DETERGENT SOLUBILIZATION OF MEMBRANE-BOUND METHANE MONOOXYGENASE REQUIRES PLASTOQUINOL ANALOGS AS ELECTRON-DONORS

Quinols can provide reducing equivalents for the membrane-bound form o f methane monooxygenase (pMMO), substituting for NADH in whole cells a nd membranes. Furthermore, quinols are effective reductants for the de tergent-solubilized enzyme, whereas NADH is ineffective. The decyl ana log of plastoquinol and duroquinol (2,3,5,6-tetramethylbenzoquinol) pr ovide the greatest methane monooxygenase activity in whole cells and m embrane suspensions, as well as detergent-solubilized samples. Lauryl maltoside is by far the best detergent for solubilization of catalytic ally active methane monooxygenase. Optimal pMMO activity in the deterg ent-solubilized fraction is obtained with a ratio of similar to 1.7 mg of detergent per milligram of membrane protein, independent of protei n concentration. The detergent-solubilized pMMO retains its sensitivit y to inhibition by cyanide, acetylene, and EDTA. It is also stimulated by exogenous copper, as in isolated membrane fractions. Reaction of t he detergent-solubilized enzyme with [C-14]acetylene results in labeli ng of a 26-kDa peptide, analogous to the behavior observed for isolate d membrane suspensions. The selectivity of pMMO for duroquinol and dec yl-plastoquinol, relative to other structurally similar quinols, sugge sts that the enzyme obtains reducing equivalents directly from a quino l (probably plastoquinol) in vivo. (C) 1995 Academic Press Inc.