Detergent solubilization of membrane-bound methane monooxygenase requires plastoquinol analogs as electron donors.
Quinols can provide reducing equivalents for the membrane-bound form of methane monooxygenase (pMMO), substituting for NADH in whole cells and membranes. Furthermore, quinols are effective reductants for the detergent-solubilized enzyme, whereas NADH is ineffective. The decyl analog of plastoquinol and duroquinol (2,3,5,6-tetramethylbenzoquinol) provide the greatest methane monooxygenase activity in whole cells and membrane suspensions, as well as detergent-solubilized samples. Lauryl maltoside is by far the best detergent for solubilization of catalytically active methane monooxygenase. Optimal pMMO activity in the detergent-solubilized fraction is obtained with a ratio of approximately 1.7 mg of detergent per milligram of membrane protein, independent of protein concentration. The detergent-solubilized pMMO retains its sensitivity to inhibition by cyanide, acetylene, and EDTA. It is also stimulated by exogenous copper, as in isolated membrane fractions. Reaction of the detergent-solubilized enzyme with [14C]acetylene results in labeling of a 26-kDa peptide, analogous to the behavior observed for isolated membrane suspensions. The selectivity of pMMO for duroquinol and decyl-plastoquinol, relative to other structurally similar quinols, suggests that the enzyme obtains reducing equivalents directly from a quinol (probably plastoquinol) in vivo.
Shiemke
A K
AK
Department of Biochemistry, Robert C. Byrd Health Sciences Center, West Virginia University School of Medicine, Morgantown 26506-9142, USA.
Cook
S A
SA
Miley
T
T
Singleton
P
P
D002462:Cell Membrane / Q000201:enzymology; D003902:Detergents; D004579:Electron Transport; D019605:Euryarchaeota / Q000201:enzymology*; D006873:Hydroquinones / Q000494:pharmacology*; D007700:Kinetics; D010105:Oxygenases / Q000302:isolation & purification* / Q000378:metabolism*; D010971:Plastoquinone / Q000031:analogs & derivatives* / Q000494:pharmacology; D012995:Solubility; D013329:Structure-Activity Relationship
D003902:Detergents; D006873:Hydroquinones; C003165:plastoquinol; D010105:Oxygenases; C021555:methane monooxygenase; D010971:Plastoquinone