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

ABSTRACT: 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.

SUBMITTER: Koo CW

PROVIDER: S-EPMC9357287 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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

Koo Christopher W CW Tucci Frank J FJ He Yuan Y Rosenzweig Amy C AC

Science (New York, N.Y.) 20220317 6586

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-el ...[more]

PMID: 35298269

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Structure and activity of particulate methane monooxygenase arrays in methanotrophs.

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Crystal structure and characterization of particulate methane monooxygenase from Methylocystis species strain M.

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

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| S-EPMC6036204 | biostudies-literature

Architecture and active site of particulate methane monooxygenase.

Project description:Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria, organisms that live on methane gas as their sole carbon source. Understanding pMMO function has important implications for bioremediation applications and for the development of new, environmentally friendly catalysts for the direct conversion of methane to methanol. Crystal structures of pMMOs from three different methanotrophs reveal a trimeric architecture, consisting of three copies each of the pmoB, pmoA, and pmoC subunits. There are three distinct metal centers in each protomer of the trimer, mononuclear and dinuclear copper sites in the periplasmic regions of pmoB and a mononuclear site within the membrane that can be occupied by copper or zinc. Various models for the pMMO active site have been proposed within these structural constraints, including dicopper, tricopper, and diiron centers. Biochemical and spectroscopic data on pMMO and recombinant soluble fragments, denoted spmoB proteins, indicate that the active site involves copper and is located at the site of the dicopper center in the pmoB subunit. Initial spectroscopic evidence for O(2) binding at this site has been obtained. Despite these findings, questions remain about the active site identity and nuclearity and will be the focus of future studies.

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Particulate methane monooxygenase contains only mononuclear copper centers.

Project description:Bacteria that oxidize methane to methanol are central to mitigating emissions of methane, a potent greenhouse gas. The nature of the copper active site in the primary metabolic enzyme of these bacteria, particulate methane monooxygenase (pMMO), has been controversial owing to seemingly contradictory biochemical, spectroscopic, and crystallographic results. We present biochemical and electron paramagnetic resonance spectroscopic characterization most consistent with two monocopper sites within pMMO: one in the soluble PmoB subunit at the previously assigned active site (CuB) and one ~2 nanometers away in the membrane-bound PmoC subunit (CuC). On the basis of these results, we propose that a monocopper site is able to catalyze methane oxidation in pMMO.

| S-EPMC6664434 | biostudies-literature

Structural conservation of the B subunit in the ammonia monooxygenase/particulate methane monooxygenase superfamily.

Project description:The ammonia monooxygenase (AMO)/particulate methane monooxygenase (pMMO) superfamily is a diverse group of membrane-bound enzymes of which only pMMO has been characterized on the molecular level. The pMMO active site is believed to reside in the soluble N-terminal region of the pmoB subunit. To understand the degree of structural conservation within this superfamily, the crystal structure of the corresponding domain of an archaeal amoB subunit from Nitrosocaldus yellowstonii has been determined to 1.8 Å resolution. The structure reveals a remarkable conservation of overall fold and copper binding site location as well as several notable differences that may have implications for function and stability.

| S-EPMC4133332 | biostudies-literature

The metal centers of particulate methane monooxygenase from Methylosinus trichosporium OB3b.

Project description:Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains approximately 2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended X-ray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 A. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.

| S-EPMC2664655 | biostudies-literature

Evidence for oxygen binding at the active site of particulate methane monooxygenase.

Project description:Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. The enzyme consists of three subunits, pmoB, pmoA, and pmoC, organized in an ?(3)?(3)?(3) trimer. Studies of intact pMMO and a recombinant soluble fragment of the pmoB subunit (denoted as spmoB) indicate that the active site is located within the soluble region of pmoB at the site of a crystallographically modeled dicopper center. In this work, we have investigated the reactivity of pMMO and spmoB with oxidants. Upon reduction and treatment of spmoB with O(2) or H(2)O(2) or pMMO with H(2)O(2), an absorbance feature at 345 nm is generated. The energy and intensity of this band are similar to those of the ?-?(2):?(2)-peroxo-Cu(II)(2) species formed in several dicopper enzymes and model compounds. The feature is not observed in inactive spmoB variants in which the dicopper center is disrupted, consistent with O(2) binding to the proposed active site. Reaction of the 345 nm species with CH(4) results in the disappearance of the spectroscopic feature, suggesting that this O(2) intermediate is mechanistically relevant. Taken together, these observations provide strong new support for the identity and location of the pMMO active site.

| S-EPMC3348976 | biostudies-literature

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