| Particulate methane monooxygenase (pMMO) is an integral membrane-bound metalloenzyme complex used by methanotrophic bacteria for the conversion of methane to methanol under ambient conditions. Understanding biological methane oxidation could aid in the rational design of new synthetic and biomimetic catalysts. The nature and location of the pMMO metal active site have been mired in controversy due to variability in the isolated metal contents, activity measurements, and spectroscopic data. Functional roles for copper and/or iron emerged and three very different active site models have been proposed; multiple trinuclear copper clusters, a diiron center, and a dinuclear copper center. Despite the dinuclear copper center being present in two crystal structures of pMMO, this did little to alleviate the active site controversy. In this work, new methods were developed to prepare metal free (apo) membrane-bound pMMO and to reconstitute apo pMMO with metal ions. Also described are protocols to clone, express, and refold metal-loaded soluble domain constructs of the pmoB subunit. Using these approaches the nature and location of the pMMO active site were answered; pMMO has copper dependent activity and oxidizes methane with a dinuclear copper active site.;The difficulty culturing methanotrophic bacteria have limited the diversity of pMMO isolations. To extend this knowledge, pMMO from Methylocystis sp. strain M has been isolated and characterized by biochemical, spectroscopic, and crystallographic methods. Membrane-bound and solubilized pMMO from Methylocystis sp. strain M have propylene epoxidation activity and contain ∼ 2 copper ions per 100 kDa pMMO protomer. Electron paramagnetic resonance (EPR) and extended x-ray absorption fine structure (EXAFS) spectroscopic data indicate that pMMO contains a type 2 Cu II center and a short (∼ 2.6 A) Cu-Cu cluster, respectively. Details of the 2.7 A resolution crystal structure, representing the highest resolution pMMO structure, resolve features of the overall architecture present in the previously solved crystal structures and provide evidence for alternative ligand placement around a conserved intramembrane metal site. Taken together, these data increase our knowledge of pMMO and support a dinuclear copper active site. |
