Characterization of O₂ uncoupling in biodegradation reactions of nitroaromatic contaminants catalyzed by rieske oxygenases

Rieske oxygenases are known as catalysts that enable the cleavage of aromatic and aliphatic C–H bonds in structurally diverse biomolecules and recalcitrant organic environmental pollutants through substrate oxygenations and oxidative heteroatom dealkylations. Yet, the unproductive O₂ activation, which is concomitant with the release of reactive oxygen species (ROS), is typically not taken into account when characterizing Rieske oxygenase function. Even if considered an undesired side reaction, this O₂ uncoupling allows for studying active site perturbations, enzyme mechanisms, and how enzymes evolve as environmental microorganisms adapt their substrates to alternative carbon and energy sources. Here, we report on complementary methods for quantifying O₂ uncoupling based on mass balance or kinetic approaches that relate successful oxygenations to total O₂ activation and ROS formation. These approaches are exemplified with data for two nitroarene dioxygenases (nitrobenzene and 2-nitrotoluene dioxygenase) which have been shown to mono- and dioxygenate substituted nitroaromatic compounds to substituted nitrobenzylalcohols and catechols, respectively.