Nitric oxide is an obligate bacterial nitrification intermediate produced by hydroxylamine oxidoreductase

Abstract

Ammonia (NH 3 )-oxidizing bacteria (AOB) emit substantial amounts of nitric oxide (NO) and nitrous oxide (N 2 O), both of which contribute to the harmful environmental side effects of large-scale agriculture. The currently accepted model for AOB metabolism involves NH 3 oxidation to nitrite (NO 2 -) via a single obligate intermediate, hydroxylamine (NH 2 OH). Within this model, the multiheme enzyme hydroxylamine oxidoreductase (HAO) catalyzes the four-electron oxidation of NH 2 OH to NO 2 -. We provide evidence that HAO oxidizes NH 2 OH by only three electrons to NO under both anaerobic and aerobic conditions. NO 2 -observed in HAO activity assays is a nonenzymatic product resulting from the oxidation of NO by O 2 under aerobic conditions. Our present study implies that aerobic NH 3 oxidation by AOB occurs via two obligate intermediates, NH 2 OH and NO, necessitating a mediator of the third enzymatic step.nitrification | nitric oxide | enzymology | bioinorganic chemistry S ynthetic nitrogenous fertilizers are necessary in agriculture to sustain the growing human population, but their use causes significant imbalance in the biogeochemical nitrogen cycle (1). The application of ammonia (NH 3 )-based fertilizers increases concentrations of nitrite (NO 2 -) and nitrate (NO 3 -) in the water table. These species pollute drinking water and drive the eutrophication of lakes and estuaries. Moreover, elevated NH 3 concentrations in soil have been linked to nitrous oxide (N 2 O) and nitric oxide (NO) emissions. N 2 O is an ozone-depleting greenhouse gas with a global warming potential ∼300× greater than that of carbon dioxide (2), and NO contributes to the production of ground-level ozone and acid rain (3, 4). Balancing human needs with environmental impact requires an intimate understanding of the biological pathways that produce these pollutants (5).Biological sources of NO and N 2 O include NH 3 -oxidizing bacteria (AOB), which mediate the oxidation of NH 3 to NO 2 -. The prevailing view of NH 3 oxidation, based largely on studies of the model AOB Nitrosomonas europaea, is that it occurs via a two-step enzymatic process (6):An integral membrane metalloenzyme, NH 3 monooxygenase (AMO), catalyzes the dioxygen (O 2 )-dependent hydroxylation of NH 3 to hydroxylamine (NH 2 OH; Eq. 1). Two electrons are required to turn over AMO. NH 2 OH is then oxidized by four electrons to NO 2 -(Eq. 2) by a multiheme enzyme, hydroxylamine oxidoreductase (HAO). Two of these electrons return to AMO, leaving two net electrons to enter the respiratory electron transport chain using O 2 as the terminal electron acceptor. Under anaerobic conditions, AOB carry out nitrifier denitrification, in which O 2 is substituted by NO 2 -as the terminal electron acceptor and is reduced to N 2 O or dinitrogen (7). The obligate intermediates of nitrifier denitrification are NO and N 2 O, both of which can escape from cells and into the atmosphere. Emissions of NO and N 2 O have also been linked to aerobic NH 3 oxidation (4, 8), which suggests that alternate ...

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

“…Hooper and Terry (1979) provided evidence that NO and N 2 O are also produced through incomplete oxidation of hydroxylamine. This mechanism is compatible with the revised, 2-intermediate model for ammonia oxidation (Caranto & Lancaster, 2017). This mechanism is compatible with the revised, 2-intermediate model for ammonia oxidation (Caranto & Lancaster, 2017).…”

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).…”

Hydroxylamine as a Potential Indicator of Nitrification in the Open Ocean

“…The prevailing view of bacterial ammonia oxidation is via a two-step enzymatic ©2019 process involving the enzyme ammonia monooxygenase and hydroxylamine oxidoreductase (HAO; Arp & Stein, 2003). Recently, Caranto and Lancaster (2017) suggested that NO is an intermediate of NH 3 oxidation to NO 2 − and not a by-product. Recently, Caranto and Lancaster (2017) suggested that NO is an intermediate of NH 3 oxidation to NO 2 − and not a by-product.…”

“…Here nitric oxide (NO) is arising from the incomplete oxidation of NH 2 OH by HAO, in which NO is a HAO by-product that is further reduced to N 2 O by the enzyme NO reductase (Stein, 2011). Their results showed that under aerobic conditions most of the NH 2 OH is converted to NO 2 − via both NO oxidation and the nonenzymatic reaction of NO with O 2 by a pure culture of the nitrifier Nitrosomonas europaea (Caranto & Lancaster, 2017). Their results showed that under aerobic conditions most of the NH 2 OH is converted to NO 2 − via both NO oxidation and the nonenzymatic reaction of NO with O 2 by a pure culture of the nitrifier Nitrosomonas europaea (Caranto & Lancaster, 2017).…”