Nitrosomonas europaea cytochrome P460 is a direct link between nitrification and nitrous oxide emission.

Abstract

Ammonia oxidizing bacteria (AOB) are major contributors to the emission of nitrous oxide (N₂O). It has been proposed that N₂O is produced by reduction of NO. Here, we report that the enzyme cytochrome (cyt) P460 from the AOB Nitrosomonas europaea converts hydroxylamine (NH₂OH) quantitatively to N₂O under anaerobic conditions. Previous literature reported that this enzyme oxidizes NH₂OH to nitrite ([Formula: see text]) under aerobic conditions. Although we observe [Formula: see text] formation under aerobic conditions, its concentration is not stoichiometric with the NH₂OH concentration. By contrast, under anaerobic conditions, the enzyme uses 4 oxidizing equivalents (eq) to convert 2 eq of NH₂OH to N₂O. Enzyme kinetics coupled to UV/visible absorption and electron paramagnetic resonance (EPR) spectroscopies support a mechanism in which an Fe^III-NH₂OH adduct of cyt P460 is oxidized to an {FeNO}⁶ unit. This species subsequently undergoes nucleophilic attack by a second equivalent of NH₂OH, forming the N-N bond of N₂O during a bimolecular, rate-determining step. We propose that [Formula: see text] results when nitric oxide (NO) dissociates from the {FeNO}⁶ intermediate and reacts with dioxygen. Thus, [Formula: see text] is not a direct product of cyt P460 activity. We hypothesize that the cyt P460 oxidation of NH₂OH contributes to NO and N₂O emissions from nitrifying microorganisms.