Substrate-Specific Coupling of O(2) Activation to Hydroxylations of Aromatic Compounds by Rieske Non-heme Iron Dioxygenases

[Image: see text] Rieske dioxygenases catalyze the initial steps in the hydroxylation of aromatic compounds and are critical for the metabolism of xenobiotic substances. Because substrates do not bind to the mononuclear non-heme Fe(II) center, elementary steps leading to O(2) activation and substrate hydroxylation are difficult to delineate, thus making it challenging to rationalize divergent observations on enzyme mechanisms, reactivity, and substrate specificity. Here, we show for nitrobenzene dioxygenase, a Rieske dioxygenase capable of transforming nitroarenes to nitrite and substituted catechols, that unproductive O(2) activation with the release of the unreacted substrate and reactive oxygen species represents an important path in the catalytic cycle. Through correlation of O(2) uncoupling for a series of substituted nitroaromatic compounds with (18)O and (13)C kinetic isotope effects of dissolved O(2) and aromatic substrates, respectively, we show that O(2) uncoupling occurs after the rate-limiting formation of Fe(III)-(hydro)peroxo species from which substrates are hydroxylated. Substituent effects on the extent of O(2) uncoupling suggest that the positioning of the substrate in the active site rather than the susceptibility of the substrate for attack by electrophilic oxygen species is responsible for unproductive O(2) uncoupling. The proposed catalytic cycle provides a mechanistic basis for assessing the very different efficiencies of substrate hydroxylation vs unproductive O(2) activation and generation of reactive oxygen species in reactions catalyzed by Rieske dioxygenases.