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

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.

The membrane-associated form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a copper/iron protein.

A protocol has been developed which permits the purification of a membrane-associated methane-oxidizing complex from Methylococcus capsulatus (Bath). This complex has approximately 5 fold higher specific activity than any purified particulate methane mono-oxygenase (pMMO) previously reported from M. capsulatus (Bath). This efficiently functioning methane-oxidizing complex consists of the pMMO hydroxylase (pMMOH) and an unidentified component we have assigned as a potential pMMO reductase (pMMOR). The complex was isolated by solubilizing intracytoplasmic membrane preparations containing the high yields of active membrane-bound pMMO (pMMO(m)), using the non-ionic detergent dodecyl-beta-D-maltoside, to yield solubilized enzyme (pMMO(s)). Further purification gave rise to an active complex (pMMO(c)) that could be resolved (at low levels) by ion-exchange chromatography into two components, the pMMOH (47, 27 and 24 kDa subunits) and the pMMOR (63 and 8 kDa subunits). The purified complex contains two copper atoms and one non-haem iron atom/mol of enzyme. EPR spectra of preparations grown with (63)Cu indicated that the copper ion interacted with three or four nitrogenic ligands. These EPR data, in conjunction with other experimental results, including the oxidation by ferricyanide, EDTA treatment to remove copper and re-addition of copper to the depleted protein, verified the essential role of copper in enzyme catalysis and indicated the implausibility of copper existing as a trinuclear cluster. The EPR measurements also demonstrated the presence of a tightly bound mononuclear Fe(3+) ion in an octahedral environment that may well be exchange-coupled to another paramagnetic species.