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PubMedDate not supplied

[Method for assaying methane monooxygenase activity by measuring methane uptake in the reaction vessel].

The reaction vessel has been designed to measure methane monooxygenase activity. An elastic membrane has been built into one of the walls of the vessel to take liquid samples, avoiding formation of the gaseous phase in the reaction volume. The methane content in the samples is measured in a gas-liquid chromatograph with a flame ionization detector in two ways: 1. by direct measurement of methane in the liquid sample, and 2. by measurement of methane in the gaseous phase after methane diffusion from the liquid sample into the gaseous space of another vessel. The method is simple, sensitive (with a lower limit of 0.1 nMole CH4), and well reproducible. This method permits measurement of the oxidation kinetics of methane and other gaseous hydrocarbons both by intact cells and cell-free preparations of methane oxidizing bacteria.

Chromatography, Gas↗
PubMed1997

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.

Journal Article↗
PubMed2001

Dioxygen Activation and Methane Hydroxylation by Soluble Methane Monooxygenase: A Tale of Two Irons and Three Proteins A list of abbreviations can be found in Section 7.

Methanotrophic bacteria are capable of using methane as their sole source of carbon and energy. The first step in methane metabolism, the oxidation of methane to methanol, is catalyzed by a fascinating enzyme system called methane monooxygenase (MMO). The selective oxidation of the very stable C-H bond in methane under ambient conditions is a remarkable feat that has not yet been repeated by synthetic catalysts and has attracted considerable scientific and commercial interest. The best studied MMO is a complex enzyme system that consists of three soluble protein components, all of which are required for efficient catalysis. Dioxygen activation and subsequent methane hydroxylation are catalyzed by a hydroxylase enzyme that contains a non-heme diiron site. A reductase protein accepts electrons from NADH and transfers them to the hydroxylase where they are used for the reductive activation of O(2). The third protein component couples electron and dioxygen consumption with methane oxidation. In this review we examine different aspects of catalysis by the MMO proteins, including the mechanisms of dioxygen activation at the diiron site and substrate hydroxylation by the activated oxygen species. We also discuss the role of complex formation between the different protein components in regulating various aspects of catalysis.

Journal Article↗
PubMed1976

Stoichiometry of methane oxidation in the methane-oxidizing strain M 102 under the influence of various CH4/O2 mixtures.

In laboratory-scale experiments with growing cells of the obligate methane-oxidizing strain M 102, an overall molar gas turnover ratio of the order given below could be postulated: 1 CH4+1--1.2 O2=0.3 CO2+water. Expectations that the optimal gas mixture of methane and oxygen should lie within the range of this stoichiometric consumption ratio have been verified in fermenter 5 1 batch culture experiments. The optimal range of methane-oxygen mixture, found under the experimental conditions described, is based on the estimated growth parameters as generation and doubling times, yield coefficients related to methane and oxygen, and the efficiency of methane metabolism as indicated in the absolute amounts of CH4, O2, and CO2 turned over. The mentioned stoichiometric relation of 1 CH4:1--1.202 did not change with varying the composition, i.e. the partial pressures of CH4 and O2 introduced as a mixture to the cells. The efficiency of methane oxidation was obviously influenced and decreased markedly when deviating from the broad optimal range of CH4/O2 mixtures. With non-growing cells, on the other hand, the stoichiometric relation showed a considerable shift (1:1.4--1.8 CH4:O2) with a clear tendency towards more O2 consumption. The oxidation potential of growing cells, seems then to have a linear interdependence to the substrate concentrations, i.e. partial pressures.

Bacteria↗
PubMed2006

Conversion of methane to methanol at the mononuclear and dinuclear copper sites of particulate methane monooxygenase (pMMO): a DFT and QM/MM study.

Methane hydroxylation at the mononuclear and dinuclear copper sites of pMMO is discussed using quantum mechanical and QM/MM calculations. Possible mechanisms are proposed with respect to the formation of reactive copper-oxo and how they activate methane. Dioxygen is incorporated into the Cu(I) species to give a Cu(II)-superoxo species, followed by an H-atom transfer from a tyrosine residue near the monocopper active site. A resultant Cu(II)-hydroperoxo species is next transformed into a Cu(III)-oxo species and a water molecule by the abstraction of an H-atom from another tyrosine residue. This process is accessible in energy under physiological conditions. Dioxygen is also incorporated into the dicopper site to form a (mu-eta(2):eta(2)-peroxo)dicopper species, which is then transformed into a bis(mu-oxo)dicopper species. The formation of this species is more favorable in energy than that of the monocopper-oxo species. The reactivity of the Cu(III)-oxo species is sufficient for the conversion of methane to methanol if it is formed in the protein environment. Since the sigma orbital localized in the Cu-O bond region is singly occupied in the triplet state, this orbital plays a role in the homolytic cleavage of a C-H bond of methane. The reactivity of the bis(mu-oxo)dicopper species is also sufficient for the conversion of methane to methanol. The mixed-valent bis(mu-oxo)Cu(II)Cu(III) species is reactive to methane because the amplitude of the sigma singly occupied MO localized on the bridging oxo moieties plays an essential role in C-H activation.

Binding Sites↗
PubMed2006

Dichloro[(3,5-dimethyl-1H-pyrazol-1-yl)methane]zinc(II) and di-mu-chloro-bis{chloro[(3,5-dimethyl-1H-pyrazol-1-yl)methane]cadmium(II)}.

The Zn atom in dichloro[(3,5-dimethyl-1H-pyrazol-1-yl)methane]zinc(II), [ZnCl2(C11H16N4)], (I), is tetrahedrally coordinated by two N atoms from one bis(3,5-dimethylpyrazolyl)methane ligand and two terminal Cl atoms. The molecule has no crystallographic symmetry. One H atom of the CH2 group of the bis(3,5-dimethylpyrazolyl)methane ligand interacts with a Cl atom of an adjacent molecule to yield intermolecular C-H...Cl contacts, thereby forming a one-dimensional zigzag chain extending along the b axis. On the other hand, in di-mu-chloro-bis{chloro[(3,5-dimethyl-1H-pyrazol-1-yl)methane]cadmium(II)}, [Cd2Cl4(C11H16N4)2], (II), each of the two crystallographically equivalent Cd atoms is pentacoordinated by two N atoms from one bis(3,5-dimethylpyrazolyl)methane ligand, and by one terminal and two bridging Cl- anions. The molecule has a crystallographic centre of symmetry located at the mid-point of the Cd...Cd line. One H atom of the CH2 group of the bis(3,5-dimethylpyrazolyl)methane ligand interacts with a Cl atom of an adjacent molecule to produce pairwise intermolecular C-H...Cl contacts, thereby affording chains of molecules running along the c axis.

Journal Article↗
PubMed1982

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.

Journal Article↗
PubMed1998

Methane and Trichloroethylene Degradation by Methylosinus trichosporium OB3b Expressing Particulate Methane Monooxygenase.

Whole-cell assays of methane and trichloroethylene (TCE) consumption have been performed on Methylosinus trichosporium OB3b expressing particulate methane monooxygenase (pMMO). From these assays it is apparent that varying the growth concentration of copper causes a change in the kinetics of methane and TCE degradation. For M. trichosporium OB3b, increasing the copper growth concentration from 2.5 to 20 muM caused the maximal degradation rate of methane (V(max)) to decrease from 300 to 82 nmol of methane/min/mg of protein. The methane concentration at half the maximal degradation rate (K(s)) also decreased from 62 to 8.3 muM. The pseudo-first-order rate constant for methane, V(max)/K(s), doubled from 4.9 x 10 to 9.9 x 10 liters/min/mg of protein, however, as the growth concentration of copper increased from 2.5 to 20 muM. TCE degradation by M. trichosporium OB3b was also examined with varying copper and formate concentrations. M. trichosporium OB3b grown with 2.5 muM copper was unable to degrade TCE in both the absence and presence of an exogenous source of reducing equivalents in the form of formate. Cells grown with 20 muM copper, however, were able to degrade TCE regardless of whether formate was provided. Without formate the V(max) for TCE was 2.5 nmol/min/mg of protein, while providing formate increased the V(max) to 4.1 nmol/min/mg of protein. The affinity for TCE also increased with increasing copper, as seen by a change in K(s) from 36 to 7.9 muM. V(max)/K(s) for TCE degradation by pMMO also increased from 6.9 x 10 to 5.2 x 10 liters/min/mg of protein with the addition of formate. From these whole-cell studies it is apparent that the amount of copper available is critical in determining the oxidation of substrates in methanotrophs that are expressing only pMMO.

Journal Article↗
PubMed2004

Methane-dependent denitrification by a semi-partitioned reactor supplied separately with methane and oxygen.

Methane (CH4) can be used as an alternative carbon source for denitrification with added oxygen (O2). However, the off-gas of denitrification reactors using a CH4-O2 mixture contains unused CH4 and O2 in proportions that make it unusable for fuel, carry explosion risks, and, if released into the atmosphere, contribute to the greenhouse effect. This study tested a novel reactor with a partition dividing the headspace completely and extending partly into the liquid layer. When CH4 and O2 were supplied separately to the liquid layer on opposite sides of the partition, the methane-dependent denitrification (MDD) activity was similar to that when the two gases were supplied as a mixture. In reactors with separate gas supplies, the off-gas from the CH4 supply side was high in CH4 and low in O2, and was usable for fuel, and that from the O2 supply side was very low in CH4, and might be released into the atmosphere. MDD activity increased with the O2 supply rate, and separate discharge of CH4 and O2 was maintained. The concentration of dissolved methane in the effluent was decreased by lowering the CH4/O2 supply ratio to 1.0 and drawing the effluent from the O2 supply side. This novel reactor enhances the safety of MDD, allows reuse of methane as fuel, and reduces methane leakage to the atmosphere.

Air Pollution↗
PubMed2006

Theoretical modeling of the hydroxylation of methane as mediated by the particulate methane monooxygenase.

We present here the results of density functional theory (DFT) calculations directed toward elucidation of the CH bond activation mechanism that might be adopted by the particulate methane monooxygenase (pMMO) in the hydroxylation of methane and related small alkanes. In these calculations, we considered three of the most probable models for the transition metal active site mediating the "oxo-transfer": (i) the trinuclear copper cluster bis(mu(3)-oxo)trinuclear copper(II, II, III) complex 1, recently proposed by Chan et al. [S.I. Chan, K.H.-C. Chen, S.S.-F. Yu, C.-L. Chen, S.S.-J. Kuo, Biochemistry 43 (2004) 4421-4430.]; (ii) the most frequently used model complex, bis(mu-oxo)Cu(III)(2) complex 2; and (iii) the mixed-valence bis(mu-oxo)Cu(II)Cu(III) complex 3. The results obtained indicate that the methane hydroxylation chemistry mediated by the trinuclear copper cluster bis(mu(3)-oxo)trinuclear copper(II, II, III) complex 1 offers the most facile pathway for methane hydroxylation, and this model yields KIE values that are in good agreement with experiment. In this mechanism, the reaction proceeds along a "singlet" potential surface and a "singlet oxene" is directly inserted across a CH bond in a concerted manner. Kinetic isotope effects (k(H)/k(D) or KIE) associated with the concerted oxene insertion process mediated by complex 1 are calculated to be 5.2 at 300K when tunneling effects are included. Overall rate constants for the methane hydroxylation by the three models have been calculated as a function of temperature, and the rates are at least 5-6 orders of magnitude more facile when the chemistry is mediated by complex 1 compared to complex 2 or complex 3.

Binding Sites↗
PubMed1999

Role of multiple gene copies in particulate methane monooxygenase activity in the methane-oxidizing bacterium Methylococcus capsulatus Bath.

Genes for the subunits of particulate methane monooxygenase, PmoABC, have been sequenced from the gamma-proteobacterial methanotroph Methylococcus capsulatus Bath. M. capsulatus Bath contains two complete copies of pmoCAB, as well as a third copy of pmoC. The two pmoCAB regions were almost identical at the nucleotide sequence level, differing in only 13 positions in 3183 bp. At the amino acid level, each translated gene product contained only one differing residue in each copy. However, the pmoC3 sequence was more divergent from the two other pmoC copies at both the far N-terminus and far C-terminus. Chromosomal insertion mutations were generated in all seven genes. Null mutants could not be obtained for pmoC3, suggesting that it may play an essential role in growth on methane. Null mutants were obtained for pmoC1, pmoC2, pmoA1, pmoA2, pmoB1 and pmoB2. All of these mutants grew on methane, demonstrating that both gene copies were functional. Copy 1 mutants showed about two-thirds of the wild-type whole-cell methane oxidation rate, while copy 2 mutants showed only about one-third of the wild-type rate, indicating that both gene copies were necessary for wild-type particulate methane monooxygenase activity. It was not possible to obtain double null mutants that were defective in both pmo copies, which may indicate that some expression of pMMO is important for growth.

Amino Acid Sequence↗
PubMed1998

Use of an oxygen-insensitive microscale biosensor for methane to measure methane concentration profiles in a rice paddy.

An oxygen-insensitive microscale biosensor for methane was constructed by furnishing a previously described biosensor with an oxygen guard. The guard consisted of a glass capillary containing heterotrophic bacteria, which consumed oxygen diffusing through the tip membrane, thus preventing it from diffusing into the methane-sensing unit. Oxygen microprofiles were measured through the oxygen guard capillary, demonstrating the principle and limitations of the method. When the tip of the guard capillary was exposed to 100% oxygen at 21 degrees C, heterotrophic oxygen consumption prevented oxygen from diffusing further than 170 mum into the capillary, whereas atmospheric levels of oxygen were consumed within 50 mum. The capacity of the oxygen guard for scavenging oxygen decreased with decreasing temperature, and atmospheric levels of oxygen caused oxygen penetration to 200 mum at 5 degrees C. The sensors could be manufactured with tip diameters as small as 25 mum, and response times were about 1 min at room temperature. Pore water profiles of methane concentrations in a rice paddy soil were measured, and a strong correlation between the depths of oxygen penetration and methane appearance was observed as a function of the light regimen; this finding confirmed the role of microbenthic photosynthesis in limiting methane emissions from surfaces of waterlogged sediments and soils.

Journal Article↗
PubMed2006

Cohn's Crenothrix is a filamentous methane oxidizer with an unusual methane monooxygenase.

135 years ago Ferdinand Cohn, the founder of bacteriology, microscopically observed a conspicuous filamentous bacterium with a complex life cycle and described it as Crenothrix polyspora. This uncultured bacterium is infamous for mass developments in drinking water systems, but its phylogeny and physiology remained unknown. We show that C. polyspora is a gammaproteobacterium closely related to methanotrophs and capable of oxidizing methane. We discovered that C. polyspora encodes a phylogenetically very unusual particulate methane monooxygenase whose expression is strongly increased in the presence of methane. Our findings demonstrate a previously unrecognized complexity of the evolutionary history and cell biology of methane-oxidizing bacteria.

Bacterial Proteins↗
PubMed2001

Methane oxidation in three Alberta soils: influence of soil parameters and methane flux rates.

Current concern over the potentially negative impacts of climate change has brought attention to anthropogenic sources of methane, a primary greenhouse gas. Two such emission sources are methane leakage at heavy oil wells and sanitary landfills. At both of these sources, substantial quantities of methane could potentially be oxidised by methanotrophic microbes living in soils. Optimisation of this phenomenon may serve as an inexpensive technique for reducing methane emissions. Soil column and batch incubation experiments were performed on a landfill loam, an agricultural loam and a sedge peat to gain a better quantitative understanding of the biological and physical processes limiting CH4 oxidation in soils that undergo the freeze-thaw cycles associated with northern climates. Moisture content emerged as a critical variable that can limit a soil's CH4 oxidation potential. For example, the oxidation rate of the agricultural soil was seen to increase by an order of magnitude after increasing its moisture content from 6% to 10% of its dry weight.

Alberta↗
PubMedDate not supplied

Biological methane oxidation: regulation, biochemistry, and active site structure of particulate methane monooxygenase.

Particulate methane monooxygenase (pMMO) is a three-subunit integral membrane enzyme that catalyzes the oxidation of methane to methanol. Although pMMO is the predominant methane oxidation catalyst in nature, it has proved difficult to isolate, and most questions regarding its molecular structure, active site composition, chemical mechanism, and genetic regulation remain unanswered. Copper ions are believed to play a key role in both pMMO regulation and catalysis, and there is some evidence that the enzyme contains iron as well. A number of research groups have solubilized and purified or partially purified pMMO. These preparations have been characterized by biochemical and biophysical methods. In addition, aspects of methane monooxygenase gene regulation and copper accumulation in methanotrophs have been studied. This review summarizes for the first time the often controversial pMMO literature, focusing on recent progress and highlighting unresolved issues.

Amino Acid Sequence↗
PubMed1981

Ruminant methane delta(13C/12C) values: relation to atmospheric methane.

The delta(13C/12C)--values of methane produced by fistulated steers, dairy cattle, and wethers, and dairy and beef cattle herds show a bimodal distribution that appears to be correlated with the plant type (C3 or C4, that is, producing either a three- or a four-carbon acid in the first step of photosynthesis) consumed by the animals. These results indicate that cattle and sheep, on a global basis, release methane with an average delta(13C/12C) value of -60 and -63 per mil, respectively. Together they are a source of atmospheric methane whose delta(13C/12C) is similar to published values for marsh gas and cannot explain the 20 per mil higher values for atmospheric methane.

Animals↗
PubMed2005

Analysis of methane monooxygenase genes in mono lake suggests that increased methane oxidation activity may correlate with a change in methanotroph community structure.

Mono Lake is an alkaline hypersaline lake that supports high methane oxidation rates. Retrieved pmoA sequences showed a broad diversity of aerobic methane oxidizers including the type I methanotrophs Methylobacter (the dominant genus), Methylomicrobium, and Methylothermus, and the type II methanotroph Methylocystis. Stratification of Mono Lake resulted in variation of aerobic methane oxidation rates with depth. Methanotroph diversity as determined by analysis of pmoA using new denaturing gradient gel electrophoresis primers suggested that variations in methane oxidation activity may correlate with changes in methanotroph community composition.

Aerobiosis↗
PubMed1970

Oxygenation of methane by methane-grown Pseudomonas methanica and Methanomonas methanooxidans.

1. Experimental conditions have been found in which small amounts of methanol (approximately 2.5mm) accumulated when washed cell suspensions of methane-grown Pseudomonas methanica and Methanomonas methanooxidans were incubated with methane+oxygen mixtures in Warburg flasks. 2. The methanol formed could be separated completely from water by fractional distillation through glass helices followed by gas chromatography using 20% polyethylene glycol 400 on a Celite 545 support. 3. By using (18)O-enriched oxygen gas the abundance of (18)O in the methanol formed from oxidation of methane was measured with a Perkin-Elmer 270 combined gas chromatograph/mass spectrometer. The results showed that the oxygen in methanol was derived exclusively from gaseous oxygen in both micro-organisms. 4. Control experiments using [(18)O]water in incubation mixtures confirmed that there was negligible incorporation of the oxygen atom from water into methanol.

Bacteria↗