Clarifying the regulation of NO/N2O production in Nitrosomonas europaea during anoxic–oxic transition via flux balance analysis of a metabolic network model

Perez-Garcia, Octavio; Villas‐Bôas, Silas G.; Swift, Simon; Chandran, Kartik; Singhal, Naresh

doi:10.1016/j.watres.2014.04.049

Cited by 46 publications

(44 citation statements)

References 32 publications

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“…Alternatively, NO could be scavenged by cyt P460 or cyt c 554 to form N 2 O (35). A decrease in the rate of NO oxidation to NO 2 -might be expected at low O 2 concentrations, and this may help explain the observed increase in N 2 O emissions at low O 2 or during oxic to anoxic transitions (41,42). The presently afforded revision of HAO's activity should facilitate understanding of the sources and rates of NO and N 2 O emissions from ammonia oxidizing bacteria.…”

Section: Hao Stoichiometrically Oxidizes Nh 2 Oh To No Under Anaerobicmentioning

confidence: 96%

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

Caranto

Lancaster

2017

Proc. Natl. Acad. Sci. U.S.A.

364

274

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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 ...

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Section: Hao Stoichiometrically Oxidizes Nh 2 Oh To No Under Anaerobicmentioning

confidence: 96%

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

Caranto

Lancaster

2017

Proc. Natl. Acad. Sci. U.S.A.

364

274

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“…This proposal is based on the precedent that [Fe(CN) 5 NO] 2− reacts with NH 2 OH to form [Fe(CN) 5 (OH 2 )] 3− . However, the characteristic 463-nm UV/visible absorption peak of Fe II cyt P460 was never observed in our experiments as an intermediate or an end product.…”

Section: Resultsmentioning

confidence: 99%

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

Caranto

Vilbert

Lancaster

2016

Proc. Natl. Acad. Sci. U.S.A.

172

212

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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 ...

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“…A metabolic network modeling study of NO x gas production during the anoxic-oxic transition by N. europaea suggests that N 2 O production is associated with cytochromemediated electron flow imbalances (20). Clearly, more experiments are needed to study the effects of IC limitation on nitrifiers and on N 2 O evolution.…”

Section: Nitrosomonas Europaea Physiology Under Limiting Carbonmentioning

confidence: 99%

“…Other studies have suggested that N 2 O is produced via reduction of nitric oxide (NO) formed either by reduction of NO 2 Ϫ or by incomplete oxidation of NH 2 OH (15)(16)(17). N 2 O production by AOB has traditionally been associated with high NH 4 ϩ /NH 3 and NO 2 Ϫ concentrations and with low oxygen conditions (18)(19)(20), and further studies are needed to confirm the mechanism of N 2 O production under IC-limiting conditions.…”

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confidence: 99%

Steady-State Growth under Inorganic Carbon Limitation Conditions Increases Energy Consumption for Maintenance and Enhances Nitrous Oxide Production in Nitrosomonas europaea

Mellbye

Giguere

Chaplen

et al. 2016

Appl Environ Microbiol

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Nitrosomonas europaea is a chemolithoautotrophic bacterium that oxidizes ammonia (NH 3 ) to obtain energy for growth on carbon dioxide (CO 2 ) and can also produce nitrous oxide (N 2 O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses of N. europaea to conditions of replete (>5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na 2 CO 3 ) supplemented with atmospheric CO 2 . IC-limited cultures oxidized 25 to 58% of available NH 3 to nitrite, depending on the dilution rate and Na 2 CO 3 concentration. IC limitation resulted in a 2.3-fold increase in cellular maintenance energy requirements compared to those for NH 3 -limited cultures. Rates of N 2 O production increased 2.5-and 6.3-fold under the two IC-limited conditions, increasing the percentage of oxidized NH 3 -N that was transformed to N 2 O-N from 0.5% (replete) up to 4.4% (0.2 mM Na 2 CO 3 ). Transcriptome analysis showed differential expression (P < 0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in a decreased expression of ammonium/ ammonia transporter and ammonia monooxygenase subunits and increased the expression of genes involved in C 1 metabolism, including the genes for RuBisCO (cbb gene cluster), carbonic anhydrase, folate-linked metabolism of C 1 moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924 to NE0927) correlated with increased production of N 2 O. Together, these data suggest that N. europaea adapts physiologically during IC-limited steady-state growth, which leads to the uncoupling of NH 3 oxidation from growth and increased N 2 O production. IMPORTANCENitrification, the aerobic oxidation of ammonia to nitrate via nitrite, is an important process in the global nitrogen cycle. This process is generally dependent on ammonia-oxidizing microorganisms and nitrite-oxidizing bacteria. Most nitrifiers are chemolithoautotrophs that fix inorganic carbon (CO 2 ) for growth. Here, we investigate how inorganic carbon limitation modifies the physiology and transcriptome of Nitrosomonas europaea, a model ammonia-oxidizing bacterium, and report on increased production of N 2 O, a potent greenhouse gas. This study, along with previous work, suggests that inorganic carbon limitation may be an important factor in controlling N 2 O emissions from nitrification in soils and wastewater treatment. During nitrification in aerobic environments, ammonia (NH 3 ) oxidation to nitrite (NO 2 Ϫ ) is initiated by ammonia-oxidizing bacteria (AOB) and archaea (1). These microorganisms are predominantly chemolithoautotrophs that derive energy for growth from NH 3 and meet their carbon (C) needs by fixing carbon dioxide (CO 2 ) (1). All known AOB fix CO 2 via the CalvinBenson-Bassham (CBB) cycle for growth, utilizing ribulose bisphosphate carboxylase/oxygenase (RuB...

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Clarifying the regulation of NO/N2O production in Nitrosomonas europaea during anoxic–oxic transition via flux balance analysis of a metabolic network model

Cited by 46 publications

References 32 publications

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

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

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

Steady-State Growth under Inorganic Carbon Limitation Conditions Increases Energy Consumption for Maintenance and Enhances Nitrous Oxide Production in Nitrosomonas europaea

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