413

Bmelectrochemistty

and Bioenergetics,

19 (1988) 413-423
Vol. 253 (1988)
- Printed in The Netherlands

A section of J. Electroanal. Chem., and constituting

Elsevier Sequoia

S.A., Lausanne

FAD used as a mediator in the electron transfer between

platinum and several biomolecules
H. Durliat, M.B. Barrau and M. Comtat
Laboratoire de Genie Chimique, UA CNRS 192, Laboratoire de Chimie Physique
Unioersith Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse Cidex (France)
(Received

30 July 1987, in revised

et Electrochimie,

form 1 February 1988)

ABSTRACT
Flavin adenine dinucleotide is used as a mediator to reduce cytochrome c, glucose oxidase and
methemoglobin
and to oxidize ferredoxin on the platinum electrode. Thin layer spectroelectrochemistry
allows for precise and simple comparison of these biomacromolecules
in the presence or absence of FAD.
Their biological activity is not modified by the electrochemical
treatment.
and electrochemical
preparation of reduced or oxidized biomolecule solutions on a preparative
scale can be considered.

INTRODUCTION

Heterogeneous
electron transfer between metals or carbon and biomolecules
is
known to be difficult, if not impossible. However, when carried out in a fast way, it
can be applied in analytical
or preparative
chemistry. Many papers are therefore
being published on the acceleration
of these transfers.
Research has been done on the use of surface activators
[l-9].
These are
electroinactive
organic molecules used in order to avoid the denaturing
adsorption
of biomolecules
and facilitate their fixation on the electrode. Other studies deal with
the modulation
of electrochemical
activity by means of electrolysis, using charged
ions or complexes in order to modify the local electrostatic
environment
at the
electrode/solution
interface [lo-141. Electrochemical
treatments
such as repetitive
potentiodynamic
perturbations
have been considered [15,16].
The development
of the use of mediators fixed in polymer films must also be
noted [17-241.
Finally, many mediators in solution are still being used either with analytical
aims in mind or in order to determine thermodynamic
data [25-291.
0302-4598/88/$03.50

0 1988 Elsevier Sequoia

S.A

414

The drawbacks in using these mediators lie in their ability to form complexes
with the biomolecules. They can also lead to biological products deprived of part of
their activity.
The study presented here deals with the use of flavin adenine dinucleotide (FAD)
as a mediator in the electron transfer between platinum and various biomolecules.
This molecule has several of the qualities necessary for a mediator. Previous
electrochemical-type
studies showed that electron exchanges between FAD and
various metals are fast. Besides, homogeneous reactions between FAD and the
molecules under study are fast too. Finally, since FAD is naturally associated with
some molecules studied in electron transfer processes, a better biocompatibility than
that observed with most organic or inorganic mediators can be expected.

EXPERIMENTAL

Apparatus
Thin layer spectroelectrochemistry
is the method used. The working electrode
was a platinum grid slotted between two glass slides separated by a 0.05 cm gap.
The auxiliary electrode was made of platinum and has a big surface area. The
reference electrode was a saturated calomel one, to which all potentials will be
referred in the following. The general setup has already been described [15]. It
allows many electrochemical methods to be carried out, particularly linear sweep
voltammetry and controlled potential electrolysis, which are more specifically used
in this study.
The volume of the solutions treatedwas about 40 mm3. The circuits included a
Tacussel potentiostat (type PRT 20 X 2) a Tacussel signal generator (type Servovit)
and a Sefram XY recorder (type Luxy-trace).
Optical measurements were carried out by means of a 8451A Hewlett Packard
spectrophotometer linked to a computerized apparatus for the data to be stored and
read out on a plotter.
Reagents
The various solutions were made from twice distilled water plus high purity salts
of various origins.
The buffered solution, pH 7.2, was a mixture of 0.2 M monopotassium and
disodium phosphate. The buffered solution, pH 5.3, was a mixture of 0.13 M
monopotassium phosphate and 0.005 M citric acid.
Horse heart cytochrome c was provided either by Sigma (type VI) or Boehringer.
The protein fraction in reduced form was oxidized by potassium ferricyanide; excess
ferricyanide and ferrocyanide formed were eliminated through a Sephadex G25
column.
Methemoglobin was of placental origin; the fraction of oxyhemoglobin present
was oxidized by potassium ferricyanide. The products are separated when applied
on a Sephadex G25 column.

415

Glucose oxidase (GO) from Aspergilh

niger, ferredoxin from Clostridium
pasteurianum and flavin adenine dinucleotide were provided by Sigma.
In the case of some experiments with controlled potential, a bigger volume cell
was also used. This parallelepipedic cell was waterproof; the working electrode was
set parallel to the larger surfaces sides. The beam could thus be introduced in order
to follow the reactions by in situ absorbance measurements. The cell was about 1 cm
thick; its volume was about 25 cm3.
Procedure
The experiments consisted of a comparison between current-potential
curves
obtained with protein alone in solution and those observed with FAD + protein
mixtures.
Before any experiment, the platinum grid was oxidized with a flame for several
minutes.
The experiments began with an electrolysis at a constant potential of - 0.1 V for
about 15 min, in order to reduce and eliminate the dissolved oxygen present in the
thin layer. The cells were designed so that the experiments could be carried out in
strictly anaerobic conditions. Unless indicated otherwise, the tracings of the current-potential curves begin with a cathodic sweep.
In order to comply with the hypothesis concerning thin layer electrochemistry,
the sweep rate of the potentials was adapted to the thickness of the cell: 3.3 mV/s,
unless specifically mentioned.
Glucose oxidase biological activity before and after electrochemical treatments
was measured by the following two reactions:
glucose + O,%gluconic

acid + H,O,

H,O, + reduced o-dianisidine + oxidized o-dianisidine + H,O

The enzyme activity was calculated from the rate of disappearance of reduced
o-dianisidine as followed by spectrophotometry at 436 nm.
For hemoglobin, the equilibrium curves corresponding to the reaction:
deoxyhemoglobin + 0, + oxyhemoglobin
were compared before and after electrolysis. The experiments consisted of lowering
the pressure in the tonometer sufficiently for all the oxyhemoglobin to be transformed into deoxyhemoglobin. Known volumes of air were then introduced in the
apparatus and the oxy- and deoxyhemoglobin concentration ratio was determined
by spectrophotometry. Experiments were carried out at 37 o C after dilution of the
samples in a pH 7.0 phosphate buffer. Comparison concerned:
(a) the psO value which corresponds to the oxygen pressure in equilibrium with a
solution containing the two forms in equimolecular quantities
(b) the Hill coefficient, i.e. the slope of the straight line [30]:
[oxyhemoglobin]
log [ deoxyhemoglobin]

=(

PO, )

416

RESULTS

AND DISCUSSION

FAD
The electrochemical
behaviour
of this molecule has been studied previously
[16,31,32]. The apparent
standard
potentials
which correspond
to two-electron
transfer are close to -0.34 V at pH 5.3 and to -0.45 V at pH 7.2. The conversion
from the oxidized to the reduced
form takes place through
a semi-quinonic
intermediate
(FAD,) which allows for an oxido-reduction
reaction between FAD
and an enzyme whose prosthetic group only allows for the exchange of one electron.
When the adsorbed molecules are transferred on graphite, the film electrodes are
very stable, and the transfer constants higher than 1 ss [33].
One must remember that this molecule does not absorb light above 500 nm.
Cytochrome c
Many studies on this biological molecule can be found in the literature. Heterogeneous electron transfer rate constants observed are very varied: 3.5 x 10e5 cm/s
with methylviologen
as a fixed mediator [34], about lop2 cm/s with bipyridine
used
as a surface activator [2,3,35]. At the mercury electrode the rate constant is from
lo-
to 1o-2 cm/s [36]. On solid metallic or semi-conducting
electrodes, the same
disparity can be observed [10,11,35-401.
For repetitive potentiodynamic
perturbations
with a platinum
electrode in contact with the cytochrome
c solution, at a potential
sweep rate of 8.3 mV/s, rate
constants of the order of 2 X 1O-5 cm/s are obtained [15].

-0.5

U/V vs

SCE

0.5

'

0.8

,
480

600

Fig. 1. I-U curve and absorption

spectra for a cytochrome
c solution;
concentration
= 0.48 mM;
pH = 7.2; sweep rate = 0.33 mV/s. (1) Cytochrome
entirely oxidized (U 2 0); (2) during the electrochemical reduction; (3) cytochrome
in the reduced form ((/ = - 0.6 V).
Fig. 2. I-(icurve(), and absorbance
at 550 nm (- - -) for a 0.50 mM cytochrome+O.lO
FAD solution. pH = 7.2; sweep rate = 0.33 mV/s. At 550 nm only cytochrome
absorbs (top).

mM

417

The standard
potential
of the oxidized cytochrome
c/reduced
cytochrome
c
V. The experiments
were performed
at pH 7.2 in a phosphate
system is -0.012
buffer.
Spectral modifications
due to preliminary
reduction of dissolved oxygen in the
presence of cytochrome c in the thin layer were never observed. A reaction between
superoxide ion and oxidized cytochrome can therefore never be obtained.
Sweep voltammetry
modifies the rate constant of the electron transfer reaction
between platinum and cytochrome in the absence of potentiodynamic
perturbations.
When cytochrome
c is the only species in solution, the current-potential
curve
corresponding
to the first sweep presents a relatively ill-defined
reduction
and
oxidation peak separated by about 0.3 V, due to the irreversibility
of the system
(Fig. 1). However, the absorption
spectra show the total transformation
of the
molecules during the tracing of the curve for potentials in the range of the solvent
electroactivity.
During the second sweep the peaks are considerable
lower, the
system becomes more irreversible (the heterogeneous
reaction rate constant is below
10P lo cm/s) and only a small fraction of -the molecules
is reduced before the
potential of -0.6V is reached. This behaviour may be due to strong adsorption
of
the molecule on platinum.
Under these conditions,
the potential
at which the
cytochrome reduction rate becomes relatively high is lower than the FAD reduction
potential. Although comparison
of the apparent standard potentials
shows a thermodynamically
unfavourable
oxidized cytochrome c-FAD reaction, the irreversibility of the cytochrome
system means that the role of flavin as a mediator can be
considered.

-90

.\- 50
?
n
:

ci

tlh
10

10

20

Fig. 3. Constant
potential
electrolysis
for a cytochrome
c solution performed
in a waterproof
cell.
Cytochrome:
0.07 mM; pH = 7.2; applied potential = -0.54 V; volume = 6 cm3. (1) Without FAD; (2)
with 0.01 mM FAD.

418

reaction is beAlong the electron transfer respiratory

chain, the spontaneous
tween FADH,
and the oxidized form of cytochrome
c. Thus the standard apparent
potential of the FADH,/FAD
system is lower than that of cyt c,,/cyt
c,,~.
If the transfer between oxidized cytochrome
c and platinum
were fast, two
independent
reductions
with two specific reduction
peaks would be observed by
thin layer electrochemistry.
The first sweep traced in a solution containing
a mixture of cytochrome
and
flavin corresponds
to the sum of the curves obtained with these species alone in
solution;
the absorbance
measurements
show that reduction
of cytochrome
takes
place before the complete reduction of flavin. Similarly, FAD oxidation takes place
at a potential lower than that of cytochrome. The flavin system is still a fast system.
In the second sweep, a single peak appears in reduction,
but the absorbance
spectrum (Fig. 2) shows the transformation
of all the cytochrome
molecules at the
potential at which FAD reduction takes place. This is still true for a cytochrome
to
FAD concentration
ratio equal to 5. The amount of electricity exchanged confirms
this result. Because of the FAD disproportionation,
the following catalytic mechanism can be proposed, where FAD, is the semi-quinonic
form:
FAD + e- + FAD,
FAD,

+ cyt ox + FAD + cyt red

During oxidation the only change is the decrease in the heterogeneous

reaction
rate of cytochrome, which corresponds
to a higher irreversibility
of the system. This
behaviour is maintained
throughout
the following sweeps.
FAD was used as a catalyst to reduce cytochrome solutions with larger volumes
than that of the thin layer cell. The results are shown in Fig. 3 for a solution
electrolyzed at -0.54 V, which corresponds
to the reduction of FAD.
For short times the cytochrome
reduction rate is low because electric energy is
used first to reduce dissolved oxygen. The rate increases later, and the cytochrome
reduction rate can be observed to be multiplied by a factor close to 3 in the presence
of flavin. FAD begins to appear in its reduced form when 90% of the cytochrome
has already been reduced.
Glucose oxidase
Electron transfer between glucose oxidase and an electrode was observed in the
presence of methylviologen
included in a polymeric film fixed on a gold electrode
[41], without a mediator on a mercury electrode [41] and with cyanuric chloride used
to fix GO on a graphite electrode [42]. On platinum,
direct transfer without loss of
enzymatic
activity after electrochemical
reduction
and reoxidation
has also been
demonstrated
[16].
Glucose oxidase is studied here at pH 5.3. The absorption
spectrum
of this
molecule is very close to that of FAD. The apparent standard potential at pH 5.3 is
close to -0.29 V [26], but the system is irreversible;
therefore the reduction and the
reoxidation
reactions take place at very different potentials.
During the tracing of
the current-potential
curves in a thin layer cell, reduction begins at about -0.3 V

419

-4

-8
I
-12 c

Fig. 4. I-U curve and absorption

spectra at various potentials
and 0.32 mM FAD. pH = 5.3; sweep rate = 0.33 mV/s. Spectra
-0.10,
-0.28,
-0.32 and -0.46 V respectively.

for a solution containing

0.32 mM GO
1 to 4 were obtained during reduction at

but the reaction is slow and only a fraction of the molecules is reduced at a potential
higher than that for solvent reduction (-0.5
V at this pH). At this potential,
electrolysis must last about 1 h for all the molecules to be transformed. The color of
the solution goes from yellow to colorless and the amount of electricity is in
agreement with the molecules transformed (oxygen was reduced preliminarily).
Reoxidation is difficult too, and 1 h electrolysis at a potential of +0.5 V is
necessary. The high difference between the oxidation and the reduction potentials is
characteristic of an irreversible system.
Figure 4 shows the results which correspond to a solution in which glucose
oxidase and FAD are present in equimolecular amounts. The current-potential
curve shows a marked reduction peak and the amount of electricity indicates the
reduction of all the glucose oxidase and FAD molecules. However, the oxidation
peak concerns only the flavin molecules and protein reoxidation remains difficult.
The reduction of glucose oxidase is similar when the FAD concentration is five
times lower, indicating that FAD acts as a mediator. On the other hand, no decrease
in the enzymatic activity induced by the electrochemical reduction is observed.
Methemoglobin
A few studies have been carried out on the mercury electrode in which this
molecule shows a half-wave potential of the order of -0.6
V and in which
electrolysis at -0.1 V gives a solution whose spectrum is identical to that of the
oxyhemoglobin to be obtained [43]. The heterogeneous transfer constant on tin
oxide in the presence of Cl- ions is 5.2 X lop6 cm/s [44]. (A constant of 3.8 x 10P
cm/s on a gold electrode modified by methylviologen [45] is obtained with sperm
whale myoglobin.)

420

Fig. 5. I-U curve and absorption

spectra for a solution containing
initially 0.7 mM MetHb, 1 mM
FAD, 1.5 mM ferricyanide+
ferrocyanide;
pH = 7.2; sweep rate = 0.33 mV/s.
Spectra 1 and 3 are
characteristic
of MetHb and deoxyhemoglobin
respectively.

Methemoglobin
is studied at pH 7.2. This molecule shows no electrochemical
reaction on the current-potential
curve. Electron exchange with platinum
is very
difficult and about 10 h are necessary to reduce a diluted solution (10 puM) by
electrolysis at -0.7 V. Electrochemical
reoxidation
could not be carried out. When
the solution is more concentrated
(0.7 mM) reduction becomes negligible.
Under our conditions,
the electrochemical
reoxidation
of deoxyhemoglobin
on
platinum electrode is not possible. To regenerate methemoglobin,
a second mediator
is necessary which will react with deoxyhemoglobin
in a fast way.
Figure 5 shows the current-potential
curve which corresponds
to a solution
containing
methemoglobin,
FAD and ferri-ferrocyanide
mixture.
At peak A, the evolution of spectra 1 to 3, with two isobestic points, shows the
reduction of all the methemoglobin
molecules into deoxyhemoglobin.
FAD is a mediator in the reduction reaction with the following mechanism:
FAD + e- -+ FAD,
4 FAD,

+ MetHb

+ 4 FAD + Hb

FAD is reduced to FADH, when the methemoglobin

has disappeared.
Peak B corresponds
to the oxidation of the adsorbed hydrogen formed at the end
of the previous sweep. Reoxidation
of FAD takes place later (peak C). When the

421

ferrocyanide
oxidation potential is reached, a new catalytic
for the reoxidation
of deoxyhemoglobin
(peak D):
[Fe(CN)4]4-+

[Fe(CN),13-+

4[Fe(CN)6]3-+

Hb -+ 4[ Fe(CN),]

phenomenon

appears

e4p + MetHb

The order of the spectra is the opposite of the one presented for the reduction
of
methemoglobin
by FAD, and the spectrum obtained
at a potential
of 0.5 V is
identical to the initial one. During the negative sweep, ferricyanide
is reduced in the
thin layer (peak E).
When the ferri-ferrocyanide
mixture is eliminated before the experiment through
a Sephadex G25 column, the phenomena
observed between -0.1 and -0.55 V are
not changed.
Due to the catalytic properties of FAD, 25 cm3 of 0.7 mM methemoglobin
in the
presence of 0.15 mM FAD were reduced in a non-optimized
parallelepipedic
cell in
a few hours [46]. The equilibrium
curve for the oxidation
of deoxyhemoglobin
is
identical in each case to the one observed previous to the electrochemical
experiment.
Ferredoxin
Papers on different types of ferredoxins are fairly numerous in the literature and
various electrochemical
methods have been used on carbon or gold electrodes in the
presence of methylviologen
[20,47-521 or on modified mercury electrodes [53-551.
A ferredoxin solution at pH 7.2 does not show any electrochemical
reaction on
the current-potential
curve at a platinum
electrode. Electrolysis
at a controlled
potential close to the potential of the reduction of the solvent leads to a very slow
reduction
of the molecules.
For the reoxidation,
a change in the absorbance
spectrum appears only when the electrode is at a potential
close to 0.0 V and
indicates a denaturation
of the molecules (the spectrum of the reoxidized molecule
is not similar to the initial one).
Figure 6 shows the results obtained
with a solution
containing
FAD and
ferredoxin.
In accordance with the values of the apparent standard potentials,
the reduction
of FAD occurs before the reduction
of ferredoxin.
Moreover,
FAD makes the
reduction of ferredoxin easier as the reaction begins at about - 0.15 V (spectrum 4)
and a few minutes of electrolysis at a potential of -0.7 V is sufficient to transform
all the molecules. No explanation
for this phenomenon
can be given so far.
The reoxidation
of ferredoxin corresponds
to the shoulder prior to the peak on
the current-potential
curve. The spectral change in inverse order can be observed to
occur as for the reduction.
Spectrum 4 corresponds
to a potential
of -0.48 V at
which oxidized FAD appears.
The catalytic phenomenon
can now be observed for the oxidation of the protein.
The peak centred at 0.2 V is probably due to an adsorption phenomenon.
Spectrum
and current-potential
curve do not change throughout
the following sweeps.

422

Fig. 6. I-U curve and absorption spectra for a 0.13 mM ferredoxin + 0.46 mM FAD solution. pH = 7.2;
sweep rate = 0.33 mV/s. Both molecules absorb light in the range of wavelengths
used. During the
reduction, FAD is reduced first up to a potential of - 0.51 V (spectrum 4); then the ferredoxin is reduced
at -0.6 V (spectrum 6). During the reoxidation,
ferredoxin is reoxidized first up to -0.48 V (spectrum
4), then FAD is oxidized up to -0.3 V (spectrum 1). Spectra 2 and 3: the ferredoxin is in the oxidized
form while FAD is partly in the oxidized and partly in the reduced form. Spectrum 5: FAD is in the
reduced form while ferredoxin is partly in the oxidized and partly in the reduced form.

The catalytic role of FAD for the electron transfer between platinum and some
biological molecules is clearly demonstrated.
This property may be used in the analytical field, where various sensors can be
proposed, and in preparative chemistry. The elimination of methemoglobin in
hemoglobin solutions by electrochemical reduction is a first example of its application.
Due to their very different molar masses, oxyhemoglobin and flavin are easy to
separate. Ultrafiltration
could be carried out, and an apparatus which would
associate an electrolyser and an ultrafiltration system for the recycling of flavin
could be elaborated.
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