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The membrane bound enzyme methane monooxygenase (pMMO), is a mixed function oxygenase that initiates the oxidative metabolism of methane in methanotropic bacteria. Copper is required for catalytic activity of pMMO, illustrated by the recovery of pMMO activity in EDTA-inhibited membranes, whereas zinc and nickel had little effect or were inhibitory. Inhibition kinetics, cosubstrate dependence, and substrate protection experiments show that acetylene acts as a suicide substrate for pMMO, binding specifically to a 26-kDa peptide. The acetylene binding capability of pMMO increased as a function of copper concentration as does the specific activity, suggesting that copper activates pMMO by binding to the inactive apo-pMMO. NADH serves as the source of electrons necessary to drive the pMMO reaction, and detergent solubilization results in the loss of this ability. However, quinols can provide reducing equivalents to detergent-solubilized pMMO. Optimal solubilized pMMO activity is obtained with a ratio of 1.7 mg of lauryl maltoside per mg of membrane protein, with decyl-plastoquinol serving as the electron source. Detergent-solubilized pMMO displayed many of the characteristics of the membrane bound form: copper stimulation, acetylene inhibition and labeling, and EDTA and cyanide inhibition. Loss of NADH reduction upon detergent solubilization is explained by dilution or destruction of the natural quinone reductant. Addition of exogenous oxidized quinones restored NADH-dependent pMMO activity in the detergent soluble fraction. The enzyme responsible for reducing the quinones is a type 2 NADH: quinone oxidoreductase (NDH-2). This enzyme was purified 83 fold and found to contain a 36-kDa protein with 0.9 mole of FAD per mole of enzyme. This NDH-2 was mildly inhibited by amytal and most reactive with coenzyme Q{dollar}\\sb0{dollar} as substrate. Decyl-Plastoquinol could not be tested with this NDH-2 due to its extreme insolubility.