Nitrosomonas europaea cytochrome P460 is a direct link between nitrification and nitrous oxide emission
Significance Nitrous oxide (N2O) is a potent ozone depleting greenhouse gas. This work identifies a means by which N2O is generated during nitrification, or biological ammonia oxidation. Fertilizer use in agriculture stimulates nitrification, thus increasing the volume of N2O emissions worldwide. The results presented herein will inform models and strategies toward optimized, sustainable agriculture. Moreover, these results highlight a rare example of biological N N bond formation. 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 (NO2 under aerobic conditions. Although we observe NO2 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 FeIII 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 NO2 results when nitric oxide (NO) dissociates from the {FeNO}6 intermediate and reacts with dioxygen. Thus, NO2 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.
Proceedings of the National Academy of Sciences