Acetylene as a suicide substrate and active site probe for methane monooxygenase from Methylococcus capsulatus (Bath)

Transcription

1 FEMS Microbiology Letters 29 (1985) Published by Elsevier FEM Acetylene as a suicide substrate and active site probe for methane monooxygenase from Methylococcus capsulatus (Bath) (Inhibitor of methane-oxidising activity) S.D. Prior and H. Dalton Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, U.K. Received 18 May 1985 Accepted 20 May SUMMARY Acetylene was shown to be an inhibitor of cell-free methane monooxygenase (MMO) activity in Methylococcus capsulatus (Bath). Inhibition was demonstrated for both the soluble and particulate forms of the enzyme and was dependent on the presence of both NADH and oxygen. Inactivation of the enzyme complex was irreversible and was due to binding of the acetylene to specific proteins of the enzyme complex. The use of radiolabelled [14C]acetylene provided a method for visualisation of the bound inhibitor: protein complex on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Acetylene was shown to bind to proteins which are associated with methane-oxidising activity and it is proposed that acetylene acts as a suicide substrate. 2. INTRODUCTION Acetylene has been shown to act as a potent inhibitor of methane-oxidising activity in Methylomonas methanica [1] Methylococcus capsulatus [2] and Methylosinus trichosporium [3] and a 'Methylosinus' type organism [4]. The first report of the inhibitory effect of acetylene on cell-free extracts of M. capsulatus [5] demonstrated that 3% acetylene in the assay mixture was sufficient to totally inhibit the conversion of methane to methanol, a reaction catalysed by the enzyme MMO [6]. In addition it was reported that of eight acetylenic compounds tested for inhibition of MMO activity, four were found to strongly inhibit methane oxidation (acetylene, propyne, but-l-yne and but-2-yne) but that inhibition efficiency decreased not only with increasing the carbon chain length but also with the shifting of the acetylenic bond way from the terminal carbon to a subterminal position. Acetylene has been shown to be an inhibitor of cell-free methane-oxidising activity in other methanotrophs, including M. trichosporium OB3b [7], M. capsulatus (Texas) [5] and both the soluble and particulate forms of MMO in M. capsulatus (Bath) [6]. The work of Stirling and Dalton [5] showed that, when acetylene inhibited the MMO activity in the soluble fraction of cell-free extracts of M. capsulatus (Bath), the concentration of acetylene in the assay diminished. They further demonstrated that the apparent loss of acetylene was dependent on the concentration of the extract present and on the presence of NADH and oxygen. Attempts to identify a product for the apparent acetylene oxidation was unsuccessful and it was /85/$ Federation of European Microbiological Societies

2 106 suggested that the inhibitor may have bound to the enzyme complex. It has recently been demonstrated that the MMO activity of M. capsulatus (Bath) can occur in either a soluble or membranebound form, and that acetylene is a potent inhibitor of both forms of the enzyme [8]. In this paper we report that both forms of the MMO enzyme are only inhibited by acetylene in the presence of NADH and oxygen and that by using radiolabelled acetylene the site of methane oxidation on the enzyme complex may be identified. 3. MATERIALS AND METHODS 3.1. Preparation of cell-free extracts of M. capsulatus (Bath) The organism was grown in continuous culture as described in [8]. Particulate MMO activity was expressed when the copper sulphate concentration in the medium was 1.2 mg.1-1, whereas soluble MMO activity was expressed at the lower concentration of 0.2 mg.1-1. Cell-free extracts were prepared as previously described [9] C-Labelled acetylene Radiolabelled experiments were performed as outlined in section 3.2 except that the evacuation steps and assay of MMO activity were omitted. Samples for SDS-PAGE were prepared as described previously [8] and run on a vertical slab gel system [10]. [14C]acetylene was obtained from New England Nuclear (Boston, M) Fluorography of SDS-PA GE gels Gels which had been stained with Coomassie Brilliant Blue were destained using methanolacetic acid-water (40 : 10 : 50 v/v) and then dehydrated by placing them in dimethyl sulphoxide (DMSO) for 1 h with one change of DMSO. The dehydrated gel was then soaked in a DMSO/PPO (33.5 g 2.5-diphenyloxazole in 100 ml DMSO) for 2 h, after which time the PPO was recrystallized by immersion of the gel in running cold water for 1 h. The gel was then dried down onto Whatman filter paper and, when dry, placed in an X-ray cassette with Kodax X-ray film. The cassette was stored at -70 C for 7 days before developing the autoradiograph Inhibition of cell-free extracts by acetylene 5-ml conical flasks containing cell-free extracts to be tested were sealed using rubber Suba-Seal stoppers. Soluble extract was added to sodium/ potassium phosphate buffer (20 mm, ph 7.0) to give a final concentration of 7 mg protein.m1-1. NADH, when present, was added to a final concentration of 5 mm. Acetylene was added by injection through the rubber seal and in all cases 30 /~1 was injected. The flasks were incubated at 45 C in a reciprocal shaking water bath (90 rev./min) for 7 min prior to removal of the gaseous phase by evacuation. In each case the flasks were evacuated and then flushed with air 4 times prior to assay. Particulate fractions were resuspended to a final concentration of 2 mg protein.ml-1 in 5 mm Pipes buffer (ph 7.0) and then treated in the same way as the soluble fractions. MMO activity was determined by measuring the epoxidation of propylene as previously described [9]. 4. RESULTS 4.1. Inhibition of methane monoxygenase activity by acetylene The effect of acetylene on MMO activity is shown in Table 1. Soluble MMO activity was not significantly inhibited in the absence of acetylene, but in the presence of acetylene alone, or acetylene plus either oxygen or NADH, there was some inhibition of MMO activity. Total inhibition of MMO activity required the presence of both NADH and oxygen. The results are also shown for MMO activity in the particulate fractions of cell extracts and are similar to the soluble system. At no time during any of the assay procedures was any product of acetylene oxidation detected by gas chromatography suggesting that the oxidation of acetylene may lead to formation of an inhibitor/ enzyme complex. The possibility that acetylene or a product of acetylene oxidation causing inhibition by irreversible binding to the enzyme was investigated using [14C]acetylene.

3 107 Table 1 Inhibition of methane monooxygenase activity by acetylene Samples were preincubated under the conditions shown for 7 min at 45 C before removal of the gaseous phase by evacuation. Samples were then assayed for MMO activity by monitoring the epoxidation of propylene by gas chromatography [9]. Control samples had specific activity as follows: soluble MMO 38.3 nmol propylene oxide formed/min/mg protein. Particulate MMO 58.1 nmol propylene oxide formed/min/mg protein. Preincubation conditions Activity (%) Soluble Particulate MMO MMO Control (no preincubation) (5 ml) NADH (5 mm) C2H 2 (30/~1) C2H (30/~1/5 ml) C 2 H 2 + NADH (30/~1/5 mm) C2H 2 +NADH~-O 2 (30/~1/5 mm/5 ml) The binding of acetylene to methane monooxygenase If acetylene were bound to the enzyme then, by using radiolabelled acetylene, it should be possible to visualize the protein: inhibitor complex by the use of PAGE and fluorography. The experiments were performed as described in section 3.3. The resultant polypeptide-binding patterns on SDS-PAGE showed that the binding of acetylene had no effect on the migration of the proteins in the gel. The fluorgraphs, when developed, showed that the [14C]acetylene remained bound to the proteins, even after boiling in SDS, and confirms the theory that acetylene, or the product of acetylene oxidation, forms a strong bond with proteins in the enzyme complex (Fig. 1). The soluble fraction of cell extracts of M. capsulatus (Bath), grown under conditions where soluble MMO activity was expressed [8], showed that the [14C]acetylene had bound to a single polypeptide of M r This polypeptide corresponded to the subunit of protein A of soluble methane monooxygenase [11]. Experiments on the purified components of soluble MMO have shown that acetylene does not inhibit the NADH : acceptor reductase activity of protein C of the enzyme, and does not therefore inhibit MMO activity by Fig. 1. [14C]acetylene binding to the active site of methane monooxygenase proteins. The figure shows the protein banding pattern on SDS-PAGE and the corresponding fluorographs which show that in each case acetylene is bound to a single protein of the MMO complex. Lane A, soluble fraction, high copper; lane B, membrane fraction, high copper showing proteins associated with particulate MMO activity. Lane C, membrane fraction, low copper; lane D, soluble fraction, low copper with the a, fl and y components of protein A. Lane 1, fluorograph of proteins corresponding to lane B. Lane 2, fluorograph of proteins corresponding to lane D and demonstrating the binding of radiolabelled acetylene to the a component of protein A. preventing the transfer of electrons from NADH to protein A via protein C (J. Green, personal communication). The cells grown under conditions where they expressed particulate MMO activity [11] showed that radiolabelled acetylene again bound to a single polypeptide but, in this instance, the polypeptide was found in the particulate fraction of cell extracts and had an M r of This corresponds to one of three proteins which are induced at high copper : biomass ratios [8,9] and which are thought to be associated with particulate MMO activity [9]. The fact that acetylene binding occurs to specific proteins which are associated with MMO activity suggests that the mode of inhibition of enzyme activity by acetylene may involve monooxygenase activity and binding to proteins may occur at the active site of the enzyme. 5. DISCUSSION Reports of acetylene acting as an inhibitor of MMO activity in cell extracts (both soluble and

4 108 particulate forms of the enzyme), have suggested that inactivation is rapid, irreversible and dependent on the presence of NADH and oxygen [3,5,7,8]. In addition, whole-cell studies have shown that cells grown on methane are inhibited by acetylene but that, when grown on methanol, conditions under which MMO activity does not appear to be essential for growth [9], acetylene does not inhibit growth of the organism [1]. There is also a report that whole-cell methane oxidation by Methylosinus sp. Type 41 was inhibited by acetylene in a competitive manner [14]. Acetylene has also been shown to inhibit growth of the chemolithotroph Nitrosomonas europaea [13] possibly by inhibition of the enzyme ammonia monooxygenase [14] and it has been suggested recently [15] that inhibition of this enzyme by acetylene involves the inhibitor acting as a 'suicide substrate' but no results were presented. Suicide substrates are compounds that cause self-inactivation of enzymes due to formation of transiently reactive intermediates as products of catalysis [16,17]. Often, inhibition of enzymes by such compounds involves the irreversible binding of the intermediate to the active site of the enzyme. The C2H 2 acetylenic group is essentially inert and it is unlikely that it would act as a potent inhibitor of MMO activity in this form. In the experiments reported here and by Stirling and Dalton [5] acetylene only caused total inhibition of MMO activity in the presence of NADH and oxygen both of which are required for functional MMO activity. The inhibition of MMO activity observed in all other assay procedures was probably due to residual amounts of acetylene that were not removed by evacuation. We therefore propose that it is not acetylene per se that is acting as an inhibitor of MMO but that acetylene binds to the enzyme at the active site, and, in the presence of NADH and oxygen, acts as a substrate of the enzyme to produce a reactive intermediate which can interact with amino acids adjacent to the active site rendering the enzyme inactive. This thesis would explain why total inactivation requires NADH and oxygen and the disappearance of acetylene during the reaction. It also explains the failure to detect a product for acetylene oxidation and why radiolabebed acetylene remains bound to proteins even after SDS treatment. The nature of the intermediate is not known; however, a possible candidate might be that hydroxylation of acetylene by the enzyme yields a ketene in the reaction shown below. NADH + H + NAD + HC=CH ~ ~., H 2C=C=O Acetylene 02 H 20 Ketene Ketene is a highly unsaturated and reactive compound which will react very readily with compounds containing hydroxyl and amino groups, such as the amino acids adjacent to the active site of the enzyme. Amino acids are acetylated via the primary amino group, e.g: R.CH (NH 2 ).COOH + CH 2 : CO ~ R.CH. (NH.CO.CH3).COOH. The soluble MMO of M. capsulatus (Bath) is composed of three subunits all of which are essential for enzyme activity in vitro and which appear to have discrete functions [18]. Protein A is an acidic, non-haem iron protein of M r , which is composed of two copies of each of three subunits M r and It is this component that appears to interact with the hydrocarbon substrate [11]. Protein B is a protein of M r with no apparrent prosthetic groups [10]. Protein C is an iron-sulphur flavoprotein of M r and which appears to be the reductase component of the enzyme [19]. The results of the experiments presented here show that acetylene interacts with protein A of the soluble MMO complex, and that it specifically binds to the a- subunit which has an M r of The interaction of reduced protein A with hydrocarbon substrates leads to a change in the ESR signal attributed to the iron centre of the protein and suggests that reduced protein A may be responsible for substrate binding [11]. If acetylene is acting as a suicide substrate it is likely that binding to protein A occurs at the iron centre and that monooxygenase activity yields a reactive intermediate, possibly ketene, which now binds with amino acids adjacent to the active site of the protein. The possibility that the reactive intermediate also binds to the iron centre is discounted since SDS treatment causes loss of iron from the protein and as such would not be expected to be radioactively

5 109 labelled when visualized on SDS-PAGE. MMO activity has only recently been reported in the particulate fractions of M. capsulatus (Bath) [8] and the enzyme involved has not yet been purified. The expression of this form of the enzyme is dependent on the copper : biomass ratio of the growth medium; particulate MMO activity is only expressed when the copper : biomass ratio is high and an increase in this activity is concomitant with increased levels of three proteins which occur in the membrane fraction and can be visualised on SDS-PAGE gels. These proteins have Mrs of 46000, and [9], the experiments detailed here show that acetylene is bound to the polypeptide of M r and we therefore suggest, for reasons outlined earlier, that the active site of methane oxidation in particulate fractions of M. capsulatus (Bath) occurs on this protein. Confirmation of this thesis will, however, require purification of the particulate MMO system. The use of radiolabelled [tnc]acetylene as a suicide substrate of MMO activity provides a relatively simple but effective method for determining the site of methane oxidation in methanotrophs and will be extended to examine a range of methanotrophs in an attempt to define any similarity in active-site proteins. ACKNOWLEDGEMENTS REFERENCES [1] Colby, J., Dalton, H. and Whittenbury, R. (1975) Biochem. J. 151, [2] Dalton, H, and Whittenbury, R. (1976) Arch. Microbiol. 109, [3] Stirling, D.I. and Dalton, H. (1979) Eur. J. Biochem. 96, [4] De Bont, J.A.M. and Mulder, E.G. (1976) Appl. Environ. Microbiol. 31, [5] Stirling, D.I. and Dalton, H. (1977) Arch. Microbiol. 114, [6] Colby, J., Stirling, D.I. and Dalton, H. (1977) Biochem. J. 165, [7] Scott, D., Brannon, J. and Higgins, I.J. (1981) J. Gen. Microbiol. 125, [8] Stanley, S.H., Prior, S.D., Leak, D.J. and Dalton, H. (1983) Biotechnol. Lett. 5, [9] Prior, S.D. and Dalton, H. (1985) J. Gen. Microbiol. 131, [10] Laemmli, U.K. (1970) Nature 227, [11] Woodland, M.P. and Dalton, H. (1984) J. Biol. Chem. 259, [12] Colby, J. and Dalton, H. (1978) Biochem. J. 171, [13] Hynes, R.K. and Knowles, R. (1978) FEMS Microbiol. Lett. 4, [14] Hynes, R.K. and Knowles, R. (1982) Can. J. Microbiol. 28, [15] Shears, J.J. and Wood, P.M. (1985) Biochem. J. 226, [16] Walsh, C.T. (1981) Tetrahedron 38, [17] Walsh, C.T. (1984) Annu. Rev. Biochem. 53, [18] Dalton, H. (1980) Adv. Appl. Microbiol. 26, [19] Lund, J., Woodland, M.P. and Dalton, H. (1985) Eur. J. Biochem. 147, The authors would like to gratefully acknowledge an SERC Studentship Grant to SDP.