HALOALKENE OXIDATION BY THE SOLUBLE METHANE MONOOXYGENASE FROM METHYLOSINUS-TRICHOSPORIUM OB3B - MECHANISTIC AND ENVIRONMENTAL IMPLICATIONS

The soluble, three-protein component methane monooxygenase purified from Methylosinus trichosporium OB3b is capable of oxidizing chlorinated, fluorinated, and brominated alkenes, including the widely distributed ground-water contaminant trichloroethylene (TCE). The oxidation rates for the chloroalkenes were observed to be comparable to that for methane, the natural substrate, and up to 7000-fold higher than those reported for other well-defined biological systems. The competitive inhibitor tetrachloroethylene was found to be the only chlorinated ethylene not turned over. However, this appears to be due to steric effects rather than electronic effects or the lack of an abstractable proton because chlorotrifluoroethylene was efficiently oxidized. As evidenced by the formation of diagnostic adducts with 4-(p-nitrobenzyl)pyridine, the halogenated alkenes were oxidized predominantly by epoxidation. Stable acidic products resulting from subsequent hydrolysis were identified as the major products. However, additional aldehydic products resulting from intramolecular halide or hydride migration were observed in 3–10% yield during the oxidation of TCE, vinylidene chloride, trifluoroethylene, and tribromoethylene. Product analysis of the hydrolysis reaction of authentic TCE epoxide showed little or no 2,2,2-trichloro-Acetaldehyde (chloral) formation, indicating that atomic migration occurred prior to product dissociation from the enzyme. The occurrence of atomic migration products shows that an intermediate in the substrate to product conversion carries significant cationic character. Such a species could be generated through interaction with a highly electron-deficient activated oxygen in the active site. The oxidation of TCE to TCE epoxide and chloral has also been reported for microsomal cytochrome P-450 [Miller, R. E., & Guengerich, F. P. (1982) Biochemistry 21, 1090–1097], suggesting that cytochrome P-450 and methane monooxygenase utilize a similar oxidizing species. A turnover-dependent inactivation of all methane monooxygenase protein components occurred during the oxidation of TCE. Radiolabeling of each of the components during turnover of [1,2-14C2]TCE showed that covalent modification by a diffusible product of the reaction had occurred. Correlation of the rates of inactivation with product formation suggests that the modifying species is a hydrolysis product of TCE epoxide. © 1990, American Chemical Society. All rights reserved.