Howard Dalton

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

SummaryThe 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.

The effect of copper ions on membrane content and methane monooxygenase activity in methanol-grown cells of Methylococcus capsulatus (Bath)

SUMMARY: Methylococcus capsulatus (Bath) was grown in continuous culture with methanol (1·0%, v/v) as sole carbon and energy source. Cells grown on methanol exhibited differences in methane monooxygenase (MMO) activity which were dependent on the concentration of copper sulphate present in the growth medium; an increase in the concentration of copper in the growth medium enhanced both in vivo and in vitro MMO activity. The MMO activity in methanol-grown Methylococcus capsulatus (Bath) was always associated with the particulate fraction of cell-free extracts; at no time was soluble MMO activity detected. In vitro MMO activity was also stimulated by the addition of copper compounds to the assay system and the stimulation was shown to be pH-dependent. The concentration of copper sulphate in the growth medium also determined the intracytoplasmic membrane content of the cells, as judged by electron microscopy of thin-sections, which could be correlated with particulate MMO activity, although it is not possible at this time to say whether the increase in MMO activity seen is due to the increased membrane content or due to the copper ions per se.

Oxidation of Hydrocarbons by Methane Monooxygenases from a Variety of Microbes

Publisher Summary Methane monooxygenase is the enzyme responsible for the initial oxygenation of methane to methanol. The methanotrophs (methane-oxidizing bacteria) are divided into two types depending on whether they assimilate carbon via the ribulose monophosphate pathway (RMP) (type I) or serine pathway (type II), or both. The range of substrates oxidized and products observed from M. capsulatus and M. trichosporium extracts imply that the monooxygenases from these two organisms essentially are similar. The monooxygenase from M. methanica does not oxidize n-alkanes with more than six carbon atoms, or alicyclic, heterocyclic, and aromatic compounds. Experiments have shown that the extracts of M. methanica would catalyze the O 2 and NADPH-dependent disappearance of bromomethane. They presented evidence that the methane monooxygenase was responsible for the catalysis by showing that the enzyme was particulate and stable to freezing but unstable at 2°C.

The Methylotrophic Bacteria

Three groups of microbes are considered here: the methane utilizers, the methanol utilizers, and the carbon monoxide utilizers. The methane utilizers (methanotrophs) appear to be composed of distinctive groups of bacteria with properties that set them apart from the majority of other microbes. The methanol utilizers (methylotrophs a term that also includes the methanotrophs) are a disparate group. While sharing certain metabolic pathways concerned with the utilizers of methanol and other C1 compounds, they can differ radically in other properties, e.g., some are prokaryotes and some eukaryotes. The carbon monoxide oxidizers are largely an unexplored group of microbes, and a generally accepted view of their biology has yet to be formulated.

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.

Nitrogen Fixation in Obligate Methanotrophs

Summary: A number of representative species of obligate methane-oxidizing bacteria were surveyed for their ability to fix N2 by growth experiments and the acetylene reduction test. Although all strains exhibited growth on nitrogen-free plates, only type II organisms and the type X methanotroph Methylococcus capsulatus (Bath) grew well in nitrogen-free liquid medium and were capable of active acetylene reduction. N2-fixation in type II methanotrophs was less senstive to O2 than in the type X methanotroph Methylococcus capsulatus (Bath) and batch cultures of type II organisms could be established at pO2 values of up to 0.2 bar. N2-fixation in Methylococcus capsulatus (Bath) was inhibited at pO2 values above 0.15 bar and the “switch-off” of nitrogenase activity by ammonia was also observed in this organism.

Growth yields of methanotrophs

SummaryThe 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 acetylene reduction technique as an assay for nitrogenase activity in the methane oxidizing bacterium methylococcus capsulatus strain bath

The use of acetylene as a convenient assay substrate for nitrogenase in methane oxidising bacteria is complicated by the observation that it is a potent inhibitor of the methane monooxygenase enzyme in both whole cells and cell-free extracts. If the cells were provided with alternative oxidisable carbon substrates other than methane then nitrogen fixing cells would reduce acetylene to ethylene. Hydrogen gas also served as an oxidisable substrate in the assay. Nitrous oxide, which is reduced by nitrogenase to N2 and H2O, was not an inhibitor of methane monooxygenase function and could be used as a convenient assay substrate for nitrogenase. Reduction of both substrates by whole cells showed similar response to oxygen in the assay system and in this respect Methylococcus resembles other free living nitrogen fixing aerobes.

The membrane-associated form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a copper/iron protein

A protocol has been developed which permits the purification of a membrane-associated methane-oxidizing complex from Methylococcus capsulatus (Bath). This complex has approximately 5 fold higher specific activity than any purified particulate methane mono-oxygenase (pMMO) previously reported from M. capsulatus (Bath). This efficiently functioning methane-oxidizing complex consists of the pMMO hydroxylase (pMMOH) and an unidentified component we have assigned as a potential pMMO reductase (pMMOR). The complex was isolated by solubilizing intracytoplasmic membrane preparations containing the high yields of active membrane-bound pMMO (pMMO(m)), using the non-ionic detergent dodecyl-beta-D-maltoside, to yield solubilized enzyme (pMMO(s)). Further purification gave rise to an active complex (pMMO(c)) that could be resolved (at low levels) by ion-exchange chromatography into two components, the pMMOH (47, 27 and 24 kDa subunits) and the pMMOR (63 and 8 kDa subunits). The purified complex contains two copper atoms and one non-haem iron atom/mol of enzyme. EPR spectra of preparations grown with (63)Cu indicated that the copper ion interacted with three or four nitrogenic ligands. These EPR data, in conjunction with other experimental results, including the oxidation by ferricyanide, EDTA treatment to remove copper and re-addition of copper to the depleted protein, verified the essential role of copper in enzyme catalysis and indicated the implausibility of copper existing as a trinuclear cluster. The EPR measurements also demonstrated the presence of a tightly bound mononuclear Fe(3+) ion in an octahedral environment that may well be exchange-coupled to another paramagnetic species.

ESR studies of protein A of the soluble methane monooxygenase from Methylococcus capsulatus (Bath)

Abstract ESR spectroscopy of protein A, proposed to be the oxygenase component of the methane monooxygenase (methane, NAD(P)H:oxygen oxidoreductase (hydroxylating), EC 1.14.13.25) from Methylococcus capsulatus (Bath), revealed a minor signal at g = 4.3, and a free radical signal at g = 2.01 in the oxidised state. Upon reduction with sodium dithionite to potentials around 0–100 mV a further rhombic signal appeared with g values less than 2. This spectrum was demonstrated to be from an intermediate reduction level, since on further reduction the signal disappeared. The redox properties of the g = 1.95 signal and its temperature dependence are described. The signal was also formed upon reduction of the protein with protein C of the methane monooxygenase complex, which is known to function as an NADH acceptor-reductase protein. The results are interpreted as indicative of a binuclear Fe centre as the redox site in the protein. The ESR spectra are consistent with a centre of the haemerythin type as opposed to an iron-sulphur cluster.