Characterization of a Peroxodiiron(III) Intermediate in the T201S Variant of Toluene/o-Xylene Monooxygenase Hydroxylase from Pseudomonas sp. OX1

Abstract

We report the observation of a novel intermediate in the reaction of a reduced toluene/o-xylene monooxygenase hydroxylase (ToMOH[subscript red]) T201S variant, in the presence of a regulatory protein (ToMOD), with dioxygen. This species is the first oxygenated intermediate with an optical band in any toluene monooxygenase. The UV−vis and Mössbauer spectroscopic properties of the intermediate allow us to assign it as a peroxodiiron(III) species, T201S[subscript peroxo], similar to H[subscript peroxo] in methane monooxygenase. Although T201S generates T201S[subscript peroxo] in addition to optically transparent ToMOH[subscript peroxo], previously observed in wild-type ToMOH, this conservative variant is catalytically active in steady-state catalysis and single-turnover experiments and displays the same regiospecificity for toluene and slightly different regiospecificity for o-xylene oxidation.