Investigation of the Quaternary Structure and of the Active Site of Methane Monooxygenase from Methane-Oxidizing Bacteria Methylococcus capsulatus (strain M) by Physico-Chemical Methods

Abstract

Methane monooxygenase (MMO, E.C. 1.14.13.25) was purified from membranes of the methaneoxidizing bacterium Methylococcus capsulatus (strain M) and separated into two components: monooxygenase and NADH-oxidoreductase. The individual components did not oxidize methane. Oxidation of the substrate was observed in the presence of two components. MMO specific activity was 3600 nmol CH,/min/mg of the protein. The degree of purification was 170. The criterion of purity was sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed that the monooxygenase and NADH-reductase consist of subunits with a molecular weight of 35 f 3 kDa and 45 f 3 kDa, respectively. From gel chromatography on Sephadex G-200, a molecular weight of 180 kDa was found for the native NADH-reductase, suggesting a tetrameric structure for this enzyme. The quaternary structure of the NADH-reductase and monooxygenase were investigated by electron microscopy. NADH-reductase is constructed from four identical subunits with molecular weight of about 45 kDa.Two-dimensional crystalline arrays of the monooxygenase have been obtained. The monooxygenase has an hexagonal arrangement of the subunits. This enzyme contains 2 atom iron per mol of protein.One mol of FAD and 2 atoms of iron were found in one mol of NADH-reductase. The visible absorption spectrum of NADH-reductase exhibits maxima at 270, 395 and 465 nm. The apparent K,,, values of the NADH-reductase for NADH and NADPH were calculated from Lineweaver-Burk plots as 3 and 50 p~, respectively, V , , = 7.0 . M/s. NADH-reductase reduces several redox compounds: 2.6-dichlorophenolindophenol, nitroblue tetrazolium, neotetrazolium, iodonitrotetrazolium and ferricyanide.Mossbauer studies of the native monooxygenase show the presence of two near high-spin ferric ions.KEY WORDS Methane monooxygenase, NADH-reductase, enzyme structure, electron microscopy, methane-oxidizing bacteria, active site of enzyme. 39Biocatal Biotransformation Downloaded from informahealthcare.com by McMaster University on 02/06/15 For personal use only. Biocatal Biotransformation Downloaded from informahealthcare.com by McMaster University on 02/06/15 For personal use only. Biocatal Biotransformation Downloaded from informahealthcare.com by McMaster University on 02/06/15 For personal use only.