Elucidating the Role of O2 Uncoupling in the Oxidative Biodegradation of Organic Contaminants by Rieske Non-heme Iron Dioxygenases

Abstract

Oxygenations of aromatic soil and water contaminants with molecular O₂ 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 O₂ 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 O₂ activation with generation of reactive oxygen species through so-called O₂ uncoupling. The ¹⁸O and ¹³C kinetic isotope effects of O₂ activation and nitroaromatic substrate hydroxylation, respectively, suggest that O₂ 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 O₂ uncoupling and hydroxylation activity cannot be assessed from simple structure–reactivity relationships. By quantifying O₂ 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.