Oxidation Reactions Performed by Soluble Methane Monooxygenase Hydroxylase Intermediates Hperoxo and Q Proceed by Distinct Mechanisms

摘要

Soluble methane monooxygenase is a bacterial enzyme that converts methane to methanol at ancarboxylate-bridged diiron center with exquisite control. Because the oxidizing power required for thisntransformation is demanding, it is not surprising that the enzyme is also capable of hydroxylating andnepoxidizing a broad range of hydrocarbon substrates in addition to methane. In this work we took advantagenof this promiscuity of the enzyme to gain insight into themechanisms of action ofHperoxo andQ, two oxidantsnthat are generated sequentially during the reaction of reduced protein with O2. Using double-mixing stopped-nflow spectroscopy, we investigated the reactions of the two intermediate species with a panel of substrates ofnvarying C-Hbond strength. Three classes of substrates were identified according to the rate-determining stepnin the reaction. We show for the first time that an inverse trend exists between the rate constant of reactionnwith Hperoxo and the C-H bond strength of the hydrocarbon examined for those substrates in which C-Hnbond activation is rate-determining.Deuteriumkinetic isotope effects revealed that reactions performed byQ,nbut probably not Hperoxo, involve extensive quantum mechanical tunneling. This difference sheds light on thenobservation that Hperoxo is not a sufficiently potent oxidant to hydroxylate methane, whereas Q can performnthis reaction in a facile manner. In addition, the reaction of Hperoxo with acetonitrile appears to proceed by andistinct mechanism in which a cyanomethide anionic intermediate is generated, bolstering the argument thatnHperoxo is an electrophilic oxidant that operates via two-electron transfer chemistry.