Previous studies of the copper enzymes, dopamine β-monooxygenase and peptidylglycine α-hydroxylating monooxygenase, have implicated Cu(II)-superoxide in substrate activation. Double isotope effects, which examine the sensitivity of the O-18 kinetic isotope effect (18O-KIE) to substrate deuteration, provide a non-perturbing probe of O2 activation. The observation of an 18O-KIE that increases following substrate deuteration indicates that activation of O2 must take place via a reversible process prior to the C-H abstraction step. While non-heme iron oxygenases have traditionally been thought to react through the intermediacy of iron-oxo species, there are instances in which iron(III)-superoxo- has been implicated. We now apply the double isotope effect method to the non-heme iron enzymes hydroxyethylphosphonate dioxygenase (HEPD) and methylphosphonate synthase (MPnS) that react with an unactivated aliphatic C-H bond of their substrate in the absence of exogenous reductant. Remarkably, these two iron enzymes reveal double isotope effects very similar to those seen earlier in the copper enzyme systems. The situations in which metallo-enzymes may employ metal-superoxo species is discussed in the context of their reaction mechanisms as well as substrate bond dissociation energies. (Supported by the National Institutes of Health, GM025765 to JPK and GM077596 to WAV)

Involvement of metal-superoxo species in iron and copper C-H activating enzymes

251st National Meeting (2016)

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