Elucidating the Role of O 2 Uncoupling in the Oxidative Biodegradation of Organic Contaminants by Rieske Non-heme Iron Dioxygenases.

Oxygenations of aromatic soil and water contaminants with molecular O 2 catalyzed by Rieske dioxygenases are frequent initial steps of biodegradation in natural and engineered environments. Many of these non-heme ferrous iron enzymes are known to be involved in contaminant metabolism, but the understanding of enzyme-substrate interactions that lead to successful biodegradation is still elusive. Here, we studied the mechanisms of O 2 activation and substrate hydroxylation of two nitroarene dioxygenases to evaluate enzyme- and substrate-specific factors that determine the efficiency of oxygenated product formation. Experiments in enzyme assays of 2-nitrotoluene dioxygenase (2NTDO) and nitrobenzene dioxygenase (NBDO) with methyl-, fluoro-, chloro-, and hydroxy-substituted nitroaromatic substrates reveal that typically 20-100% of the enzyme's activity involves unproductive paths of O 2 activation with generation of reactive oxygen species through so-called O 2 uncoupling. The 18 O and 13 C kinetic isotope effects of O 2 activation and nitroaromatic substrate hydroxylation, respectively, suggest that O 2 uncoupling occurs after generation of Fe III -(hydro)peroxo species in the catalytic cycle. While 2NTDO hydroxylates ortho -substituted nitroaromatic substrates more efficiently, NBDO favors meta -substituted, presumably due to distinct active site residues of the two enzymes. Our data implies, however, that the O 2 uncoupling and hydroxylation activity cannot be assessed from simple structure-reactivity relationships. By quantifying O 2 uncoupling by Rieske dioxygenases, our work provides a mechanistic link between contaminant biodegradation, the generation of reactive oxygen species, and possible adaptation strategies of microorganisms to the exposure of new contaminants.