Membrane-Associated Quinoprotein Formaldehyde Dehydrogenase from Methylococcus capsulatus Bath

A membrane-associated, dye-linked formaldehyde dehydrogenase (DL-FalDH) was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The enzyme was the major formaldehyde-oxidizing enzyme in cells cultured in high (above 1 micromol of Cu per mg of cell protein) copper medium and expressing the membrane-associated methane monooxygenase. Soluble NAD(P)(+)-linked formaldehyde oxidation was the major activity in cells cultured in low-copper medium and expressing the soluble methane monooxygenase (Tate and Dalton, Microbiology 145:159-167, 1999; Vorholt et al., J. Bacteriol. 180:5351-5356, 1998). The membrane-associated enzyme is a homotetramer with a subunit molecular mass of 49,500 Da. UV-visible absorption, electron paramagnetic resonance, and electrospray mass spectrometry suggest the redox cofactor of the DL-FalDH is pyrroloquinoline quinone (PQQ), with a PQQ-to-subunit stochiometry of approximately 1:1. The enzyme was specific for formaldehyde, oxidizing formaldehyde to formate, and utilized the cytochrome b(559/569) complex as the physiological electron acceptor.     

Substrate specificity of soluble methane monooxygenase. Mechanistic implications

Following the example set by studies of the mechanistic aspects of the substrate specificity of various cytochrome P-450 enzymes, we have undertaken a parallel investigation of the soluble methane monooxygenase from Methylococcus capsulatus (Bath). Soluble methane monooxygenase is a multicomponent enzyme with a broad substrate specificity. Using substrates previously tested with cytochrome P-450 enzymes and using purified enzyme preparations, this work indicates that soluble methane monooxygenase has a similar oxidative reaction mechanism to cytochrome P-450 enzymes. The evidence suggests that soluble methane monooxygenase oxidizes substrates via a nonconcerted reaction mechanism (hydrogen abstraction preceding hydroxylation) with radical or carbocation intermediates. Aromatic hydroxylation proceeds by epoxidation followed by an NIH shift.     

Electron transfer reactions in the soluble methane monooxygenase of Methylococcus capsulatus (Bath)

Aerobic stopped-flow experiments have confirmed that component C is the methane monooxygenase component responsible for interaction with NADH. Reduction of component C by NADH is not the rate-limiting step for component C in the methane monooxygenase reaction. Removal and reconstitution of the redox centres of component C suggest a correlation between the presence of the FAD and Fe2S2 redox centres and NADH: acceptor reductase activity and methane monooxygenase activity respectively, consistent with the order of electron flow: NADH----FAD----Fe2S2----component A. This order suggests that component C functions as a 2e-1/1e-1 transformase, splitting electron pairs from NADH for transfer to component A via the one-electron-carrying Fe2S2 centre. Electron transfer has been demonstrated between the reductase component, component C and the oxygenase component, component A, of the methane monooxygenase complex from Methylococcus capsulatus (Bath) by three separate methods. This intermolecular electron transfer step is not rate-determining for the methane monooxygenase reaction. Intermolecular electron transfer was independent of component B, the third component of the methane monooxygenase. Component B is required to switch the oxidase activity of component A to methane mono-oxygenase activity, suggesting that the role of component B is to couple substrate oxidation to electron transfer, via the methane monooxygenase components.     

Oxidation of deuterated compounds by high specific activity methane monooxygenase from Methylosinus trichosporium. Mechanistic implications

Hydrocarbon oxidations catalyzed by methane monooxygenase purified to high specific activity from the type II methanotroph Methylosinus trichosporium OB3b were compared to the same reactions catalyzed by methane monooxygenase from the type I methanotroph Methylococcus capsulatus Bath and liver microsomal cytochrome P-450. The two methane monooxygenases produced nearly identical product distributions, in accord with physical studies of the enzymes which have shown them to be very similar. The products obtained from the oxidation of a series of deuterated substrates by the M. trichosporium methane monooxygenase were very similar to those reported for the same reaction catalyzed by liver microsomal cytochrome P-450, suggesting that the enzymes use similar mechanisms. However, differences in the product distributions and other aspects of the reactions indicated the mechanisms are not identical. Methane monooxygenase epoxidized propene in D2O and d6-propene in H2O without exchange of substrate protons or deuterons with solvent, in contrast to cytochrome P-450 (Groves, J. T., Avaria-Neisser, G. E., Fish, K. M., Imachi, M., and Kuczkowski, R. L. (1986) J. Am. Chem. Soc. 108, 3837-3838), suggesting that the mechanism of epoxidation of olefins by methane monooxygenase differs at least in part from that of cytochrome P-450. Hydroxylation of alkanes by methane monooxygenase revealed close similarities to hydroxylations by cytochrome P-450. Allylic hydroxylation of 3,3,6,6-d4-cyclohexene occurred with approximately 20% allylic rearrangement in the case of methane monooxygenase, whereas 33% was reported for this reaction catalyzed by cytochrome P-450 (Groves, J. T., and Subramanian, D. V. (1984) J. Am. Chem. Soc. 106, 2177-2181). Similarly, hydroxylation of exo,exo,exo,exo-2,3,5,6-d4-norbornane by methane monooxygenase occurred with epimerization, but to a lesser extent than reported for cytochrome P-450 (Groves, J. T., McClusky, G. A., White, R. E., and Coon, M. J. (1978) Biochem. Biophys. Res. Commun. 81, 154-160). A large intramolecular isotope effect, kH,exo/kD,exo greater than or equal to 5.5, was calculated for this reaction. However, the intermolecular kinetic isotope effect on Vm for methane oxidation was small, suggesting that steps other than C-H bond breakage were rate limiting in the overall enzymatic reaction. Similar isotope effects have been observed for cytochrome P-450. These observations indicate a stepwise mechanism of hydroxylation for methane monooxygenase analogous to that proposed for cytochrome P-450.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath). A novel regulatory protein of enzyme activity

An understanding of the mechanism of biological methane oxidation has been hampered by the lack of purified proteins. We describe here a purification protocol for the previously uncharacterized protein B of the soluble methane monooxygenase from the obligate methanotroph Methylococcus capsulatus (Bath). Soluble methane monooxygenase is a multicomponent enzyme consisting of a hydroxylase component, protein A, a reductase component, protein C, and protein B. All three proteins are required for monooxygenase activity. Protein B proves to be a low molecular weight (16,000) single subunit protein devoid of prosthetic groups. The protein is a powerful regulator of soluble methane monooxygenase activity, possessing the capacity to convert the enzyme from an oxidase to an oxygenase. Proteins A and C together catalyze the reduction of molecular oxygen to water, a reaction prevented by protein B. The uncoupling of soluble methane monooxygenase in this manner displays a number of novel features. First, the product of the uncoupled reaction is water, and second, the uncoupling is independent of substrate. Free hydrogen peroxide is not an intermediate in the reduction of oxygen by the incomplete methane monooxygenase enzyme complex. Finally, electron transfer can occur between protein C and protein A in the absence of protein B and protein B prevents the steady-state transfer of electrons in the absence of an oxidizable substrate, such as methane. It is demonstrated that oxygen reduction occurs at the active site of the hydroxylase component, protein A. A unifying mechanism, describing the interaction of the three proteins of soluble methane monooxygenase, is proposed.     

Detection of methanotrophic bacteria in environmental samples with the PCR.

We designed PCR primers by using the DNA sequences of the soluble methane monooxygenase gene clusters of Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), and these primers were found to be specific for four of the five structural genes in the soluble methane monooxygenase gene clusters of several methanotrophs. We also designed primers for the gram-negative methylotroph-specific methanol dehydrogenase gene moxF. The specificity of these primers was confirmed by hybridizing and sequencing the PCR products obtained. The primers were then used to amplify methanotroph DNAs in samples obtained from various aquatic and terrestrial environments. Our sequencing data suggest that a large number of different methanotrophs are present in peat samples and also that there is a high level of variability in the mmoC gene, which codes for the reductase component of the soluble methane monooxygenase, while the mmoX gene, which codes for the alpha subunit of the hydroxylase component of this enzyme complex, appears to be highly conserved in methanotrophs.     

Optimization of solubilization and purification procedures for the hydroxylase component of membrane-bound methane monooxygenase from Methylococcus capsulatus strain M

The hydroxylase component of membrane-bound (particulate) methane monooxygenase (pMMO) from Methylococcus capsulatus strain M was isolated and purified to homogeneity. The pMMO molecule comprises three subunits of molecular masses 47, 26, and 23 kD and contains three copper atoms and one iron atom. In solution the protein exists as a stable oligomer of 660 kD with possible subunit composition (alpha beta gamma)6. Mass spectroscopy shows high homology of the purified protein with methane monooxygenase from Methylococcus capsulatus strain Bath. Pilot screening of crystallization conditions has been carried out.     

Screening of obligate methanotrophs for soluble methane monooxygenase genes

A 5.8 kb fragment of chromosomal DNA from Methylococcus capsulatus (Bath) containing genes encoding the soluble methane monooxygenase enzyme complex was used as a probe for the detection of soluble monooxygenase genes in a number of representative strains of obligate methanotrophs. Only type II methanotrophs of the genus Methylosinus were found to contain homologues to the Methylococcus gene probe. This probe was also used successfully to detect soluble methane monooxygenase genes in a variety of methanotrophs by colony hybridizations.     

Redox properties of the hydroxylase component of methane monooxygenase from Methylococcus capsulatus (Bath). Effects of protein B, reductase, and substrate.

The reduction potentials of the hydroxylase component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath) have been investigated through potentiometric titrations. The potentials were determined by EPR spectroscopic quantitation of the mixed valent hydroxylase as a function of added sodium dithionite in the presence of appropriate mediators. The reduction of the oxidized Fe(III).Fe(III) form to the mixed valent Fe(II).Fe(III) form occurs at 48 mV versus NHE while the potential for the formation of the fully reduced Fe(II).Fe(II) species from the mixed valent form was determined to be -135 mV. Addition of the substrate propylene to the hydroxylase did not have a major effect on the reduction potentials. Introduction of the protein B and the reductase components, however, completely inhibited reduction of the hydroxylase at potentials as far negative as -200 mV. Addition of propylene to all three methane monooxygenase components greatly facilitated hydroxylase reduction. Under these conditions, the fully reduced form of the protein was obtained at potentials of greater than 150 mV. This high redox potential indicates that the oxidized form of the protein is highly reactive, as required for methane oxidation. The present results reveal aspects of how both protein B and substrate can regulate electron transfer into and out of the hydroxylase component of methane monooxygenase.     

Inhibition of Methane Oxidation by Methylococcus capsulatus with Hydrochlorofluorocarbons and Fluorinated Methanes

The inhibition of methane oxidation by cell suspensions of Methylococcus capsulatus (Bath) exposed to hydrochlorofluorocarbon 21 (HCFC-21; difluorochloromethane [CHF(inf2)Cl]), HCFC-22 (fluorodichloromethane [CHFCl(inf2)]), and various fluorinated methanes was investigated. HCFC-21 inhibited methane oxidation to a greater extent than HCFC-22, for both the particulate and soluble methane monooxygenases. Among the fluorinated methanes, both methyl fluoride (CH(inf3)F) and difluoromethane (CH(inf2)F(inf2)) were inhibitory while fluoroform (CHF(inf3)) and carbon tetrafluoride (CF(inf4)) were not. The inhibition of methane oxidation by HCFC-21 and HCFC-22 was irreversible, while that by methyl fluoride was reversible. The HCFCs also proved inhibitory to methanol dehydrogenase, which suggests that they disrupt other aspects of C(inf1) catabolism in addition to methane monooxygenase activity.     

Microbial Oxidation of Hydrocarbons: Properties of a Soluble Methane Monooxygenase from a Facultative Methane-Utilizing Organism, Methylobacterium sp. Strain CRL-26

Methylobacterium sp. strain CRL-26 grown in a fermentor contained methane monooxygenase activity in soluble fractions. Soluble methane monooxygenase catalyzed the epoxidation/hydroxylation of a variety of hydrocarbons, including terminal alkenes, internal alkenes, substituted alkenes, branched-chain alkenes, alkanes (C₁ to C₈), substituted alkanes, branched-chain alkanes, carbon monoxide, ethers, and cyclic and aromatic compounds. The optimum pH and temperature for the epoxidation of propylene by soluble methane monooxygenase were found to be 7.0 and 40°C, respectively. Among various compounds tested, only NADH₂ or NADPH₂ could act as an electron donor. Formate and NAD⁺ (in the presence of formate dehydrogenase contained in the soluble fraction) or 2-butanol in the presence of NAD⁺ and secondary alcohol dehydrogenase generated the NADH₂ required for the methane monooxygenase. Epoxidation of propylene catalyzed by methane monooxygenase was not inhibited by a range of potential inhibitors, including metal-chelating compounds and potassium cyanide. Sulfhydryl agents and acriflavin inhibited monooxygenase activity. Soluble methane monooxygenase was resolved into three components by ion-exchange chromatography. All three compounds are required for the epoxidation and hydroxylation reactions.     

Solubilisation of methane monooxygenase from Methylococcus capsulatus (Bath)

The membrane-bound (particulate) form of methane monooxygenase from Methylococcus capsulatus (Bath) has been solubilised using the non-ionic detergent dodecyl-beta-D-maltoside. A wide variety of detergents were tested and found to solubilise membrane proteins but did not yield methane monooxygenase in a form that could be subsequently activated. After solubilisation with dodecyl-beta-D-maltoside, enzyme activity was recovered using either egg or soya-bean lipids. Attempts to further purify the solubilized methane monooxygenaser protein into its component polypeptides were unsuccessful and resulted in complete loss of enzyme activity. The major polypeptides present in the solubilised enzyme had molecular masses of 49 kDa, 23 kDa and 22 kDa which were similar to those seen in crude extracts [Prior, S. D. & Dalton H. (1985) J. Gen. Microbiol. 131, 155-163]. Studies on substrate and inhibitor specificities indicated that the membrane-associated and solubilised forms of methane monooxygenase were quite similar to each other but differed substantially from the well-characterised soluble methane monooxygenase found in cells grown in a low copper regime and synthesised independently of the particulate methane monooxygenase.     

Identification of the gene encoding the regulatory protein B of soluble methane monooxygenase

Abstract Purification of the regulatory protein B of the soluble methane monooxygenase complex from Methylococcus capsulatus (Bath) has revealed that the organism contains two forms of this protein, one of which appears to be a carboxy-terminal truncate. Protein sequencing has confirmed the identity of these two proteins and allowed the identification of the gene encoding protein B on the methane monooxygenase gene cluster.     

Oxidation of hydroxylamine by cytochrome P-460 of the obligate methylotroph Methylococcus capsulatus Bath.

An enzyme capable of the oxidation of hydroxylamine to nitrite was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The absorption spectra in cell extracts, electron paramagnetic resonance spectra, molecular weight, covalent attachment of heme group to polypeptide, and enzymatic activities suggest that the enzyme is similar to cytochrome P-460, a novel iron-containing protein previously observed only in Nitrosomonas europaea. The native and subunit molecular masses of the M. capsulatus Bath protein were 38,900 and 16,390 Da, respectively; the isoelectric point was 6.98. The enzyme has approximately one iron and one copper atom per subunit. The electron paramagnetic resonance spectrum of the protein showed evidence for a high-spin ferric heme. In contrast to the enzyme from N. europaea, a 13-nm blue shift in the soret band of the ferrocytochrome (463 nm in cell extracts to 450 nm in the final sample) occurred during purification. The amino acid composition and N-terminal amino acid sequence of the enzyme from M. capsulatus Bath was similar but not identical to those of cytochrome P-460 of N. europaea. In cell extracts, the identity of the biological electron acceptor is as yet unestablished. Cytochrome c-555 is able to accept electrons from cytochrome P-460, although the purified enzyme required phenazine methosulfate for maximum hydroxylamine oxidation activity (specific activity, 366 mol of O2 per s per mol of enzyme). Hydroxylamine oxidation rates were stimulated approximately 2-fold by 1 mM cyanide and 1.5-fold by 0.1 mM 8-hydroxyquinoline.     

Three-dimensional structure determination of a protein supercomplex that oxidizes methane to formaldehyde in Methylococcus capsulatus (Bath)

The oxidation of methane to methanol in methanotrophs is catalyzed by the enzyme methane monooxygenase (MMO). Two distinct forms of this enzyme exist, a soluble cytoplasmic MMO (sMMO) and a membrane-bound particulate form (pMMO). The active protein complex termed pMMO-C was purified recently from Methylococcus capsulatus (Bath). The complex consists of pMMO hydroxylase and an additional component pMMO-R, which was proposed to be the reductase for the pMMO complex. Further study of this complex has led here to the proposal that the pMMO-R is in fact methanol dehydrogenase, the subsequent enzyme in the methane oxidation pathway by methanotrophs. We describe here the biochemical and biophysical characterization of a stable purified complex of pMMO hydroxylase (pMMO-H) with methanol dehydrogenase (MDH) and report the first three-dimensional (3D) structure, determined by cryoelectron microscopy and single particle analysis to approximately 16 A resolution. The 3D structure reported here provides the first insights into the supramolecular organization of pMMO with MDH. These studies of pMMO-MDH complexes have provided further understanding of the structural basis for the particular functions of the enzymes in this system which might also be of relevance to the complete process of methane oxidation by methanotrophs under high copper concentration in the environment.     

Inhibition of membrane-bound methane monooxygenase and ammonia monooxygenase by diphenyliodonium: implications for electron transfer

Diphenyliodonium (DPI) is known to irreversibly inactivate flavoproteins. We have found that DPI inhibits both membrane-bound methane monooxygenase (pMMO) from Methylococcus capsulatus and ammonia monooxygenase (AMO) of Nitrosomonas europaea. The effect of DPI on NADH-dependent pMMO activity in vitro is ascribed to inactivation of NDH-2, a flavoprotein which we proposed catalyzes reduction of the quinone pool by NADH. DPI is a potent inhibitor of type 2 NADH:quinone oxidoreductase (NDH-2), with 50% inhibition occurring at approximately 5 micro M. Inhibition of NDH-2 is irreversible and requires NADH. Inhibition of NADH-dependent pMMO activity by DPI in vitro is concomitant with inhibition of NDH-2, consistent with our proposal that NDH-2 mediates reduction of pMMO. Unexpectedly, DPI also inhibits pMMO activity driven by exogenous hydroquinols, but with approximately 100 micro M DPI required to achieve 50% inhibition. Similar concentrations of DPI are required to inhibit formate-, formaldehyde-, and hydroquinol-driven pMMO activities in whole cells. The pMMO activity in DPI-treated cells greatly exceeds the activity of NDH-2 or pMMO in membranes isolated from those cells, suggesting that electron transfer from formate to pMMO in vivo can occur independent of NADH and NDH-2. AMO activity, which is known to be independent of NADH, is affected by DPI in a manner analogous to pMMO in vivo: approximately 100 micro M is required for 50% inhibition regardless of the nature of the reducing agent. DPI does not affect hydroxylamine oxidoreductase activity and does not require AMO turnover to exert its inhibitory effect. Implications of these data for the electron transfer pathway from the quinone pool to pMMO and AMO are discussed.     

Growth yields of methanotrophs

Summary The growth yield ofMethylococcus capsulatus (Bath) on methane was dependent on the availability of copper in the growth medium. In nitrate mineral salts medium the carbon conversion efficiency increased by 38%, concomitant with the transition from soluble to particulate methane monooxygenase, after transfer from low to high copper medium. An increase in growth efficiency was also observed with ammonia as nitrogen source but not when methanol replaced methane as carbon source. The high growth efficiency is attributed to a reduced NADH requirement for methane oxidation. This could only arise if methanol dehydrogenase was capable of electron transfer, either directly or indirectly to the particulate methane monooxygenase (MMO). The carbon conversion efficiency from methanol with nitrate as nitrogen source was as high as theoretically predicted. It is suggested that the previously low yields of methanotrophs grown on methanol resulted from the use, as nitrogen source, of ammonia which was oxidised by the MMO still present under these growth conditions. The term ‘methanotroph’ is used throughout to distinguish those organisms capable of growth on methane from ‘methylotrophs’ capable of growth on reduced C, compounds other than methane     

Insights into the obligate methanotroph Methylococcus capsulatus

Completion of the genome sequence of Methylococcus capsulatus Bath is an important event in molecular microbiology, and an achievement for which the authors deserve congratulation. M. capsulatus, along with other methanotrophs, has been the subject of intense biochemical and molecular study because of its role in the global carbon cycle: the conversion of biogenic methane to carbon dioxide. The methane monooxygenase enzymes that are central to this process also have high biotechnological potential. Analysis of the genome sequence will potentially accelerate elucidation of the regulation of methane-dependent metabolism in obligate methanotrophs, and help explain the cause of obligate methanotrophy, the phenomenon making most methanotrophs unable to grow on any substrates other than methane and a very small number of other one-carbon compounds.     

The functional role of cytochrome b5 reincorporated into hepatic microsomal fractions

Incorporation of detergent-solubilized cytochrome b5 into phenobarbital-induced rabbit liver microsomal fractions decelerates hexobarbital-dependent reduction of ferric cytochrome P-450; this is accompanied by retardation of NADPH utilization and H2O2 formation in the assay media. Integration of manganese-substituted cytochrome b5 into the microsomal preparations fails to affect these parameters. Analysis of the cytochrome P-450 reduction kinetics in the presence of increasing amounts of cytochrome b5 reveals a gradual augmentation of the amplitude of slow-phase electron transfer at the expense of the relative contribution of the fast phase; finally, a slow, apparently monophasic reaction persists. This defect in enzymatic reduction is not due to detergent effects and also does not seem to reflect cytochrome b5-induced perturbation of anchoring of NADPH-cytochrome c(P-450) reductase to cytochrome P-450. Experiments with the highly purified cytochrome P-450 isozyme LM2, in which amino acid residue(s) close to the heme edge had undergone suicidal inactivation through covalent attachment of chloramphenicol metabolite(s) do not exclude the possibility that cytochrome b5 and reductase might compete for a common electron transmission site on the terminal acceptor. Hence, the inhibitory action of cytochrome b5 on the reduction of ferric cytochrome P-450 is tentatively attributed to partial substitution of the former pigment for reductase in direct transport of the first electron to the monooxygenase.