Publications

• Detergent solubilization of membrane-bound methane monooxygenase requires plastoquinol analogs as electron donors.

  Shiemke A.K., Cook S.A., Miley T., Singleton P.

  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 plastoquino ... >> More

  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. << Less

  Arch. Biochem. Biophys. 321:421-428(1995) [PubMed] [EuropePMC]

• Characterization and structural analysis of an active particulate methane monooxygenase trimer from Methylococcus capsulatus (Bath).

  Kitmitto A., Myronova N., Basu P., Dalton H.

  The oxidation of methane to methanol in methanotrophs is catalyzed by the enzyme methane monooxygenase (MMO). Two distinct forms of this enzyme exist, a soluble cytoplasmic MMO (sMMO) and a membrane-bound particulate form (pMMO). We describe here the biochemical characterization of a stable and ac ... >> More

  The oxidation of methane to methanol in methanotrophs is catalyzed by the enzyme methane monooxygenase (MMO). Two distinct forms of this enzyme exist, a soluble cytoplasmic MMO (sMMO) and a membrane-bound particulate form (pMMO). We describe here the biochemical characterization of a stable and active purified pMMO hydroxylase (pMMO-H) and report a three-dimensional (3D) structure, determined by electron microscopy and single-particle analysis at 23 A resolution. Both biochemical and structural data indicate that pMMO hydroxylase is trimeric, with each monomer unit comprised of three polypeptides of 47, 26, and 23 kDa. Comparison of the recent crystal structure [Lieberman, R. L., and Rosenzweig, A. C. (2005) Nature 434, 177] of an uncharacterized pMMO-H complex with the three-dimensional (3D) structure determined here yielded a good match between the principal features and the organization of the enzyme monomers into trimers. The data presented here advance our current understanding of particulate methane monooxygenase function by the characterization of an active form of the enzyme and the corresponding 3D structure. << Less

  Biochemistry 44:10954-10965(2005) [PubMed] [EuropePMC]