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. 2016 Dec 20;113(51):14704-14709.
doi: 10.1073/pnas.1611051113. Epub 2016 Nov 16.

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

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

Jonathan D Caranto et al. Proc Natl Acad Sci U S A. .
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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 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 [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.

Conflict of interest statement

The authors declare no conflict of interest.


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 FeIII–OH2 cyt P460 (black), FeIII–NH2OH cyt P460 (blue), and {FeNO}6 cyt P460 generated via treatment with PROLI-NONOate (red line) or oxidation of FeIII–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.

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