Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol ⅐ min ؊1 ⅐ mg of protein ؊1 . Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 M) to 95 M. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/ protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio.Methanotrophs are a group of gram-negative bacteria that utilize methane or methanol as the sole source of carbon and energy (1,20). The initial oxidation of methane to methanol is catalyzed by methane monooxygenase (MMO). In some methanotrophs, two different MMOs can be expressed, depending on the copper concentration during growth (11, 37, 39): a soluble cytoplasmic MMO (sMMO) and a membrane-associated, or particulate, MMO (pMMO). In cells cultured under low copper/biomass ratios (Յ0.9 nmol of Cu/mg of cell protein), the sMMO is expressed (20, 28). Cells cultured under higher copper/biomass ratios express pMMO, and there is no detectable sMMO expression (35,43). While sMMO is a wellcharacterized enzyme that consists of a hydroxylase component composed of three polypeptides and a hydroxo-bridged binuclear iron cluster-an NADH-dependent reductase component composed of one polypeptide containing both FAD and [Fe 2 S 2 ] cofactors and a regulatory polypeptide (18,26,27,31,47)-information on the molecular properties of pMMO is limited due to the instability of pMMO in cell-free fractions.Purification of the pMMO has been reported from Methylococcus capsulatus Bath (2,25,33, 52) and M. trichosporium

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