Techniques for Investigating Hydroxylamine Disproportionation by Hydroxylamine Oxidoreductases

Pacheco, A. Andrew; McGarry, Jennifer; Kostera, Joshua; Corona, Angel

doi:10.1016/b978-0-12-381294-0.00020-1

Cited by 25 publications

(22 citation statements)

References 12 publications

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“…NO 2 -was quantified using the Griess diazotization assay (Cayman Chemical) and measuring the resulting absorbance at 542 nm (e 542 = 50 mM -1 ·cm -1 ). The combined NO 2 -and NO 3 -concentrations were determined by treating samples with nitrate reductase and cofactor solution (Cayman Chemical) at room temperature for 1 h followed by NO NO production under anaerobic conditions was monitored by using a catalase NO-binding assay previously described by Pacheco and coworkers (32). To determine the stoichiometry of NH 2 OH oxidation to NO, anaerobic solutions containing 150 nM HAO, 8 μM catalase, and 25 μM DCPIP in 1 mL of degassed 250 mM Na 2 HPO 4 , pH 8.0, were prepared in an anaerobic glovebox.…”

Section: Methodsmentioning

confidence: 99%

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

Caranto

Lancaster

2017

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

367

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: Methodsmentioning

confidence: 99%

“…To test for NO as an HAO product, an NO sequestration system developed by Pacheco and coworkers was used (32). In this assay, catalase rapidly binds NO (k on = 1.3 × 10 7 M -1 ·s -1 ) (33) with concomitant appearance of defined UV-visible (UV-vis) absorption features (SI Appendix, Fig.…”

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

confidence: 99%

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

Caranto

Lancaster

2017

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

367

274

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“…However, hydroxylamine measurements after one day of incubation showed undetectable levels of this compound in the cultures; only after three weeks of incubation a minimal production of hydroxylamine was detected (Table 2). Thus, it is possible that either hydroxylamine is not an intermediate, or that given the inherent instability of hydroxylamine it is disproportionated as soon as it is formed (Pacheco et al, 2011) leading to loss of nitrogen as a gas product.…”

Section: Resultsmentioning

confidence: 99%

Ammonia-oxidizing microbial communities in reactors with efficient nitrification at low-dissolved oxygen

et al. 2015

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Ammonia-oxidizing microbial communities involved in ammonia oxidation under low dissolved oxygen (DO) conditions (<0.3 mg/L) were investigated using chemostat reactors. One lab-scale reactor (NS_LowDO) was seeded with sludge from a full-scale wastewater treatment plant (WWTP) not adapted to low-DO nitrification, while a second reactor (JP_LowDO) was seeded with sludge from a full-scale WWTP already achieving low-DO nitrifiaction. The experimental evidence from quantitative PCR, rDNA tag pyrosequencing, and fluorescence in situ hybridization (FISH) suggested that ammonia-oxidizing bacteria (AOB) in the Nitrosomonas genus were responsible for low-DO nitrification in the NS_LowDO reactor, whereas in the JP_LowDO reactor nitrification was not associated with any known ammonia-oxidizing prokaryote. Neither reactor had a significant population of ammonia-oxidizing archaea (AOA) or anaerobic ammonium oxidation (anammox) organisms. Organisms isolated from JP_LowDO were capable of autotrophic and heterotrophic ammonia utilization, albeit without stoichiometric accumulation of nitrite or nitrate. Based on the experimental evidence we propose that Pseudomonas, Xanthomonadaceae, Rhodococcus, and Sphingomonas are involved in nitrification under low-DO conditions.

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“…Nitric oxide was assayed as described previously, 22 by conducting the same reaction as described above in the presence of 5 μ M catalase (Sigma). Catalase binds nitric oxide tightly, leading to a characteristic change in the UV–vis spectrum that is readily quantified.…”

Section: Methodsmentioning

confidence: 99%

Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) Does Not Disproportionate Hydroxylamine to Ammonia and Nitrite, Despite a Strongly Favorable Driving Force

et al. 2014

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Cytochrome c nitrite reductase (ccNiR) from Shewanella oneidensis, which catalyzes the six-electron reduction of nitrite to ammonia in vivo, was shown to oxidize hydroxylamine in the presence of large quantities of this substrate, yielding nitrite as the sole free nitrogenous product. UV–visible stopped-flow and rapid-freeze-quench electron paramagnetic resonance data, along with product analysis, showed that the equilibrium between hydroxylamine and nitrite is fairly rapidly established in the presence of high initial concentrations of hydroxylamine, despite said equilibrium lying far to the left. By contrast, reduction of hydroxylamine to ammonia did not occur, even though disproportionation of hydroxylamine to yield both nitrite and ammonia is strongly thermodynamically favored. This suggests a kinetic barrier to the ccNiR-catalyzed reduction of hydroxylamine to ammonia. A mechanism for hydroxylamine reduction is proposed in which the hydroxide group is first protonated and released as water, leaving what is formally an NH2+ moiety bound at the heme active site. This species could be a metastable intermediate or a transition state but in either case would exist only if it were stabilized by the donation of electrons from the ccNiR heme pool into the empty nitrogen p orbital. In this scenario, ccNiR does not catalyze disproportionation because the electron-donating hydroxylamine does not poise the enzyme at a sufficiently low potential to stabilize the putative dehydrated hydroxylamine; presumably, a stronger reductant is required for this.

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Techniques for Investigating Hydroxylamine Disproportionation by Hydroxylamine Oxidoreductases

Cited by 25 publications

References 12 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

Ammonia-oxidizing microbial communities in reactors with efficient nitrification at low-dissolved oxygen

Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) Does Not Disproportionate Hydroxylamine to Ammonia and Nitrite, Despite a Strongly Favorable Driving Force

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