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 methanotrophi c bacteria catalyzes the O-2-dependent conversion of methane to methanol, a s well as the adventitious oxidation of many other hydrocarbons. In past st udies, it was reported that the oxidation reaction of methylcubane, a radic al clock substrate, catalyzed by MMO from Methylococcus capsulatus (Bath) g ave only cubylmethanol as the product rather than methylcubanol(s) or rearr anged 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., Newco mb, M., Putt, D. A., Upadhyaya, S. P., and Xiong, Y. (1996) J. Am. Chem. So c. 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 m ethylcubane with the reconstituted MMO system from Methylosinus trichospori um OB3b yields both cubylmethanol and methylcubanols, with methyl hydroxyla tion favored over cubyl hydroxylation. This unexpected regioselectivity ind icates steric effects on the reaction in agreement with past product distri bution 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 da ta: This result suggests the formation of a radical intermediate in the rea ction, thus supporting a radical-based mechanism for this form of MMO.