Characterization of the second prosthetic group of the flavoenzyme NADH–acceptor reductase (component C) of the methane mono-oxygenase from Methylococcus capsulatus (Bath)

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Abstract
A new, 2-step purification is described that routinely yields 100 mg quantities of component C for biochemical studies. Chemical analyses show component C purified by this procedure to contain 2 g-atoms of Fe, 2 mol of acid-labile sulfide (S*) and 1 mol of FAD/mol of protein. The Fe-S* core of component C was extruded by treating the protein with p-methoxybenzenethiol in hexamethyl phosphoramide/50 mM-Tris/HCl buffer, pH 8.5 (4:1, vol/vol), under anaerobic conditions. The spectral properties of the extruded core suggest that component C contains 1 mol of [2Fe-2S*(S-Cys)4] center/mol of protein. EPR spectroscopy confirms the presence of an Fe-S* center in component C. Component C catalyzes the reduction by NADH of ferricyanide, 2,6-dichlorophenol-indophenol or horse heart cytochrome c, with specific activities of 50-230 units/mg of protein. The optimum pH for the NADH-acceptor reductase activity is 8.5-9.0, and the apparent Km values for NADH and NADPH are 0.05 mM and 15.5 mM, respectively. Unlike methane mono-oxygenase activity, NADH-acceptor reductase activity of component C is not inhibited by 8-hydroxyquinoline or by acetylene.

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