Rieske oxygenases (ROs) use an active site nonheme mononuclear Fe center (nhmFe) and a nearby 2Fe2S Rieske cluster (RC) to catalyze a diverse repertoire of oxidative chemistries mimicking those of cytochrome P450 and methane monooxygenase in both type and scope.   Members of the RO family also catalyze regio and stereospecific cis-dihydroxylation of aromatic compounds, a reaction for which they are the only known biological catalyst. The molecular mechanism of Rieske cis-diol dioxygenases remains mysterious, although it is known that one electron from each reduced metal center is utilized during a single turnover reaction to activate O2 and form the product. The reaction occurs in a highly regulated stepwise fashion, so that both metal centers must be reduced and substrate bound before O2 can bind and be activated.  At the end of the reaction cycle, the cis-diol product is bound in the active site and is only released upon re-reduction of the metals centers.  The reaction steps that occur in the middle of the reaction cycle as product is formed are largely unresolved.  Our group and others have proposed mechanisms in which the electrons from the nhmFe and RC are transferred to O2 as it binds, resulting in either an Fe(III)-(hydro)peroxo or an Fe(V)-oxo-hydroxo complex as the reactive species.  These proposals are supported by the observation that hydrogen peroxide added to the fully oxidized RO in the presence of substrate can support cis-diol formation. Moreover, both crystallographic and spectroscopic studies have revealed an Fe(III)-peroxo intermediate. Here the product forming reaction cycle steps are examined using slow alternative substrates and oxygen analogs in combination with transient kinetic and product yield studies.  The results support the formation of a novel reactive species that can account for the unique cis-diol forming chemistry of the enzyme.

Oxygen activation by Rieske cis-diol dioxygenases: Chemistry at the mononuclear active site iron

248th National Meeting (2014)

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