Báo cáo khoa học: Cofactor-independent oxygenation reactions catalyzed by soluble methane monooxygenase at the surface of a modified gold electrode pot

Cofactor-independent oxygenation reactions catalyzed by soluble methane monooxygenase at the surface of a modified gold electrode Yann Astier 1 *, Suki Balendra 2 , H. Allen O. Hill 1 , Thomas J. Smith 2† and Howard Dalton 2 1 Chemistry Department, University of Oxford, UK; 2 Department of Biological Sciences, University of Warwick, UK Soluble methane monooxygenase (sMMO) is a three-com- ponent enzyme that catalyses dioxygen- and NAD(P)H- dependent oxygenation of methane and numerous other substrates. Oxygenation occurs at the binuclear iron active centre in the hydroxylase component (MMOH), to which electrons are passed from NAD(P)H via the reductase component (MMOR), along a pathway that is facilitated and controlled by the third component, protein B (MMOB). We previously demonstrated that electrons could be passed to MMOH from a hexapeptide-modified gold electrode and thus cyclic voltammetry could be used to measure the redox potentials of the MMOH active site. Here we have shown that the reduction current is enhanced by the presence of catalase or if the reaction is performed in a flow-cell, prob- ably because oxygen is reduced to hydrogen peroxide, by MMOH at the electrode surface and the hydrogen peroxide then inactivates the enzyme unless removed by catalase or a continuous flow of solution. Hydrogen peroxide production appears to be inhibited by MMOB, suggesting that MMOB is controlling the flow of electrons to MMOH as it does in the presence of MMOR and NAD(P)H. Most importantly, in the presence of MMOB and catalase, the electrode-asso- ciated MMOH oxygenates acetonitrile to cyanoaldehyde and methane to methanol. Thus the electochemically driven sMMO showed the same catalytic activity and regulation by MMOB as the natural NAD(P)H-driven reaction and may have the potential for development into an economic, NAD(P)H-independent oxygenation catalyst. The signifi- cance of the production of hydrogen peroxide, which is not usually observed with the NAD(P)H-driven system, is also discussed. Keywords: electrochemical oxygenation; regulatory protein; soluble methane monooxygenase. Soluble methane monooxygenase (sMMO) catalyses the bacterial oxidation of methane to methanol using NAD(P)H as cofactor [1]. sMMO consists of three compo- nents: the hydroxylase (MMOH), the reductase (MMOR) and a regulatory protein (known as MMOB or protein B). The active site of the enzyme is located on the a subunit of MMOH and consists of a binuclear iron species located in a hydrophobic pocket approximately 12 A ˚ beneath the protein surface [2]. MMOH, which has an (abc) 2 quater- nary structure, interacts with the MMOR component from which it receives reductant to drive the reaction [3]: CH 4 þ NADH þ H þ þ O 2 ! CH 3 OH þ NAD þ þ H 2 O The regulatory protein (MMOB) plays several roles in the catalytic process including modulating the rate of electron transfer between MMOR and MMOH [4,5], altering the redox potential of the binuclear iron site [6], optimizing the interaction between MMOR and MMOH [7], controlling the access of large substrates to the active site [8] and affecting the regioselectivity of substrate oxygenation [9,10]. The 15.9-kDa MMOB also exists in a naturally occurring truncated form in which 12 amino acids are lost from the N-terminus. This truncated form (MMOB tru ) is completely inactive within the sMMO complex [11]. The sMMO reaction can also be driven using hydrogen peroxide via the peroxide shunt reaction [10,12], in which case the only protein component required is MMOH and neither NADH nor O 2 is needed to catalyze substrate oxygenation. Indeed MMOB, which facilitates substrate oxygenation in the whole-complex sMMO reaction, actually inhibits the per- oxide shunt [10]. Conversely MMOB tru , which has no stimulatory effect on the whole-complex reaction, has little effect on the peroxide shunt reaction [13]. The inability of MMOB tru to facilitate a catalytically competent sMMO complex is supported by electrochemical evidence in which MMOB was able to cause a negative shift in redox potential of MMOH at a modified gold electrode whereas MMOB tru was ineffective [6]. In these experiments it was demonstrated that the modified electrode could serve as the source of electrons to MMOH, thus obviating the need for NADH and MMOR although only direct electron transfer to the MMOH from the electrode was measured. Correspondence to H. Dalton, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. Fax: + 44 24 76523568, Tel.: + 44 24 76523552, E-mail: hdalton@bio.warwick.ac.uk Abbreviations: sMMO, soluble methane monooxygenase; MMOB, regulatory component of sMMO; MMOBtru, truncated form of MMOB; MMOH, hydroxylase component of sMMO; MMOR, reductase component of sMMO; SCE, saturated calomel electrode. Enzymes: methane monooxygenase (EC 1.14.13.25). Note: a web site is also available at http://www.bio.warwick.ac.uk/ dalton/ Note: Y. Astier and S. Balendra made equal contributions to this study. *Present address: Oxford Biosensors, Begbroke Science Park, Yarnton OX5 1PF, UK. Present address: Biomedical Research Centre, Sheffield Hallam University, Howard Street, Sheffield S1 1WB, UK. (Received 23 September 2002, accepted 3 December 2002) Eur. J. Biochem. 270, 539–544 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03411.x Here we report, for the first time since attempts were made in the mid-1970s, that such an electron transfer can be effectively coupled to the oxidation of hydrocarbon sub- strate that is facilitated by the MMOB protein. Materials and methods Chemicals and enzymes The MMOH and MMOR components of sMMO were purified from Methylococcus capsulatus (Bath) as described previously [7,14]. The regulatory protein used in all experi- ments was a catalytically active mutant with increased stability, in which glycine 13 was replaced by a glutamine. Expression in Escherichia coli, affinity purification and removal of the N-terminal affinity tag were effected as previously reported [11]. MMOB tru can be prepared from wild-type MMOB purified from M. capsulatus (Bath) by leaving a purified sample (containing a mixture of MMOB and MMOB tru ) at room temperature for 2–3 days. To ensure we had a homogeneous sample of MMOB tru we made a construct for expression of MMOB tru in E. coli as a fusion to glutathione S-transferase and prepared the recombinant truncate by the same method used for the full-length protein. Like the recombinant intact MMOB [11], the recombinant MMOB tru had two additional vector- encoded amino acids (Gly-Ser) at the N-terminus that had no effect on its catalytic properties. Analytical grade acetonitrile and glycolic acid nitrile solution (70% in water) were supplied by Fluka. Analytical grade HCl was purchased from BDH, methanol (HPLC grade) from Prolab, and oxygen and methane (both at 99% purity) from BOC. The water used in electrochemistry experiments was conductivity water from Nanopure (resis- tivity 18.2 MW cm). The hexapeptide, Lys-Cys-Thr-Cys- Cys-Ala, used to modify the gold electrode, was synthesized using a Pioneer Peptide synthesiser (Applied Biosystems) and purified by means of HPLC using a Jupiter RPC18 column. Electrochemical measurements Voltammetric experiments were performed using a potenti- ostat/galvanostat PGSTAT12 (Autolab) controlled by a Dell GX110MT computer fitted with GPES software. The surface of the 4-mm diameter gold electrode (Oxford Electrodes) was modified by cycling the electrode at reducing potentials in the presence of the hexapeptide, as described previously [6], to enable electron transfer to MMOH. The counter electrode was a platinum wire (99% pure). Except where otherwise stated all potentials are reported in reference to a saturated calomel electrode (SCE) supplied by Radiometer Analytical and all experiments were performed at 22 °Cin25m M Mops pH 7.0. Cyclic voltammetry of MMOH (25 l M ) was performed at 2, 5, 10, 20 and 50 mVÆs )1 between 0 and )0.6 V. Voltammetry to investigate the effects of catalase, MMOB and MMOB tru and the substrate acetonitrile on electron transfer to MMOH was also performed between 0 and )0.6 V, at the scan speed and protein and substrate concentrations stated for each experiment. Where necessary, the solution contacting the electrodes was ventilated by means of a stream of air from an electric fan. The effect of the substrate methane on the electrochemical properties of the MMOH/MMOB/catalase system was investigated by enclosing the electrode assembly in a PVC chamber in which a 1 : 1 oxygen/methane atmosphere was maintained. Detection of the products of substrate oxygenation by the adsorbed MMOH was achieved by holding the potential at )0.5 V for 30 min in the presence of substrate and then removing the liquid (50 lL) from the electrode surface for analysis by GC/MS using a Hewlett-Packard HP 5890A gas chromatograph coupled to a Trio 1000 mass spectrometer. Products were identified by reference to authentic samples. For the flow cell experiments, eight 1-mm diameter gold electrodes were cast in epoxy resin and polished to mirror finish. All electrodes were modified with hexapeptide and four had MMOH adsorbed as well. A potential of ) 500 mV vs. the 1 M KCl Ag/AgCl electrode (equivalent to approximately )520 mV relative to the SCE) was applied to all electrodes. The counter electrode was a platinum mesh and a silver wire was used a reference. Each electrode was monitored individually by a potentiostat. A peristaltic pump, P500, from Pharmacia, was used to maintain a flow of 0.1 M Tris/H 2 SO 4 , pH 7.4, at the electrode surfaces. Results Direct electrochemistry of the MMOH on the hexapeptide- modified gold electrode from 0 to )0.6 V at various scan rates between 2 mVÆs )1 and 50 mVÆs )1 indicated the redu- cibility of the binuclear iron centre and its reoxidation by dissolved oxygen (Fig. 1). The reduction current peak intensities varied linearly with the square root of the scan Fig. 1. Cyclic voltammetry of MMOH in solution showing current (I) vs. applied potential (E). Scans were performed at scanning rates (v)of2 (solid line), 5 (dotted line), 10 (broken line), 20 (broken/dotted line) and 50 (dashed line) mVÆs )1 ,from0to)0.6 V, using 25 lL of MMOH solution (25 l M ). Peaks currents were linear with respect to the square root of the scan rate, consistent with a diffusion-controlled electron transfer (insert). 540 Y. Astier et al.(Eur. J. Biochem. 270) Ó FEBS 2003 rate (see inset to Fig. 1), which agreed with our earlier observation [6] and was indicative of a diffusional reaction. During cyclic voltammetry the reduction current changed from a diffusion-limited current to one in which the current indicated (by being directly proportional to the scan rate) that the MMOH protein was absorbed onto the electrode. This was confirmed after repeated cycling for 40 min (Fig. 2). The signal remained unchanged after rinsing the electrode and undertaking cyclic voltammetry in protein- free 0.1 M Tris/H 2 SO 4 buffer, pH 7.4. Thus it was possible to evaluate the rate of electron transfer to MMOH from the equation: i/nFA ¼ k f C o where i is the current intensity in amperes, n is the number of electrons exchanged (n ¼ 2for the sMMO system), F is the Faraday constant, A is the electrode area in cm 2 , C o is the enzyme concentration in the thin layer in molÆcm )3 and k f is the potential-dependent electron transfer constant in s )1 . Using the values of the current (from which the background current had been subtracted), we can estimate that k f for electron transfer to MMOH is (3.0 ± 0.6) · 10 )5 s )1 . When the experiment was repeated in a flow cell in which the liquid phase was flowed over the electrode, the reduction current proved to be flow-rate dependent. In the presence of adsorbed MMOH the current was 10 times greater than the control in the absence of adsorbed protein, suggesting that renewing the solution at the electrode surface increased the reduction current significantly. One possibility might be that an inhibitory product was formed by MMOH but that it was readily removed in the flow-cell experiment. As dioxygen is a substrate in sMMO-catalyzed reactions, it was possible that the product that was inhibiting current flow in the static cell experiments was a reduced oxygen species. Addition of catalase to the static cell with the MMOH-adsorbed electrode resulted in a catalase-depend- ent increase in reduction current that appeared to be diffusion controlled, as evidenced by the increase in current when the solution was ventilated (Fig. 3). These data strongly suggested that hydrogen peroxide was responsible for the lack of catalytic current in the absence of catalase, presumably because the hydrogen peroxide inactivated MMOH. Addition of MMOB to the MMOH-activated electrode produced an increase in current intensity that was propor- tional to the concentration of MMOB up to a molar ratio of 2 : 1 MMOB:MMOH (Fig. 4). The electron transfer rate k f was approximately doubled to (6.2 ± 1.0) · 10 )5 s )1 .Ifthe inactive truncate MMOB tru wasaddedthennoeffectonthe Fig. 2. Cyclic voltammetry during MMOH adsorption. Repeated cyclic voltammetry was performed at 5 mVÆs )1 from 0 to )0.6 V, using 25 lL of MMOH solution (25 l M ). The arrow indicates the evolution of the reduction waves over 40 min. Fig. 3. MMOH activation by catalase and oxygen. MMOH was adsorbed onto the electrode as shown in Fig. 2 and then cycled from 0 to )0.6 V at 2 mVÆs )1 . The arrow shows the effect of increasing catalase concentrations (0, 2.4, 4.8 and 7.2 l M , respectively). The effect of ventilating the reaction at the highest catalase concentration was also investigated as shown. Fig. 4. Effect of MMOB on the electrochemistry of MMOH. Adsorbed MMOH (prepared as in Fig. 2) was cycled from 0 to )0.6 V at 5 mVÆs )1 . Aliquots of MMOB solution (8 l M ) were added to give MMOB : MMOH molar ratios of 0, 0.5, 1, 1.3, 2, 2.5 and 3.5; the arrow shows the effect of increasing concentration of MMOB. Ó FEBS 2003 Electrochemical oxygenation using sMMO (Eur. J. Biochem. 270) 541 electron transfer was detected, the constant k f being the same as that observed with MMOH alone (data not shown). When an excess of catalase was added to the MMOH/ MMOB complex the current remained unchanged (Fig. 5). We assume from this that the presence of MMOB suppressed the production of hydrogen peroxide by MMOH. In the presence of MMOB tru , however, catalase produced a marked increase in the negative current (data not shown) indicating that MMOB in its fully active form prevents hydrogen peroxide formation but its inactive form (MMOB tru ) is unable exert such an effect. In the presence of catalase, a large increase in reduction current was observed upon addition of the substrate acetonitrile (Fig. 5), consis- tent with the substrate-induced passage of electrons to MMOH during turnover. Acetonitrile produced only a modest increase in reduction current when MMOB was replaced by the inactive MMOB tru (data not shown). When the substrates methane or acetonitrile were added to the MMOH/MMOB (1 : 2) complex in the absence of catalase and the electrode was held at a negative potential for 30 min to enable any oxidation products to accumulate (as described in materials and methods), no such products were observed. However, when catalase was added to 50 l M , then product (methanol or cyanoaldehyde) was readily observed by gas chromatography that was con- firmed by mass spectrometry. No product was observed with MMOH alone or when MMOB tru was added instead of MMOB. The reactions inferred to occur at the MMOH-activated electrode under the various conditions tested are summar- ized in Table 1. Discussion For the first time we have demonstrated that it is possible to use an electrochemical method to drive the oxidation of methane by two components of the sMMO complex (MMOH and MMOB) in the absence of reductant (NADH or NADPH) and MMOR. As in the natural sMMO reaction [11], substrate oxygenation was facilitated by MMOB but not the truncated form MMOB tru . Unlike the natural system, however, the conditions for a successful reaction in the electrochemically driven sMMO require that catalase is also present (Table 1). Fromthesedataitisapparentthathydrogenperoxideis produced during the electrochemical oxidation and so it is relevant to ask how the hydrogen peroxide is formed and what role, if any, it plays in the oxygenation reaction. When catalase was added to the hexapeptide-modified electrode to which MMOH had been absorbed we observed a large increase in the negative current compared to the control in which no catalase was present (Fig. 3). We conclude that during the reduction cycle hydrogen peroxide was formed that could be either removed by catalase or its effect diminished by constant removal in the flow cell. We have also observed production of hydrogen peroxide when the complete sMMO complex was exposed to a high concentration of oxygen (Y. Jiang and H. Dalton, unpublished observations). Here again, addition of catalase would restore full activity through its ability to remove the toxic hydrogen peroxide. Thus it appears that hydrogen peroxide is formed when MMOH is either over-reduced (as seen in the electrochemical experiments) or over- oxidized when exposed to excess oxygen. This release of hydrogen peroxide under these extreme conditions may Table 1. Reactions at the MMOH-activated electrode. Components Effect of catalase Reaction(s) inferred a MMOH Catalytic current increased O 2 +2H + +2e – fi H 2 O 2 MMOH + MMOB None None MMOH + MMOB tru Catalytic current increased O 2 +2H + +2e – fi H 2 O 2 MMOH + MMOB + substrate Catalytic current increased; (a) O 2 +2H + +2e – fi H 2 O 2 oxygenated product observed b (b) substrate + O 2 +2H + +2e – fi oxygenated product + H 2 O MMOH + MMOB tru + substrate No product observed in the presence of catalase Oxygenation reaction (b) did not occur a As detailed in the text, oxygenated products were detected by GC-MS and production of hydrogen peroxide was inferred from an increase in catalytic current upon addition of catalase. b As hydrogen peroxide from reaction (a) inactivated the enzyme, oxygenated product from reaction (b) accumulated only when catalase was present. Fig. 5. Effect of catalase, MMOB and acetonitrile on the electro- chemistry of the MMOH/MMOB complex. Adsorbed MMOH/ MMOB (1 : 2 molar ratio) complex (prepared as in Fig. 4) was cycled from 0 to )0.6Vat5mVÆs )1 (solid line). Data were also collected after addition of catalase (to 50 l M ; dotted line) and subsequent addition of acetonitrile (to 95 l M ; broken line). 542 Y. Astier et al.(Eur. J. Biochem. 270) Ó FEBS 2003 arise as a result of interaction of reductant or oxidant with intermediate P in the catalytic cycle, which is believed to contain a binuclear iron site-associated peroxo species [5]. Addition of MMOB permits an increase in electron flow to MMOH and also appears to regulate and shutdown the production of hydrogen peroxide as there was no change in the current intensity when catalase is added to the MMOH/MMOB complex. It is possible that a confor- mational change in the MMOH protein is necessary to stop overproduction of hydrogen peroxide and that this is induced by MMOB, although not by MMOB tru . Earlier studies by small-angle X-ray scattering spectroscopy (SAXS) have also shown that such a conformational change is induced by the active MMOB and not MMOB tru [7]. The most interesting result arises when substrate (either methane or acetonitrile) is added to the system. Product was only observed when MMOH/MMOB and catalase were present (Table 1). In the absence of catalase or MMOB no product was formed. It is possible to rationalize these observations by proposing a possible new role for MMOB and hydrogen peroxide. As indicated above we suggest that MMOB normally serves to shut down hydrogen peroxide formation in the absence of oxidizable substrate. In the presence of substrate, however, catalase is essential for the formation of product as hydrogen peroxide is toxic. We postulate that one role for MMOB is to act as a sensor for substrate. In the absence of substrate, MMOB can stimulate electron transfer from the electrode to MMOH but it does not result in hydrogen peroxide formation because there is no substrate present. When substrate is present we observe formation of both hydrogen peroxide and product. The paradox that needs to be resolved here is if hydrogen peroxide production is shut down by MMOB why is it observed when substrate is added? Here the sensor role of MMOB may come into play. We postulate that a complex is formed between MMOB/MMOH and substrate that is different from that in the absence of substrate. In this substrate-associated complex the active site now permits an interaction between the substrate and the active oxygen species to generate product. Previous data have led to the conclusion that the species responsible for oxygenation of substrate is the diferryl intermediate Q, which forms after intermediate P during single-turnover kinetics [15,16]. As, under appropriate conditions, hydrogen peroxide is known to oxidize methane directly to form methanol [17], the data presented here permit the alternative conclusion that intermediate Q may be the result of a side reaction and hydrogen peroxide per se is effecting the oxidation of substrate. Indeed the reactivity of hydrogen peroxide may well be far greater in its nonaquated form within the highly hydrophobic pocket adjacent to the binuclear iron centre [2,18] than aquated hydrogen peroxide in the bulk solution. The need for catalase in the electrochemically driven reaction is therefore to protect the protein from inactivation by excess hydrogen peroxide that has escaped from the enzyme and become aquated, whilst methane oxidation may be catalysed by nonaquated hydrogen peroxide at the active site. A further complication arises when one considers that hydrogen peroxide is able to drive methane oxidation to methanol by MMOH alone via the peroxide shunt reaction [10]. Why is hydrogen peroxide not inhibitory under these circumstances? The answer, we believe, is quite straightfor- ward. In the electrochemical experiments hydrogen peroxide is being generated as an intermediate in low levels. It is only formed under two circumstances: (a) when MMOH is absorbed to the electrode in the absence of full-length MMOB and (b) when the MMOH, active MMOB and substrate are all present. In the first instance this is a simple unregulated formation of hydrogen peroxide due to over- reduction of the enzyme at the electrode. In the second instance the hydrogen peroxide is formed as a catalytic intermediate. The peroxide shunt reaction is based entirely upon Le Chatelier’s principle. Free hydrogen peroxide is inhibitory, but to drive the reaction high concentrations are needed to form intermediate P. The catalytic efficiency of the peroxide shunt reaction is not high, the maximum rate of methane oxygenation being only approximately 10% of that observed with the whole sMMO system; moreover, the enzyme becomes inactivated as the peroxide shunt reaction proceeds [10]. MMOB (and not MMOB tru ) is inhibitory to the peroxide shunt reaction [10] but is required for the whole-complex sMMO reaction and for the electrochemical reaction here. These observations can be explained if it is presumed that MMOB has two effects during the catalytic cycle of sMMO, a stimulatory role before compound P formation and an inhibitory role thereafter. Thus, the stimulatory effect of MMOB predominates when the oxidant is dioxygen and electrons (from MMOR or the modified gold electrode) are required for formation of compound P. In the peroxide shunt reaction, however, the natural pathway to compound P is bypassed and so only the later, inhibitory effect of MMOB is observed. Consistent with this hypothesis, fast- reaction kinetics have shown that MMOB accelerates compound P formation by approximately 1000-fold [19], whilst steady-state experiments have shown that MMOB stimulates whole-complex sMMO activity at low molar ratios with respect to MMOH, but begins to inhibit it when the MMOB : MMOH molar ratio exceeds 2 [20]. In conclusion we observe that methanol is formed from methane by MMOH and MMOB when absorbed to an electrode surface, via a reaction that may involve hydrogen peroxide as an important intermediate. Conformational changes in MMOH induced by MMOB appear to be critical in the catalytic cycle. Acknowledgements This work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and British Gas PLC through a Hub studentship (to S.B). We thank Susan Slade for expert technical assistance during protein purification. References 1. Colby, J. & Dalton, H. (1978) Resolution of the methane mono- oxygenase of Methylococcus capsulatus (Bath) into three compo- nents. Biochem. J. 171, 461–468. 2. Rosenzweig, A.C., Frederick, C.A., Lippard, S.J. & Nordlund, P. (1993) Crystal structure of a bacterial nonheme iron hydroxylase that catalyzes the biological oxidation of methane. Nature 366, 537–543. Ó FEBS 2003 Electrochemical oxygenation using sMMO (Eur. J. Biochem. 270) 543 3. Lund, J., Woodland, M.P. & Dalton, H. (1985) Electron transfer reactions in the soluble methane monooxygenase of Methylo- coccus capsulatus (Bath). Eur. J. Biochem. 147, 297–305. 4. Green, J. & Dalton, H. (1985) Protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath). J. Biol. Chem. 260, 15795–15801. 5. Liu, K.E., Valentine, A.M., Qiu, D., Edmondson, D.E., Apple- man, E.H., Spiro, T.G. & Lippard, S.J. (1995) Characterisation of a diiron (III) peroxo intermediate in the reaction cycle of methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath). J. Am. Chem. Soc. 117, 4997–4998. 6. Kazlauskaite, H., Hill, H.A.O., Wilkins, P.C. & Dalton, H. (1996) Direct electrochemistry of the hydroxylase of soluble methane monooxygenase from Methylococcus capsulatus (Bath). Eur. J. Biochem. 241, 552–556. 7. Gallagher, S.C., Callaghan, A.J., Zhao, J., Dalton, H. & Trewhella, J. (1999) Global conformational changes control the reactivity of methane monooxygenase. Biochemistry 38, 6752–6760. 8. Wallar, B.J. & Lipscomb, J.D. (2001) Methane monooxygenase component B mutants alter the kinetics of steps throughout the catalytic cycle. Biochemistry 40, 2220–2233. 9. Froland,W.A.,Andersson,K.K.,Lee,S K.,Liu,Y.&Lipscomb, J.D. (1992) Methane monooxygenase component B and reductase alter the regioselectivity of the hydroxylase component-catalysed reactions. J. Biol. Chem. 267, 17588–17597. 10. Jiang, Y., Wilkins, P.C. & Dalton, H. (1993) Activation of the hydroxylase of sMMO from Methylococcus capsulatus (Bath) by hydrogen peroxide. Biochim. Biophys. Acta 1163, 105–112. 11. Lloyd, J.S., Bhambra, A., Murrell, J.C. & Dalton, H. (1997) Inactivation of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath) by pro- teolysis can be overcome by a Gly to Gln modification. Eur. J. Biochem. 248, 72–79. 12. Andersson,K.K.,Froland,W.A.,Lee,S K.&Lipscomb,J.D. (1991) Dioxygen independent oxygenation of hydrocarbons by methane monooxygenase hydroxylase component. New J. Chem. 15, 411–415. 13. Callaghan, A.J., Smith, T.J., Slade, S.E. & Dalton, H. (2002) Residues near the N-terminus of protein B control autocatalytic proteolysis and the activity of soluble methane mono-oxygenase. Eur. J. Biochem. 269, 1835–1843. 14. Pilkington, S.J. & Dalton, H. (1990) Soluble methane mono- oxygenase from Methylococcus capsulatus Bath. Methods Enzymol. 188, 181–190. 15. Lee, S K., Nesheim, J.C. & Lipscomb, J.D. (1993) Transient in- termediates of the methane monooxygenase catalytic cycle. J. Biol. Chem. 268, 21569–21577. 16. Shu, L., Nesheim, J.C., Kauffmann, K., Mu ¨ nck, E., Lipscomb, J.D.&Que,L.(1997)AnFe IV 2 O 2 diamond core structure for the key intermediate Q of methane monooxygenase. Science 257, 515–518. 17. Nagiev, T.M., Faradzhev, E.G. & Mamedov, E.M. (2001) Oxi- dation of methane to methanol by hydrogen peroxide under pressure. Russ. J. Phys. Chem. 75, 43–48. 18. George, A.R., Wilkins, P.C. & Dalton, H. (1996) A computational investigation of the possible substrate binding sites in the hydro- xylase of soluble methane monooxygenase. J. Mol. Catal. B 2, 103–113. 19. Liu, Y., Nesheim, J.C., Lee, S K. & Lipscomb, J.D. (1995) Gating effects of component B on oxygen activation by the methane monooxygenase hydroxylase component. J. Biol. Chem. 270, 24662–24665. 20. Gassner, G.T. & Lippard, S.J. (1999) Component interactions in the soluble methane monooxygenase from Methylococcus capsu- latus (Bath). Biochem. 38, 12768–12785. 544 Y. Astier et al.(Eur. J. Biochem. 270) Ó FEBS 2003 . Cofactor-independent oxygenation reactions catalyzed by soluble methane monooxygenase at the surface of a modified gold electrode Yann Astier 1 *,. electrochemical oxygenation; regulatory protein; soluble methane monooxygenase. Soluble methane monooxygenase (sMMO) catalyses the bacterial oxidation of methane to