Activity of methanotrophic bacteria in Green Bay sediments

Abstract Sediment pore water samples obtained from a 19 m station in Green Bay in Lake Michigan were examined for levels of ambient dissolved methane and copper, and for the potential for in situ methane oxidation by methanotrophs found within surface sediments. The in situ methane concentration in the upper oxic sediment layer ranged from 20–150 μmol · 1⁻¹ at this station. The activity of methanotrophs and the kinetics of methane oxidation in these sediments were demonstrated by the uptake of radiolabeled methane. K_s values varied between 4.1–9.6 nmol · cm³ of sediment slurry. High V_max values (12.7–35.2 nmol · cm⁻³ · h⁻¹) suggest a large population of methanotrophs in the sediments. An average methane flux to the oxic sediments of 0.24 mol · m⁻² · year⁻¹ was calculated from the pore water methane gradients. Pore water concentrations of copper in the upper sediment layer ranged from 10–120 nmol · 1⁻¹. Based upon the copper concentration, other measured parameters, and equilibrium conditions defined by WATEQF4, an estimate for dissolved free Cu²⁺ concentration of 5–38 nmol · 1⁻¹ pore water was obtained. Several factors control the rate of methane oxidation, including oxygen, methane, and the bioavailability of free Cu²⁺.     

Anaerobic oxidation of methane in sediments of Lake Constance, an oligotrophic freshwater lake

Anaerobic oxidation of methane (AOM) with sulfate as terminal electron acceptor has been reported for various environments, including freshwater habitats, and also, nitrate and nitrite were recently shown to act as electron acceptors for methane oxidation in eutrophic freshwater habitats. Radiotracer experiments with sediment material of Lake Constance, an oligotrophic freshwater lake, were performed to follow 14CO2 formation from 14CH4 in sediment incubations in the presence of different electron acceptors, namely, nitrate, nitrite, sulfate, or oxygen. Whereas 14CO2 formation without and with sulfate addition was negligible, addition of nitrate increased 14CO2 formation significantly, suggesting that AOM could be coupled to denitrification. Nonetheless, denitrification-dependent AOM rates remained at least 1 order of magnitude lower than rates of aerobic methane oxidation. Using molecular techniques, putative denitrifying methanotrophs belonging to the NC10 phylum were detected on the basis of the pmoA and 16S rRNA gene sequences. These findings show that sulfate-dependent AOM was insignificant in Lake constant sediments. However, AOM can also be coupled to denitrification in this oligotrophic freshwater habitat, providing first indications that this might be a widespread process that plays an important role in mitigating methane emissions.     

Diversity and distribution of methanotrophic archaea at cold seeps

In this study we investigated by using 16S rRNA-based methods the distribution and biomass of archaea in samples from (i) sediments above outcropping methane hydrate at Hydrate Ridge (Cascadia margin off Oregon) and (ii) massive microbial mats enclosing carbonate reefs (Crimea area, Black Sea). The archaeal diversity was low in both locations; there were only four (Hydrate Ridge) and five (Black Sea) different phylogenetic clusters of sequences, most of which belonged to the methanotrophic archaea (ANME). ANME group 2 (ANME-2) sequences were the most abundant and diverse sequences at Hydrate Ridge, whereas ANME-1 sequences dominated the Black Sea mats. Other seep-specific sequences belonged to the newly defined group ANME-3 (related to Methanococcoides spp.) and to the Crenarchaeota of marine benthic group B. Quantitative analysis of the samples by fluorescence in situ hybridization (FISH) showed that ANME-1 and ANME-2 co-occurred at the cold seep sites investigated. At Hydrate Ridge the surface sediments were dominated by aggregates consisting of ANME-2 and members of the Desulfosarcina-Desulfococcus branch (DSS) (ANME-2/DSS aggregates), which accounted for >90% of the total cell biomass. The numbers of ANME-1 cells increased strongly with depth; these cells accounted 1% of all single cells at the surface and more than 30% of all single cells (5% of the total cells) in 7- to 10-cm sediment horizons that were directly above layers of gas hydrate. In the Black Sea microbial mats ANME-1 accounted for about 50% of all cells. ANME-2/DSS aggregates occurred in microenvironments within the mat but accounted for only 1% of the total cells. FISH probes for the ANME-2a and ANME-2c subclusters were designed based on a comparative 16S rRNA analysis. In Hydrate Ridge sediments ANME-2a/DSS and ANME-2c/DSS aggregates differed significantly in morphology and abundance. The relative abundance values for these subgroups were remarkably different at Beggiatoa sites (80% ANME-2a, 20% ANME-2c) and Calyptogena sites (20% ANME-2a, 80% ANME-2c), indicating that there was preferential selection of the groups in the two habitats. These variations in the distribution, diversity, and morphology of methanotrophic consortia are discussed with respect to the presence of microbial ecotypes, niche formation, and biogeography.     

Sulfur bacteria promote dissolution of authigenic carbonates at marine methane seeps

Carbonate rocks at marine methane seeps are commonly colonized by sulfur-oxidizing bacteria that co-occur with etch pits that suggest active dissolution. We show that sulfur-oxidizing bacteria are abundant on the surface of an exemplar seep carbonate collected from Del Mar East Methane Seep Field, USA. We then used bioreactors containing aragonite mineral coupons that simulate certain seep conditions to investigate plausible in situ rates of carbonate dissolution associated with sulfur-oxidizing bacteria. Bioreactors inoculated with a sulfur-oxidizing bacterial strain, Celeribacter baekdonensis LH4, growing on aragonite coupons induced dissolution rates in sulfidic, heterotrophic, and abiotic conditions of 1773.97 (±324.35), 152.81 (±123.27), and 272.99 (±249.96) μmol CaCO₃ • cm⁻² • yr⁻¹, respectively. Steep gradients in pH were also measured within carbonate-attached biofilms using pH-sensitive fluorophores. Together, these results show that the production of acidic microenvironments in biofilms of sulfur-oxidizing bacteria are capable of dissolving carbonate rocks, even under well-buffered marine conditions. Our results support the hypothesis that authigenic carbonate rock dissolution driven by lithotrophic sulfur-oxidation constitutes a previously unknown carbon flux from the rock reservoir to the ocean and atmosphere.Subject terms: Microbial ecology, Water microbiology, Biogeochemistry, Biogeochemistry, Biofilms     

Spatial variations of methanotrophic consortia at cold methane seeps: implications from a high-resolution molecular and isotopic approach

Anaerobic methane‐oxidizing microbial communities in sediments at cold methane seeps are important factors in controlling methane emission to the ocean and atmosphere. Here, we investigated the distribution and carbon isotopic signature of specific biomarkers derived from anaerobic methanotrophic archaea (ANME groups) and sulphate‐reducing bacteria (SRB) responsible for the anaerobic oxidation of methane (AOM) at different cold seep provinces of Hydrate Ridge, Cascadia margin. The special focus was on their relation to in situ cell abundances and methane turnover. In general, maxima in biomarker abundances and minima in carbon isotope signatures correlated with maxima in AOM and sulphate reduction as well as with consortium biomass. We found ANME‐2a/DSS aggregates associated with high abundances of sn‐2,3‐di‐O‐isoprenoidal glycerol ethers (archaeol, sn‐2‐hydroxyarchaeol) and specific bacterial fatty acids (C_16:1ω5c, cyC_17:0ω5,6) as well as with high methane fluxes (Beggiatoa site). The low to medium flux site (Calyptogena field) was dominated by ANME‐2c/DSS aggregates and contained less of both compound classes but more of AOM‐related glycerol dialkyl glycerol tetraethers (GDGTs). ANME‐1 archaea dominated deeper sediment horizons at the Calyptogena field where sn‐1,2‐di‐O‐alkyl glycerol ethers (DAGEs), archaeol, methyl‐branched fatty acids (ai‐C_15:0, i‐C_16:0, ai‐C_17:0), and diagnostic GDGTs were prevailing. AOM‐specific bacterial and archaeal biomarkers in these sediment strata generally revealed very similar δ¹³C‐values of around ?100. In ANME‐2‐dominated sediment sections, archaeal biomarkers were even more ¹³C‐depleted (down to ?120), whereas bacterial biomarkers were found to be likewise ¹³C‐depleted as in ANME‐1‐dominated sediment layers (δ¹³C: ?100). The zero flux site (Acharax field), containing only a few numbers of ANME‐2/DSS aggregates, however, provided no specific biomarker pattern. Deeper sediment sections (below 20 cm sediment depth) from Beggiatoa covered areas which included solid layers of methane gas hydrates contained ANME‐2/DSS typical biomarkers showing subsurface peaks combined with negative shifts in carbon isotopic compositions. The maxima were detected just above the hydrate layers, indicating that methane stored in the hydrates may be available for the microbial community. The observed variations in biomarker abundances and ¹³C‐depletions are indicative of multiple environmental and physiological factors selecting for different AOM consortia (ANME‐2a/DSS, ANME‐2c/DSS, ANME‐1) along horizontal and vertical gradients of cold seep settings.     

Production of methanol from methane by methanotrophic bacteria

Methanotrophs can oxidize methane to carbon dioxide through sequential reactions catalyzed by a series of enzymes including methane monooxygenase, methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase. When suspensions of methanotrophic bacteria of Methylosinus trichosporium IMV 3011 were incubated at 32°C with methane and oxygen, there was an extracellular accumulation of methanol from methane oxidation in response to carbon dioxide addition. Maximal accumulation of methanol was achieved with 40% carbon dioxide in the mixed reaction gases. A continuous experiment was performed in a continuous ultrafiltration reactor. The optimum gas mixture containing 20% (v v^-1) methane, 20% oxygen, 20% nitrogen and 40% carbon dioxide was used to provide substrates and to maintain the transmembrane pressure. The product (methanol) was removed in the eluate buffer. The initial methanol concentration in the eluate buffer was 8.22 μmol L^-1. The bioreactor was operated continuously for 198 h without obvious loss of productivity.     

Production of methanol from methane by methanotrophic bacteria

Methanotrophs can oxidize methane to carbon dioxide through sequential reactions catalyzed by a series of enzymes including methane monooxygenase, methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase. When suspensions of methanotrophic bacteria of Methylosinus trichosporium IMV 3011 were incubated at 32°C with methane and oxygen, there was an extracellular accumulation of methanol from methane oxidation in response to carbon dioxide addition. Maximal accumulation of methanol was achieved with 40% carbon dioxide in the mixed reaction gases. A continuous experiment was performed in a continuous ultrafiltration reactor. The optimum gas mixture containing 20% (v v^?1) methane, 20% oxygen, 20% nitrogen and 40% carbon dioxide was used to provide substrates and to maintain the transmembrane pressure. The product (methanol) was removed in the eluate buffer. The initial methanol concentration in the eluate buffer was 8.22 μmol L^?1. The bioreactor was operated continuously for 198 h without obvious loss of productivity.     

Sulfate-reducing bacteria in littoral sediment of Lake Constance

Abstract The viable population of sulfate-reducing bacteria (SRB) in littoral sediments of Lake Constance was investigated using enrichment and enumeration techniques. Enrichment studies established that most types of SRB grew best in media with low salt concentrations (max. 0.4 g Cl⁻/1), consistent with the low salinity of the freshwater habitat. Enumerations were based on an adequate medium with the following electron donors: H₂, lactate, acetate, propionate, butyrate, caprylate, succinate, benzoate, or S₂O₃²⁻ for thiosulfate-disproportionating bacteria. Cultures were incubated for 6 weeks to obtain maximum counts. A maximum cell density of 6.3 × 10⁶ cells per ml sediment was estimated, which is the highest number of SRB ever reported for anoxic sediments. A comparison with measured sulfate reduction rates showed that the enumeration techniques were about 10–100-fold more efficient than those previously used. The population of SRB had a characteristic structure consisting of 87.7% H₂-utilizing SRB (physiologically resembling the classical Desulfovibrio species); 12.0% propionate utilizers (tentatively identified as Desulfobulbus species); 0.3% long chain fatty acid-oxidizing Desulfovibrio sapovorans species. Acetate-utilizing SRB ( Desulfotomaculum acetoxidans ) constituted ≤ 0.05% of the total estimated population. Moreover, the latter species was only present as inactive spores. Benzoate-degrading SRB were not detected.     

Cultivation of methanotrophic bacteria in opposing gradients of methane and oxygen

In sediments, methane-oxidizing bacteria live in opposing gradients of methane and oxygen. In such a gradient system, the fluxes of methane and oxygen are controlled by diffusion and consumption rates, and the rate-limiting substrate is maintained at a minimum concentration at the layer of consumption. Opposing gradients of methane and oxygen were mimicked in a specific cultivation set-up in which growth of methanotrophic bacteria occurred as a sharp band at either c. 5 or 20 mm below the air-exposed end. Two new strains of methanotrophic bacteria were isolated with this system. One isolate, strain LC 1, belonged to the Methylomonas genus (type I methantroph) and contained soluble methane mono-oxygenase. Another isolate, strain LC 2, was related to the Methylobacter group (type I methantroph), as determined by 16S rRNA gene and pmoA sequence similarities. However, the partial pmoA sequence was only 86% related to cultured Methylobacter species. This strain accumulated significant amounts of formaldehyde in conventional cultivation with methane and oxygen, which may explain why it is preferentially enriched in a gradient cultivation system.     

Fate of methane bubbles released by pockmarks in Lake Constance

In the eastern part of Lake Constance, the second largest pre-alpine lake in Europe, about five hundred pockmarks (morphological depressions on the lake floor) were recently discovered of which ~40 % release methane bubbles. The carbon isotopic composition of the escaping gas indicated that the methane is of biogenic origin. In our study, we investigated the fate of the released methane bubbles, i.e., the dissolution, oxidation or transport of the bubbles to the surface. At a littoral pockmark site (PM12, 12 m water depth) and a profundal pockmark (PM80, 80 m water depth), we analysed the dissolved methane concentrations and the methane isotopic carbon signature in the water column. At PM80, higher methane concentrations (up to 1,523 nM), compared to the control site and the surface waters (225 ± 72 nM), were recorded only on some occasions and only in the bottom water, despite the fact that the released bubbles were dissolving within the hypolimnion based on bubble modeling. The isotope data suggest that most of the dissolved methane is oxidized below 40 m water depth. The isotopic signature of the methane in the surface water at PM80, however, differed from that of the methane in the hypolimnion; therefore, the surface methane at this profundal site is most likely an export product from the littoral zone. Assuming an initial bubble diameter of 5 mm, we calculated that these small bubbles would reach the surface, but approximately 96 % of the methane would have dissolved from the bubble into the hypolimnion. At PM12, we observed higher concentrations of dissolved methane (312 ± 52 nM) with no significant differences between seasons or between control sites versus pockmark site. In the shallow water, divers estimated the bubble size to be 10–15 mm, which from a release depth of 12 m would barely dissolved into the water column. The isotopic signature also indicated that there had been almost no methane oxidation in the shallow water column. Thus, the water depth of bubble release as well as the initial bubble size determine whether the methane enters the atmosphere largely unhindered (shallow site) or if the released methane is incorporated into the profundal water column.     

Viable methanotrophic bacteria enriched from air and rain can oxidize methane at cloud-like conditions

Atmospheric methane is degraded by both photooxidation and, in topsoils, by methanotrophic bacteria, but this may not totally account for the global sink of this greenhouse gas. Topsoils are a prominent source of airborne bacteria, which can degrade some organic atmospheric compounds at rates similar to photooxidation. Although airborne methanotrophs would have direct access to atmospheric methane, their presence and activity in the atmosphere has not been investigated so far. We enriched airborne methanotrophs from air and rainwater and showed that they oxidized methane at atmospheric concentration. The majority of seven OTUs, detected using pmoA gene clone libraries, were affiliated to the type II methanotrophic genera Methylocystis and Methylosinus. Furthermore, 16S rRNA gene clone libraries revealed the presence of OTUs affiliated with the genera Hyphomicrobium and Variovorax, members of which can stimulate methane oxidation by yet unidentified mechanisms. Simulating cloud-like conditions revealed that although both low pH and the presence of common cloud-borne organics negatively affected methane oxidation, airborne methanotrophs were able to degrade atmospheric methane in most cases. We demonstrate here for the first time that viable methanotrophic bacteria are present in air and rain and thus expand our knowledge on the global distribution of methanotrophs to include the atmosphere. The fact that they can degrade methane to below atmospheric concentrations when inoculated into artificial cloud water leads to an important possible effect of these organisms: the atmosphere may not only function as a medium for microbial dissemination, but also as a site of active microbial methane turnover.     

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.     

Mass spectrometric measurements of methane and oxygen utilization by methanotrophic bacteria

Abstract A mass spectrometer with membrane inlet was used to measure methane and oxygen utilization rates at various methane concentrations in Methylosinus trichosporium and a locally isolated strain of a methane-oxidizing coccus (OU-4-1). The apparent K _m for methane was found to be 2 μM for M. trichosporium and 0.8 μM for strain OU-4-1. These K _m-values are 10–30 times lower than most previously reported values. The ratio of oxygen to methane utilization rates was 1.7 for M. trichosporium and 1.5 for strain OU-4-1 corresponding to a growth yield of 0.38 and 0.63 g dry weight/g methane, respectively.     

Consumption of methane and CO2 by methanotrophic microbial mats from gas seeps of the anoxic Black Sea

The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH(4) and CO(2) assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO(2) reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average delta(13)C carbon isotopic signature of -67.1 per thousand, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (-66.4 per thousand +/- 3.9 per thousand [mean +/- standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (-72.9 per thousand +/- 2.2 per thousand; n = 7). Incorporation of (14)C from radiolabeled CH(4) or CO(2) revealed one hot spot for methanotrophy and CO(2) fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with (14)CH(4) or (14)CO(2) revealed that there was interconversion of CH(4) and CO(2.) The level of CO(2) reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.     

Response and adaptation of different methanotrophic bacteria to low methane mixing ratios

Described genera of methanotrophic bacteria are present in most upland soils, but it is not known whether these are sufficiently oligotrophic to oxidize methane at its trace atmospheric mixing ratio of 1.75 ppmv. Members of the genera Methylocystis, Methylosinus, Methylocaldum and Methylobacter were isolated from different upland soils and compared with type strains for growth and activity under low methane mixing ratios. The specific affinity (a0s) varied by about one order of magnitude among different methanotrophs. It was highest in some Methylocystis spp., suggesting that these were the most oligotrophic. In direct tests, the threshold mixing ratio of methane required by most methanotrophs for growth ranged from 100 to greater than 1000 ppmv. However, two Methylocystis strains grew at only 10-100 ppmv of methane and one oxidized atmospheric methane for >3 months with little or no decline in the absolute rate. The results show that some cultivated methanotrophic bacteria are much more oligotrophic than others, and may contribute to atmospheric methane oxidation in soils. However, it is likely that these need additional energy sources for long-term survival, and that uncultivated groups of methanotrophic bacteria are primarily responsible for the process in soils possessing high methane oxidation rates.     

A carbonate corrosion experiment at a marine methane seep: The role of aerobic methanotrophic bacteria

Methane seeps are typified by the formation of authigenic carbonates, many of which exhibit corrosion surfaces and secondary porosity believed to be caused by microbial carbonate dissolution. Aerobic methane oxidation and sulfur oxidation are two processes capable of inducing carbonate corrosion at methane seeps. Although the potential of aerobic methanotrophy to dissolve carbonate was confirmed in laboratory experiments, this process has not been studied in the environment to date. Here, we report on a carbonate corrosion experiment carried out in the REGAB Pockmark, Gabon-Congo-Angola passive margin, in which marble cubes were deployed for 2.5 years at two sites (CAB-B and CAB-C) with apparent active methane seepage and one site (CAB-D) without methane seepage. Marble cubes exposed to active seepage (experiment CAB-C) were found to be affected by a new type of microbioerosion. Based on 16S rRNA gene analysis, the biofilms adhering to the bioeroded marble mostly consisted of aerobic methanotrophic bacteria, predominantly belonging to the uncultured Hyd24-01 clade. The presence of abundant ¹³C-depleted lipid biomarkers including fatty acids (n-C_16:1ω8c, n-C_18:1ω8c, n-C_16:1ω5t), various 4-mono- and 4,4-dimethyl sterols, and diplopterol agrees with the dominance of aerobic methanotrophs in the CAB-C biofilms. Among the lipids of aerobic methanotrophs, the uncommon 4α-methylcholest-8(14)-en-3β,25-diol is interpreted to be a specific biomarker for the Hyd24-01 clade. The combination of textural, genetic, and organic geochemical evidence suggests that aerobic methanotrophs are the main drivers of carbonate dissolution observed in the CAB-C experiment at the REGAB pockmark.     

Growth characteristics of small and large free-living and attached bacteria in Lake Constance

The growth characteristics of small (0.2–1.0m) and large (1.0–3.0 (m) free-living and attached bacteria were studied in Lake Constance by comparing the spatial and seasonal dynamics of their biomass turnover time (ratio of biomass/production). The biomass of small free-living bacteria usually turned over significantly faster than that of large free-living bacteria throughout the water column. The turnover of attached bacterial biomass was characterized by large fluctuations. Occasionally, in aphotic water layers, it was as long as that of large free-living bacteria, but when large amounts of decaying organic particles were present, it was shorter than that of small free-living cells. Biomass turnover times of free-living bacteria were in the same range as their generation times, which were estimated from the increase in bacterial abundance in 3m prefiltered samples. The biomass turnover time of actively metabolizing bacteria was comparable to the generation time of actively metabolizing cells. These results indicate that the biomass turnover time is a useful indicator of the growth of different bacterial fractions, as it reflects their different amounts of participation in microbial processes of aquatic ecosystems.     

Toward delineating the structure and function of the particulate methane monooxygenase from methanotrophic bacteria

The particulate methane monooxygenase (pMMO) is a complex membrane protein complex that has been difficult to isolate and purify for biochemical and biophysical characterization because of its instability in detergents used to solubilize the enzyme. In this perspective, we summarize the progress recently made toward obtaining a purified pMMO-detergent complex and characterizing the enzyme in pMMO-enriched membranes. The purified pMMO is a multi-copper protein, with ca. 15 copper ions sequestered into five trinuclear copper clusters: two for dioxygen chemistry and alkane hydroxylation (catalytic or C-clusters) and three to provide a buffer of reducing equivalents to re-reduce the C-clusters following turnover (electron transfer or E-clusters). The enzyme is functional when all the copper ions are reduced. When the protein is purified under ambient aerobic conditions in the absence of a hydrocarbon substrate, only the C-clusters are oxidized; there is an apparent kinetic barrier for electron transfer from the E-cluster copper ions to the C-clusters under these conditions. Evidence is provided in support of both C-clusters participating in the dioxygen chemistry, but only one C-cluster supporting alkane hydroxylation. Acetylene modification of the latter C-cluster in the hydrophobic pocket of the active site lowers or removes the kinetic barrier for electron transfer from the E-clusters to the C-clusters so that all the copper ions could be fully oxidized by dioxygen. A model for the hydroxylation chemistry when a hydrocarbon substrate is bound to the active site of the hydroxylation C-cluster is presented. Unlike soluble methane monooxygenase (sMMO), pMMO exhibits limited substrate specificity, but the hydroxylation chemistry is highly regioselective and stereoselective. In addition, the hydroxylation occurs with total retention of configuration of the carbon center that is oxidized. These results are consistent with a concerted mechanism involving direct side-on insertion of an active singlet "oxene" from the activated copper cluster across the "C-H" bond in the active site. Finally, in our hands, both the purified pMMO-detergent complex and pMMO-enriched membranes exhibit high NADH-sensitive as well as duroquinol-sensitive specific activity. A possible role for the two reductants in the turnover of the enzyme is proposed.