Ammonia oxidizing bacteria (AOB) are major contributors to the emission of nitrous oxide (N 2 O). It has been proposed that N 2 O is produced by reduction of NO. Here, we report that the enzyme cytochrome (cyt) P460 from the AOB Nitrosomonas europaea converts hydroxylamine (NH 2 OH) quantitatively to N 2 O under anaerobic conditions. Previous literature reported that this enzyme oxidizes NH 2 OH to nitrite (NO − 2 ) under aerobic conditions. Although we observe NO − 2 formation under aerobic conditions, its concentration is not stoichiometric with the NH 2 OH concentration. By contrast, under anaerobic conditions, the enzyme uses 4 oxidizing equivalents (eq) to convert 2 eq of NH 2 OH to N 2 O. Enzyme kinetics coupled to UV/visible absorption and electron paramagnetic resonance (EPR) spectroscopies support a mechanism in which an Fe III -NH 2 OH adduct of cyt P460 is oxidized to an {FeNO} 6 unit. This species subsequently undergoes nucleophilic attack by a second equivalent of NH 2 OH, forming the N-N bond of N 2 O during a bimolecular, rate-determining step. We propose that NO − 2 results when nitric oxide (NO) dissociates from the {FeNO} 6 intermediate and reacts with dioxygen. Thus, NO − 2 is not a direct product of cyt P460 activity. We hypothesize that the cyt P460 oxidation of NH 2 OH contributes to NO and N 2 O emissions from nitrifying microorganisms. possesses a global warming potential nearly 300-fold greater than carbon dioxide (1). Atmospheric N 2 O concentrations have increased ∼120% since the preindustrial era, largely due to the widespread use of fertilizers required to produce sustenance for humans and livestock. N 2 O is a byproduct of the microbial metabolism of fertilizer components, including ammonia (NH 3 ) and nitrate (NO − 3 ); consequently, agricultural soils account for an estimated 60-75% of global N 2 O emissions. The metabolic pathway by which microorganisms oxidize NH 3 , nitrification, occurs in two phases, both of which are mediated by autotrophic microorganisms. In the first, NH 3 -oxidizing bacteria (AOB) or archaea (AOA) oxidize NH 3 to nitrite (NO − 2 ). In the second, NO − 2 is subsequently oxidized to NO − 3 by NO − 2 -oxidizing bacteria. NH 3 -oxidizing microbes contribute substantially to global N 2 O emissions, whereas NO − 2 -oxidizing bacteria produce negligible N 2 O (2, 3). AOB are proposed to emit N 2 O either as a byproduct of the nitrification pathway or as a product of the nitrifier denitrification pathway (i.e., the reduction of NO − 2 ) (4-6). Nitrification of NH 3 to NO − 2 occurs in two steps (7,8). The first step is catalyzed by NH 3 monooxygenase, which uses copper (Cu) and dioxygen (O 2 ) to hydroxylate NH 3 to hydroxylamine (NH 2 OH) (9). In AOB, the second step is thought to be the four-electron oxidation of NH 2 OH to NO − 2 by NH 2 OH oxidoreductase (HAO). HAO is a multiheme enzyme with eight c-type hemes per subunit: seven are electron transfer cofactors, and the eighth is the so-called P460 active site that contains a unique tyrosine cross-link to the ...

A vital role has been identified for the heme-lysine cross-link unique to cytochromes P460: preventing enzyme deactivation during catalysis by the obligate nitrification metabolite nitric oxide.

One amino acid makes the difference between a metalloenzyme and a metalloprotein in two otherwise effectively identical cytochrome P460s.