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

Ammonia oxidizing bacteria (AOB) are major contributors to the emission of nitrous oxide (NO). It has been proposed that NO is produced by reduction of NO. Here, we report that the enzyme cytochrome (cyt) P460 from the AOB Nitrosomonas europaea converts hydroxylamine (NHOH) quantitatively to NO under anaerobic conditions. Previous literature reported that this enzyme oxidizes NHOH 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 NHOH concentration. By contrast, under anaerobic conditions, the enzyme uses 4 oxidizing equivalents (eq) to convert 2 eq of NHOH to NO. Enzyme kinetics coupled to UV/visible absorption and electron paramagnetic resonance (EPR) spectroscopies support a mechanism in which an Fe-NHOH adduct of cyt P460 is oxidized to an {FeNO} unit. This species subsequently undergoes nucleophilic attack by a second equivalent of NHOH, forming the N-N bond of NO during a bimolecular, rate-determining step. We propose that [Formula: see text] results when nitric oxide (NO) dissociates from the {FeNO} 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 NHOH contributes to NO and NO emissions from nitrifying microorganisms.