DESCRIPTION: The PI proposes to investigate the 3D structure, active site architecture, catalytic mechanism and mechanism of activation of soluble methane monooxygenase from the Type II methanotroph Methylosinus trichosporum OB3b. This enzyme catalyzes the first step in the oxidation of methane to CO2 by methanogenic bacteria. In this way, the atmospheric egress of nearly all the enormous quantity of biogenic methane (a potent greenhouse gas) generated by anaerobic bacteria is prevented. MMO also adventitiously catalyzes the oxidation of many other hydrocarbons leading to applications in synthesis and biodegradation. Work to date suggests that the reaction is catalyzed by a cofactor not found in other oxygenases, implying a new strategy for oxygen activation. The proposed studies build on the very significant progress that has been made in the previous funding period, and focus on: elucidation of the physical and electronic structure of dinuclear iron cluster of the active site as well as the mechanism of oxygen activation catalyzed by this center; spectroscopic and kinetic characterization of the several reaction intermediates that have been identified in the catalytic sequence; elucidation of the protein-protein interactions between the three polypeptides of the enzyme that modulate its catalytic activity.