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.

Keywords: bioinorganic chemistry; enzymology; nitric oxide; nitrification; nitrous oxide.

Fig. 1.

Ferric P460 cofactors in cyt P460 [A, Protein Data Bank (PDB) ID code 2JE2] and HAO (B, PDB ID code 4N4N).

Fig. 2.

(A) Stoichiometry of N₂O production by cyt P460 determined with GC. Data points are averages of triplicate trials with 5 μM ferric cyt P460 in anaerobic 50 mM Hepes, pH 8.0, at 25 °C overnight. Error bars represent 1 SD of three trials. For the red triangles, the concentration of PMS is held at 1 mM, whereas the NH₂OH concentration is varied; for the blue circles, the NH₂OH concentration is held at 1 mM, whereas PMS concentration varies. (B) Steady-state NH₂OH oxidase activity plot for cyt P460. The assay conditions were 1 μM cyt P460, 6 μM PMS, and 100 μM DCPIP with various NH₂OH concentrations in anaerobic 50 mM Hepes, pH 8.0, at 25 °C. Each data point is the average of three trials, with error bars representing one SD.

Fig. 3.

UV/visible absorption spectra of Fe^III–OH₂ cyt P460 (black), Fe^III–NH₂OH cyt P460 (blue), and {FeNO}⁶ cyt P460 generated via treatment with PROLI-NONOate (red line) or oxidation of Fe^III–NH₂OH (black dashed line). (Inset) Magnification of the Q-bands.

Fig. 4.

EPR spectra of species on the proposed cyt P460 NH₂OH oxidase pathway. Cyt P460 at 170 μM (A) was treated with 100 mM NH₂OH (B), with 2 mM NH₂OH and 2 mM DCPIP (C), or with 45 mM NH₂OH and 2 mM DCPIP (D) and incubated for 10 min. Black traces are spectra simulated with the parameters listed in SI Appendix, Table ST2. Spectra were collected at 10 K and 633 μW or at 20 K and 63 μW. A 5% impurity of an {FeNO}⁷ species is indicated by a single asterisk. An Mn²⁺ EPR signal is indicated by double asterisks. dχ″/dH, derivative of magnetic susceptibility vs. magnetic induction.

Fig. 5.

Proposed Cyt P460 NH₂OH oxidase mechanism. RDS, rate-determining step.