Sa. Cook et Ak. Shiemke, EVIDENCE THAT COPPER IS A REQUIRED COFACTOR FOR THE MEMBRANE-BOUND FORM OF METHANE MONOOXYGENASE, Journal of inorganic biochemistry, 63(4), 1996, pp. 273-284
The membrane bound enzyme methane monooxygenase (pMMO) is a mixed func
tion oxygenase that initiates the oxidative metabolism of methane in m
ethanotrophic bacteria. In this paper, we show that copper is required
for catalytic activity of pMMO. Addition of copper restores pMMO acti
vity to EDTA-inhibited membranes, whereas nickel and zinc have substan
tially less effect than copper, or are inhibitory. Acetylene acts as a
n inhibitor of pMMO, binding specifically and irreversibly to a 26-kDa
membrane protein. The inactivation of pMMO by acetylene and the incor
poration 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 suic
ide substrate for pMMO. The specific activity and acetylene binding ca
pability of pMMO were both found to increase as a function of copper c
oncentration. 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 b
inding site of apo-pMMO.