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 (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 ...

Genome-Scale, Constraint-Based Modeling of Nitrogen Oxide Fluxes during Coculture of Nitrosomonas europaea and Nitrobacter winogradskyi

“…Recent studies have suggested that the sole product of HAO oxidation of NH 2 OH is NO with a yield of three electrons, further suggesting that oxidation to NO 2 − is either abiotic or carried out by an unknown enzymatic step (37, 38). While this study suggests that abiotic oxidation of NO is a significant NO sink, a true yield of four electrons resulting from NO oxidation to NO 2 − by an unknown NO oxidase would result in increased biomass production by N. europaea .…”

“…While production of N oxides by N. europaea has been well studied both through experimentation and modeling (4, 18, 22, 37, 38), production of N oxides by N. winogradskyi is more cryptic. Early studies reported both production of NO and N 2 O and consumption of NO by strains of N. winogradskyi and Nitrobacter vulgaris (40, 41), but only NO consumption, not production, was shown in one later study on N. winogradskyi (42).…”

Nitrous oxide production by ammonia oxidizers: Physiological diversity, niche differentiation and potential mitigation strategies

“…Until recently, it was commonly accepted that hydroxylamine is oxidized to NO − 2 by hydroxylamine dehydrogenase (previously referred to as hydroxylamine oxidoreductase) in AOB (Arp & Stein, 2003). Caranto, Vilbert, and Lancaster (2016) suggested that under aerobic conditions, NO could be rapidly oxidized to NO − 2 by NO − 2 reductase, encoded by nirK, as this enzyme can act reversibly. This mechanism failed to explain fully a number of experimental observations and is now challenged by enzymatic studies that suggest that the direct product of the hydroxylamine dehydrogenase reaction is not NO − 2 but nitric oxide (NO; Caranto & Lancaster, 2017).…”

Complete nitrification: insights into the ecophysiology of comammox Nitrospira

“…In agreement with these data from pure culture studies, mesocosm experiments revealed an increased N 2 O yield in fertilized soil when ammonia oxidation was dominated by AOB (Hink et al 2018). In AOB, enzymatic N 2 O production is catalyzed by nitric oxide reductases (NOR) that convert NO derived from nitrite reduction (Kozlowski et al 2016a), and cytochrome P460, a periplasmic metalloenzyme shown to directly convert NH 2 OH to N 2 O under anaerobic conditions (Caranto et al 2016). Similar to AOA, the lack of NOR homologs in complete nitrifiers indicates that Nitrospira do not produce N 2 O via nitrifier denitrification.…”