Bioscience Reports i, 521-532 (1981)
Printed in Great Britain
521
Investigation of electron-transfer reactions of proteins
by electrochemical methods
Review
Mark 3. EDDOWES and H. Allen O. HILL
Inorganic Chemistry Laboratory, South Parks Road,
Oxford OXl 3QR, U.K.
Introduction
T h e r e are f o u r main classes of proteins involved in the essential
role of biological electron transfer: the cytochromes) the ferredoxins,
the copper 'blue' proteins, and flavoproteins. X-ray diffraction studies
have provided us (Adman, 1979) with structures of representatives of
each of these four classes.
Some features are common to all four.
The prosthetic group, be i t haem, i r o n - s u l p h u r c l u s t e r ) d i s t o r t e d
t e t r a h e d r a l copper site) or flavin, appears to be constructed so that
there is a m i n i m u m of l o c a l s t r u c t u r a l change c o n c o m i t a n t w i t h
electron transfer. It is at, but not on, the surface of the protein and
therefore not in direct communication with the solvent. Much interest
has c e n t e r e d (Chance et al., 1979) on the 'pathway' of electron
transfer.
Even after many investigations which have involved studies
of the kinetics of electron transfer) studies of the effects of chemical
modification) and the c o m p a r i s o n of p r o t e i n s having d e l e t i o n s or
replacements in their primary structures, no wholly convincing view of
the elusive pathway has appeared.
In the study of the e l e c t r o n - t r a n s f e r reactions of redox proteins, it
might have been e x p e c t e d t h a t e l e c t r o c h e m i c a l m e t h o d s ( A l b e r y ,
1975), at f i r s t s i ght the most direct method of investigating redox
properties, would have played a significant part. In principle, they are
c a p a b l e of p r o v i d i n g u s e f u l i n f o r m a t i o n a b o u t s t a n d a r d el ect rode
potentials, stoichiometries) or kinetics. However, t h e r e have been few
reports of rapid, direct electron transfer between el ect rodes and redox
proteins in solution.
Consequently, the application of e l e c t r o c h e m i c a l
m e t h o d s t o t h e s t u d y of r e d o x p r o t e i n s has g e n e r a l l y r e q u i r e d
(Szentrimay et al.) 1977; P r i n c e e t al., 1981) t h e use of s m a l l molecule redox mediators to carry charge between the el ect rode and
the protein. Nevertheless) in certain cases - notably with c y t o c h r o m e
c, c y t o c h r o m e c3, and ferredoxins - rapid, direct electron t ransfer has
been reported. In these cases we note that adsorption of the protein
at the elec t r ode surface occurs.
Electrochemical Methods
Of the many electrochemical methods available, the most immediately informative are those that investigate the current passed as a
consequence of an applied potential.
These potentiostatic methods
usually employ (Sawyer & Roberts, 1974) a t h r e e - e l e c t r o d e c e l l so
t h a t the w o r k i n g electrode, at which the reaction of interest takes
place, can be set at a defined potential with respect to the known
9
The Biochemical Society
522
EDDOWES & HILL
0--0--0--0--0--0--
0.0
i/#A
/
-1.0
0
f
o
/
-2.0 -
/
o
..--.-0-- O--O --O --O"O "~O'o"O0
-3.0
l
-0.4
l
-0.2
I
I
I
0.0
0.2
0.4
E/V
Fig. 1.
Typical voltammogram for the reduction of
ferricytochrome o at a rotating-disc, s u r f a c e modified gold electrode.
p o t e n t i a l of a r e f e r e n c e e l e c t r o d e .
P o l a r o g r a p h y , in which the
current-vs.-potential relationship at a dropping m e r c u r y e l e c t r o d e is
determined, is perhaps the most well known of these methods.
Here
th e dropping mercury el ect r ode not only acts as a donor of electrons,
but also serves to replenish the solution at the surface of the drop as
it grows and then falls off. On the other hand, a variety of transient
t e c h n i q u e s ( M a c d o n a l d , 1977), in which the current response to a
change in potential is determined, have been employed in the study of
reactions at stationary electrodes. The most commonly used method is
cyclic v o l t a m m e t r y , in which t h e c u r r e n t r e s u l t i n g f r o m a c y c l i c ,
triangular potential-vs.-time function is recorded. Though the current
voltage curves obtained f r o m t h e s e t r a n s i e n t t e c h n i q u e s a r e m o s t
useful in characterizing el ect r ode processes, details of the kinetics are
probably more readily derived from v o l t a m m e t r y at a r o t a t i n g disc
electr o d e (Levich, 1962; Albery, 1975). The rotation of the e l e c t r o d e
causes the solution to be drawn towards and across the surface of t he
electrod% thereby continuously replenishing the e l e c t r o a c t i v e material.
Consider, for exampl% the voltammogram ( A l b e r y e t al., 1981) of
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 o at a r o t a t i n g surface-modified gold
electr o d e (Fig. l ) .
As the p o t e n t i a l is m a d e m o r e n e g a t i v e , t h e
c u r r e n t , which had been e s s e n t i a l l y z e r o , begins to pass as the
potential approaches t h e f o r m a l p o t e n t i a l of t h e c y t o c h r o m e .
It
i n c r e a s e s s t e e p l y and then levels off as it reaches a limiting value
imposed by the transport of the c y t o c h r o m e to the e l e c t r o d e surface.
E L E C T R O N - T R A N S F E R REACTIONS OF PROTEINS
523
The current, for a purely transport-linked process, is given by (Levich,
1962)
i = 1.55#nFAD2/3~)-I/6c WI/2
( 1 -+exp[nF/RT(E-E0)
]~~_exp[nF/RT(E-E~
] )
where n is the number of electrons, F is the Faraday, A is the area
of the e l e c t r o d e , D is the diffusion coefficient, ~) is the kinematic
viscosity, C is the bulk concentration, W is the rotation speed, and E ~
is the formal potential. Since it is possible to control both the rate
of the electron-transfer step at the electrode surface by selecting the
e l e c t r o d e p o t e n t i a l and the transport rate by selecting the rotation
speed, much information can be gained about the detailed kinetics and
mechanism of electrode reactions. From such e x p e r i m e n t s (Albery et
al., 1981) it is possible to derive the diffusion c o e f f i c i e n t and t h e
f o r m a l p o t e n t i a l and, by a detailed consideration of the relationship
between the limiting c u r r e n t and t h e r o t a t i o n speed, w h e t h e r t h e
reaction at the electrode corresponds to simple diffusion followed by
electron transfer or whether other steps are involved.
Cytochrome
c
The electrochemical reduction of cytochrome c at platinum (Kono
& Nakamura, 1958) and gold (Heineman et al., 1975) electrodes has
been found to be very slow at electrode potentials around the standard
electrode potential of the protein. Electron transfer b e t w e e n t h e s e
e l e c t r o d e s and c y t o c h r o m e c does undoubtedly occur (Tarasevich &
Bogdanovskaya, 1976), but the rates are so slow t h a t t h e e l e c t r o d e
p r o c e s s is not r e a d i l y d e t e c t e d using c o n v e n t i o n a l v o l t a m m e t r i c
techniques in which the current due to the electron-transfer process is
used as a direct measure of the reaction rate. In these cases it has
therefore been necessary to employ spectrophotometric measurements,
t a k e n over a r e l a t i v e l y long period of t i m e , r a t h e r than current
measurements, to follow the progress of the reaction.
R e c e n t l y it has been shown (Eddowes & Hill, 1977, 1979) that
c y t o c h r o m e c u n d e r g o e s a rapid and r e v e r s i b l e e l e c t r o n - t r a n s f e r
reaction at a gold electrode which is surface-modified by an adsorbed
layer of 4,~'-bipyridyl. From the d.c. and a.c. cyclic voltammetry of
cytochrome c at this modified elelctrode surface, a half-wave potential
is derived which coincides with the known standard electrode potential
of c y t o c h r o m e c , i n d i c a t i n g t h a t the rate of the surface-electrontransfer step is sufficiently rapid that Nernstian concentrations of the
two redox states are obtained at the surface and that the overall rate
of electron transfer is essentially diffusion-controlled. The s u r f a c e m o d i f y i n g a d s o r b a t e , 4 , # ' - b i p y r i d y l ( n o t to be c o n f u s e d with the
derived quaternary salts, the viologens, which are frequently used as
mediators), does not behave as a conventional mediator since it is not
electroactive in the potential region of the observed electron-transfer
process. It appears to act by modifying the electrode surface, thereby
producing a suitable i n t e r f a c e at which e l e c t r o n t r a n s f e r b e t w e e n
cytochrome c and the electrode can take place. The electron-transfer
reaction of c y t o c h r o m e c at the modified electrode bears (Eddowes et
524
EDDOWES & HILL
I) Diffusion of reactant
t0 electrode
~
2)Adsorpti0n of>
5
5
/
/
5
/
/
/
/
31 Electrl0n
transfer
(•
5) Diffusion away
of product
Bulk solution
~
5
/
/
/
/
5
/
4) Desorpfion
of product
/
/
/
Electrode
surface
Equoti0ns of flux describing the rates of the individual steps:Rote
k o([Ox]
- [Ox]o) -Transport of reactant(Ox) to electrode surface.
= k~]l-e)[Ox]o
-Adsorption of reactant.
= k, teox
- Electron transfer to give adsorbed product (Red).
= kdes0Red
- Desorption of product
= kD([Red]o-[Red].) -Transport of product inf0 bulk solution
=
Fig. 2.
R e a c t i o n scheme for the reduction of
ferricytochrome c at the s u r f a c e - m o d i f i e d gold
electrode.
al., 1979a) some striking similarities to that between cytochrome c
and its physiological redox partners (Takemori et al., 1962~ Davies et
a l . , 1964; Mochan, 1970; Staudenmayer et al., 1977; Ahmed et al.,
1978; Kang et al., 1978). That is, both are inhibited by modification
of the cytochrome-c lysine residues and by the competitive inhibitor
poly-L-lyslne. This suggests that there may be an interaction between
c y t o c h r o m e c and the modified electrode surface analagous to that
between cytochrome c and its physiological redox partners, and hence
that cytochrome c binds to the electrodes prior to electron transfer.
The cyclic voltammetry current-voltage curves described above,
though demonstrating that the electrode reaction is fast, do not give
information about its mechanism.
However, detailed k i n e t i c studies
have been carried out (Albery et al., 1981) with the rotating disc
technique which confirm that cytochrome c binds to the electrode
rapidly and reversibly and that this binding interaction is an essential
feature of the overall electrode reaction. They show that the reaction
follows the scheme in Fig. 29 with a binding constant, K = 5 x I03
mol-tdm 3, and with kon = 2.5 x 105 mol-ldm3s - I and kof f = 50 s- I .
E L E C T R O N - T R A N S F E R REACTIONS OF PROTEINS
525
etecfron
transfer
f ......... -&-..........
towering in overo[[
/
AG*due to adsorpti0n
I
~,
I
/
e[ecfron
r~n~re,
III
._.!:.x,,o0
/
I
1
I
1
0Xads
Redods
With Adsorption,
Rote= Z exp( -AGet
-AGods+ AGdes')[Ox]
RT
Without Adsorption
/-AGet\
R= e- Z exp k- ?)Eoxl
Fig. 3.
Free-energy profiles for electron-transfer
reactions with and without adsorption p r i o r to
electron transfer~ illustrating the rate enhancement
due to adsorption.
The rate given for the process
involving adsorption is the maximum rate in which the
reactant concentration is sufficiently low for there
to be negligible self-inhibition and where the rates
of adsorption and desorption are sufficiently rapid
compared with electron transfer for pre-equilibrium
of the adsorbed reactant to be achieved.
The f i r s t - o r d e r electron-transfer rate constant between the electrode
and cytochrome c bound at the surface is 50 s- I at E = E 0.
The
results illustrate the importance of favourable binding interactions in
the enhancement of reaction rates. Analysis of the rate data in terms
of transition-state theory indicates that the binding of the protein to
the surface is responsible for an increase of the rate by a factor of
roughly 105 above t h a t which would be e x p e c t e d in its absence.
Therefore without a favourable binding interaction at the electrode the
o v e r a l l rate of the e l e c t r o d e r e a c t i o n w i l l be slow and this may
account for the slow rates of electron transfer encountered at other
electrodes.
This feature of the mechanism, which is analagous to the
involvement of binding in general enzyme catalysis (Page & 3encks,
1971; 3encks, 1980), is illustrated in Fig. 3.
526
EDDOWES & HILL
K i n e t i c studies of the redox reactions of cytochrome c with its
physiological redox partners~ cytochrome c oxidase (Takemori et al.,
1962; Staudenmayer et al., 1977), cytochrome c reductase (Ahmed et
al., I978), and cytochrome c peroxidase (Kang et at., t978) have
shown the importance of the s-amino groups of the cytochrome-c
lysine residues in determining its strength of binding to, and rate of
reaction withy its physiological redox partners. This binding is thought
to be due to hydrogen bonding between cytochrome-c amino groups and
carboxylate groups of the partner proteins. These cytochrome-c lysine
residues also appear to be important in the reaction of cytochrome c
at the modified gold electrode surface, and we have suggested
(Eddowes et a l , 1979a~ Albery et a l , 1981) that the hydrogen bonding
between the exposed nitrogen lone pairs of the adsorbed %#'-bipyridyl
and the c-amino groups of the ]ysine residues may be responsible for
the observed binding between cytochrome c and the electrode~
At the mercury electrode, well-defined current-voltage curves are
also obtained (Betso et al., 1972) for cytochrome c, indicating that
rapid reduction of the haem-iron prosthetic group occurs.
Combined
electrochemical and spectrophotometric experiments confirm that the
reduction process observed polarographicatty is associated w i t h the
haem iron. A t low protein concentrations the polarographic behaviour
of cytochrome c appears to be essentially reversible in the e l e c t r o chemical sense, that is, the rate of the electron-transfer process at
the electrode surface is fast compared with the rate of mass transport
of c y t o c h r o m e c to the electrode.
However, at higher p r o t e i n
concentrations, there is an increasing deviation from reversibility with
increasing concentration, as demonstrated by the negative shift in the
observed half-wave potential.
This shows that the overall electrode
process is no longer simply diffusion-controlled but is limited to a
significant extent by the kinetics of the electron-transfer process at
the electrode surface.
That is, the apparent electrochemical rate
constant decreases with increasing c y t o c h r o m e - c concentration~ and
t h e r e f o r e cytochrome c self-inhibits its electron transfer reaction at
the mercury electrode.
It has long been suggested (Betso et al.,
1972) t h a t the s e l f - i n h i b i t i o n and lowering of the apparent mass
transport rate may result from adsorption of the protein at the
electrode, and i t has been shown (Scheller et al., 1976a~b) by a
variety of experiments that the protein does adsorb at the electrode
surface.
We emphasize that the polarographic current-voltage curves
reported in the literature do not give s u f f i c i e n t l y d e t a i l e d k i n e t i c
i n f o r m a t i o n for any firm conclusions to be drawn about the role of
adsorption in the overall electron-transfer reaction. HoweveG they do
c l e a r l y i n d i c a t e t h a t a d s o r p t i o n does o c c u r and leads to apparent
self-inhibition of the r e a c t i o n .
This is m o s t r e a d i l y i n t e r p r e t e d
( E d d o w e s & Hill, in press) in terms of a multi-step process at the
electrode surface involving adsorption as illustrated in Fig. 2.
This m e c h a n i s m is analagous to that observed (Smith & Conrad,
1956; Minnaer% 1961; Orii et al., 1962; Yonetani & Ray, 1966; Nicholls
& Mochan, 1971; Nicholls, 1974) in the physiological redox reactions of
cytochrome c which proceed via tightly bound complexes.
According
to this m e c h a n i s m s e l f - i n h i b i t i o n is simply due to an increasing
coverage of the surface with i n c r e a s i n g c y t o c h r o m e c o n c e n t r a t i o n
which blocks the binding s i t e s on t h e e l e c t r o d e surfacer thereby
ELECTRON-TRANSFER
REACTIONS OF PROTEINS
527
lowering the a p p a r e n t e l e c t r o c h e m i c a l r a t e c o n s t a n t .
Such a lowering
of the a p p a r e n t r a t e c o n s t a n t is a n a ! a g o u s t o t h e d e c r e a s e in t h e
a p p a r e n t f i r s t - o r d e r r a t e c o n s t a n t observed for M i c h a e l i s - M e n t e n - t y p e
kinetics at high s u b s t r a t e c o n c e n t r a t i o n s .
T h a t is~ a t s a t u r a t i n g
concentrations
of t h e s u b s t r a t e the r a t e is limited by the t u r n o v e r
r a t e of the e n z y m e oG in the case of c y t o c h r o m e - c r e d u c t i o n at t h e
m e r c u r y e l e c t r o d e , by t h e e l e c t r o n - t r a n s f e r
r a t e to the adsorbed
protein as it a p p r o a c h e s full c o v e r a g e of the adsorption sites on t h e
electrode surface.
In addition~ r a p i d e l e c t r o n t r a n s f e r b e t w e e n c y t o c h r o m e c and a
tin-doped indium oxide e l e c t r o d e , as indicated by essentially r e v e r s i b l e
d . c . c y c l i c v o l t a m m e t r i c waves~ has been r e p o r t e d (Yeh & Kuwana,
1977). Again we e m p h a s i z e t h a t such c u r r e n t - v o l t a g e c u r v e s give no
i n f o r m a t i o n a b o u t the mechanism of the e l e c t r o d e r e a c t i o n and can
only i n d i c a t e t h a t t h e s u r f a c e e l e c t r o n - t r a n s f e r
p r o c e s s is r a p i d
c o m p a r e d with the r a t e of t r a n s p o r t to the e l e c t r o d e .
HoweveG the
m e a s u r e d t r a n s p o r t r a t e is lower than t h a t e x p e c t e d f r o m the known
diffusion
coefficient9 indicating some additional, p o t e n t i a l - i n d e p e n d e n t
r a t e - l i m i t i n g step.
In view of this and the a p p a r e n t n e e d f o r r a p i d
and r e v e r s i b l e binding indicated by the studies discussed above, it is
likely t h a t adsorption is also a crucial f e a t u r e of the e l e c t r o n - t r a n s f e r
r e a c t i o n at the tin-doped indium oxide e l e c t r o d e .
Cytochrome c 3
E l e c t r o c h e m i c a l studies of the m u l t i - h a e m protein9 c y t o c h r o m e c 3
from D e s u 2 f o v i b r i o
(sulphate-reducing) bacteria9 have been carried
out.
As in t h e c a s e of t h e m i t o c h o n d r i a l protein, rapid e l e c t r o n
t r a n s f e r b e t w e e n c y t o c h r o m e c 3 at b o t h t h e m e r c u r y ( N i k i e t al.,
1979) and # 9 # ' - b i p y r i d y l - m o d i f i e d e l e c t r o d e s (Eddowes et al.9 1979b)
has been r e p o r t e d .
At the m e r c u r y e l e c t r o d e ~ w e l l - d e f i n e d p o l a r o graphic and c y c l i c v o l t a m m e t r i c waves are observed.
In this c a s e it
was noted t h a t the protein adsorbs at t h e e l e c t r o d e s u r f a c e .
The
o c c u r r e n c e of a d s o r p t i o n a g a i n s u g g e s t s t h a t t h e overall e l e c t r o n
t r a n s f e r r e a c t i o n p r o c e e d s via the mechanism described above (Fig. 2)9
involving rate enhancement
due t o t h e f o r m a t i o n of a stabilized
p r e c u r s o r complex.
F u r t h e r and m o r e d e t a i l e d k i n e t i c s t u d i e s a r e
required to d e t e r m i n e w h e t h e r or not this is indeed the case.
Similarly9 at the 4 , # ' - b i p y r i d y l - m o d i f i e d e l e c t r o d e ~ c y t o c h r o m e c 3
t a k e s p a r t in a r a p i d and e s s e n t i a l l y 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 t r a n s f e r reaction9 as d e m o n s t r a t e d by its d . c . and a . c . c y c l i c v o l t ammetry.
However9 detailed studies9 n e c e s s a r y for a full elucidation
of the mechanism~ have not been c a r r i e d out~ but in the light of the
observations described above it appears most likely t h a t again binding
plays an i m p o r t a n t role.
Ferredoxins
S e v e r a l r e p o r t s of r a p i d e l e c t r o n t r a n s f e r b e t w e e n the m e r c u r y
e l e c t r o d e and f e r r e d o x i n s f r o m a v a r i e t y of s o u r c e s h a v e b e e n
published ( W i e t z m a n n et al., 1971; Dalton & Zubieta, 1973; Hill et al.,
1977; Ikeda e t al., 1979).
Whilst in earlier studies the p o l a r o g r a p h i c
wave observed around -550 mV vs. S.C.E. ( - 3 0 0 mV vs. N.H.E.) was
528
EDDOWES & HILL
a t t r i b u t e d s i m pl y to the reduction of the iron-sulphur cluster, more
detailed studies (Kakutani et al., 19g0) i n d i c a t e t h a t t h e e l e c t r o c h e m i c a l behaviour is more complex. These later studies Show that
ferredoxins adsorb strongly at the m e r c u r y - e l e c t r o d e s u r f a c e .
They
also suggest that the observed e l e c t r o n - t r a n s f e r reaction is not simply
the reduction of the prosthetic group but that the protein decomposes~
with loss of the iron-sulphur cluster, when adsorbed on mercury. It
has been suggested that the observed c u r r e n t m ay be due to s o m e
n o n - p h y s i o l o g i c a l l y relevant c a t a l y t i c process and reduction processes
involving adsorbed sulphur of cysteine residues. However, at pH >g it
a p p e a r s t h a t t h e o b s e r v e d (Kakutani et al., 1980) e l e c t r o c h e m i s t r y
does represent the reduction of the prosthetic group. The elucidation
of t h e e x a c t nature of the redox processes involved requires further
detailed studies.
The e l e c t r o c h e m i c a l studies of ferredoxin emphasize the importance
of c a r r y i n g out s p e c t r o s c o p i c or o t h e r e x p e r i m e n t s in o r d e r t o
d e t e r m i n e w h e t h e r or not any r e d o x process observed involves the
redox c e n t r e of interest.
Electrochemical techniques~ t h o u g h giving
i n f o r m a t i o n about a redox process, cannot, on their own, show what
r ed o x - active group or groups are involved. It is t h e r e f o r e e s s e n t i a l ,
particularly in the study of redox proteins, that supportive evidence be
obtained to show which redox p r o c e s s o c c u r s and t h a t t h e p r o t e i n
retains its structural integrity.
Discussion
The e l e c t r o c h e m i c a l studies described above clearly illustrate the"
importance of binding in the enhancement of t h e r a t e s of e l e c t r o n
t r a n s f e r b e t w e e n e l e c t r o d e s and r e d o x p r o t e i n s in s o l u t i o n .
Its
relationship to the familiar role of binding in e n z y m e c a t a l y s i s has
b een b r i e f l y discussed and we now return to consider this aspect in
more detail.
Theoretical considerations of the e v o l u t i o n of e n z y m e
e f f i c i e n c y ha ve been d e s c r i b e d ( A l b e r y & Knowles~ 1976; Fersht,
1980). Assuming that the upper limit of an e n z y m e - c a t a l y s e d r e a c t i o n
is the rate of diffusion-controlled encounteG 108 to 109 M- l s-l~ then
an enzyme p e r f e c t l y evolved to m a x i m i z e r a t e will h a v e k c a t / K M
s u f f i c i e n t l y high that diffusional transport rather than the catalysed
step is rate-limiting~ with the constraint t hat K M be g r e a t e r than the
n o r m a l in vivo s u b s t r a t e c o n c e n t r a t i o n so t h a t s e l f - i n h i b i t i o n is
minimized. A suitably rapid r at e might be achieved by either a large
kca t or a small KM.
Ways by which kca t might be increased have
been described:
ensuring t hat the ligand e n v i r o n m e n t of t h e r e d o x
c e n t r e is such that t her e are no large atomic movements consequent
upon electron transfer (Vallee & Williams, 1968); ensuring t hat binding
b e t w e e n t h e p r o t e i n s o c c u r s in such a m a n n e r t h a t the distance
required for 'injection' of the electron is short (Salemme9 1977); or
providing a pathway between the two redox cent res that offers little
impediment to electron transfer (Kraut~ 1981).
A furt her method of
increasing kca t is to have a sufficient potential d i f f e r e n c e between the
two sites to give a rapid e l e c t r o n - t r a n s f e r rate.
(This is the variable
most easily altered
in t h e e l e c t r o c h e m i c a l e x p e r i m e n t s .
The
E L E C T R O N - T R A N S F E R REACTIONS OF PROTEINS
529
relationship between the observed first-order constant and the applied
potential is
k = k0exp[nF/RT(E-E0)]
where K 0 is the value at E = E~
The rate of the overall secondorder electron-transfer-rate process may also be enhanced by having a
large binding energy that ensures that K M is small.
Where considerable r a t e e n h a n c e m e n t is achieved t h r o u g h binding~ the need for
providing redox centres in which the ligand environment ensures facile
e l e c t r o n t r a n s f e r may be less important.
In such cases the overall
electron-transfer reaction rates might be expected to be rapid o n l y
where there is considerable binding and i t is interesting to note that
in both the p h y s i o l o g i c a l and n o n - p h y s i o l o g i c a l e l e c t r o n - t r a n s f e r
reactions of redox proteins9 where rapid rates are observed~ there is
also considerable evidence for the formation of t i g h t l y bound precursor
complexes.
The m a x i m i z a t i o n of r a t e is~ of cours% not the only
criterion for assessing e n z y m e e f f i c i e n c y .
In b i o l o g i c a l e l e c t r o n t r a n s p o r t processes i t is important that electrons follow a specific
route and hence that any redox component react only with its specific
e l e c t r o n donor and a c c e p t o r and not w i t h o t h e r redox species.
Selectivity may readily be achieved through binding i n t e r a c t i o n s t h a t
ensure that only where there is considerable rate enhancement due to
binding will a reaction occur at an appreciable rate.
In addition~ the
principles outlined above of f e r a simple mechanism for the control of
a reaction rate via feedback inhibition~ in which a feedback inhibitor
influences the strength of binding and hence the reaction rate.
The
e f f i c i e n c y of an enzyme might b e t t e r be judged9 not only by its ability
to a c c e l e r a t e t h e r a t e of a r e a c t i o n but also by its d e g r e e of
selectivity and ability to control the rate. Whilst an increase in kca t
might lead to accel er a t i on of rates it does not afford the same degree
of control and specificity that can be achieved through binding. The
a c h i e v e m e n t of e f f i c i e n c y in e l e c t r o n - t r a n s p o r t chains~ rather than
requiring intrinsically facile e l e c t r o n - t r a n s f e r c e n t r e s 7 may~ f o r t h e
purposes of selectivity and/or regulation~ require the opposite.
It has t h e r e f o r e been suggested that binding may be a prerequisite
f o r t h e a c h i e v e m e n t of rapid e l e c t r o n - t r a n s f e r reactions involving
redox proteins in general. This implies that rapid e l e c t r o n t r a n s f e r
between electrodes and redox proteins might only be achieved where
binding of the protein to the el ect rode occurs.
Such a p r o p o s a l is
supported by the experimental results described above which show that~
in the few cases where rapid electron transfer between electrodes and
r e d o x p r o t e i n s is observed~ it is accompanied by adsorption.
This
obviously represents a severe constraint on the utilization of e l e c t r o c h e m i c a l m e t h o d s in t h e s t u d y of r e d o x - p r o t e i n e l e c t r o n - t r a n s f e r
reactions. With further developments in the field of surface-modified
e l e c t r o d e s it should p r o v e p o s s i b l e to produce electrodes with the
required surface properties to e n a b l e a p r o t e i n to bind and h e n c e
undergo rapid electron transfer.
However~ we emphasize that strong binding at an el ect rode need
not necessarily lead to enhancement of the overall r a t e of e l e c t r o n
transfer.
For this to be achieved it is essential that t here is rapid
530
EDDOWES & HILL
exchange between adsorbed and solution species. Indeed, if the binding
were too tight then this might lead to effective inhibition of electron
transfer to freely diffusing protein. Furthermore, we have suggested
(Eddowes et al., 1979a) that the orientation of the bound protein at
the electrode surface may also be of crucial importance, that is, that
the nature of the binding can a f f e c t the first-order electron-transfer
rate constant. In the physiological redox reactions of cytochrome e it
is the lysine residues in the vicinity of the exposed haem edge that, in
the main, determine the strength of binding and hence the overall rate
of reaction, and we have proposed that a preferred orientation of the
bound protein, which brings the exposed haem edge a d j a c e n t to either
the physiological redox p a r t n e r or t h e e l e c t r o d e s u r f a c e , m i g h t
f a c i l i t a t e the electron-transfer reaction by minimizing the distance
over which the electron must be transferred and allowing electron
transfer via the exposed haem edge.
In conclusion it may be said that the inherent chemical properties
of redox proteins may introduce severe constraints on the achievement
of rapid, direct electron transfer between electrodes and proteins in
solution.
These may be overcome by the use of suitable surfacemodified electrodes which take account of the special requirements of
the p r o t e i n .
The d e v e l o p m e n t of such electrodes should enable
electrochemical methods to be used in the study of redox proteins in
general and allow the use of enzymes as electrocatalysts. Some idea
of what might be possible if this can be achieved is evident (Hill et
al., 1991) in the coupling of a terminal oxidase to an electrode via a
short electron-transport chain.
Acknowledgements
We thank the Science Research Council and the National Research
Development Corporation for support.
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