NO Reductase Activity of the Tetraheme Cytochromec554ofNitrosomonas europaea

Upadhyay, Anup K.; Hooper, and Alan B.; Hendrich, Michael P.

doi:10.1021/ja055183+

Cited by 56 publications

(47 citation statements)

References 68 publications

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“…NO production by HAO has been previously observed in the absence of O 2 but was attributed to incomplete NH 2 OH oxidation when oxidant or O 2 concentrations were insufficient (17,18,31,35). As discussed above, the detection of NO as the HAO product was likely obscured by several rapid, nonenzymatic NO reaction pathways, especially when O 2 was present.…”

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

confidence: 79%

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

Caranto

Lancaster

2017

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

365

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: 79%

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

Caranto

Lancaster

2017

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

365

274

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“…These results are contradictory to a prior study of N. europaea ATCC 19718 incubated with ammonia, nitrite, and NO 2 in which nirK, norB, and ncyA transcription significantly increased relative to that in aerobically grown cells (4); however, the prior study examined mRNA levels at 24 to 48 h after switching from aerobic to anaerobic conditions. The present results suggest that steadystate N-oxide metabolism by N. eutropha is likely controlled by a combination of proteins such as hydroxylamine oxidoreductase (HaoAB), cytochromes c554 (CycA) and c M 552 (CycB), nitrite reductase and related proteins (NcgABC to NirK), nitrosocyanin (NcyA), cytochrome c=-beta (CytS), and cytochrome P460 (CytL) (2,8,25,27), all of which were expressed and unchanged from condition to condition. Prior measurements of changes in expression of these genes and proteins in nitrosomonads likely reflected the transient environmental conditions experienced in muchshorter-term batch and continuous culture incubations, including expression of norB in N. europaea ATCC 19718 (4).…”

Section: Discussionmentioning

confidence: 76%

Effects of Nitrogen Dioxide and Anoxia on Global Gene and Protein Expression in Long-Term Continuous Cultures of Nitrosomonas eutropha C91

Kartal

Wessels

Biezen

et al. 2012

Appl Environ Microbiol

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f Nitrosomonas eutropha is an ammonia-oxidizing betaproteobacterium found in environments with high ammonium levels, such as wastewater treatment plants. The effects of NO 2 on gene and protein expression under oxic and anoxic conditions were determined by maintaining N. eutropha strain C91 in a chemostat fed with ammonium under oxic, oxic-plus-NO 2 , and anoxicplus-NO 2 culture conditions. Cells remained viable but ceased growing under anoxia; hence, the chemostat was switched from continuous to batch cultivation to retain biomass. After several weeks under each condition, biomass was harvested for total mRNA and protein isolation. Exposure of N. eutropha C91 to NO 2 under either oxic or anoxic conditions led to a decrease in proteins involved in N and C assimilation and storage and an increase in proteins involved in energy conservation, including ammonia monooxygenase (AmoCAB). Exposure to anoxia plus NO 2 resulted in increased representation of proteins and transcripts reflective of an energy-deprived state. Several proteins implicated in N-oxide metabolism were expressed and remained unchanged throughout the experiment, except for NorCB nitric oxide reductase, which was not detected in the proteome. Rather, NorY nitric oxide reductase was expressed under oxic-plus-NO 2 and anoxic-plus-NO 2 conditions. The results indicate that exposure to NO 2 results in an energy-deprived state of N. eutropha C91 and that anaerobic growth could not be supported with NO 2 as an oxidant. N itrosomonas eutropha is a betaproteobacterial ammonia-oxidizing nitrifier with a niche preference for environments with a high ammonium flux, concentration, or load, such as wastewater treatment plants (WWTPs) (13). Although chemolithotrophic ammonia-oxidizing bacteria (AOB) are considered obligate aerobes, N. eutropha strain N904 was capable of anaerobic ammonia oxidation using nitrite as a terminal electron acceptor with externally provided nitrogen dioxide gas (NO 2 ) as an oxidant (18). Anaerobic incubation of Nitrosomonas europaea ATCC 19718 with ammonia, NO 2 , and nitrite significantly increased levels of transcripts encoding copper-containing nitrite reductase (nirK), cytochrome c-dependent nitric oxide reductase (norB), and the red copper protein nitrosocyanin (ncyA) relative to transcript levels in aerobically growing cultures without NO 2 (4). Although dinitrogen gas (N 2 ), not nitrous oxide (N 2 O), was considered to be the main product of anaerobic metabolism in N. eutropha N904 (18, 23), recognizable N 2 O reductase genes have never been identified in genomes of ammonia-oxidizing bacteria, including N. eutropha C91.Aside from anaerobic respiration, N. eutropha N904 generates and utilizes NO for aerobic growth; removal of NO by intensive aeration, chemical chelation, or consumption by a cocultivated denitrifier significantly reduced ammonia-oxidizing activity by N. eutropha N904 (29). Furthermore, addition of NO 2 to N. eutropha N904 cultures transitioning from anaerobic to aerobic cultivation (24) and addition of nitrite to ...

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“…Enzymes such as P450 nor [79,97], flavor-diiron proteins [98], and cytochrome c 554 [99] can reduce NO to N 2 O using only one high-spin heme or a dinuclear iron center as the active site. While not directly implicated in denitrification, these enzymes were proposed to have a role in detoxification acting as NO scavengers and protecting the organisms from intracellular NO accumulation or anaerobic exposure to NO.…”

Section: Nitric Oxide Reductasementioning

confidence: 99%