Purification and properties of the methane mono-oxygenase enzyme system from Methylosinus trichosporium OB3b.
1. A three-component enzyme system that catalyses the oxidation of methane to methanol has been highly purified from Methylosinus trichosporium. 2. The components are (i) a soluble CO-binding cytochrome c, (ii) a copper-containing protein and (iii) a small protein; the mol. wts. are 13 000, 47 000 and 9400 respectively. The cytochrome component cannot be replaced by similar cytochrome purified from Pseudomonas extorquens or by horse heart cytochrome c. 3. The stoicheiometry suggests a mono-oxygenase mechanism and the specific activity with methane as substrate is 6 micronmol/min per mg of protein. 4. Other substrates rapidly oxidized are ethane, n-propane, n-butane and CO. Dimethyl ether is not a substrate. 5. The purified enzyme system utilizes ascorbate or, in the presence of partially purified M. trichosporium methanol dehydrogenase, methanol as electron donor but not NADH or NADPH. 6. Activity is highly sensitive to low concentrations of a variety of chelating agents, cyanide, 2-mercaptoethanol and dithiothreitol. 7. Activity is highly pH-dependent (optimum 6.9-7.0) and no component of the enzyme is stable to freezing. 8. The soluble CO-binding cytochrome c shows oxidase acitivity and the relationship between this and the oxygenase activity is discussed.
D001204:Ascorbate Oxidase / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology*; D002248:Carbon Monoxide; D002474:Cell-Free System; D003300:Copper / Q000032:analysis; D003574:Cytochrome c Group / Q000302:isolation & purification; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000032:analysis; D008697:Methane; D008970:Molecular Weight; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013057:Spectrum Analysis; D014461:Ultracentrifugation
D003574:Cytochrome c Group; D003300:Copper; D002248:Carbon Monoxide; D007501:Iron; D001204:Ascorbate Oxidase; D010105:Oxygenases; D008697:Methane