title: Methane monooxygenase from Methylosinus trichosporium OB3b. Purification and properties of a three-component system with high specific activity from a type II methanotroph.
abstract: Methane monooxygenase has been purified from the Type II methanotroph Methylosinus trichosporium OB3b. As observed for methane monooxygenase isolated from Type I methanotrophs, three protein components are required: a 39.7-kDa NADH reductase containing 1 mol each of FAD and a [2Fe-2S] cluster, a 15.8-kDa protein factor termed component B that contains no metals or cofactors, and a 245-kDa hydroxylase which appears to contain an oxo- or hydroxo-bridged binuclear iron cluster. Through the use of stabilizing reagents, the hydroxylase is obtained in high yield and exhibits a specific activity 8-25-fold greater than reported for previous preparations. The component B and reductase exhibit 1.5- and 4-fold greater specific activity, respectively. Quantitation of the hydroxylase oxo-bridged cluster using EPR and Mössbauer spectroscopies reveals that the highest specific activity preparations (approximately 1700 nmol/min/mg) contain approximately 2 clusters/mol. In contrast, hydroxylase preparations exhibiting a wide range of specific activities below 500 nmol/min/mg contain approximately 1 cluster/mol on average. Efficient turnover coupled to NADH oxidation requires all three protein components. However, both alkanes and alkenes are hydroxylated by the chemically reduced hydroxylase under single turnover conditions in the absence of component B and the reductase. Neither of these components catalyzes hydroxylation individually nor do they significantly affect the yield of hydroxylated product from the chemically reduced hydroxylase. Hydroxylase reduced only to the mixed valent [Fe(II).Fe(III)] state is unreactive toward O2 and yields little hydroxylated product on single turnover. This suggests that the catalytically active species is the fully reduced form. The data presented here provide the first evidence based on catalysis that the site of the monooxygenation reaction is located on the hydroxylase. It thus appears likely that the oxo-bridged iron cluster is capable of catalyzing oxygenase reactions without the intervention of other cofactors. This is a novel function for this type of cluster and implies a new mechanism for the generation of highly reactive oxygen capable of insertion into unactivated carbon-hydrogen bonds.
authors.affiliation: Department of Biochemistry, Medical School, University of Minnesota, Minneapolis 55455.
authors.forename: B G
authors.initials: BG
authors.lastname: Fox
authors.forename: W A
authors.initials: WA
authors.lastname: Froland
authors.forename: J E
authors.initials: JE
authors.lastname: Dege
authors.forename: J D
authors.initials: JD
authors.lastname: Lipscomb
mesh_terms: D002474:Cell-Free System; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004578:Electron Spin Resonance Spectroscopy; D019605:Euryarchaeota / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D009097:Multienzyme Complexes; D010105:Oxygenases; C021555:methane monooxygenase