Methane Monooxygenase Activity Research Articles

Substrate Specificities of the Soluble and Particulate Methane Mono-oxygenases of Methylosinus trichosporium OB3b

Summary: The intracellular location of methane mono-oxygenase (MMO) (soluble or particulate) in Methylosinus trichosporium OB3b is dependent on the availability of copper in the growth medium. Raising the Cu2+ concentration from 1 μM to 5 μM effected a transition from soluble to particulate MMO activity, and changes in major cell polypeptides were observed on SDS-polyacrylamide gels. Organisms containing soluble MMO oxidized a wide range of substrates including n-alkanes, n-alkenes, aromatic and alicyclic compounds. By contrast, organisms containing particulate MMO did not oxidize aromatic or alicyclic compounds. These observations provide further evidence that the two types of MMO are fundamentally different.

In vivo 13C NMR Investigations of Methanol Oxidation by the Obligate Methanotroph Methylosinus trichosporium OB3b

In vivo 13C NMR has been used to observe metabolism of exogenously supplied methanol by suspensions of Methylosinus trichosporium OB3b grown under a variety of conditions. Formaldehyde, formate and bicarbonate ions were the only metabolites of methanol to be detected. Accumulation of formaldehyde was observed only with suspensions grown under conditions which yield particulate, membrane-bound, methane mono-oxygenase (MMO). Ethyne abolished MMO activity, partially inhibited methanol oxidation in whole organisms, and prevented growth of the organism on methanol (1%, v/v) in batch culture. Oxidation of ethanol, a substrate of methanol dehydrogenase, was not affected by ethyne. Ethyne caused accumulation of formaldehyde in all suspensions of the organism incubated with methanol, although oxidation of exogenously added formaldehyde was not affected. These observations are consistent with the proposal that in M. trichosporium OB3b both MMO and methanol dehydrogenase oxidize exogenously supplied methanol and suggest that the further oxidation of formaldehyde is stimulated by the consumption of reducing equivalents by MMO.

In vivo Studies of Primary Alcohols, Aldehydes and Carboxylic Acids as Electron Donors for the Methane Mono-oxygenase in a Variety of Methanotrophs

C2 to C4 primary alcohols and their corresponding aldehydes were oxidized by type I, type II and type X obligate methanotrophs. Reducing equivalents from each oxidation step could be utilized, in vivo, to stimulate methane mono-oxygenase activity. As neither oxidation step produces NADH directly, these observations are presented as evidence for reverse electron transport in methanotrophs. In type II methanotrophs, 5 mm-acetate, propionate and butyrate also stimulate methane mono-oxygenase activity apparently by inducing the breakdown of poly-β-hydroxybutyrate. subsequent metabolism of β-hydroxybutyrate giving rise to NADH.

Copper stress underlies the fundamental change in intracellular location of methane mono-oxygenase in methane-oxidizing organisms: Studies in batch and continuous cultures

The intracellular location of methane mono-oxygenase (MMO) activity in the methanotroph Methylococcus capsulatus (Bath) has been shown to depend primarily on the availability of copper. MMO activity was observed in the particulate fraction of cell extracts under conditions of copper excess but switched to a soluble location in response to copper stress. The two activities could be differentiated by sensitivity to a range of inhibitors and by major changes in the polypeptide banding patterns on denaturing polyacrylamide gels. MMO activity concomitant with the oxidation of ethanol was only observed in cells with particulate MMO activity but could be lost independently in response to copper stress. Examination of other methanotrophs indicated that the copper effect could explain a similar switch in intracellular location observed in Methylosinus trichosporium OB3b but that some methanotrophs do not have the capacity to overcome copper stress in this way.

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 (C(1) to C(8)), 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 degrees C, respectively. Among various compounds tested, only NADH(2) or NADPH(2) 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 NADH(2) 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.

The Effect of Growth Conditions on Intracytoplasmic Membranes and Methane Mono-oxygenase Activities in Methylosinus trichosporium OB3b

The distribution of methane mono-oxygenase (MMO) activities between particulate and soluble fractions of cell-free extracts of Methylosinus trichosporium OB3b was dependent upon growth conditions. Particulate activity was associated with the presence of intracytoplasmic membranes observed only under oxygen-limiting conditions in shake flask cultures. Particulate and soluble activities showed substantially different sensitivities to a range of potential inhibitors. The particulate enzyme was inhibited by metal-chelating agents, thiol reagents and amytal, whereas the soluble MMO was not inhibited by these compounds; both activities were sensitive to KCN, ethyne and 8-hydroxyquinoline. NAD(P)H was the only suitable electron donor. The activities were unstable at 0° C but the soluble enzyme could be partially stabilized by several compounds. The particulate and soluble MMO activities are compared with previously reported particulate and soluble MMO enzymes from this species and other methane oxidizers.

Ethane oxidation by methane-oxidizing bacteria.

The relationship between the rates of methane and ethane oxidation by washed suspensions of methane-oxidizing bacteria has been investigated. Considerable differences between bacterial strains were observed. Two closely related Methylomonas strains which differed in their oxidizing capacity were further investigated. The low ethane oxidation rate of one strain could be strongly stimulated by the addition of oxidizable co-substrates and the presence of ethane stimulated formate oxidation. The other strain had a much higher ethane oxidation rate and stimulation by co-substrates was negligible. Differences between the levels of dissimilative enzymes in cell-free extracts could not be detected. Attempts to produce extracts with methane mono-oxygenase activity failed. When cells were made permeable with chitosan the results suggested that strains with a low ethane oxidizing capacity obtain the required reductant for the moo-oxygenase from endogenous respiration. In strains with a high ethane oxidation rate, the reductant appears to be derived from oxidation of ethanol or acetaldehyde.

Characterization of the second prosthetic group of the flavoenzyme NADH–acceptor reductase (component C) of the methane mono-oxygenase from Methylococcus capsulatus (Bath)

1. A new two-step purification is described that routinely yields 100mg quantities of component C for biochemical studies. 2. Chemical analyses show component C purified by this procedure to contain 2 g-atoms of iron, 2 mol of acid-labile sulphide (S) and 1 mol of FAD per mol of protein. 3. The Fe-S core of component C was extruded by treating the protein with p-methoxybenzenethiol in hexamethyl phosphoramide/50mM-Tris/HCl buffer, pH 8.5 (4:1, v/v), under anaerobic conditions. The spectral properties of the extruded core suggest that component C contains 1 mol of [2Fe-2S(S-Cys)4] centre per mol of protein. 4. E.p.r. spectroscopy confirms the presence of a Fe-S centre in component C. 5. Component C catalyses the reduction by NADH of ferricyanide, 2,6-dichlorophenol-indophenol or horse heart cytochrome c, with specific activities of 50--230 units/mg of protein. 6. The optimum pH for the NADH-acceptor reductase activity is 8.5--9.0, and the apparent Km values for NADH and NADPH are 0.05mM and 15.5mM respectively. 7. Unlike methane mono-oxygenase activity, NADH-acceptor reductase activity of component C is not inhibited by 8-hydroxyquinoline or by acetylene.

Microbial oxidation of methane and methanol: isolation of methane-utilizing bacteria and characterization of a facultative methane-utilizing isolate.

A methane-utilizing organism capable of growth both on methane and on more complex organic substrates as a sole source of carbon and energy, has been isolated and studied in detail. Suspensions of methane-grown cells of this organism oxidized C-1 compounds (methane, methanol, formaldehyde, formate); hydrocarbons (ethane, propane); primary alcohols (ethanol, propanol); primary aldehydes (acetaldehyde, propionaldehyde); alkenes (ethylene, propylene); dimethylether; and organic acids (acetate, malate, succinate, isocitrate). Suspensions of methanol-or succinate-grown cells did not oxidize methane, ethane, propane, ethylene, propylene, or dimethylether, suggesting that the enzymatic systems required for oxidation of these substrates are induced only during growth on methane. Extracts of methane-grown cells contained a particulate reduced nicotinamide adenine dinucleotide-dependent methane monooxygenase activity. Oxidation of methanol, formaldehyde, and primary alcohols was catalyzed by a phenazine methosulfate-linked, ammonium ion-requiring methanol dehydrogenase. Oxidation of primary aldehydes was catalyzed by a phenazine methosulfate-linked, ammonium ion-independent aldehyde dehydrogenase. Formate was oxidized by a nicotinamide adenine dinucleotide-specific formate dehydrogenase. Extracts of methane-grown, but not succinate-grown, cells contained the key enzymes of the serine pathway, hydroxypyruvate reductase and malate lyase, indicating that the enzymes of C-1 assimilation are induced only during growth on C-1 compounds. Glucose-6-phosphate dehydrogenase was induced during growth on glucose. Extracts of methane-grown cells contained low levels of enzymes of the tricarboxylic acid cycle, including alpha-keto glutarate dehydrogenase, relative to the levels found during growth on succinate.

Effect of metal-binding agents and other compounds on methane oxidation by two strains of Methylococcus capsulatus

Inhibition studies of methane mono-oxygenase activity in whole cell suspensions of Methylococcus capsulatus (Texas) and M. capsulatus (Bath) were performed and the results compared. The inhibition pattern for M. capsulatus (Bath) was not only substantially different from the pattern obtained with M. capsulatus (Texas) but also very limited in the number of potent inhibitors specific for methane oxidation. To confirm the whole cell results of M. capsulatus (Bath) similar experiments were done using cell-free extracts. It was found that only acetylene (100% inhibition) and 8-hydroxyquinoline (71%) significantly inhibited methane oxidation, verifying the restricted inhibition pattern found with the whole cell suspensions. Eight acetylenic compounds were tested for specific inhibition of methane oxidation by whole cells and cell-free extracts of M. capsulatus (Bath). Only two compounds (acetylene and propyne) gave 100% inhibition in both cases with three other compounds (but-1-yne, but-2-yne and propyn-1-ol) giving weaker inhibitions. The inhibition pattern of methane oxidation by whole cell suspensions and cell-free extracts of M. capsulatus (Bath) is discussed and reasons for the prominent results are suggested.

Ammonia oxidation by the methane oxidising bacterium Methylococcus capsulatus strain bath

Soluble extracts of Methylococcus capsulatus (Bath) that readily oxidise methane to methanol will also oxidise ammonia to nitrite via hydroxylamine. The ammonia oxidising activity requires O2, NADH and is readily inhibited by methane and specific inhibitors of methane mono-oxygenase activity. Hydroxylamine is oxidised to nitrite via an enzyme system that uses phenazine methosulphate (PMS) as an electron acceptor. The estimated Kmvalue for the ammonia hydroxylase activity was 87 mM but the kinetics of the oxidation were complex and may involve negative cooperativity.