Electron-transfer reactions of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath)

Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to afford methanol and water, the first step of carbon assimil ation in methanotrophic bacteria. This enzyme comprises three protein compo nents: a hydroxylase (MMOH) that contains a dinuclear nonheme iron active s ite; a reductase (MMOR) that facilitates electron transfer from NADH to the diiron site of NMOH; and a coupling protein (NMOB). MMOR uses a noncovalen tly bound FAD cofactor and a [2Fe-2S] cluster to mediate electron transfer. The gene encoding MMOR was cloned from Methylococcus capsulatus (Bath) and expressed in Escherichia coli in high yield. Purified recombinant MMOR was indistinguishable from the native protein in all aspects examined, includi ng activity, mass, cofactor content, and EPR spectrum of the [2Fe-2S] clust er. Redox potentials for the FAD and [2Fe-2S] cofactors, determined by redu ctive titrations in the presence of indicator dyes, are FAD(ox/sq), - 176 /- 7 mV; FAD(sq/hq), -266 +/- 15 mV; and [2Fe-2S](ox/red), -209 +/- 14 mV. The midpoint potentials of MMOR are not altered by the addition of MMOH, MM OB, or both MMOH and MMOB. The reaction of MMOR with NADH was investigated by stopped-flow UV-visible spectroscopy, and the kinetic and spectral prope rties of intermediates are described. The effects of pH on the redox proper ties of MMOR are described and exploited in pH jump kinetic studies to meas ure the rate constant of 130 +/- 17 s(-1) for electron transfer between the FAD and [2Fe-2S] cofactors in two-electron-reduced MMOR. The thermodynamic and kinetic parameters determined significantly extend our understanding o f the sMMO system.