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Particulate methane monooxygenase (pMMO) is a copper-dependent integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. Studies of isolated pMMO have been hindered by loss of enzymatic activity upon its removal from the native membrane. To characterize pMMO in a membrane-like environment, we reconstituted pMMOs from Methylococcus (Mcc.) capsulatus (Bath) and Methylomicrobium (Mm.) alcaliphilum 20Z into bicelles. Reconstitution into bicelles recovers methane oxidation activity lost upon detergent solubilization and purification without substantial alterations to copper content or copper electronic structure, as observed by electron paramagnetic resonance (EPR) spectroscopy. These findings suggest that loss of pMMO activity upon isolation is due to removal from the membranes rather than caused by loss of the catalytic copper ions. A 2.7 Å resolution crystal structure of pMMO from Mm. alcaliphilum 20Z reveals a mononuclear copper center in the PmoB subunit and indicates that the transmembrane PmoC subunit may be conformationally flexible. Finally, results from extended X-ray absorption fine structure (EXAFS) analysis of pMMO from Mm. alcaliphilum 20Z were consistent with the observed monocopper center in the PmoB subunit. These results underscore the importance of studying membrane proteins in a membrane-like environment and provide valuable insight into pMMO function.

Two distinct [Cu-O-Cu]^(2+) sites with methane monooxygenase activity are identified in the zeolite Cu-MOR, emphasizing that this Cu-O-Cu active site geometry, having a ∠Cu-O-Cu ∼140°, is particularly formed and stabilized in zeolite topologies. Whereas in ZSM-5 a similar [Cu-O-Cu]^(2+) active site is located in the intersection of the two 10 membered rings, Cu-MOR provides two distinct local structures, situated in the 8 membered ring windows of the side pockets. Despite their structural similarity, as ascertained by electronic absorption and resonance Raman spectroscopy, the two Cu-O-Cu active sites in Cu-MOR clearly show different kinetic behaviors in selective methane oxidation. This difference in reactivity is too large to be ascribed to subtle differences in the ground states of the Cu-O-Cu sites, indicating the zeolite lattice tunes their reactivity through second-sphere effects. The MOR lattice is therefore functionally analogous to the active site pocket of a metalloenzyme, demonstrating that both the active site and its framework environment contribute to and direct reactivity in transition metal ion-zeolites.

Methanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. When Methylosinus trichosporium OB3b was grown in the presence of 1 μM CuCl 2 , expression of mmoX , encoding a subunit of the hydroxylase component of sMMO, was very low. mmoX expression increased, however, when methanobactin from Methylocystis sp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated with M. trichosporium OB3b. If M. trichosporium OB3b was grown in the absence of CuCl 2 , the mmoX expression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure of Methylosinus trichosporium OB3b to SB2-Mb had no effect on expression of mbnA , encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl 2 . mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl 2 . These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin “piracy” may be commonplace.

Pulping wastewater still contains massive refractory organics after biotreatment, with high colority, low biodegradability, and lasting biotoxicity. To eliminate refractory organics in pulping wastewater, a methanotrophic co-metabolic system in a gas cycle Sequencing Batch Biofilm Reactor (gcSBBR) seeded by soil at a ventilation opening of coal mine was quickly built on the 92nd day. The removal rate of COD, colority and TOC was 53.28%, 50.59% and 51.60%, respectively. Analysis of 3D-EEM indicated that glycolated protein-like, melanoidin-like or lignocellulose-like, and humic acid-like decreased by 7.85%, 5.02% and 1.74%, respectively. Moreover, this system exhibited high activity of soluble methane monooxygenase (sMMO) and mmoX encoding sMMO reached up to 7.89 × 105 copies/μL. Methanotrophs, namely, Methylocaldum (8.28%), Methylococcus (6.06%) and Methylomonas (0.07%), were detected by 16S rRNA sequencing. And other bacteria were dominated by Denitratisoma, Anaerolineaceae_uncultured and Methylophilaceae_uncultured. Refractory organics was biodegraded through the synergy among microorganisms, and a postulated synergy pathway was put forward.

The conversion of methane to methanol is important to economic utilization of natural/shale gas. Methanol is a valuable liquid fuel and raw material for various synthetic hydrocarbon products. Its industrial production is currently based on a two-step process that is energy-intensive and environmentally unfriendly, requiring high pressure and temperature. The biological oxidation of methane to methanol, based on methane monooxygenase activity of methanotrophic bacteria, is desirable because the oxidation is highly selective under mild conditions, but conversion rate and yield and stability of catalytic activity should be improved up to an industrially viable level. Since methanotrophic bacteria produce methanol as only a precursor of formaldehyde that is then used to synthesize various essential metabolites, the direct use of bacteria seems unsuitable for selective production of a large amount of methanol. There are two types of methane monooxygenase: soluble (sMMO) and particulate (pMMO) enzyme. sMMO consisting of three components (reductase, hydroxylase, and regulatory protein) features an (αβγ)2 dimer architecture with a di-iron active site in hydroxlase. pMMO, a trimer (pmoA, pmoB, and pmoC) in an α 3 β 3 γ 3 polypeptide arrangement is a copper enzyme with a di-copper active site located in the soluble domain of pmoB subunit. Since the membrane transports electrons well and delivers effectively methane with increased solubility in the lipid bilayer, pMMO seems more rationally designed enzyme in nature than sMMO. The engineering/evolution/modification of MMO enzymes using various biological and chemical techniques could lead to an optimal way to reach the ultimate goal of technically and economically feasible and environmentally friendly oxidation of methane. For this, multidisciplinary efforts from chemical engineering, protein engineering, and bioprocess research sectors should be systematically combined.

Methanotrophs are aerobic microorganisms that utilize methane as substrates for growth and PHB Biosynthesis. Copper plays an important role in cell growth and PHB biosynthesis. The effect of initial copper concentration on cultivation of M. trichosporium IMV 3011 on methane was investigated. With the addition of 30μmol/L CuSO4•5H2O, PMMO activity improved to 4 times of that without copper addition. The highest density of cultivated cells is 0.48g dry wtL-1, which is 2 times of that without copper addition. The lag time shortened to 15.87h, and the growth rate increased to 0.082h-1.The PHB content increased to 8.3%. It is found that certain initial copper concentration is beneficial to expression of high particulate methane monooxygenase activity, which may contribute to the synthesis of PHB in the cell.

Biocover soil has been demonstrated to have high CH 4 oxidation capacity and is considered as a good alternative cover material to mitigate CH 4 emission from landfills, yet the response of CH 4 oxidation activity of biocover soils to the variation of CH 4 loading during landfill stabilization is poorly understood. Compared with a landfill cover soil (LCS) collected from Hangzhou Tianziling landfill cell, the development of CH 4 oxidation activity of waste biocover soil (WBS) was investigated using simulated landfill systems in this study. Although a fluctuation of influent CH 4 flux occurred during landfill stabilization, the WBS covers showed a high CH 4 removal efficiency of 94–96% during the entire experiment. In the LCS covers, the CH 4 removal efficiencies varied with the fluctuation of CH 4 influent flux, even negative ones occurred due to the storage of CH 4 in the soil porosities after the high CH 4 influent flux of ∼137 g m −2 d −1 . The lower concentrations of O 2 and CH 4 as well as the higher concentration of CO 2 were observed in the WBS covers than those in the LCS covers. The highest CH 4 oxidation rates of the two types of soil covers both occurred in the bottom layer (20–30 cm). Compared to the LCS, the WBS showed higher CH 4 oxidation activity and methane monooxygenase activity over the course of the experiment. Overall, this study indicated the WBS worked well for the fluctuation of CH 4 influent flux during landfill stabilization.

Methanotrophs have promising applications in the epoxidation of some alkenes and some chlorinated hydrocarbons and in the production of a biopolymer, poly-beta-hydroxybutyrate (poly-3-hydroxybutyrate; PHB). In contrast with methane monooxygenase (MMO) activity and ability of PHB synthesis of four kinds of methanotrophic bacteria Methylosinus trichosporium OB3b, M. trichosporium IMV3011, Methylococcus capsulatus HD6T, Methylomonas sp. GYJ3, and the mixture of the four kinds of strains, M. trichosporium OB3b is the highest of the four in the activity of propene epoxidation (10.72 nmol/min mg dry weight of cell [dwc]), the activity of naphthalene oxidation (22.7 mmol/mg dwc), and ability in synthesis of PHB(11% PHB content in per gram dry weight of cell in 84 h). It could be feasible to improve the MMO activity by mixing four kinds of methanotrophs. The MMO activity dramatically decreased when the cellular PHB accumulated in the second stage. The reason for this may be the dilution of the MMO system in the cells with increasing PHB contents. It has been found that the PHB contents at the level of 1-5% are beneficial to the cells for maintenance of MMO epoxidation activity when enough PHB have been accumulated. Moreover, it was also found that high particulate methane monooxygenase activity may contribute to the synthesis of PHB in the cell, which could be used to improve the yield of PHB in methanotrophs.

Background Methane-utilizing bacteria (methanotrophs) are capable of growth on methane and are attractive systems for bio-catalysis. However, the application of natural methanotrophic strains to large-scale production of value-added chemicals/biofuels requires a number of physiological and genetic alterations. An accurate metabolic model coupled with flux balance analysis can provide a solid interpretative framework for experimental data analyses and integration. Results A stoichiometric flux balance model of Methylomicrobium buryatense strain 5G(B1) was constructed and used for evaluating metabolic engineering strategies for biofuels and chemical production with a methanotrophic bacterium as the catalytic platform. The initial metabolic reconstruction was based on whole-genome predictions. Each metabolic step was manually verified, gapfilled, and modified in accordance with genome-wide expression data. The final model incorporates a total of 841 reactions (in 167 metabolic pathways). Of these, up to 400 reactions were recruited to produce 118 intracellular metabolites. The flux balance simulations suggest that only the transfer of electrons from methanol oxidation to methane oxidation steps can support measured growth and methane/oxygen consumption parameters, while the scenario employing NADH as a possible source of electrons for particulate methane monooxygenase cannot. Direct coupling between methane oxidation and methanol oxidation accounts for most of the membrane-associated methane monooxygenase activity. However the best fit to experimental results is achieved only after assuming that the efficiency of direct coupling depends on growth conditions and additional NADH input (about 0.1–0.2 mol of incremental NADH per one mol of methane oxidized). The additional input is proposed to cover loss of electrons through inefficiency and to sustain methane oxidation at perturbations or support uphill electron transfer. Finally, the model was used for testing the carbon conversion efficiency of different pathways for C1-utilization, including different variants of the ribulose monophosphate pathway and the serine cycle. Conclusion We demonstrate that the metabolic model can provide an effective tool for predicting metabolic parameters for different nutrients and genetic perturbations, and as such, should be valuable for metabolic engineering of the central metabolism of M. buryatense strains. Electronic supplementary material The online version of this article (doi:10.1186/s12934-015-0377-3) contains supplementary material, which is available to authorized users.

The effect of chelated and soil-adsorbed copper on whole-cell soluble methane monooxygenase activity in Methylosinus trichosporium OB3b was measured to assess the bioavailability of these forms of copper. Strongly binding chelating agents (Log K > 16) added to the growth medium at a concentration of 20 μM were able to reduce copper bioavailability. As these chelating agents limited equilibrium binding of copper by cell-produced copper binding ligands and limited accumulation by passive uptake mechanisms, it appears the ability of the cells' copper uptake system to sequester copper in a bioavailable form is compromised by these competing ligands. Copper sorbed as surface complexes on montmorillonite clay, γ-alumina, ferrihydrite, and goethite were bioavailable. However, with increasing concentration of surface sites, bioavailability decreased, indicating the competitive advantage for copper binding provided by high concentrations of binding sites limited the ability of the cells' copper uptake system to ac...

Soluble methane monooxygenase (sMMO) activity in Methylosinus trichosporium OB3b was found to be more strongly affected as copper-to-biomass ratios changed in a newly developed medium, M2M, which uses pyrophosphate for metal chelation, than in nitrate mineral salts (NMS), which uses EDTA. When M2M medium was amended with EDTA, sMMO activity was similar to that in NMS medium, indicating that EDTA-bound copper had lower bioavailability than pyrophosphate-bound copper. EDTA did not limit the association of copper with the cells; rather, copper was sequestered in a form which did not affect sMMO activity.

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.

Using cell recycle, the concentration of M. trichosporium OB3b was increased to more than 12g/L in continuous culture and biomass productivity was enhanced up to 0.708g/L/h. The optimal temperature and pH for soluble MMO activity were 35°C and 6.2–6.4. The Optimal concentrations of methanol and sodium formate, as cofactor regenerators, were 0.5mM and 10 mM, respectively, at which the soluble MMO activity was about 8 times enhanced.

Reconstituted whole-cell preparations of lyophilized Methylosinus trichosporium OB3b were used to demonstrate soluble methane monooxygenase activity in a two-phase (biphasic) matrix consisting of a buffered aqueous phase and 2,2,4-trimethylpentane (isooctane). The rate of conversion of gaseous propylene to propylene oxide, a non-metabolized liquid, was used as the primary measure of enzyme activity. Appreciable soluble methane monooxygenase activity was detected when the volume of the aqueous phase represented at least 1% of the total volume, although the initial rate of product formation did increase as the volume of the aqueous phase increased. In comparison to the aqueous system, the specific rate and yields in the biphasic system were much less sensitive to increases in the concentrations of formate and protein (the methane monooxygenase). However, there was some evidence that the enzyme system was more stable in the biphasic matrix, since the rate of propylene oxide formation remained linear for an extended period of time. V(app.) in the biphasic system decreased by a factor of 0.6 relative to the same parameter in the aqueous system. Conversely, Km(app.) for propylene was 1.6 times greater in the biphasic system. Hence, the apparent catalytic efficiency in the aqueous system was four times that in the biphasic system, as indicated by a decrease in the corresponding ratios of V(app.) to Km(app.).

The effect of copper supplementation on growth, methane monooxygenase activity and lipid composition of Methylococcus capsulatus (Bath) was studied. Copper increased biomass yield, methane monooxygenase activity and phospholipid content from 7.7 to 10.2% of dry weight. Cells from copper-deficient and copper supplemented cultures contained the same major fatty acids but in the presence of copper only the contents of C16:0 and the three C16:1 isomers were increased while the contents of C14:0 and cyclic C17:0 remained unchanged. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol and cardiolipin were analysed amongst the polar lipids. PE was the main component (about 60 mol-%) but the most notable copper-induced increment occurred in the proportion of PC, from about 10 to 16 mol-%. Concomitantly with this increment the fatty acids of PC were changed so that the mol-% of C16: 1 isomers were increased at the expense of other acids. Similar trends were seen also in the fatty acid compositions of other polar lipid fractions. It is therefore concluded that phosphatidylcholine would be one of the key factors when the role of membranous lipids in methane monooxygenase activity is to be considered.

A straightforward method is described for screening methanotrophic colonies for soluble methane monooxygenase (sMMO) activity on solid media. Such activity results in the development of a colored complex between 1-naphthol, which is formed when sMMO reacts with naphthalene, and o-dianisidine (tetrazotized). Methanotrophic colonies expressing sMMO turned deep purple when exposed successively to naphthalene and o-dianisidine. The method was evaluated within the contexts of two potential applications. The first was for the enumeration of Methylosinus trichosporium OB3b in a methane-amended, unsaturated soil column dedicated to vinyl chloride treatment. The second application was for the isolation and enumeration of sMMO-bearing methanotrophs from sanitary landfill soils. The technique was effective in both applications.

Methylococcus capsulatus (Bath) possesses methane monooxygenase (MMO) which catalyses the epoxidation of propylene to propylene oxide. MMO activity could be maintained in whole cells by storage in unagitated vessels for several days. However if these cells were agitated and aerated in the absence of a carbon and energy source then 80% of the propylene-oxidizing activity Was lost within 24 h. It was shown that this loss of activity was due to the inability of the cells to provide energy to drive the oxidation process rather than the loss of MMO activity per se. If propylene oxide was added to these aerated cells then the rate of inactivation was increased and 50% of the activity was lost over a 10 min period. The addition of an exogenous energy source caused a doubling in the rate of inactivation. These marked increases in the rates of inactivation in the presence of propylene oxide were found to be caused by the loss of the methane monooxygenase activity per se rather than a further loss of the energy-pro...

Addition of copper to the medium for Methylomonas albus BG8 increased cell yield and methane monooxygenase activity. Intracytoplasmic membrane was formed only in cells grown with copper supplementation. Additionally, the abundances of two major membrane proteins were affected by copper in the growth medium. These findings indicate that effects of copper on the physiology of methanotrophic bacteria are not limited to those on types II and X.

Copper plays a key role in the physiology of methanotrophs. One way that these bacteria meet their high copper requirement is by the biosynthesis and release of high affinity copper binding compounds called methanobactins. Recent advances in methanobactin characterization include the first crystal structure, detailed spectroscopic analyses, and studies of metal ion specificity. Methanobactin may function in copper uptake, regulation of methane monooxygenase expression, protection against copper toxicity, and particulate methane monooxygenase activity. Methanobactin can extract copper from insoluble minerals and could be important for mineral weathering. Many methanobactin properties are reminiscent of iron siderophores, suggesting a similar mechanism of handling. Methanobactin-like compounds have also been identified in yeast mitochondria, suggesting that these molecules are a more universal phenomenon.

Methylosinus trichosporium OB3b biosynthesizes a broad specificity soluble methane monooxygenase that rapidly oxidizes trichloroethylene (TCE). The selective expression of the soluble methane monooxygenase was followed in vivo by a rapid colorimetric assay. Naphthalene was oxidized by purified soluble methane monooxygenase or by cells grown in copper-deficient media to a mixture of 1-naphthol and 2-naphthol. The naphthols were detected by reaction with tetrazotized o-dianisidine to form purple diazo dyes with large molar absorptivities. The rate of color formation with the rapid assay correlated with the velocity of TCE oxidation that was determined by gas chromatography. Both assays were used to optimize conditions for TCE oxidation by M. trichosporium OB3b and to test several methanotrophic bacteria for the ability to oxidize TCE and naphthalene.

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.

Addition of nitrapyrin, allylthiourea, C(inf2)H(inf2), and CH(inf3)F to freshwater sediment slurries inhibited CH(inf4) oxidation and nitrification to similar extents. Dicyandiamide and allylsulfide were less inhibitory for CH(inf4) oxidation than for nitrification. Allylsulfide was the most potent inhibitor of nitrification, and the estimated 50% inhibitory concentrations for this process and CH(inf4) oxidation were 0.2 and 121 (mu)M, respectively. At a concentration of 2 (mu)M allylsulfide, growth and CH(inf4) oxidation activity of Methylosinus trichosporium OB3b were not inhibited. Allylsulfide at 200 (mu)M inhibited the growth of M. trichosporium by approximately 50% but did not inhibit CH(inf4) oxidation activity. Nitrite production by cells of M. trichosporium was not significantly affected by allylsulfide, except at a concentration of 2 mM, when growth and CH(inf4) oxidation were also inhibited by about 50%. Methane monooxygenase activity present in soluble fractions of M. trichosporium was not inhibited significantly by allylsulfide at either 200 (mu)M or 2 mM. These results suggest that the partial inhibition of CH(inf4) oxidation in sediment slurries by high allylsulfide concentrations may be caused by an inhibition of the growth of methanotrophs rather than an inhibition of methane monooxygenase activity specifically. We conclude that allylsulfide is a promising tool for the study of interactions of methanotrophs and nitrifiers in N cycling and CH(inf4) turnover in natural systems.

Quinols can provide reducing equivalents for the membrane-bound form of methane monooxygenase (pMMO), substituting for NADH in whole cells and membranes. Furthermore, quinols are effective reductants for the detergent-solubilized enzyme, whereas NADH is ineffective. The decyl analog of plastoquinol and duroquinol (2,3,5,6-tetramethylbenzoquinol) provide the greatest methane monooxygenase activity in whole cells and membrane suspensions, as well as detergent-solubilized samples. Lauryl maltoside is by far the best detergent for solubilization of catalytically active methane monooxygenase. Optimal pMMO activity in the detergent-solubilized fraction is obtained with a ratio of approximately 1.7 mg of detergent per milligram of membrane protein, independent of protein concentration. The detergent-solubilized pMMO retains its sensitivity to inhibition by cyanide, acetylene, and EDTA. It is also stimulated by exogenous copper, as in isolated membrane fractions. Reaction of the detergent-solubilized enzyme with [14C]acetylene results in labeling of a 26-kDa peptide, analogous to the behavior observed for isolated membrane suspensions. The selectivity of pMMO for duroquinol and decyl-plastoquinol, relative to other structurally similar quinols, suggests that the enzyme obtains reducing equivalents directly from a quinol (probably plastoquinol) in vivo.

Batch culture conditions were established for the formation of H(2)-driven whole-cell soluble or particulate methane monooxygenase (sMMO or pMMO) activity in the obligate methanotroph, Methylosinus trichosporum Ob3b, to expand its potential uses in groundwater bioremediation and the production of specific chemicals. Addition of either Ni and H(2) to a nitrate-containing minimal salts growth medium or Ni and Mo to a nitrate-lacking growth medium (induces a nitrogenase that generates intracellular H(2)) markedly enhanced both the hydrogenase and the accompanying washed-cell H(2)-driven MMO activities of shake-flask cultured cells. For sMMO containing cells, H(2) provided in vitro reducing power for the oxidation of chlorinated solvents such as chloroform and trichloroethylene. Cell cultivations under N(2)-fixing conditions in a 5-L bioreactor, however, required an initial nitrate concentration of at least 1 to 2 mM to achieve high biomass yields (5 to 7 g of dry cell wt/L) for cells producing H(2)-driven sMMO or pMMO activity. Elevation of the initial medium nitrate concentration to 20 mM shortened the culture time for pMMO producing cells by 40%, yet still generated an equivalent growth yield. High nitrate also shortened the culture time for sMMO containing cells by approximately 25%, but it lowered their biomass yield by 26%. Upon storage for 5 weeks at room temperature, washed resting-state cells retained 90% and 70% of their H(2)-driven sMMO and pMMO activity, respectively. This makes their practical use quite feasible. (c) 1995 John WileySons, Inc.

The role of the storage lipid poly-β-hydroxybutyrate (PHB) in trichloroethylene transformation by methanotrophic mixed cultures was investigated. Naphthalene oxidation rates were used to assay for soluble methane monooxygenase activity. The PHB content of methanotrophic cells grown in reactors varied diurnally as well as from day to day. A positive correlation between the amount of PHB in the cells and the naphthalene oxidation rate as well as between PHB and the trichloroethylene transformation rate and capacity was found. Addition of β-hydroxybutyrate increased the naphthalene oxidation rates significantly. PHB content in cells could be manipulated by incubation at different methane-to-nitrogen ratios. A positive correlation between the naphthalene oxidation rate and the PHB content after these incubations could be seen. Both the PHB content and the naphthalene oxidation rates decreased with time in resting methanotrophic cells exposed to oxygen. However, this decrease in the naphthalene oxidation rate cannot be explained by the decrease in the PHB content alone. Probably a deactivation of the methane monooxygenase itself is also involved.

An anaerobic procedure was developed for the purification of the flavin:NADH oxidoreductase (flavoprotein) component of methane monooxygenase to homogeneity. The molecular weight of the flavoprotein determined by gel filtration was about 40,000, and by sedimentation equilibrium analysis, about 38,000. The purified flavoprotein is a monomeric protein with a sedimentation constant ( S 20W ) value of about 2.1 S. The absorption spectrum of the flavoprotein has a peak at 460 nm and shoulder at 395 nm. The fluorescent excitation and emission spectra of the fluorescent component of flavoprotein had peaks at 450, 370, and 530 nm, respectively. A FAD was identified as a prosthetic group of flavoprotein by thin-layer chromatography. The flavoprotein contained about 1 mol of FAD and 2 mol each of iron and acid-labile sulfide per mole of protein. The flavoprotein was directly reduced by NADH under anaerobic conditions. The formation of neutral flavin semiquinone was detected during anaerobic titration of flavoprotein by NADH and also as a free radical signal at a g value of 2.004 by EPR spectroscopy. The iron sulfur cluster has g values of 2.04, 1.96, and 1.87, yielding a g average of 1.96, characteristic of a Fe 2 S 2 center. Antibody prepared against the flavoprotein reacted with flavoprotein and inhibited methane monooxygenase 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.

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.

1 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. 2 Removal and reconstitution of the redox centres of component C suggests 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. 3 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. 4 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 monooxygenase activity, suggesting that the role of component B is to couple substrate oxidation to electron transfer, via the methane monooxygenase components.

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°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.

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.

SUMMARY More than 100 Gram-negative, strictly aerobic, methane-utilizing bacteria were isolated. All used only methane and methanol of the substrates tested for growth. The organisms were classified into five groups on the basis of morphology, fine structure, and type of resting stage formed (exospores and different types of cysts) and into subgroups on other properties. Methods of enrichment, isolation and culture are described.

1. Methane mono-oxygenase of Methylococcus capsulatus (Bath) catalyses the oxidation of various substituted methane derivatives including methanol. 2. It is a very non-specific oxygenase and, in some of its catalytic properties, apparently resembles the analogous enzyme from Methylomonas methanica but differs from those found in Methylosinus trichosporium and Methylomonas albus. 3. CO is oxidized to CO2. 4. C1-C8 n-alkanes are hydroxylated, yielding mixtures of the corresponding 1- and 2-alcohols; no 3- or 4-alcohols are formed. 5. Terminal alkenes yield the corresponding 1,2-epoxides. cis- or trans-but-2-ene are each oxidized to a mixture of 2,3-epoxybutane and but-2-en-1-ol with retention of the cis or trans configuration in both products; 2-butanone is also formed from cis-but-2-ene only. 6. Dimethyl ether is oxidized. Diethyl ether undergoes sub-terminal oxidation, yielding ethanol and ethanal in equimolar amounts. 7. Methane mono-oxygenase also hydroxylates cyclic alkanes and aromatic compounds. However, styrene yields only styrene epoxide and pyridine yields only pyridine N-oxide. 8. Of those compounds tested, only NADPH can replace NADH as electron donor.

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 α-keto glutarate dehydrogenase, relative to the levels found during growth on succinate.

Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Zinc is a known inhibitor of pMMO, but the details of zinc binding and the mechanism of inhibition are not understood. Metal binding and activity assays on membrane-bound pMMO from Methylococcus capsulatus (Bath) reveal that zinc inhibits pMMO at two sites that are distinct from the copper active site. The 2.6 Å resolution crystal structure of Methylocystis species strain Rockwell pMMO reveals two previously undetected bound lipids, and metal soaking experiments identify likely locations for the two zinc inhibition sites. The first is the crystallographic zinc site in the pmoC subunit, and zinc binding here leads to the ordering of 10 previously unobserved residues. A second zinc site is present on the cytoplasmic side of the pmoC subunit. Parallels between these results and zinc inhibition studies of several respiratory complexes suggest that zinc might inhibit proton transfer in pMMO.