Probing the mechanism of C-H activation:: Oxidation of methylcubane by soluble methane monooxygenase from Methylosinus trichosporium OB3b

The soluble form of methane monooxygenase (MMO) isolated from methanotrophic bacteria catalyzes the O-2-dependent conversion of methane to methanol, as well as the adventitious oxidation of many other hydrocarbons. In past studies, it was reported that the oxidation reaction of methylcubane, a radical clock substrate, catalyzed by MMO from Methylococcus capsulatus (Bath) gave only cubylmethanol as the product rather than methylcubanol(s) or rearranged products characteristic of a radical formed on the methyl group [Choi, S.-Y., Eaten, P. E., Hollenberg, P. F., Liu, K. E., Lippard, S. J., Newcomb, M., Putt, D. A., Upadhyaya, S. P., and Xiong, Y. (1996) J. Am. Chem. Soc. 118, 6547-6555]. Such a substrate radical intermediate would be expected if the mechanism of MMO involves hydrogen atom abstraction as indicated by many previous mechanistic studies. Here it is shown that the reaction of methylcubane with the reconstituted MMO system from Methylosinus trichosporium OB3b yields both cubylmethanol and methylcubanols, with methyl hydroxylation favored over cubyl hydroxylation. This unexpected regioselectivity indicates steric effects on the reaction in agreement with past product distribution studies. In addition, the apparent majority product of the reaction is tentatively assigned as one of the possible rearranged products for this radical probe, on the basis of gas chromatography and mass spectrometry data: This result suggests the formation of a radical intermediate in the reaction, thus supporting a radical-based mechanism for this form of MMO.