(1) Background: Particulate methane monooxygenase (pMMO) has a strong dependence on the natural electron transfer path and is prone to denaturation, which results in its redox activity centers being unable to transfer electrons with bare electrodes directly and making it challenging to observe an electrochemical response; (2) Methods: Using methanobactin (Mb) as the electron transporter between gold electrodes and pMMO, a bionic interface with high biocompatibility and stability was created. The Mb-AuNPs-modified functionalized gold net electrode as a working electrode, the kinetic behaviors of pMMO bioelectrocatalysis, and the effect of Mb on pMMO were analyzed. The CV tests were performed at different scanning rates to obtain electrochemical kinetics parameters. (3) Results: The values of the electron transfer coefficient (α) and electron transfer rate constant (ks) are relatively large in test environments containing only CH4 or O2. In contrast, in the test environment containing both CH4 and O2, the bioelectrocatalysis of pMMO is a two-electron transfer process with a relatively small α and ks; (4) Conclusions: It was inferred that Mb formed the complex with pMMO. More importantly, Mb not only played a role in electron transfer but also in stabilizing the enzyme structure of pMMO and maintaining a specific redox state. Furthermore, the continuous catalytic oxidation of natural substrate methane was realized.

The vast majority of processes in the carbon and nitrogen cycles are driven by microorganisms. The nitrite-dependent anaerobic oxidation of methane (N-DAMO) process links carbon and nitrogen cycles, offering a novel approach for the simultaneous reduction of methane emissions and nitrite pollution. However, there is currently no comprehensive summary of the current status of the N-DAMO process in natural aquatic environments. Therefore, our study aims to fill this knowledge gap by conducting a comprehensive review of the global research trends in N-DAMO processes in various aquatic environments (excluding artificial bioreactors). Our review mainly focused on molecular identification, global study sites, and their interactions with other elemental cycling processes. Furthermore, we performed a data integration analysis to unveil the effects of key environmental factors on the abundance of N-DAMO bacteria and the rate of N-DAMO process. By combining the findings from the literature review and data integration analysis, we proposed future research perspectives on N-DAMO processes in global aquatic environments. Our overarching goal is to advance the understanding of the N-DAMO process and its role in synergistically reducing carbon emissions and removing nitrogen. By doing so, we aim to make a significant contribution to the timely achievement of China\'s carbon peak and carbon neutrality targets.

Particulate methane monooxygenase (pMMO) is a multi-subunit membrane metalloenzyme used by methanotrophic bacteria to convert methane to methanol. A major hurdle to studying pMMO is the lack of a recombinant expression system, precluding investigation of individual residues by mutagenesis and hampering a complete understanding of its mechanism. Here, we developed an Escherichia coli lysate-based cell-free protein synthesis (CFPS) system that can be used to express pMMO in vitro in the presence of nanodiscs. We used a SUMO fusion construct to generate the native PmoB subunit and showed that the SUMO protease (Ulp1) cleaves the protein in the reaction mixture. Using an affinity tag to isolate the complete pMMO complex, we demonstrated that the complex forms without the need for exogenous translocon machinery or chaperones, confirmed by negative stain electron microscopy. This work demonstrates the potential for using CFPS to express multi-subunit membrane-bound metalloenzymes directly into lipid bilayers.

A high NH4+ load is known to inhibit bacterial methane oxidation. This is due to a competition between CH4 and NH3 for the active site of particulate methane monooxygenase (pMMO), which converts CH4 to CH3OH. Here, we combined global proteomics with amino acid profiling and nitrogen oxides measurements to elucidate the cellular acclimatization response of Methylocystis sp. strain SC2 to high NH4+ levels. Relative to 1 mM NH4+, a high (50 mM and 75 mM) NH4+ load under CH4-replete conditions significantly increased the lag phase duration required for proteome adjustment. The number of differentially regulated proteins was highly significantly correlated with an increasing NH4+ load. The cellular responses to increasing ionic and osmotic stress involved a significant upregulation of stress-responsive proteins, the K+ \"salt-in\" strategy, the synthesis of compatible solutes (glutamate and proline), and the induction of the glutathione metabolism pathway. A significant increase in the apparent Km value for CH4 oxidation during the growth phase was indicative of increased pMMO-based oxidation of NH3 to toxic hydroxylamine. The detoxifying activity of hydroxlyamine oxidoreductase (HAO) led to a significant accumulation of NO2- and, upon decreasing O2 tension, N2O. Nitric oxide reductase and hybrid cluster proteins (Hcps) were the candidate enzymes for the production of N2O. In summary, strain SC2 has the capacity to precisely rebalance enzymes and osmolyte composition in response to increasing NH4+ exposure, but the need to simultaneously combat both ionic-osmotic stress and the toxic effects of hydroxylamine may be the reason why its acclimatization capacity is limited to 75 mM NH4+. IMPORTANCE In addition to reducing CH4 emissions from wetlands and landfills, the activity of alphaproteobacterial methane oxidizers of the genus Methylocystis contributes to the sink capacity of forest and grassland soils for atmospheric methane. The methane-oxidizing activity of Methylocystis spp. is, however, sensitive to high NH4+ concentrations. This is due to the competition of CH4 and NH3 for the active site of particulate methane monooxygenase, thereby resulting in the production of toxic hydroxylamine with an increasing NH4+ load. An understanding of the physiological and molecular response mechanisms of Methylocystis spp. is therefore of great importance. Here, we combined global proteomics with amino acid profiling and NOx measurements to disentangle the cellular mechanisms underlying the acclimatization of Methylocystis sp. strain SC2 to an increasing NH4+ load.

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.

In this focused review, we portray the recently reported 2.5 Å cyro-EM structure of the particulate methane monooxygenase (pMMO) from M. capsulatus (Bath). The structure of the functional holo-pMMO near atomic resolution has uncovered the sites of the copper cofactors including the location of the active site in the enzyme. The three coppers seen in the original X-ray crystal structures of the enzyme are now augmented by additional coppers in the transmembrane domain as well as in the water-exposed C-terminal subdomain of the PmoB subunit. The cryo-EM structure offers the first glimpse of the catalytic machinery capable of methane oxidation with high selectivity and efficiency. The findings are entirely consistent with the biochemical and biophysical findings previously reported in the literature, including the chemistry of hydrocarbon hydroxylation, regeneration of the catalyst for multiple turnovers, and the mechanism of aborting non-productive cycles to ensure kinetic competence.

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.

Particulate methane monooxygenase (pMMO) is a membrane protein embedded in the intracytoplasmic membrane of methane-oxidizing bacteria. Structural analysis of pMMO showed the existence of a hydrophilic region exposed outside of the bacterial membrane. To obtain information regarding the role of this hydrophilic region in the enzymatic function of pMMO, trypsin proteolysis of the membrane-bound form of pMMO from Methylosinus trichosporium OB3b was performed at 4 °C. The polypeptides produced by this hydrolysis were analyzed by polyacrylamide gel electrophoresis and MALDI-TOF/TOF. Furthermore, the influence of this tryptic digestion on the methane hydroxylation and propene epoxidation enzymatic activities of pMMO was investigated. Among the three subunits of pMMO, PmoB and PmoC were hydrolyzed by trypsin, but PmoA was not. With 10 mg L-1 trypsin, both terminal regions or the C-terminal region of PmoC polypeptide was selectively hydrolyzed. Furthermore, the stability of pMMO was decreased by this digestion. These results indicate that PmoC plays a role in maintaining the stability of pMMO in vitro. On the other hand, the digestion of PmoB with 100 mg L-1 trypsin produced several polypeptides, indicating that trypsin digestion occurs at several sites of the hydrophilic region of PmoB. Hydrolysis led to a decrease in pMMO activity towards methane hydroxylation and propene epoxidation. These results indicate that the hydrophilic region of PmoB is critically important for the enzymatic function of pMMO, which is consistent with the models of the functional mechanism of pMMO proposed so far.

Septic systems represent a source of greenhouse gases generated by microbial processes as wastewater constituents are degraded. Both aerobic and anerobic wastewater transformation processes can generate nitrous oxide and methane, both of which are potent greenhouse gases (GHGs). To understand how microbial communities in the surface soils above shallow drainfields contribute to methane and nitrous oxide consumption, we measured greenhouse gas surface flux and below-ground concentrations and compared them to the microbial communities present using functional genes pmoA and nosZ. These genes encode portions of particulate methane monooxygenase and nitrous oxide reductase, respectively, serving as a potential sink for the respective greenhouse gases. We assessed the surface soils above three drainfields served by a single household: an experimental layered passive N-reducing drainfield, a control conventional drainfield, and a reserve drainfield not in use but otherwise identical to the control. We found that neither GHG flux, below-ground concentration or soil properties varied among drainfield types, nor did methane oxidizing and nitrous oxide reducing communities vary by drainfield type. We found differences in pmoA and nosZ communities based on depth from the soil surface, and differences in nosZ communities based on whether the sample came from the rhizosphere or surrounding bulk soils. Type I methanotrophs (Gammaproteobacteria) were more abundant in the upper and middle portions of the soil above the drainfield. In general, we found no relationship in community composition for either gene based on GHG flux or below-ground concentration or soil properties (bulk density, organic matter, above-ground biomass). This is the first study to assess these communities in the surface soils above an experimental working drainfield, and more research is needed to understand the dynamics of greenhouse gas production and consumption in these systems.

Particulate methane monooxygenase (pMMO) is a characteristic membrane-bound metalloenzyme of methane-oxidizing bacteria that can catalyze the bioconversion of methane to methanol. However, in order to achieve pMMO-based continuous methane-to-methanol bioconversion, the problems of reducing power in vitro regeneration and pMMO stability need to be overcome. Methanobactin (Mb) is a small copper-chelating molecule that functions not only as electron carrier for pMMO catalysis and pMMO protector against oxygen radicals, but also as an agent for copper acquisition and uptake. In order to improve the activity and stability of pMMO, methanobactin-Cu (Mb-Cu)-modified gold nanoparticle (AuNP)-pMMO nanobiohybrids were straightforwardly synthesized via in situ reduction of HAuCl4 to AuNPs in a membrane fraction before further association with Mb-Cu. Mb-Cu modification can greatly improve the activity and stability of pMMO in the AuNP-pMMO nanobiohybrids. It is shown that the Mb-Cu-modified AuNP-pMMO nanobiohybrids can persistently catalyze the conversion of methane to methanol with hydroquinone as electron donor. The artificial heterogeneous nanobiohybrids exhibited excellent reusability and reproducibility in three cycles of catalysis, and they provide a model for achieving hydroquinone-driven conversion of methane to methanol.