Oxygen kinetic isotope effects in soluble methane monooxygenase

Soluble methane monooxygenase (sMMO) contains a nonheme, carboxylate-bridge d diiron site that activates dioxygen in the catalytic oxidation of hydroca rbon substrates, Oxygen kinetic isotope effects (KIEs) have been determined under steady-state conditions for the sMMO-catalyzed oxidation of CH3CN, a liquid substrate analog. Kinetic studies of the steady-state sMMO reaction revealed a competition between fully coupled oxygenase activity, which pro duced glycolonitrile (HOCH2CN) and uncoupled oxidase activity that led to w ater formation. The oxygen KIE was measured independently for both the oxyg enase and oxidase reactions, and values of 1.0152 +/- 0.0007 and 1.0167 +/- 0.0010 were obtained, respectively. The isotope effects and separate dioxy gen binding studies do not support irreversible formation of an enzyme-diox ygen Michaelis complex. Additional mechanistic implications are discussed i n the context of previous data obtained from single turnover and steady-sta te kinetic studies.