Evidence for a Transient Peroxynitro Acid in the Reaction Catalyzed by Nitronate Monooxygenase with Propionate 3-Nitronate.

Nitronate monooxygenase is a flavin-dependent enzyme that catalyzes the denitrification of propionate 3-nitronate (P3N) and other alkyl nitronates. The enzyme was previously known as 2-nitropropane dioxygenase, until its reclassification in 2010 by the IUBMB. Physiologically, the K monooxygenase from fungi protects the organism from the environmental occurrence of P3N, which shuts down the Krebs cycle by inactivating succinate dehydrogenase and fumarase. The inhibition of these enzymes yields severe neurological disorders or death. Here, we have used for the first time steady-state and rapid kinetics, viscosity and pH ciicl-ls, iim uuic-icauiveu duauiudjict; i-penroscopy ui me enzyme in turnover wim fP3N ana me suostrate analogue ethyl nitronate (EN) to elucidate the mechanism of the reaction. A transient increase in absorbance at ~300 nm, never reported before, was seen during steady-state turnover of the enzyme with P3N and oxygen, with no concomitant changes between 400 and 600 nm. The transient species was not detected when oxygen was absent. Anaerobic reduction of the enzyme with P3N yielded anionic flavosemiquinone and was fast (e.g., 5:1900 s '). Steady-state kinetics demonstrated that oxygen reacts before the release of the product of P3N oxidation from the enzyme. No pH effects were seen with P3N on km/Kcatk/Koxygen and kcat; in contrast, with EN, the kczt/Km and fccat decreased with increasing pH denning two plateaus and a pKa ∼8.0. Solvent viscosity at the pH optima suggested product release as being partially controlling the overall rate of turnover with the physiological substrate and its analogue. A mechanism that satisfies the kinetic results is proposed. [ABSTRACT FROM AUTHOR]