DIRECT EVIDENCE FOR A SOLUBLE METHANE MONOOXYGENASE FROM TYPE-I METHANOTROPHIC BACTERIA - PURIFICATION AND PROPERTIES OF A SOLUBLE METHANE MONOOXYGENASE FROM METHYLOMONAS SP. GYJ3

The hydroxylase and reductase components of a soluble methane monooxyg enase from type I methanotrophs-Methylomonas sp. GYJ3-were purified by a multiple-step LC procedure, The hydroxylase (approximately 240 kDa, determined by an HPLC-size exclusion chromatography method) has three subunits with molecular masses of 56, 43, and 27 kDa, suggesting that the enzyme has an (alpha beta gamma)(2) subunit structure, The HPLC m ethod was developed to purify the hydroxylase component, and the purif ied protein has a specific activity of 541 nmol propene oxide . mg(-1) protein . min(-1), which is two times the specific activity of the pr otein purified by the two-step LC procedure, The iron content in the h ydroxylase purified by the two-step LC procedure is 2.1 mol of Fe per mole of protein, but the iron content in the protein by the HPLC proce dure is 3.78 mol of Fe per mole of protein, The diversity of iron cont ents in this protein is due mainly to the use of different purificatio n methods, The reductase has a molecular mass of 42 kDa, The UV-VIS sp ectrum of the protein is similar to that of proteins from other methan otrophs, suggesting that the protein contains a FAD cofactor and a [2F e-2S] center, The partially purified component B stimulated the MMO ac tivity of the hydroxylase and reductase system by 40-fold. (C) 1997 Ac ademic Press.