m o l e c u l e s o f t h e first a d s o r p t i o n l a y e r l e a d s to a p a r t i a l l y b l o c k e d surface. T h e l i m i t i n g current o f an e l e c t r o d e reaction o c c u r r i n g at " c o n d u c t i n g i s l a n d s " s e p a r a t e d b y a n a v e r a g e d i s t a n c e r , w h i c h c o r r e s p o n d s a p p r o x i m a t e l y to t h e t h i c k n e s s o f t h e d i f f u s i o n l a y e r δ is g i v e n b y the f o l l o w i n g relationship

. t u m

(47):

nFDc δ+/(δ/Γ)

m ( A ;

Figure 5. Model of partially blocked electrode surface.


228

BIOLOGICAL R E D O X COMPONENTS

T h i s equation requires a linear concentration dependence o f the l i m i t i n g c u r r e n t w i t h a s m a l l e r s l o p e as c o m p a r e d w i t h a n u n c o v e r e d e l e c t r o d e . I n contrast to t h e s m a l l e r m o l e c u l e s , t h e r e d u c t i o n o f pro t e i n s m a y p r o c e e d o n l y i n r e l a t i v e l y l a r g e sites w h e r e t h e d i s t a n c e s b e t w e e n t h e m are greater than the thickness o f the diffusion layer. T h e l i n e a r d e p e n d e n c e o f the l i m i t i n g current o n the root o f the m e r c u r y c o l u m n h e i g h t also c o r r e s p o n d s to this m o d e l . T h e average distance d o f d i s c h a r g i n g m o l e c u l e s to the electrode w i l l g r o w w i t h i n c r e a s i n g surface pressure, P , a n d c o n s e q u e n t l y the Downloaded by UNIV OF ARIZONA on November 11, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch010

E1/2 i s s h i f t e d . T h e w o r k t o b e d o n e a g a i n s t t h e p r e s s u r e i s c o m p e n - , s a t e d b y t h e i n c r e a s e o f t h e e l e c t r i c a l e n e r g y (48a):

Ê1/2 = -P(S - S )/anF

(2)

0

w h e r e S = the surface area o f a m o l e c u l e a n d S = the area o f a pore i n the adsorption layer. 0

B e c a u s e t h e d e s o r p t i o n rate o f the s e c o n d l a y e r is rather l o w a n d c a n n o t a c c o u n t for t h e r a t e o f t h e e l e c t r o d e p r o c e s s o b s e r v e d , t h e electrons must travel through the adsorption layer. T h e control o f the e l e c t r o d e process b y a s l o w i n t e r m o l e c u l a r e l e c t r o n transfer w o u l d explain the decrease o f the l i m i t i n g current, b u t it contradicts the diffusion limitation o f the p r o t e i n - e l e c t r o d e reactions. A l s o , the i n e f f e c t i v e n e s s o f i n e r t p r o t e i n s (12) a r g u e s a g a i n s t a c o n t r o l b y s p e c i f i c i n t e r m o l e c u l a r e l e c t r o n transfer i n t h e c y t o c h r o m e c p o l y l a y e r .

Reversible Multilayer Electrode Process T h e m u l t i - h e m e p r o t e i n f e r r o c y t o c h r o m e c h a s at 2 6 8 Κ a r e s i s t i v i t y o f o n l y 5 7 Ω · c m ( 1 5 ) . I t w a s t h e first h e m e p r o t e i n s h o w i n g a p o l a r o g r a p h i c a l l y reversible p o l y l a y e r o x i d a t i o n - r e d u c t i o n process (34). R e c e n t l y , B i a n c o a n d H a l a d j i a n (36) e s t a b l i s h e d , w i t h t h e t h r e e heme-containing protein cytochrome c , the same type of electrochem i c a l b e h a v i o r . F o r t h e f e r r e d o x i n o f Clostridium pasteurianum (48b) ( f o u r F e - S c e n t e r s ) , a d i f f u s i o n - c o n t r o l l e d r e d u c t i o n at t h e D M E a l s o was obtained. 3

7

I n t h e first c y c l 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 is a v e r y fast e l e c t r o d e p r o c e s s . I n c o n t r a s t , n o o x i d a t i o n p e a k i s o b s e r v e d w i t h adsorbed cytochrome c on the D M E . Therefore the adsorbed layer, consisting o f ferrous c y t o c h r o m e c , b e c o m e s 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 i n r e p e t i t i v e scans. H o w e v e r , t h e e l e c t r o n transfer t h r o u g h this i n a c t i v e e l e c t r o d e c o a t i n g to m o l e c u l e s o f t h e s e c o n d l a y e r is a p o l a r o g r a p h i c a l l y reversible process. I n d i l u t e c y t o c h r o m e c solution, t h e r e a c t i n g m o l e c u l e s o f t h e s e c o n d l a y e r m a y b e i n a fast a d s o r p t i o n e x c h a n g e . T h e e l e c t r o d e r e a c t i o n t h r o u g h a p o l y l a y e r f o r m e d at h i g h 3

3

3

3


10.



229

p r o t e i n c o n c e n t r a t i o n s is a r a t h e r s l o w p r o c e s s . B o t h t h e a n o d i c a n d t h e cathodic peaks i n the cyclic voltammograms decrease i n repetitive scans (49).


W i t h cytochrome C3, the addition o f cytochrome c suppresses the l i m i t i n g c u r r e n t , b u t t h e h a l f - w a v e p o t e n t i a l is n o t a l t e r e d ( 5 0 ) . T h i s result supports t h e theory that t h e s u b s t i t u t i o n o f c y t o c h r o m e C 3 m o l e cules b y c y t o c h r o m e c gives rise to t h e formation o f a p a r t i a l l y b l o c k e d e l e c t r o d e (see E q u a t i o n 1). T h u s , f r o m t h e first l a y e r o f c y t o c h r o m e C 3 a r a p i d i n t e r m o l e c u l a r e l e c t r o n t r a n s f e r h a s t o o c c u r so t h a t t h e m a s s t r a n s p o r t is t h e s l o w e s t s t e p . B e s i d e s t h e i n t e r m o l e c u l a r e l e c t r o n transfer, t h e c h a r g e i n j e c t i o n f r o m t h e e l e c t r o d e i n t o t h e first a d s o r p t i o n l a y e r m a y c o n t r o l t h e e l e c trode process. T h e d e c i s i v e r o l e o f this process is u n d e r l i n e d b y t h e finding that m o d i f i c a t i o n o f t h e e l e c t r o d e surface b y means o f p o l y m e r i c m e t h y l v i o l o g e n (51), or covalent grafting w i t h m e t h y l vio l o g e n d e r i v a t i v e s ( 5 2 ) , o r t h e a d s o r p t i o n o f 4 , 4 ' - b i p y r i d y l ( 5 3 , 54a, 54b), l e a d s t o a q u a s i - r e v e r s i b l e e l e c t r o d e r e a c t i o n for t h e m u l t i l a y e r s o f f e r r e d o x i n o r c y t o c h r o m e c. T h e c o n d u c t i n g l a y e r s o f t h e s e c o m p o u n d s p o s s e s s i n g a c o n j u g a t e d π - e l e c t r o n s y s t e m a r e as l i k e l y t o p r e v e n t t h e i r r e v e r s i b l e p r o t e i n a d s o r p t i o n as t o f a c i l i t a t e t h e e l e c t r o n t r a n s f e r t o t h e first p r o t e i n l a y e r . A s i g n i f i c a n t rate o f t h e e l e c t r o d e r e a c t i o n o f i n t a c t g l u c o s e o x i d a s e , b o t h f r o m Pénicillium notatum a n d f r o m Aspergillus niger, w a s f o u n d at t h e g o l d m i n i g r i d e l e c t r o d e m o d i f i e d b y p o l y m e r i c m e t h y l v i o l o g e n (55). T h e potential step s p e c t r o e l e c t r o c h e m i c a l ex periments were performed i n a thin-layer cell monitoring the change i n a b s o r b a n c e at 4 6 0 n m v s . t i m e ( F i g u r e 6). O n a p p l i c a t i o n o f a p o t e n tial o f - 4 0 0 m V v s . S C E , the absorbance decreases c o n t i n u o u s l y r e a c h i n g , after 1 0 m i n , t h e v a l u e o f g l u c o s e o x i d a s e r e d u c e d b y g l u c o s e . T h e o r i g i n a l a b s o r b a n c e is a c h i e v e d b y a n o d i c o x i d a t i o n at 2 0 0 m V vs. S C E . N o change o f the total spectrum b e t w e e n 600 a n d 300 n m w a s f o u n d after t h r e e c y c l e s o f c o m p l e t e r e d u c t i o n a n d o x i d a t i o n o f t h e same glucose oxidase s a m p l e . T h i s result shows that the native struc ture o f the e n z y m e m a y b e retained. Y e h a n d K u w a n a (56) d e m o n s t r a t e d a d i f f u s i o n - c o n t r o l l e d , r e v e r s i b l e e l e c t r o n transfer from t h e t i n - s u p p l e m e n t e d i n d i u m o x i d e e l e c trode to c y t o c h r o m e c. Whereas t h e p o s i t i o n o f the a n o d i c a n d c a t h o d i c peak potential c o r r e s p o n d e d to the redox potential, t h e diffusion c o e f f i c i e n t d e t e r m i n e d f r o m t h e p e a k c u r r e n t s w a s t o o s m a l l b y a factor of about two. I n d i r e c t e v i d e n c e for a fast e l e c t r o n - e x c h a n g e b e t w e e n t h e p r o t e i n prosthetic group a n d t h e electrode w a s o b t a i n e d b y catalysis o f the e l e c t r o c h e m i c a l substrate conversion b y several e n z y m e s (bioelectro catalysis). T h e e l e c t r o n - e x c h a n g e w a s a c c e l e r a t e d b y t h e interac-



230


Figure 6. Optically transparent thin-layer electrochemical cell results of glucose oxidase at the methyl viologen-modified gold minigrid electrode. Solutions contain 0.1 M phosphate buffer, pH 5.8 ana 0.1 m M glucose oxidase from P é n i c i l l i u m notatum with a 0.18-mm optical pathLength. The gold minigrid electrode has a 60% optical transparency with 120 wireslin.

t i o n o f t h e e n z y m e w i t h a s e m i c o n d u c t i n g g e l (57-60) or w i t h o r g a n i c m e t a l s (61-63). H o w e v e r , t h e f o r m a t i o n o f t h e r e d u c e d p r o t e i n w a s not demonstrated d i r e c t l y . D e v i a t i o n s f r o m t h e r e v e r s i b l e b e h a v i o r a l s o o c c u r at m o d i f i e d electrodes w i t h proteins that have a s l o w i n t e r m o l e c u l a r electronexchange (low k value). n

W e s t u d i e d t h e effect o f m o d i f y i n g a g o l d e l e c t r o d e b y a d s o r p t i o n o f 4 , 4 ' - b i p y r i d y l o n the b e h a v i o r o f intact g l u c o s e oxidase. P r e l i m i n a r y r e s u l t s i n d i c a t e d t h a t g l u c o s e o x i d a s e i s r e d u c i b l e o n l y i n t h e first c y c l e b u t n o t a n o d i c a l l y r e o x i d i z e d at t h e m o d i f i e d p l a n e g o l d e l e c trode. T h u s , addition o f glucose removes the cathodic peak but does not b r i n g about a c o r r e s p o n d i n g o x i d a t i o n s i g n a l . C y t o c h r o m e P - 4 5 0 (29b), m e t h e m o g l o b i n , a n d m e t m y o g l o b i n e x h i b i t at t h e b i p y r i d y l m o d i f i e d g o l d electrode o n l y a reduction peak, b u t no reoxidation sig nal was found. A s w i t h t h e mercury electrode, the peak potentials shifted i n the cathodic d i r e c t i o n . T h e s e results demonstrate that b o t h the m o d i f i c a t i o n o f the e l e c trode surface a n d changes o f the p r o t e i n m o i e t y d r a s t i c a l l y influence the character o f the e l e c t r o d e process.


10.



231

Biological Significance E l e c t r o n transfer proteins p l a y a d o m i n a n t r o l e i n the catalysis o f e l e c t r o n transport a n d the c o u p l e d energy c o n v e r s i o n i n the respiratory c h a i n . I n c o n t r a s t to t h e s m a l l m o l e c u l e e l e c t r o n transfer, t h e m e c h a n i s m o f i n t e r m o l e c u l a r a n d i n t r a m o l e c u l a r e l e c t r o n transfer o f b i o l o g i c a l m a c r o m o l e c u l e s i n d e t a i l (2) is n o t u n d e r s t o o d f u l l y . T h i s fact is p a r t i c u l a r l y p e r p l e x i n g i n l i g h t o f the extensive research i n this area. O n e a p p r o a c h to t h e e l u c i d a t i o n o f t h e s e c o m p l e x s y s t e m s w a s t h e


kinetic study o f isolated oxidoreductases

with small molecule

re

a g e n t s . T h e g o a l o f s u c h r e s e a r c h is t h e e l u c i d a t i o n o f r e a c t i o n p a t h w a y s a v a i l a b l e to t h e p r o t e i n s . T h e s e c o n d p r i n c i p l e w a s t h e c l a s s i c a l s t u d y o f i n t a c t or p a r t i a l l y d i s r u p t e d p r e p a r a t i o n s o f e l e c t r o n t r a n s f e r chains. I n the last f e w years i n c r e a s i n g attention was p a i d to the d i r e c t e l e c t r o n transfer b e t w e e n the e l e c t r o d e a n d proteins. T h e r e v e r s i b l e c a t h o d i c r e d u c t i o n o f several layers o b v i o u s l y r e q u i r e s a r a p i d transfer o f e l e c t r o n s t o t h e first p r o t e i n l a y e r c o u p l e d w i t h a fast i n t e r m o l e c u l a r e l e c t r o n transfer. T h e s e c o n d s t e p o f t h i s p r o c e s s is m o r e s i m i l a r to t h a t o f the e l e c t r o n transfer i n b i o l o g i c a l r e d o x c h a i n s t h a n to the

outer

sphere c o l l i s i o n reactions w i t h l o w m o l e c u l a r w e i g h t partners. T h e r e fore, 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 a p p e a r s to b e t h e m o s t d i r e c t m e t h o d u s e d to i n v e s t i g a t e t h e r e d o x p r o p e r t i e s o f p r o t e i n s . T h i s h y p o t h e s i s is i n a g r e e m e n t w i t h r e s u l t s (64) t h a t e s t a b l i s h e d analogies

between

the

reaction o f cytochrome

c at t h e

bipyridyl-

c o v e r e d g o l d e l e c t r o d e a n d its r e a c t i o n w i t h c y t o c h r o m e o x i d a s e . T h e m o d i f i c a t i o n o f c y t o c h r o m e l y s i n e residues or the a d d i t i o n o f p o l y - L l y s i n e influences b o t h the e l e c t r o d e reaction a n d the o x i d a t i o n b y the oxidase. T h e p r o p o s e d analogy o f the e l e c t r o d e process a n d p r o t e i n reac t i o n s is t h e b a s i s o f t h e a p p l i c a t i o n o f t h e M a r c u s t h e o r y ( 6 5 ) . F o r o u t e r s p h e r e e l e c t r o n transfer, t h e r a t e c o n s t a n t o f t h e s e l f - e x c h a n g e

reac

t i o n , fen, a l l o w s o n e to d r a w c o n c l u s i o n s o n t h e rate c o n s t a n t , k , o f t h e el

electrode process:

(3) 3

U s i n g t h e k v a l u e o f 1 0 · 1 / M · s (66) for c y t o c h r o m e c t h e e s t i m a t e d value o f k predicts a p o l a r o g r a p h i c a l l y reversible electrode process. O n t h e o t h e r h a n d , t h e rate c o n s t a n t s o f s e l f - e x c h a n g e w i t h m e t m y o g l o b i n (4 χ 1 0 · 1 / M · s) (67) a n d m e t h e m o g l o b i n (3 x 1 0 · 1 / M · s) (67) g i v e a Κι for a q u a s i - r e v e r s i b l e a n d o n e i r r e v e r s i b l e e l e c t r o d e r e a c t i o n , r e s p e c t i v e l y . T h e e l e c t r o c h e m i c a l b e h a v i o r o f t h e h e m o p r o t e i n s at n

el

2

3


232


m o d i f i e d e l e c t r o d e s e x h i b i t e d t h e p r o p o s e d p a r a l l e l i s m t o t h e rate o f the homogeneous redox reaction.

Technological Aspects


I n b i o t e c h n o l o g y , the d i r e c t e l e c t r o n transfer b e t w e e n the e n z y m e m o l e c u l e s a n d t h e m e t a l e l e c t r o d e offers n e w p r o s p e c t s b e c a u s e t h e e l e c t r o d e m a y substitute b o t h cofactor a n d electron-transferases. P i o n e e r i n g experiments (68, 69) w e r e c a r r i e d out u s i n g glucose oxidase i n fuel cells operating o n glucose and oxygen. M a n y carbon electrodes were fabricated a n d tested where the e n z y m e was covalently b o u n d to the electrode, e m b e d d e d i n a carbon powder, or en trapped i n a p o l y a c r y l a m i d e g e l - p l a t i n u m gauze matrix. T h e l o w cur rent d e n s i t y w a s a t t r i b u t e d t o difficulty i n e l e c t r o n transfer b e t w e e n the e n z y m e a n d the c o n d u c t i n g e l e c t r o d e surface. A l s o , d u r i n g c o v a l e n t g r a f t i n g o f g l u c o s e o x i d a s e at t h e e l e c t r o d e s u r f a c e n o e l e c t r o n e x c h a n g e w i t h t h e p r o t e i n p r o s t h e t i c g r o u p w a s f o u n d (70-72). T h e s e r e s u l t s are i n a g r e e m e n t w i t h t h e finding ( 7 3 ) t h a t n o e x c h a n g e c u r r e n t between the platinum-indicator electrode and the dissolved protein was m e a s u r a b l e i n the absence o f m e d i a t o r s . N e v e r t h e l e s s , reagentless e n z y m e electrodes, w h i c h reuse t h e cofactor n i c o t i n e a d e n i n e d i n u c l e o t i d e ( N A D ) , w e r e e s t a b l i s h e d b y t h i s p r i n c i p l e (74-76). W i t h g l u cose oxidase or lactic dehydrogenase, the substrate-dependent regen e r a t i o n c u r r e n t w a s u s e d as t h e m e a s u r i n g s i g n a l ( 7 7 , 7 8 ) . O n the other h a n d , t h e c h e m i c a l modification o f the electrode surface r e s u l t e d i n a considerable acceleration o f the electrode reac tion. T h i s a p p r o a c h was u s e d i n bio-fuel cells u s i n g hydrogenase a n d l a c c a s e ( 5 7 , 5 9 ) a n d i n sensors for g l u c o s e (63) a n d l a c t a t e (61) o n t h e basis o f g l u c o s e oxidase or c y t o c h r o m e b . T h e s a m e a p p r o a c h m a y b e a p p l i e d to the i n d i r e c t e l e c t r o c h e m i c a l conversion o f substrate to ob t a i n a d e s i r e d l e v e l o f p r o d u c t , for e x a m p l e , l a c t a t e , i n v i v o . 2

I n g e n e r a l , c o f a c t o r - d e p e n d e n t s y s t e m s s h o u l d b e d e v i s e d for t h e cofactorless e l e c t r o c h e m i c a l substrate or energy c o n v e r s i o n u s i n g the direct protein-electrode reaction.

Conclusions B o t h i n the m e c h a n i s m o f b i o l o g i c a l e l e c t r o n transfer c h a i n s a n d i n 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 b e t w e e n t h e a c t i v e site o f p r o teins a n d electrodes, m a n y basic p r o b l e m s are u n s o l v e d . T h e d e v e l o p m e n t a n d a p p l i c a t i o n o f m o d i f i e d electrodes a n d the d e r i v a t i z a t i o n o f proteins w i l l c o n t r i b u t e c o n s i d e r a b l y to the e l u c i d a t i o n o f the m e c h a n i s m o f b i o l o g i c a l redox reactions. I n a d d i t i o n to the theoretical i m p o r t a n c e o f these e l e c t r o d e reac t i o n s , t h e m a j o r s t i m u l u s for d i r e c t i n g t h e efforts o f s c i e n t i s t s t o t h i s


10. SCHELLER AND STRNAD


233

study is their advantageous applications in biotechnology. The appli cation of enzymes as catalysts of organic electrode reactions attracts increasing attention. Furthermore, the substrate specificity of enzymes offers much promise for designing specific sensors for quantitative analysis.


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RECEIVED for review June 2, 1981.


Electrode Reactions of Protein Prosthetic Groups - American Chemical

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