MEMBRANE-ASSOCIATED METHANE MONOOXYGENASE FROM METHYLOCOCCUS-CAPSULATUS (BATH)

An active preparation of the membrane-associated methane monooxygenase (pMMO) from Methylococcus capsulatus Bath was isolated by ion-exchang e and hydrophobic interaction chromatography using dodecyl beta-D-malt oside as the detergent, The active preparation consisted of three majo r polypeptides with molecular masses of 47,000, 27,000, and 25,000 Da, Two of the three polypeptides (those with molecular masses of 47,000 and 27,000 Da) were identified as the polypeptides induced when cells expressing the soluble MMO are switched to culture medium in which the pMMO is expressed. The 27,000-Da polypeptide was identified as the ac etylene-binding protein, The active enzyme complex contained 2.5 iron atoms and 14.5 copper atoms per 99,000 Da. The electron paramagnetic r esonance spectrum of the enzyme show;ed evidence for a type 2 copper c enter (g(perpendicular to) = 2.057, g parallel to = 2.24, and [A paral lel to] = 172 G), a weak high-spin iron signal (g = 6.0), and a broad low-field (g = 12.5) signal, Treatment of the pMMO with nitric oxide p roduced the ferrous-nitric oxide derivative observed in the membrane f raction of cells expressing the pMMO. When duroquinol was used as a re ductant, the specific activity of the purified enzyme was 11.1 nmol of propylene oxidized . min(-1). mg of protein(-1) , which accounted for approximately 30% of the cell-free propylene oxidation activity, The activity was stimulated by ferric and cupric metal ions in addition to the cytochrome b-specific inhibitors myxothiazol and 2-heptyl-4-hydro xyquinoline-N-oxide.