Oxidation Reactions Performed by Soluble Methane Monooxygenase Hydroxylase Intermediates Hperoxo 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.