Rieske oxygenases (ROs) use a nonheme mononuclear Fe and a 2Fe2S Rieske cluster located 12 Å apart in adjacent subunits to catalyze a range of oxidative reactions that rivals those of cyt P450 and methane monooxygenase. Like the latter two enzymes, two electrons transferred from NAD(P)H to the RO metal centers are required for reaction. Our past studies have focused on Rieske dioxygenases (RDO) that insert both atoms of oxygen from O2 into an aromatic substrate to form a non-aromatic cis-dihydrodiol product. During a single turnover (STO) reaction, one electron is utilized from each metal center. Pre-steady-state STO studies and computational approaches have shown that, contrary to expectation, the reactive oxygen species is an Fe(III)-O2●- rather than a (two electron reduced) peroxo or a high valent HO-Fe(V)=O that could derive from it. Nevertheless, the large range of catalytic chemistry suggests that one or both of the latter two more potent oxidants might be utilized by some members of the RO class. Here we examine the reaction of a Rieske monooxygenase (RMO) that catalyzes aromatic hydroxylation. Pre-steady state kinetic studies show that rate constant for electron transfer from the Rieske cluster to the mononuclear Fe is dependent on the electronic characteristics of the substrate, suggesting that the initial attack on the substrate by an active oxygen species occurs before electron transfer. This observation is consistent with the formation of a one electron reduced reactive oxygen species such as Fe(III)- O2●-, as in the RDO case. In contrast to other ROs reactions, the product complex with the iron can be directly detected by its distinctive chromophore for the RMO reaction. Pre-steady state kinetic measurements show that product release occurs in several steps involving complexation with the iron and conformational changes. The relationship of these intermediates to regulation of catalysis will be discussed.

Intermediates in aromatic hydroxylation catalyzed by a Rieske monooxygenase

2019 Fall National Meeting

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