Membrane-bound Methane Monooxygenase Research Articles

Methylacidimicrobium thermophilum AP8, a Novel Methane- and Hydrogen-Oxidizing Bacterium Isolated From Volcanic Soil on Pantelleria Island, Italy.

The Favara Grande is a geothermal area located on Pantelleria Island, Italy. The area is characterized high temperatures in the top layer of the soil (60°C), low pH (3–5) and hydrothermal gas emissions mainly composed of carbon dioxide (CO2), methane (CH4), and hydrogen (H2). These geothermal features may provide a suitable niche for the growth of chemolithotrophic thermoacidophiles, including the lanthanide-dependent methanotrophs of the phylum Verrucomicrobia. In this study, we started enrichment cultures inoculated with soil of the Favara Grande at 50 and 60°C with CH4 as energy source and medium containing sufficient lanthanides at pH 3 and 5. From these cultures, a verrucomicrobial methanotroph could be isolated via serial dilution and floating filters techniques. The genome of strain AP8 was sequenced and based on phylogenetic analysis we propose to name this new species Methylacidimicrobium thermophilum AP8. The transcriptome data at μmax (0.051 ± 0.001 h−1, doubling time ~14 h) of the new strain showed a high expression of the pmoCAB2 operon encoding the membrane-bound methane monooxygenase and of the gene xoxF1, encoding the lanthanide-dependent methanol dehydrogenase. A second pmoCAB operon and xoxF2 gene were not expressed. The physiology of strain AP8 was further investigated and revealed an optimal growth in a pH range of 3–5 at 50°C, representing the first thermophilic strain of the genus Methylacidimicrobium. Moreover, strain AP8 had a KS(app) for methane of 8 ± 1 μM. Beside methane, a type 1b [NiFe] hydrogenase enabled hydrogen oxidation at oxygen concentrations up to 1%. Taken together, our results expand the knowledge on the characteristics and adaptations of verrucomicrobial methanotrophs in hydrothermal environments and add a new thermophilic strain to the genus Methylacidimicrobium.

Native top-down mass spectrometry provides insights into the copper centers of membrane-bound methane monooxygenase

Aerobic methane oxidation is catalyzed by particulate methane monooxygenase (pMMO), a copper-dependent, membrane metalloenzyme composed of subunits PmoA, PmoB, and PmoC. Characterization of the copper active site has been limited by challenges in spectroscopic analysis stemming from the presence of multiple copper binding sites, effects of detergent solubilization on activity and crystal structures, and the lack of a heterologous expression system. Here we utilize nanodiscs coupled with native top-down mass spectrometry (nTDMS) to determine the copper stoichiometry in each pMMO subunit and to detect post-translational modifications (PTMs). These results indicate the presence of a mononuclear copper center in both PmoB and PmoC. pMMO-nanodisc complexes with a higher stoichiometry of copper-bound PmoC exhibit increased activity, suggesting that the PmoC copper site plays a role in methane oxidation activity. These results provide key insights into the pMMO copper centers and demonstrate the ability of nTDMS to characterize complex membrane-bound metalloenzymes.

Characterization of the role of copCD in copper uptake and the 'copper-switch' in Methylosinus trichosporium OB3b.

Methanotrophs or methane-oxidizing bacteria exhibit a unique 'copper-switch' where expression of two forms of methane monooxygenase (MMO) is controlled by the availability of copper. In the absence of copper, a cytoplasmic or soluble methane monooxygenase (sMMO) is expressed. In the presence of copper, a membrane-bound or particulate methane monooxygenase (pMMO) is expressed. These two forms of MMO have very different properties, and elucidation of the basis of the copper-switch is of significant interest as methanotrophs are becoming increasingly popular for the valorization of methane. Recently, it was suggested via characterization of a mutant of Methylosinus trichosporium OB3b that expresses sMMO in the presence of copper (smmoC mutant) that the copper-switch may be based on copCD. These genes encode for a periplasmic copper-binding protein and an inner membrane protein, respectively, and are used by other bacteria for copper uptake. Specific knockouts of copCD in M. trichosporium OB3b wild type, however, show that these genes are not part of the copper-switch in methanotrophs, nor do they appear to be critical for copper uptake. Rather, it appears that the constitutive expression of sMMO in the smmoC mutant of M. trichosporium OB3b may be due to multiple lesions as smmoC was generated via random chemical mutagenesis.

A four-helix bundle stores copper for methane oxidation.

Methane-oxidising bacteria (methanotrophs) require large quantities of copper for the membrane-bound (particulate) methane monooxygenase (pMMO)1,2. Certain methanotrophs are also able to switch to using the iron-containing soluble MMO (sMMO) to catalyse methane oxidation, with this switchover regulated by copper3,4. MMOs are Nature’s primary biological mechanism for suppressing atmospheric levels of methane, a potent greenhouse gas. Furthermore, methanotrophs and MMOs have enormous potential in bioremediation and for biotransformations producing bulk and fine chemicals, and in bioenergy, particularly considering increased methane availability from renewable sources and hydraulic fracturing of shale rock5,6. We have discovered and characterised a novel copper storage protein (Csp1) from the methanotroph Methylosinus trichosporium OB3b that is exported from the cytosol, and stores copper for pMMO. Csp1 is a tetramer of 4-helix bundles with each monomer binding up to 13 Cu(I) ions in a previously unseen manner via mainly Cys residues that point into the core of the bundle. Csp1 is the first example of a protein that stores a metal within an established protein-folding motif. This work provides a detailed insight into how methanotrophs accumulate copper for the oxidation of methane. Understanding this process is essential if the wide-ranging biotechnological applications of methanotrophs are to be realised. Cytosolic homologues of Csp1 are present in diverse bacteria thus challenging the dogma that such organisms do not use copper in this location.

Methanobactin from Methylocystis sp. strain SB2 affects gene expression and methane monooxygenase activity in Methylosinus trichosporium OB3b.

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 CuCl2, 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 CuCl2, 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 CuCl2. mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl2. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin "piracy" may be commonplace.

Getting the metal right

Controversy has raged over the identity of the metal cofactor of membrane-bound methane monooxygenase, a methane-oxidizing enzyme. A study suggests that the answer is a cluster of two copper ions.

Controlled Oxidation of Hydrocarbons by the Membrane-Bound Methane Monooxygenase: The Case for a Tricopper Cluster

[Figure: see text]. The growing need for inexpensive methods to convert methane to methanol has sparked considerable interest in methods that catalyze this process. The integral membrane protein particulate methane monooxygenase (pMMO) mediates the facile conversion of methane to methanol in methanotrophic bacteria. Most evidence indicates that pMMO is a multicopper enzyme, and these copper ions support redox, dioxygen, and oxo-transfer chemistry. However, the exact identity of the copper species that mediates the oxo-transfer chemistry remains an area of intense debate. This highly complex enzyme is notoriously difficult to purify because of its instability outside the lipid bilayer and tendency to lose its essential metal cofactors. For this reason, pMMO has resisted both initial identification and subsequent isolation and purification for biochemical and biophysical characterization. In this Account, we describe evidence that pMMO is a multicopper protein. Its unique trinuclear copper cluster mediates dioxygen chemistry and O-atom transfer during alkane hydroxylation. Although a recent crystal structure did not show this tricopper cluster, we provide compelling evidence for such a cluster through redox potentiometry and EPR experiments on the "holo" enzyme in pMMO-enriched membranes. We also identify a site in the structure of pMMO that could accommodate this cluster. A hydrophobic pocket capable of harboring pentane, the enzyme's largest known substrate, lies adjacent to this site. In addition, we have designed and synthesized model tricopper clusters to provide further chemical evidence that a tricopper cluster mediates the enzyme's oxo-transfer chemistry. These biomimetic models exhibit similar spectroscopic properties and chemical reactivity to the putative tricopper cluster in pMMO. Based on computational analysis using density functional theory (DFT), triangular tricopper clusters are capable of harnessing a "singlet oxene" upon activation by dioxygen. An oxygen atom is then inserted via a concerted process into the C-H bond of an alkane in the transition state during hydroxylation. The turnover frequency and kinetic isotope effect predicted by DFT show excellent agreement with experimental data.

Purification and properties of membrane-bound methane hydroxylase from Methylococcus capsulatus (Strain M)

Membrane fraction of Methylococcus capsulatus (strain M) were treated with [14C]acetylene, an affinity label binding to the active center of membrane-bound methane monooxygenase (MMO). High-purity particulate form of methane hydroxylase (pMH) was obtained by ion exchange and hydrophobic chromatography. According to SDS-PAGE data, the enzyme contained three polypeptides with molecular weights of 47 (alpha), 27 (beta), and 25 (gamma) kDa in the ratio 1:1:1. The radiolabel was contained in the beta-subunit of pMH. The protein contained 1 or 2 atoms of nonheme iron and 2-4 atoms of copper per a minimum molecular weight of 99 kDa. This protein did not oxidize methane or propylene in the presence of NADH but was able to oxidize low quantities of methane in the presence of duroquinol. It was established that methanol dehydrogenase (MD) and NADH oxidoreductase (NADH-OR) are peripheral membrane proteins. Possible causes of low activity of high-purity methane hydroxylase are discussed.

Methanotrophy below pH 1 by a new Verrucomicrobia species

Mud volcanoes, mudpots and fumaroles are remarkable geological features characterized by the emission of gas, water and/or semi-liquid mud matrices with significant methane fluxes to the atmosphere (10(-1) to 10(3) t y(-1)). Environmental conditions in these areas vary from ambient temperature and neutral pH to high temperatures and low pH. Although there are strong indications for biological methane consumption in mud volcanoes, no methanotrophic bacteria are known that would thrive in the hostile conditions of fumaroles (temperatures up to 70 degrees C and pH down to 1.8). The first step in aerobic methane oxidation is performed by a soluble or membrane-bound methane mono-oxygenase. Here we report that pmoA (encoding the beta-subunit of membrane-bound methane mono-oxygenase) clone libraries, made by using DNA extracted from the Solfatara volcano mudpot and surrounding bare soil near the fumaroles, showed clusters of novel and distant pmoA genes. After methanotrophic enrichment at 50 degrees C and pH 2.0 the most distant cluster, sharing less than 50% identity with any other described pmoA gene, was represented in the culture. Finally we isolated an acidiphilic methanotrophic bacterium Acidimethylosilex fumarolicum SolV belonging to the Planctomycetes/Verrucomicrobia/Chlamydiae superphylum, 'outside' the subphyla of the Alpha- and Gammaproteobacteria containing the established methanotrophs. This bacterium grows under oxygen limitation on methane as the sole source of energy, down to pH 0.8--far below the pH optimum of any previously described methanotroph. A. fumarolicum SolV has three different pmoA genes, with two that are very similar to sequences retrieved from the mudpot. Highly homologous environmental 16S rRNA gene sequences from Yellowstone Park show that this new type of methanotrophic bacteria may be a common inhabitant of extreme environments. This is the first time that a representative of the widely distributed Verrucomicrobia phylum, of which most members remain uncultivated, is coupled to a geochemically relevant reaction.

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.

Regulation of methane oxidation in the facultative methanotroph Methylocella silvestris BL2

The molecular regulation of methane oxidation in the first fully authenticated facultative methanotroph Methylocella silvestris BL2 was assessed during growth on methane and acetate. Problems of poor growth of Methylocella spp. in small-scale batch culture were overcome by growth in fermentor culture. The genes encoding soluble methane monooxygenase were cloned and sequenced, which revealed that the structural genes for soluble methane monooxygenase, mmoXYBZDC, were adjacent to two genes, mmoR and mmoG, encoding a sigma54 transcriptional activator and a putative GroEL-like chaperone, located downstream (3') of mmoC. Transcriptional analysis revealed that the genes were all cotranscribed from a sigma54-dependent promoter located upstream (5') of mmo X. The transcriptional start site was mapped. Transcriptional analysis of soluble methane monooxygenase genes and expression studies on fermentor grown cultures showed that acetate repressed transcription of sMMO in M. silvestris BL2. The possibility of the presence of a particulate, membrane-bound methane monooxygenase enzyme in M. silvestris BL2 and the copper-mediated regulation of soluble methane monooxygenase was investigated. Both were shown to be absent. A promoter probe vector was constructed and used to assay transcription of the promoter of the soluble methane monoxygenase genes of M. silvestris BL2 grown under various conditions and with different substrates. These data represent the first insights into the molecular physiology of a facultative methanotroph.

Aerobic Methanotrophic Communities in the Bottom Sediments of Lake Baikal

The results of the first methodical investigation into the aerobic methanotrophic communities inhabiting the bottom sediments of Lake Baikal are reported. Use of the radioisotopic method revealed methane consumption in 12 10- to 50-cm-long sediment cores. The maximum methane consumption rates (495-737 microl/(dm3 day) were recorded in sediments in the regions of hydrothermal vents and oil and gas occurrence. Methane consumption was most active in the surface layers of the sediments (0-4 cm); it decreased with the sediment depth and became negligible or absent at depths below 20 cm. The number of methanotrophic bacteria usually ranged from 100 to 1000 cells/cm3 of sediment and reached 1 million cells/cm3 in the regions of oil and gas occurrence. The 17 enrichment cultures obtained were represented mainly by morphotype II methanotrophs. Phylogenetic analysis of the enrichment cultures in terms of the amino acid sequence of the alpha subunit of the membrane-bound methane monooxygenase revealed the predominance of methanotrophs of the genus Methylocystis. The results obtained suggest the presence of an active aerobic methanotrophic community in Lake Baikal.

New Thermophilic Methanotrophs of the Genus Methylocaldum

Two pure cultures of obligate methanotrophs, strains H-11 and 0-12, growing in the temperature range from 30 to 61 degrees C with an optimum at 55 degrees C were isolated from samples of silage and manure. Based on the results of analysis of the 16S rRNA genes, membrane-bound methane monooxygenase, and phenotypic properties, the isolates were assigned to the genus Methylocaldum. Significant temperature-dependent variations in morphology and phospholipid and fatty acid composition were revealed. Both strains assimilated methane carbon via the ribulose monophosphate, serine, and ribulose bisphosphate pathways. The activity of hexulose phosphate synthase was independent of the cultivation temperature; however, the activities of hydroxypyruvate reductase and ribulose bisphosphate carboxylase were higher in cells grown at 55 degrees C that in cells grown at 37 degrees C, indicating the important roles of the serine and ribulose bisphosphate pathways in the thermoadaptation of the strains under study. NH4+ assimilation occurred through reductive amination of alpha-ketoglutarate and via the glutamate cycle. The relationship between the physiological-biochemical peculiarities of the isolates and their thermophilic nature is discussed.

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.

A continuous-wave electron-nuclear double resonance (X-band) study of the Cu2+ sites of particulate methane mono-oxygenase of Methylococcus capsulatus (strain M) in membrane and pure dopamine beta-mono-oxygenase of the adrenal medulla.

All methanotrophic bacteria express a membrane-bound (particulate) methane mono-oxygenase (pMMO). In the present study, we have investigated pMMO in membrane fragments from Methylococcus capsulatus (strain M). pMMO contains a typical type-2 Cu(2+) centre with the following EPR parameters: g(z) 2.24, g(x,y) 2.06, A(Cu)(z) 19.0 mT and A(Cu)(x,y) 1.0 mT. Simulation of the Cu(2+) spectrum yielded a best match by using four equivalent nitrogens (A(N)=1.5 mT, 42 MHz). Incubation with ferricyanide neither changed nor increased the amount of EPR-active Cu(2+), in contrast with other reports. The EPR visible copper seems not to be part of any cluster, as judged from the microwave power saturation behaviour. Continuous-wave electron-nuclear double resonance (CW ENDOR; 9.4 GHz, 5-20 K) experiments at g( perpendicular) of the Cu(II) spectrum show a weak coupling to protons with an A(H) of 2.9 MHz that corresponds to a distance of 3.8 A (1 A identical with 0.1 nm), assuming that it is a purely dipolar coupling. Incubation in (2)H(2)O leads to a significant decrease in these (1)H-ENDOR intensities, showing that these protons are exchangeable. This result strongly suggests that the EPR visible copper site of pMMO is accessible to solvent, which was confirmed by the chelation of the Cu(2+) by diethyldithiocarbamic acid. The (1)H and (14)N hyperfine coupling constants confirm a histidine ligation of the EPR visible copper site in pMMO. The hyperfine structure in the ENDOR or EPR spectra of pMMO is not influenced by the inhibitors azide, cyanide or ammonia, indicating that they do not bind to the EPR visible copper. We compared pMMO with the type-2 Cu(2+) enzyme, dopamine beta-mono-oxygenase (DbetaM). For DbetaM, it is assumed that the copper site is solvent-accessible. CW ENDOR shows similar weakly coupled and (2)H(2)O-exchangeable protons (2.9 MHz), as observed in pMMO, as well as the strongly coupled nitrogens (40 MHz) from the co-ordinating N of the histidines in DbetaM. In conclusion, the resting EPR visible Cu in pMMO is not part of a trinuclear cluster, as has been suggested previously.

A continuous-wave electron–nuclear double resonance (X-band) study of the Cu2+ sites of particulate methane mono-oxygenase of Methylococcus capsulatus (strain M) in membrane and pure dopamine β-mono-oxygenase of the adrenal medulla

All methanotrophic bacteria express a membrane-bound (particulate) methane mono-oxygenase (pMMO). In the present study, we have investigated pMMO in membrane fragments from Methylococcus capsulatus (strain M). pMMO contains a typical type-2 Cu2+ centre with the following EPR parameters: gz 2.24, gx,y 2.06, ACuz 19.0mT and ACux,y 1.0mT. Simulation of the Cu2+ spectrum yielded a best match by using four equivalent nitrogens (AN = 1.5mT, 42MHz). Incubation with ferricyanide neither changed nor increased the amount of EPR-active Cu2+, in contrast with other reports. The EPR visible copper seems not to be part of any cluster, as judged from the microwave power saturation behaviour. Continuous-wave electron—nuclear double resonance (CW ENDOR; 9.4GHz, 5–20K) experiments at g⊥ of the Cu(II) spectrum show a weak coupling to protons with an AH of 2.9MHz that corresponds to a distance of 3.8Å (1Å≡0.1nm), assuming that it is a purely dipolar coupling. Incubation in 2H2O leads to a significant decrease in these 1H-ENDOR intensities, showing that these protons are exchangeable. This result strongly suggests that the EPR visible copper site of pMMO is accessible to solvent, which was confirmed by the chelation of the Cu2+ by diethyldithiocarbamic acid. The 1H and 14N hyperfine coupling constants confirm a histidine ligation of the EPR visible copper site in pMMO. The hyperfine structure in the ENDOR or EPR spectra of pMMO is not influenced by the inhibitors azide, cyanide or ammonia, indicating that they do not bind to the EPR visible copper. We compared pMMO with the type-2 Cu2+ enzyme, dopamine β-mono-oxygenase (DβM). For DβM, it is assumed that the copper site is solvent-accessible. CW ENDOR shows similar weakly coupled and 2H2O-exchangeable protons (2.9MHz), as observed in pMMO, as well as the strongly coupled nitrogens (40MHz) from the co-ordinating N of the histidines in DβM. In conclusion, the resting EPR visible Cu in pMMO is not part of a trinuclear cluster, as has been suggested previously.

Evidence That a Type-2 NADH:Quinone Oxidoreductase Mediates Electron Transfer to Particulate Methane Monooxygenase in Methylococcus capsulatus

NADH readily provides reducing equivalents to membrane-bound methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) in isolated membrane fractions, but detergent solubilization disrupts this electron-transfer process. Addition of exogenous quinones (especially decyl-plastoquinone and duroquinone) restores the NADH-dependent pMMO activity. Results of inhibitor and substrate dependence of this activity indicate the presence of only a type-2 NADH:quinone oxidoreductase (NDH-2). A 100-fold purification of the NDH-2 was achieved using lauryl-maltoside solubilization followed by ion exchange, hydrophobic-interaction, and gel-filtration chromatography. The purified NDH-2 has a subunit molecular weight of 36 kDa and exists as a monomer in solution. UV–visible and fluorescence spectroscopy identified flavin adenine dinucleotide (FAD) as a cofactor present in stoichiometric amounts. NADH served as the source of electrons, whereas NADPH could not. The purified NDH-2 enzyme reduced coenzyme Q0, duroquinone, and menaquinone at high rates, whereas the decyl analogs of ubiquinone and plastoquinone were reduced at ∼100-fold lower rates. Rotenone and flavone did not inhibit the NDH-2, whereas amytal caused partial inhibition but only at high concentrations.

Utilization of methane and carbon dioxide by symbiotrophic bacteria in gills of Mytilidae (Bathymodiolus) from the Rainbow and Logachev hydrothermal fields on the Mid-Atlantic Ridge

Bivalve mollusks Bathymodiolus asoricus and Bathymodiolus puteoserpentis collected from the Rainbow and Logachev hydrothermal fields during dives of Mir 1 and Mir 2 deep-sea manned submersibles were studied. Rates of methane oxidation and carbon dioxide assimilation in mussel gill tissue were determined by radiolabel analysis. During oxidation of 14Ch4, radiocarbon was detected in significant quantities not only in carbon dioxide but also in dissolved organic matter, most notably 14C-formate and 14C-acetate, occurring in a 2:1 ratio. Activities of hexulose-phosphate synthase, phosphoribulokinase, and ribulose 1,5-bisphosphate carboxylase were shown in the soluble fraction of gill tissue cells. At the same time, no activity of hydroxypyruvate reductase--the key enzyme of the serine pathway of C1-assimilation--was detected. The results of PCR amplification using genetic probes for membrane-bound methane monooxygenase (pmoA) and methanol dehydrogenase (mxaF) attest to the presence of the genes of these enzymes in the total DNA extracted from gill samples. However, no appropriate PCR responses were obtained with the mmoX primer system, which is a marker for soluble methane monooxygenase. All samples studied showed amplification with primers for the genera Methylobacter and Methylosphaera. At the same time, no genes specific to the genera Methylomonas, Methylococcus, Methylomicrobium, or Methylosinus and Methylocystis were detected. Electron microscopic examinations revealed the presence of two groups of endosymbiotic bacteria in the mussel gill tissue. The first group was represented by large cells possessing a complex system of cytoplasmic membranes, typical of methanotrophs of morphotype I. The other type of endosymbionts, having much smaller cells and lacking intracellular membrane structures, is likely to be constituted by sulfur bacteria.

Structural and functional model of methane hydroxylase of membrane-bound methane monooxygenase from Methylococcus capsulatus (Bath)

Computer analysis of a wide range of primary sequences showed that α-, β-, and γ-peptides of membrane-bound methane hydroxylase contained 2, 7, and 6 transmembrane helices respectively. Conservative amino acid residues participating in complex formation were revealed. The α- and γ-peptides are suggested to contain mononuclear copper ions with the ligand environment mainly consisting of His residues. The Cu sites are located in the hydrophilic region and are responsible for ESR signals. The active site of β-peptide in which the activation of O2 and oxidation of CH4 occur is localized in the hydrophobic region close to the membrane surface. This site is formed by the amino acid residues of four transmembrane helices and one loop between them and is suggested to be a binuclear Cu—Fe or Fe—Fe center. The Cu site of α-peptide transfers electrons to the active site of β-peptide, and the Cu site of γ-peptide is either involved in this process or only stabilizes the protein structure.

Quinols as electron donors for detergent solubilized and membrane-bound methane monooxygenase

Quinols as electron donors for detergent solubilized and membrane-bound methane monooxygenase