Characterization of the second prosthetic group of the flavoenzyme NADH-acceptor reductase (component C) of the methane mono-oxygenase from Methylococcus capsulatus (Bath).
1. A new two-step purification is described that routinely yields 100mg quantities of component C for biochemical studies. 2. Chemical analyses show component C purified by this procedure to contain 2 g-atoms of iron, 2 mol of acid-labile sulphide (S) and 1 mol of FAD per mol of protein. 3. The Fe-S core of component C was extruded by treating the protein with p-methoxybenzenethiol in hexamethyl phosphoramide/50mM-Tris/HCl buffer, pH 8.5 (4:1, v/v), under anaerobic conditions. The spectral properties of the extruded core suggest that component C contains 1 mol of [2Fe-2S(S-Cys)4] centre per mol of protein. 4. E.p.r. spectroscopy confirms the presence of a Fe-S centre in component C. 5. Component C catalyses 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. 6. The optimum pH for the NADH-acceptor reductase activity is 8.5--9.0, and the apparent Km values for NADH and NADPH are 0.05mM and 15.5mM respectively. 7. Unlike methane mono-oxygenase activity, NADH-acceptor reductase activity of component C is not inhibited by 8-hydroxyquinoline or by acetylene.
D004578:Electron Spin Resonance Spectroscopy; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D005420:Flavoproteins / Q000302:isolation & purification*; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification*; D008667:Metalloproteins / Q000302:isolation & purification*; D008697:Methane; D008772:Methylococcaceae / Q000201:enzymology*; D010105:Oxygenases; D013053:Spectrophotometry
D005420:Flavoproteins; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D005182:Flavin-Adenine Dinucleotide; D010105:Oxygenases; D008697:Methane