Enzymatic Electrocatalysis Studies of Escherichia Coli Pyruvate

Mole´culaire, UMR 7591, Universite´ Paris 7, Denis Diderot, 75251 Paris Cedex 05, France. Received October 14, 1997. In Final Form: January 14, 1998...
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Langmuir 1998, 14, 1692-1696

Enzymatic Electrocatalysis Studies of Escherichia Coli Pyruvate Oxidase, Incorporated into a Biomimetic Supported Bilayer Olivier Pierrat,† Christian Bourdillon,† Jacques Moiroux,‡ and Jean-Marc Laval*,† Laboratoire de Technologie Enzymatique, UPRESA 6022, Universite´ de Technologie de Compie` gne, B.P. 20 529, 60205 Compie` gne Ce´ dex, France, and Laboratoire d’Electrochimie Mole´ culaire, UMR 7591, Universite´ Paris 7, Denis Diderot, 75251 Paris Cedex 05, France Received October 14, 1997. In Final Form: January 14, 1998 The peripheral Escherichia coli pyruvate oxidase (Pox) was incorporated into a mixed [(alkanethiols + disulfides)/phospholipids] bilayer, supported on a plane gold electrode. The activity of the enzyme was measured by the coupling between enzymatic and electrochemical reactions with ferrocenemethanol in solution (enzymatic electrocatalysis). Cyclic voltammetry allowed the rapid determination of the kinetic parameters of the membrane enzyme in situ. The experimental kinetic data, obtained by cyclic voltammetry, were well-correlated to a kinetic simulation, using a model assuming a ping-pong mechanism for Pox. The apparent Michaelis constants of Pox for pyruvate and ferricinium methanol and the constant for the rate of oxidation of Pox by ferricinium methanol were estimated.

Introduction Models of biological membranes are of two kinds: (i) the nonsupported bilayers, formed at the liquid/liquid or air/liquid interface, such as proteoliposomes,1 black lipid membranes (BLMs), folded bilayers, and tip-dip bilayers,2 and (ii) the supported bilayer, formed at the liquid/solid interface. The supported bilayer can be prepared either by the Langmuir-Blodgett technique3 or by the selfassembling method involving the fusion of lipid vesicles directly on a hydrophilic support4 or on a first selfassembled monolayer of molecules.4-6 Supported bilayers are stable compared to nonsupported bilayers and hence, they can be used for studying various phenomena such as catalysis, diffusion, adhesion, and molecular recognition or for building up biosensors. Previous work from our laboratory consisted in immobilizing redox enzyme (i.e., glucose oxidase, hydrogenase) on the surface of a supported bilayer and measuring enzymatic activity by the electroactive current with electroactive amphiphile molecules as redox mediators between the enzyme and the gold surface.5 In a previous paper, we reported the step-bystep self-assembly of a biomimetic structure onto a gold electrode surface, involving a membrane enzyme.7 In our model of membranes, a mixed self-assembled monolayer (SAM) on a plane gold electrode was formed from dibenzyl disulfide (DBDS) and octadecyl mercaptan (OM) solutions (both products are assumed to form thiolates on a gold † ‡

Universite´ de Technologie de Compie`gne. Universite´ Paris 7.

(1) Scotto, A. W.; Goodwyn, D.; Zakim, D. Biochemistry 1987, 26, 833. (2) Erlich, B. E. In The heart and cardiovascular system, 2nd ed.; Fozzard, H. A., et al., Eds.; Raven Press: New York, 1992; Chapter 25, p 551. (3) (a) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105. (b) Lindholm-Sethson, B. Langmuir 1996, 12, 3305. (4) Brian, A. A.; McConnell, H. M. Proc. Natl. Acad. Sci. 1984, 81, 6159. (5) Bourdillon, C.; Majda, M. J. Am. Chem. Soc. 1990, 112, 1795. (6) (a) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307. (b) Plant, A. L. Langmuir 1993, 9, 2764. (7) Pierrat, O.; Lechat, N.; Bourdillon, C.; Laval, J. M. Langmuir 1997, 13, 4112.

surface8) and a successive monolayer of phospholipid (dimyristoylphosphatidylcholine, DMPC) was fused onto the first hydrophobic OM + DBDS monolayer to form the hybrid (OM + DBDS)/DMPC bilayer. Then, the membrane enzyme pyruvate oxidase (Pox) of Escherichia coli (pyruvate:ubiquinone-8 oxidoreductase, EC 1.2.2.2) was incorporated into the gold-supported bilayer by hydrophobic interactions via small amphipatic R-helices.9 We managed to characterize each step of the construction of the self-assembly by electrochemistry and by surface plasmon resonance (SPR). Our main results were as follows:7 (i) fusion of DMPC was as effective on the OM + DBDS electrode as on the OM electrode and resulted in the formation of a DMPC monolayer on the hydrophobic monolayer; (ii) DBDS formed minor defects in the OM monolayer which allowed an apparent reversibility of the ferrocenemethanol electrochemistry at the gold surface; (iii) incorporation of Pox resulted in the measurement of a catalytic current with ferricinium methanol as a new electron acceptor of the enzyme. In the study presented in this paper, we determined the kinetics of Pox in this two-dimensional reconstituted membrane system, using cyclic voltammetry, with ferrocenemethanol in solution. By appropriate electrochemical measurements as previously reported for immobilized glucose oxidase,10 we managed to well-correlate our experimental data to a kinetic simulation on Minim 3.0.8, using a model assuming a ping-pong mechanism for Pox. The apparent Michaelis constant of Pox for ferricinium methanol (KM,Fc) and the kinetic constant k3 for the oxidation of Pox by ferricinium methanol, were estimated. The kinetic behavior of the enzyme in the biomimetic structure was compared to that of the sodium dodecyl sulfate (SDS)-activated enzyme in solution.11 The results (8) (a) Biebuyck, H. A.; Whitesides, G. M. Langmuir 1993, 9, 1766. (b) Folkers, J. P.; Laibinis, P. E.; Whitesides, G. M.; Deutch, J. J. Phys. Chem. 1994, 98, 563. (9) Grabau, C.; Chang, Y. Y.; Cronan, J. E. J. Biol. Chem. 1989, 264, 12510. (10) Bourdillon, C.; Demaille, C.; Gueris, J.; Moiroux, J.; Saveant, J. M. J. Am. Chem. Soc. 1993, 115, 12264. (11) Mather, M.; Gennis, R. B. J. Biol. Chem. 1985, 260, 16148.

S0743-7463(97)01120-7 CCC: $15.00 © 1998 American Chemical Society Published on Web 02/28/1998

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demonstrated that cyclic voltammetry was a very efficient and original technique to determine the kinetic parameters of a membrane enzyme in situ, and this method will certainly be useful for the kinetic study of other membrane oxidoreductase enzymes. Experimental Section Dibenzyl disulfide (DBDS), ferrocenemethanol (FcMeOH), and octadecyl mercaptan (OM) were from Aldrich. L-R-Dimyristoylphosphatidylcholine (DMPC) was from Sigma. Only OM was recrystallized in ethanol before use. Water was purified by a Milli-Q System (Millipore). E. coli pyruvate oxidase (Pox) was purified from the mutant strain YYC 458, generously given by the L. P. Hager laboratory (University of Illinois, ChampaignUrbana), according to the procedure described before.7 The preparation of gold substrates (surface 0.54 cm2), OM + DBDS self-assembled monolayers (SAMs), and the DMPC monolayer and the incorporation of Pox into the (OM + DBDS)/DMPC bilayer were performed as mentioned previously.7 Briefly, freshly cleaned gold substrates were immersed overnight in 10 mM DBDS in ethanol/water (4:1) and then immersed in 0.25-1 mM OM in ethanol/water (4:1) for 1 h. (OM + DBDS)/DMPC bilayers were obtained by fusion of DMPC vesicles at 30 °C during 60 min on the OM + DBDS monolayer. The (OM + DBDS)/DMPC electrode was immersed into a solution of 20 µg of Pox/mL of reaction medium12 containing 200 mM sodium pyruvate (0.1 M sodium phosphate buffer, pH 6, 200 µM thiamine pyrophosphate, and 20 mM Mg(NO3)2) for 30 min before the overall biomimetic structure was transferred into the electrochemical cell. Electrochemical measurements were carried out in an anaerobic electrochemical cell with three electrodes: the working (OM + DBDS)/(DMPC + Pox) covered-gold electrode, a saturated KCl calomel electrode (SCE) as the reference electrode, and a platinum foil auxiliary electrode. Cyclic voltammetry was performed at 30 °C in 4 mL of the reaction medium12 of Pox (0.1 M sodium phosphate buffer, pH 6, 200 µM thiamine pyrophosphate, and 20 mM Mg(NO3)2) containing 10-4 M ferrocenemethanol and sodium pyruvate, using an EG&G potentiostat Model 273 (Princeton Applied Research), at 25 mV/s. The enzymatic activity of Pox incorporated into the (OM + DBDS)/DMPC bilayer was measured using ferricinium methanol, produced by the electrochemical reaction, as the electron acceptor and the mediator between the enzyme and the gold surface. Kinetic parameters of the enzyme were obtained by alternating the different concentrations of the substrate of the enzyme in the electrochemical cell: pyruvate concentrations varying between 0 and 50 mM were tested in disorder in the electrochemical cell solution via a continuous stirring tank reactor (CSTR). Each time a different pyruvate concentration was introduced in the electrochemical cell, a large volume up to 10 times the volume of the electrochemical cell was used to discard all traces of the previous solution. One concentration of pyruvate (23 mM) was tested several times, between the other concentrations, to estimate the loss of enzyme activity with time and, if necessary, to apply a correction factor. The time course of the experiment did not exceed 1.5 h. Minim 3.0.8 Macintosh application for nonlinear parameter estimation was used for the determination of the kinetic constants.13

Results and Discussion Our previous experiments showed that ferricinium methanol (Fc+MeOH) is a new artificial electron acceptor of E. coli pyruvate oxidase (Pox) incorporated in the (OM + DBDS)/DMPC bilayer.7 Figure 1 is a schematic representation of the catalytic cycle of Pox with ferrocenemethanol (FcMeOH) in solution as the redox mediator between the enzyme and the electrode surface. Enzymatic electrocatalysis results from the coupling between the oxidation of pyruvate by Pox and the electrochemical oxidation of FcMeOH at the gold surface. In our system, (12) Bertagnolli, B. L.; Hager, L. P. J. Biol. Chem. 1991, 266, 10168. (13) Purves, R. D. J. Pharm. Pharmacol. 1993, 45, 934.

Figure 1. Schematic representation of the catalytic cycle of pyruvate oxidase, with ferrocenemethanol in solution as redox mediator between the enzyme and the gold electrode surface.

the enzymatic reaction was triggered by the electrochemical production of Fc+MeOH at the electrode surface (ferricinium methanol is absent in the bulk solution). To study Pox kinetics, we were particularly interested in the reversible electrochemistry of ferrocenemethanol for two reasons:10 (i) the optimal efficiency of the catalysis is reached when the mediator couple remains electrochemically reversible in the potential scan rate range of interest, and (ii) the local cosubstrate concentrations at each value of potential can be easily calculated according to the Nernst law. The cyclic voltammetry of ferrocenemethanol in solution, on the (OM + DBDS)/(DMPC + Pox) coveredgold electrode, is illustrated in Figure 2a. This typical cyclic voltammogram showed the occurrence of the catalytic reaction. In the absence of pyruvate in the electrochemical cell, the enzymatic catalysis was ineffective and the peak-to-peak separation of 60 mV, at 25 mV/s, indicated the reversible electrochemistry of FcMeOH (Figure 2a). In the presence of 23 mM sodium pyruvate in solution (Figure 2a), the enzymatic activity of Pox was detected by an increase in the anodic current and a decrease in the cathodic current. The catalytic current (icat) was calculated at each potential, from Figure 2a, by subtracting the current without catalysis (in the absence of pyruvate) from the current with catalysis (in the presence of 23 mM pyruvate). Figure 2b represents the values of icat typically obtained from Figure 2a, that is, for one concentration of pyruvate in solution (23 mM pyruvate). The icat value reached a maximum value (icat max) for potentials above 250 mV. We showed that icat was not influenced by changing the scan rate (data not shown). In the absence of pyruvate, the anodic peak current i°p, remains proportional to the square root of the scan rate (ν) and we calculated a diffusion coefficient for ferrocenemethanol of about 7 × 10-6 cm2‚s-1, in agreement with the literature data.14 For each pyruvate and ferrocenemethanol concentration tested in the cell, icat was calculated, at each potential value, from a single cyclic voltammogram similar to the one in Figure 2a. The icat values, obtained at different concentrations of pyruvate and with 10-4 M ferrocenemethanol in solution, were plotted as a function of the potential (Figure 3). The characteristic rate constants of the catalytic process may be derived from the cyclic voltammograms as follows. We assumed that this catalytic process can be described by the same equations as those previously proposed11,12,15 for a ping-pong mechanism. In this case, it consists of the (14) (a) Ohsawa, Y.; Aoyagui, S. J. Electroanal. Chem. 1982, 136, 353. (b) Bond, A. M.; McLennan, E. A.; Stojanovic, R. S.; Thomas, F. G. Anal. Chem. 1987, 59, 2853. (15) O’Brien, T. A.; Kluger, R.; Pike, D. C.; Gennis, R. B. Biochim. Biophys. Acta 1980, 613, 10.

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Figure 3. Catalytic current as a function of potential, obtained on a (OM + DBDS)/(DMPC + Pox)-covered gold electrode. Cyclic voltammetry was performed in a reaction medium of Pox,12 at 30 °C, 25 mV/s, in the presence of different pyruvate concentrations: 3.5 mM ([); 7 mM (9); 23 mM (2); 50 mM (×). The catalytic current was obtained, at each pyruvate concentration, in the same way as indicated in Figure 2 for 23 mM pyruvate.

At the electrode surface, the Fc+MeOH/FcMeOH couple obeys the Nernst law as discussed earlier and

C°Fc -F (E - E0) 1 + exp RT

[Fc+MeOH]0 )

Figure 2. Pox activity in the gold-supported (OM + DBDS)/ DMPC bilayer, by enzymatic electrocatalysis with 10-4 M ferrocenemethanol in solution: (a) Cyclic voltammetry in the absence (thin line) or in the presence (thick line) of 23 mM pyruvate in solution (30 °C, 25 mV/s). Electrolyte solution was the reaction medium of the enyme.12 (b) Catalytic current (icat) as a function of the potential value. icat was obtained at each potential from the difference in Figure 2a between current with pyruvate (thick curve) and current without pyruvate (thin curve).

FcMeOH a Fc+MeOH + ek1

(2)

E-FAD-TPP-P a E-FAD-TPP-L

(3a)

E-FAD-TPP-L f E-FAD-TPP-HE + CO2

(3b)

-1

E-FAD-TPP-HE f E-FADH2-TPP + acetate

(

)

∂[Fc+MeOH] ∂x

(3c)

Reactions 3a, 3b, and 3c were grouped into one single reaction (eq 3): k2

E-FAD-TPP-P 98 E-FADH2-TPP + acetate + CO2 (3) k3

E-FADH2-TPP + 2Fc+MeOH 98 E-FAD-TPP + 2FcMeOH (4) (E, apoenzyme; FAD, flavin adenine dinucleotide; P, pyruvate; TPP, thiamine pyrophosphate; L-TPP, lactylTPP; HE-TPP, hydroxyethyl-TPP). Reaction 3 can be taken as irreversible, since FADH2 is rapidly oxidized by Fc+MeOH.

(I)

+

0

2FSk3Γ°E[Fc+MeOH]0 k-1 + k2 1 1 + k3[Fc+MeOH]0 + k2 k1k2[P]

(

(1)

} E-FAD-TPP-P E-FAD-TPP + P {\ k

)

where [Fc+MeOH]0 is the ferricinium concentration at the electrode surface and C°Fc is the ferrocenemethanol concentration in the bulk solution (10-4 M). The standard potential E° was estimated at 0.188 V, as the mean value between the potential at the anodic peak and the potential at the cathodic peak. Assuming that E-FAD-TPP, E-FAD-TPP-P, and E-FADH2-TPP obey the steady-state approximation within the surface film and taking account of the fact that pyruvate is in large excess, the current i flowing through the electrode is10

i ) -FSD

following sequence of reactions:

(

)

(Γ°E is the surface concentration of active enzyme monomer present at the electrode surface, D is the diffusion coefficient, S is the electrode surface, and P is the bulk concentration of pyruvate). The first term corresponds to the current in the absence of pyruvate, and the second term corresponds to the catalysis. Therefore, the final catalytic current expression can be written as follows:

icat ) 1+

2FSk2Γ°E KM,Fc [Fc+MeOH]0

+

KM,P

(II)

[P]

where KM,P ) (k-1 + k2)/k1 and KM,Fc ) k2/k3 (KM,P and KM,Fc are the apparent Michaelis constants of Pox for pyruvate and ferricinium methanol, respectively. The icat values obtained experimentally in Figure 3 were plotted as a function of the ferricinium methanol concentration at the electrode surface deduced from eq I (Figure 4). Simulation curves of the catalytic current were plotted using expression (II) (Figure 4). A good correlation coefficient between experimental and simulated data (R2

E. coli Pyruvate Oxidase in a Bilayer

Langmuir, Vol. 14, No. 7, 1998 1695 Table 1. Estimation of Kinetic Parameters and Coverage of Pyruvate Oxidase in the Biomimetic Supported (OM + DBDS)/DMPC Bilayera

Figure 4. Catalytic current as a function of ferricinium methanol concentration at the gold surface. Experimental points shown in this figure were obtained from those of Figure 3, according to eq I (see results and discussion). Cyclic voltammetry was performed in a reaction medium of Pox,12 at 30 °C, 25 mV/s, in the presence of different concentrations of pyruvate: 3.5 mM ([); 7 mM (9); 23 mM (2); 50 mM (×). The kinetic simulation on Minim 3.0.8, according to eq II (see Results and Discussion), is represented by the continuous line curves.

) 0.995) demonstrated that the ping-pong mechanism provides a good approximation of the kinetics of catalysis of E. coli Pox, at least for the range of ferricinium methanol and pyruvate concentrations chosen for the experiments. However, a certain divergence from the simple ping-pong mechanism for the highest concentration of pyruvate (50 mM) and the lowest concentrations of ferricinium methanol was noticed (data not shown). Results from ref 11 were in good agreement with our findings, if one considers that a different redox cosubstrate of the enzyme, i.e., ferricyanide, was used: below 50 mM pyruvate, the lines of constant ferricyanide concentrations in the doublereciprocal plot are parallel, a pattern expected for an enzyme with a pong-pong mechanism, whereas, above 50 mM pyruvate, the lines curve downward and the doublereciprocal plot becomes intersecting, indicating the simple ping-pong mechanism was insufficient to explain the overall catalytic process of Pox. The ping-pong mechanism is common among flavoenzymes catalyzing reduction/ oxidation reactions, such as glucose oxidase. It remains unclear, for E. coli Pox, whether the ping-pong mechanism predominates under in vivo conditions of substrate concentrations. In our experiment and that of ref 11, artificial acceptors of Pox were used. We are still trying to puzzle out which mechanism the enzyme should have with the natural membrane electron acceptor of the enzyme, that is, ubiquinone-40. From the least-squares parameter estimation with the Gauss-Newton-Marquardt method on Minim 3.0.8, we obtained the apparent Michaelis constants of Pox for pyruvate (KM,P) and ferricinium methanol (KM,Fc) in this two-dimensional membrane system. With around 40 experimental data points, as in Figure 4, the least-squares method gave one solution for the set of the kinetic parameters. The estimated values and the standard variations are listed in Table 1. Our value of KM,Fc (0.3 mM) is close to that of 0.1 mM obtained for dichloroindophenol (DCIP) (16) but is 10 times lower than that of ferricyanide (3 mM) (calculated from the kinetic curves of ref 11). The KM,Fc was 3 times greater than the concentration in ferrocenemethanol used in the electrochemical cell. The low solubility of ferrocenemethanol in aqueous solution (0.4 mM at 30 °C) greatly limited the concentrations we could test in cyclic voltammetry. In the range of pyruvate concentrations in which the Pox kinetics followed a ping-pong mechanism, we calculated from the kinetic curves of ref 11 a KM,P of 30 mM, in agreement with our results.

parameters

values

KM,Fc, M KM,P, M k2Γ°E, mol‚s-1‚cm-2 k3, M-1 s-1 kred, M-1 s-1 Γ°E, mol cm-2

(3.3 ( 0.5) × 10-4 (25 ( 3) × 10-3 (2.1 ( 0.5) × 10-10 (6.3 ( 0.2) × 105 (8.3 ( 0.2) × 103 (1 ( 0.3) × 10-12

a Pox kinetic constants were obtained by a simulation on Minim 3.0.8, using a model (eq II: see Results and Discussion) assuming a ping-pong mechanism for the enzyme. According to the usage in the enzyme kinetics, k3 ) k2/kM,Fc and kred ) k1k2/(k-1 + k2) ) k2/ KM,P. In this table, k3 and kred are expressed per active FAD monomer. k2 was taken at 207 s-1/FAD17 for the determination of k3, kred and Γ°E.

Under the condition of maximal rate, eq II can be rewritten: icat max) 2FSk2Γ°E, where S is taken to 0.54 cm2, considering a roughness factor of 1.4. As the icat max obtained by simulation on Minim 3.0.8 was 22 × 10-6A, we estimated k2Γ°E at (2.1 ( 0.5) × 10-10 mol‚s-1‚cm-2 (Table 1). If one considers a ping-pong mechanism for Pox, the k2 values should be the same whether the activity is measured with ferricyanide or ferricinium methanol as the artificial electron acceptor or with the Warburg apparatus, for measuring carbonic gas production. The latter was used in ref 17 and found 6000 CO2 units produced per 30 min per mg of pure enzyme. These literature data allowed the estimation of the k2 value at 207 s-1/FAD at 30 °C. Considering this k2, we deduced an approximate value for k3 and Γ°E (Table 1). This first estimation of k3 for the pyruvate oxidase of E. coli (6.3 × 105 M-1‚s-1/FAD) indicated an efficient electron transfer by ferrocenemethanol, not very far from that previously observed with glucose oxidase at the same pH (2 × 106 M-1‚s-1).10 Taking a diameter of 10 nm for the Pox tetramer (5.3 nm Stokes radius18), we estimated that Γ°E corresponds to 13% of the maximal theoretical coverage (packed enzyme monolayer). Conclusion A study of the kinetics of E. coli pyruvate oxidase incorporated into a plane gold electrode-supported bilayer was performed by enzymatic electrocatalysis with a new artificial electron acceptor of the enzyme, ferricinium methanol. This method of determination of the Pox kinetics is very efficient compared to the traditional spectrophotometric measurements with ferricyanide, since only a few cyclic voltammograms were necessary to get the kinetic data. The peripheral membrane enzyme Pox can be incorporated in the mixed supported bilayer in a way similar to that for lipid vesicles, and the enzyme kinetics in the biomimetic structure were comparable to those of the SDS-activated enzyme in solution.11 In the chosen range of pyruvate and ferricinium methanol concentrations, we were able to well-correlate the experimental results to a kinetic simulation, using the model of the ping-pong mechanism described by ref 19. However, a divergence from this ping-pong mechanism for high pyruvate concentrations (50 mM) was noticed, as reported previously.11 Kinetics simulations give the Michaelis (16) Cunningham, C. C.; Hager, L. P. J. Biol. Chem. 1971, 246, 1583. (17) Williams, F. R.; Hager, L. P. Arch. Biochem. Biophys. 1966, 116, 168. (18) Raj, T.; Russel, P.; Flygare, W. H.; Gennis, R. B. Biochim. Biophys. Acta 1977, 481, 42. (19) Houghton, R. L.; Swoboda, B. E. P. Biochem. Soc. Trans. 1973, 1, 665.

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constants of Pox for pyruvate (KM,P ) 25 mM) and for ferricinium methanol (KM,Fc) 0.3 mM). The KM,P value is similar to the one deduced from literature data.11 The rate constant k3 (6.3 × 105 M-1‚s-1/FAD) indicates that ferricinium methanol is an efficient electron acceptor of Pox. This study was an integral part of our laboratory work, which aims at the reconstitution of an electron transport chain in phospholipid bilayers supported on a plane gold electrode or on microporous aluminum oxide. Future experiments on Pox will carry on with its kinetic

Pierrat et al.

study by enzymatic electrocatalysis but with the membrane ubiquinone-40 as a natural electron acceptor of the enzyme. Acknowledgment. We are grateful to Drs. Bertagnolli and Hager (University of Illinois, Champaign-Urbana) for the generous gift of E. coli mutant YYC 458. This work was partially supported by a grant from DRET (No. 95-159). LA971120W