Methane Monooxygenase Activity Research Articles

Methane-Oxidizing Activity Enhances Sulfamethoxazole Biotransformation in a Benthic Constructed Wetland Biomat.

Ammonia monooxygenase and analogous oxygenase enzymes contribute to pharmaceutical biotransformation in activated sludge. In this study, we hypothesized that methane monooxygenase can enhance pharmaceutical biotransformation within the benthic, diffuse periphytic sediments (i.e., "biomat") of a shallow, open-water constructed wetland. To test this hypothesis, we combined field-scale metatranscriptomics, porewater geochemistry, and methane gas fluxes to inform microcosms targeting methane monooxygenase activity and its potential role in pharmaceutical biotransformation. In the field, sulfamethoxazole concentrations decreased within surficial biomat layers where genes encoding for the particulate methane monooxygenase (pMMO) were transcribed by a novel methanotroph classified as Methylotetracoccus. Inhibition microcosms provided independent confirmation that methane oxidation was mediated by the pMMO. In these same incubations, sulfamethoxazole biotransformation was stimulated proportional to aerobic methane-oxidizing activity and exhibited negligible removal in the absence of methane, in the presence of methane and pMMO inhibitors, and under anoxia. Nitrate reduction was similarly enhanced under aerobic methane-oxidizing conditions with rates several times faster than for canonical denitrification. Collectively, our results provide convergent in situ and laboratory evidence that methane-oxidizing activity can enhance sulfamethoxazole biotransformation, with possible implications for the combined removal of nitrogen and trace organic contaminants in wetland sediments.

Recovery of particulate methane monooxygenase structure and activity in a lipid bilayer.

Bacterial methane oxidation using the enzyme particulate methane monooxygenase (pMMO) contributes to the removal of environmental methane, a potent greenhouse gas. Crystal structures determined using inactive, detergent-solubilized pMMO lack several conserved regions neighboring the proposed active site. We show that reconstituting pMMO in nanodiscs with lipids extracted from the native organism restores methane oxidation activity. Multiple nanodisc-embedded pMMO structures determined by cryo-electron microscopy to 2.14- to 2.46-angstrom resolution reveal the structure of pMMO in a lipid environment. The resulting model includes stabilizing lipids, regions of the PmoA and PmoC subunits not observed in prior structures, and a previously undetected copper-binding site in the PmoC subunit with an adjacent hydrophobic cavity. These structures provide a revised framework for understanding and engineering pMMO function.

X-ray Crystal Structures of Methane Monooxygenase Hydroxylase Complexes with Variants of Its Regulatory Component: Correlations with Altered Reaction Cycle Dynamics.

Full activity of soluble methane monooxygenase (sMMO) depends upon the formation of a 1:1 complex of the regulatory protein MMOB with each alpha subunit of the (αβγ)2 hydroxylase, sMMOH. Previous studies have shown that mutations in the core region of MMOB and in the N- and C-termini cause dramatic changes in the rate constants for steps in the sMMOH reaction cycle. Here, X-ray crystal structures are reported for the sMMOH complex with two double variants within the core region of MMOB, DBL1 (N107G/S110A), and DBL2 (S109A/T111A), as well as two variants in the MMOB N-terminal region, H33A and H5A. DBL1 causes a 150-fold decrease in the formation rate constant of the reaction cycle intermediate P, whereas DBL2 accelerates the reaction of the dinuclear Fe(IV) intermediate Q with substrates larger than methane by three- to fourfold. H33A also greatly slows P formation, while H5A modestly slows both formation of Q and its reactions with substrates. Complexation with DBL1 or H33A alters the position of sMMOH residue R245, which is part of a conserved hydrogen-bonding network encompassing the active site diiron cluster where P is formed. Accordingly, electron paramagnetic resonance spectra of sMMOH:DBL1 and sMMOH:H33A complexes differ markedly from that of sMMOH:MMOB, showing an altered electronic environment. In the sMMOH:DBL2 complex, the position of M247 in sMMOH is altered such that it enlarges a molecular tunnel associated with substrate entry into the active site. The H5A variant causes only subtle structural changes despite its kinetic effects, emphasizing the precise alignment of sMMOH and MMOB required for efficient catalysis.

Copper stimulation on methane-supported perchlorate reduction in a membrane biofilm reactor

The present study demonstrated that the perchlorate reduction rate in a methane-based membrane biofilm reactor was significantly enhanced from 14.4 to 25.6 mg-Cl/L/d by increasing copper concentration in the feeding medium from 1 to 10 μM, indicating a stimulatory effect of copper on the methane-supported perchlorate reduction process. Batch tests further confirmed that the increased copper concentration enhanced both methane oxidation and perchlorate reduction rates, which was supported by an increasing trend of functional genes (pmoA for methanotrophs and pcrA for specific perchlorate reducers) abundances through quantitative polymerase chain reaction (qPCR). Both 16S rRNA gene sequencing and functional genes (pmoA and pcrA) sequencing jointly revealed that the biofilm supplied with a higher copper concentration exhibited a more diverse microbial community. The methane-supported perchlorate reduction was accomplished through a synergistic association of methanotrophs (Methylocystis, Methylomonas, and Methylocystaceae) and perchlorate reducers (Dechloromonas, Azospira, Magnetospirillum, and Denitratisoma). Acetate may function as the key syntrophic linkage between methanotrophs and perchlorate reducers. It was proposed that the increased copper concentration improved the activity of particulate methane monooxygenase (pMMO) for methane oxidation or promoted the biosynthesis of intracellular carbon storage compounds polyhydroxybutyrate (PHB) in methanotrophs for generating more acetate available for perchlorate reduction.

Methane oxidation to methanol over copper-containing zeolite

Methane oxidation to methanol over copper-containing zeolite

Selection of methanotrophic platform for methanol production using methane and biogas

To develop biotechnological process for methane to methanol conversion, selection of a suitable methanotrophic platform is an important aspect. Systematic approach based on literature and public databases was developed to select representative methanotrophs Methylotuvimicrobium alcaliphilum, Methylomonas methanica, Methylosinus trichosporium and Methylocella silvestris. Selected methanotrophs were further investigated for methanol tolerance and methanol production on pure methane as well as biogas along with key enzyme activities involved in methane utilization. Among selected methanotrophs M.alcaliphilum showed maximum methanol tolerance of 6% v/v along with maximum methanol production of 307.90mg/L and 247.37mg/L on pure methane and biogas respectively. Activity of methane monooxygenase and formate dehydrogenase enzymes in M.alcaliphilum was significantly higher up to 98.40nmol/min/mg cells and 0.87 U/mg protein, respectively. Biotransformation trials in 14 L fermentor resulted in increased methanol production up to 418 and 331.20mg/L, with yield coefficient 0.83 and 0.71mg methanol/mg of pure methane and biogas respectively. The systematic selection resulted in haloalkaliphilic strain M.alcaliphilum as one of the potential methanotroph for bio-methanol production.

Nanozymes: From New Concepts, Mechanisms, and Standards to Applications.

Nanozymes are nanomaterials with intrinsic enzyme-like characteristics that have been booming over the past decade because of their capability to address the limitations of natural enzymes such as low stability, high cost, and difficult storage. Along with the rapid development and ever-deepening understanding of nanoscience and nanotechnology, nanozymes hold promise to serve as direct surrogates of traditional enzymes by mimicking and further engineering the active centers of natural enzymes. In 2007, we reported the first evidence that Fe3O4 nanoparticles (NPs) have intrinsic peroxidase-mimicking activity, and since that time, hundreds of nanomaterials have been found to mimic the catalytic activity of peroxidase, oxidase, catalase, haloperoxidase, glutathione peroxidase, uricase, methane monooxygenase, hydrolase, and superoxide dismutase. Uniquely, a broad variety of nanomaterials have been reported to simultaneously exhibit dual- or multienzyme mimetic activity. For example, Fe3O4 NPs show pH-dependent peroxidase-like and catalase-like activities; Prussian blue NPs simultaneously possess peroxidase-, catalase-, and superoxide dismutase-like activity; and Mn3O4 NPs mimic all three cellular antioxidant enzymes including superoxide dismutase, catalase, and glutathione peroxidase. Taking advantage of the physiochemical properties of nanomaterials, nanozymes have shown a broad range of applications from in vitro detection to replacing specific enzymes in living systems. With the emergence of the new concept of "nanozymology", nanozymes have now become an emerging new field connecting nanotechnology and biology. Since the landmark paper on nanozymes was published in 2007, we have extensively explored their catalytic mechanism, established the corresponding standards to quantitatively determine their catalytic activities, and opened up a broad range of applications from biological detection and environmental monitoring to disease diagnosis and biomedicine development. Here we mainly focus on our progress in the systematic design and construction of functionally specific nanozymes, the standardization of nanozyme research, and the exploration of their applications for replacing natural enzymes in living systems. We also show that, by combining the unique physicochemical properties and enzyme-like catalytic activities, nanozymes can offer a variety of multifunctional platforms with a broad of applications from in vitro detection to in vivo monitoring and therapy. For instance, targeting antibody-conjugated ferromagnetic nanozymes simultaneously provide three functions: target capture, magnetic separation, and nanozyme color development for target detection. We finally will address the prospect of nanozyme research to become "nanozymology". We expect that nanozymes with unique physicochemical properties and intrinsic enzyme-mimicking catalytic properties will attract broad interest in both fundamental research and practical applications and offer new opportunities for traditional enzymology.

Application and development of methanotrophs in environmental engineering

Bioconversion by aerobic methanotrophs, with their relatively high selectivity for specific substance (i.e., methane), is a more efficient and cost-effective methanol production method compared to chemical processes. Aerobic methanotrophs oxidize methane to oxidize to methanol, then formaldehyde, followed by formate, and finally carbon dioxide via enzymatic reaction pathways. One of the intermediates of this process, methanol can be recovered by inhibiting enzymatic catalysis of methanol dehydrogenase while maintaining the activity of methane monooxygenase (MMO). Then, the biologically converted methanol is available for use in chemical or alternative fuel production, which, in turn, contributes to reducing greenhouse gas emissions, consequently completing allowable carbon resource circulation. Furthermore, methanotrophs decompose persistent organic pollutants, including halogenated hydrocarbons through MMO reaction. However, many studies have addressed the restrictions associated with methanotrophic oxidation, e.g., slow species growth rate, organic substance uptake limitations, and difficulty in controlling MMO and methanol dehydrogenase activities. Thus, there are additional incubation conditions for methanotrophs that require optimization such as methane and oxygen gas fractions, gas transfer rate to cells, and bioreactor design.

Influence of copper ions removal from membrane-bound form of particulate methane monooxygenase from Methylosinus trichosporium OB3b on its activity for methane hydroxylation

The influence of metal removal with a chelating reagent, ethylenediaminetetraacetic acid (EDTA), and metal reconstitution on the activity of particulate methane monooxygenase (pMMO) from Methylosinus trichosporium OB3b toward methane oxidation to methanol was investigated. For this study, a membrane fraction containing pMMO and bacterial cell membrane components was prepared. pMMO activity was assessed with two different reductants, nicotinamide adenine dinucleotide (NADH) and 2,3,5,6-tetramethyl hydroquinone (duroquinol). The partial removal of metal ions with EDTA resulted in the selective inhibition of NADH-driven activity. The NADH-driven activity was restored by exogenous copper ions, but not by other divalent metal cations. Furthermore, both NADH- and duroquinol-driven activities were lost completely by increasing the amount of metal removed. Duroquinol-driven activity of the metal-deficient membrane fraction increased with increasing the amount of copper ions added, while NADH-driven activity increased when more than 5 mol of copper ions per mol of pMMO monomer was added. These results suggest that NADH-driven pMMO activity requires not only the catalytic copper center of pMMO but also copper ions outside the catalytic site.

From micelles to bicelles: Effect of the membrane on particulate methane monooxygenase activity

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.

Effects of copper on expression of methane monooxygenases, trichloroethylene degradation, and community structure in methanotrophic consortia.

Copper plays a key role in regulating the expression of enzymes that promote biodegradation of contaminants in methanotrophic consortia (MC). Here, we utilized MC isolated from landfill cover to investigate cometabolic degradation of trichloroethylene (TCE) at nine different copper (Cu2+) concentrations. The results demonstrated that an increase in Cu2+ concentration from 0 to 15μM altered the specific first-order rate constant k 1,TCE, the expression levels of methane monooxygenase (pmoA and mmoX) genes, and the specific activity of soluble methane monooxygenase (sMMO). High efficiency TCE degradation (95%) and the expression levels of methane monooxygenase (MMO) were detected at a Cu2+ concentration of 0.03μM. Notably, sMMO-specific activity ranged from 74.41 nmol/(mgcellh) in 15μM Cu2+ to 654.99 nmol/(mgcellh) in 0.03μM Cu2+, which contrasts with cultures of pure methanotrophs in which sMMO activity is depressed at high Cu2+ concentrations, indicating a special regulatory role for Cu2+ in MC. The results of MiSeq pyrosequencing indicated that higher Cu2+ concentrations stimulated the growth of methanotrophic microorganisms in MC. These findings have important implications for the elucidation of copper-mediated regulatory mechanisms in MC.

A novel methanotrophic co-metabolic system with high soluble methane monooxygenase activity to biodegrade refractory organics in pulping wastewater

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.

Engendering Methane Monooxygenase and Hydrogen Peroxide Oxidase Activity into a Designed Dimetal Protein by Increasing Protein Dynamics

Engendering Methane Monooxygenase and Hydrogen Peroxide Oxidase Activity into a Designed Dimetal Protein by Increasing Protein Dynamics

Effect of landfill cover layer modification on methane oxidation.

Levels of methane (CH4) oxidation in materials used for landfill cover attained in the laboratory are not often replicated in the field due to effects from the surrounding environment. This study investigates the three dominant factors affecting CH4 oxidation in the cover layer, namely, the thickness of cover layer, the methanotroph spraying manner, and the osmotic coefficient of the cover material. Results show that improved CH4 emission performance of the cover layer can be realized if methanotroph are introduced, meaning that a thinner cover layer is required. The highest CH4 emission reduction can be realized by spraying methanotroph into the top, middle, and bottom layers of a 30-cm thick cover layer with an osmotic coefficient of 7.76×10-5cms-1. Comparing results on cover layer thickness, methane monooxygenase (MMO) activity was much lower with increasing thickness meaning that the thicker cover could reduce O2 availability, thus inhibiting MMO activity. This suggests that MMO may be responsible for differences in CH4 emission reduction and/or oxidation making the osmotic coefficient an important factor for cover layer material.

Coupled effects of methane monooxygenase and nitrogen source on growth and poly-β-hydroxybutyrate (PHB) production of Methylosinus trichosporium OB3b

The coupled effects of nitrogen source and methane monooxygenase (MMO) on the growth and poly-β-hydroxybutyrate (PHB) accumulation capacity of methanotrophs were explored. The ammonia-supplied methanotrophs expressing soluble MMO (sMMO) grew at the highest rate, while N2-fixing bacteria expressing particulate MMO (pMMO) grew at the lowest rate. Further study showed that more hydroxylamine and nitrite was formed by ammonia-supplied bacteria containing pMMO, which might cause their slightly lower growth rate. The highest PHB content (51.0%) was obtained under nitrogen-limiting conditions with the inoculation of nitrate-supplied bacteria containing pMMO. Ammonia-supplied bacteria also accumulated a higher content of PHB (45.2%) with the expression of pMMO, while N2-fixing bacteria containing pMMO only showed low PHB production capacity (32.1%). The maximal PHB contents of bacteria expressing sMMO were low, with no significant change under different nitrogen source conditions. The low MMO activity, low cell growth rate and low PHB production capacity of methanotrophs continuously cultivated with N2 with the expression of pMMO were greatly improved in the cyclic NO3−N2 cultivation regime, indicating that long-term deficiency of nitrogen sources was detrimental to the activity of methanotrophs expressing pMMO.

Genome-scale metabolic reconstructions and theoretical investigation of methane conversion in Methylomicrobium buryatense strain 5G(B1).

BackgroundMethane-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.ResultsA 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.ConclusionWe 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 materialThe online version of this article (doi:10.1186/s12934-015-0377-3) contains supplementary material, which is available to authorized users.

Cerium regulates expression of alternative methanol dehydrogenases in Methylosinus trichosporium OB3b.

Methanotrophs have multiple methane monooxygenases that are well known to be regulated by copper, i.e., a "copper switch." At low copper/biomass ratios the soluble methane monooxygenase (sMMO) is expressed while expression and activity of the particulate methane monooxygenase (pMMO) increases with increasing availability of copper. In many methanotrophs there are also multiple methanol dehydrogenases (MeDHs), one based on Mxa and another based on Xox. Mxa-MeDH is known to have calcium in its active site, while Xox-MeDHs have been shown to have rare earth elements in their active site. We show here that the expression levels of Mxa-MeDH and Xox-MeDH in Methylosinus trichosporium OB3b significantly decreased and increased, respectively, when grown in the presence of cerium but the absence of copper compared to the absence of both metals. Expression of sMMO and pMMO was not affected. In the presence of copper, the effect of cerium on gene expression was less significant, i.e., expression of Mxa-MeDH in the presence of copper and cerium was slightly lower than in the presence of copper alone, but Xox-MeDH was again found to increase significantly. As expected, the addition of copper caused sMMO and pMMO expression levels to significantly decrease and increase, respectively, but the simultaneous addition of cerium had no discernible effect on MMO expression. As a result, it appears Mxa-MeDH can be uncoupled from methane oxidation by sMMO in M. trichosporium OB3b but not from pMMO.

Spectroscopic definition of the copper active sites in mordenite: selective methane oxidation.

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.

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.

Effects of Zinc on Particulate Methane Monooxygenase Activity and Structure

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.