Oxidation reactions performed by soluble methane monooxygenase hydroxylase intermediates H(peroxo) and Q proceed by distinct mechanisms.

Soluble methane monooxygenase is a bacterial enzyme that converts methane to methanol at a carboxylate-bridged diiron center with exquisite control. Because the oxidizing power required for this transformation is demanding, it is not surprising that the enzyme is also capable of hydroxylating and epoxidizing a broad range of hydrocarbon substrates in addition to methane. In this work we took advantage of this promiscuity of the enzyme to gain insight into the mechanisms of action of H(peroxo) and Q, two oxidants that are generated sequentially during the reaction of reduced protein with O(2). Using double-mixing stopped-flow spectroscopy, we investigated the reactions of the two intermediate species with a panel of substrates of varying C-H bond strength. Three classes of substrates were identified according to the rate-determining step in the reaction. We show for the first time that an inverse trend exists between the rate constant of reaction with H(peroxo) and the C-H bond strength of the hydrocarbon examined for those substrates in which C-H bond activation is rate-determining. Deuterium kinetic isotope effects revealed that reactions performed by Q, but probably not H(peroxo), involve extensive quantum mechanical tunneling. This difference sheds light on the observation that H(peroxo) is not a sufficiently potent oxidant to hydroxylate methane, whereas Q can perform this reaction in a facile manner. In addition, the reaction of H(peroxo) with acetonitrile appears to proceed by a distinct mechanism in which a cyanomethide anionic intermediate is generated, bolstering the argument that H(peroxo) is an electrophilic oxidant that operates via two-electron transfer chemistry.