Particulate Methane Monooxygenase

DESCRIPTION (provided by applicant): Methanotrophic bacteria utilize methane as their sole source of carbon and energy. In the first step of their metabolic pathway, methane monooxygenase (MMO) enzyme systems oxidize methane to methanol. All methanotrophs produce a membrane-bound, copper-containing enzyme called particulate methane monooxygenase (pMMO). Although pMMO is the predominant methane oxidation catalyst in nature, it has proved difficult to isolate, and most investigators have instead opted to study soluble methane monooxygenase (sMMO), a diiron carboxylate-bridged enzyme that is more tractable, but less universal, than pMMO. The structure and mechanism of pMMO remains one of the major unsolved problems in bioinorganic chemistry. In addition to methane, pMMO oxidizes a variety of alkanes and alkenes, including trichloroethylene (TCE), a toxic and carcinogenic groundwater pollutant. Therefore, methanotrophs are excellent candidates for bioremediation of contaminated soil and drinking water. Since pMMO is the primary biocatalytic system in these processes, an understanding of its structure and mechanism could help to engineer microorganisms with different substrate selectivities for future bioremediation applications. Methanotrophs also play a key role in the global methane cycle, preventing large quantities of methane from reaching the atmosphere where it is a potent greenhouse gas. Global warming due to increasing greenhouse gas emissions can have detrimental effects on human health. Some biochemical and biophysical studies of pMMO have been conducted, but most questions regarding the molecular structure and active site remain unanswered. The evidence to date points to a primary catalytic role for copper ions as well as the possible existence of a functional iron center. The exact stoichiometry, location, and ligand environment of the metal ions in the pMMO complex have not been elucidated. The specific aims of the proposed research are to solubilize and purify active pMMO from multiple strains of methanotrophic bacteria, to characterize the metal centers in pMMO, to obtain well diffracting crystals of pMMO, and to determine its X-ray structure.