Evidence that copper is a required cofactor for the membrane-bound form of methane monooxygenase

The membrane bound enzyme methane monooxygenase (pMMO) is a mixed function oxygenase that initiates the oxidative metabolism of methane in methanotrophic bacteria. In this paper, we show that copper is required for catalytic activity of pMMO. Addition of copper restores pMMO activity to EDTA-inhibited membranes, whereas nickel and zinc have substantially less effect than copper, or are inhibitory. Acetylene acts as an inhibitor of pMMO, binding specifically and irreversibly to a 26-kDa membrane protein. The inactivation of pMMO by acetylene and the incorporation of radiolabeled acetylene occurred at approximately the same rate. Furthermore, the inhibition of pMMO and concomitant irreversible binding of acetylene required enzyme activity, oxygen, and NADH, and was attenuated by pMMO substrates, suggesting that acetylene is a suicide substrate for pMMO. The specific activity and acetylene binding capability of pMMO were both found to increase as a function of copper concentration. This suggests that the increased availability of copper leads to the regeneration of active sites in copper-depleted pMMO, and that exogenously added copper is probably targeted to the substrate binding site of apo-pMMO.