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STUDIES ON THE OXIDATION OF AMMONIA BY NITROSOMONAS.

ABSTRACT: 1. Free-energy calculations for pH7 showed that the oxidation of ammonia to hydroxylamine is endergonic and that the oxidations of hydroxylamine to nitrite and hydrazine to nitrogen are exergonic. It is suggested that the oxidation of ammonia requires the expenditure of energy. 2. The anaerobic dehydrogenation of hydrazine to nitrogen by extracts of the autotrophic nitrifying micro-organism, Nitrosomonas, in the presence of methylene blue as electron acceptor, was less rapid than the anaerobic dehydrogenation of hydroxylamine to nitric oxide. The inhibition by hydrazine of the dehydrogenation of hydroxylamine was attributed to substrate competition. 3. Whole cells in air did not produce nitrite from hydrazine. They produced nitrite from low concentrations of hydroxylamine more rapidly than from equimolar concentrations of ammonia; this result is consistent if hydroxylamine is an intermediate of the oxidation of ammonia. 4. The production of nitrite from hydroxylamine by whole cells was slightly inhibited by hydrazine, but the production of nitrite from ammonia was greatly inhibited and small amounts of hydroxylamine were formed. These results suggested that the dehydrogenation of hydroxylamine supplied energy required for the oxidation of ammonia and that hydroxylamine appeared because the energy production was replaced by that of the dehydrogenation of hydrazine. 5. The oxidation of hydroxylamine by whole cells was not inhibited by thiourea, but micromolar concentrations of the metal-binding agent markedly inhibited the oxidation of ammonia to hydroxylamine, suggesting that the oxidation of ammonia involved copper. A possible mechanism for the activation of ammonia is suggested.

SUBMITTER: ANDERSON JH

PROVIDER: S-EPMC1206795 | biostudies-other | 1965 Jun

REPOSITORIES: biostudies-other

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Project description:Ammonia-oxidizing microorganisms perform the first step of nitrification, the oxidation of ammonia to nitrite. The bacterium Nitrosomonas europaea is the best-characterized ammonia oxidizer to date. Exposure to hypoxic conditions has a profound effect on the physiology of N. europaea, e.g., by inducing nitrifier denitrification, resulting in increased nitric and nitrous oxide production. This metabolic shift is of major significance in agricultural soils, as it contributes to fertilizer loss and global climate change. Previous studies investigating the effect of oxygen limitation on N. europaea have focused on the transcriptional regulation of genes involved in nitrification and nitrifier denitrification. Here, we combine steady-state cultivation with whole-genome transcriptomics to investigate the overall effect of oxygen limitation on N. europaea Under oxygen-limited conditions, growth yield was reduced and ammonia-to-nitrite conversion was not stoichiometric, suggesting the production of nitrogenous gases. However, the transcription of the principal nitric oxide reductase (cNOR) did not change significantly during oxygen-limited growth, while the transcription of the nitrite reductase-encoding gene (nirK) was significantly lower. In contrast, both heme-copper-containing cytochrome c oxidases encoded by N. europaea were upregulated during oxygen-limited growth. Particularly striking was the significant increase in transcription of the B-type heme-copper oxidase, proposed to function as a nitric oxide reductase (sNOR) in ammonia-oxidizing bacteria. In the context of previous physiological studies, as well as the evolutionary placement of N. europaea's sNOR with regard to other heme-copper oxidases, these results suggest sNOR may function as a high-affinity terminal oxidase in N. europaea and other ammonia-oxidizing bacteria.IMPORTANCE Nitrification is a ubiquitous microbially mediated process in the environment and an essential process in engineered systems such as wastewater and drinking water treatment plants. However, nitrification also contributes to fertilizer loss from agricultural environments, increasing the eutrophication of downstream aquatic ecosystems, and produces the greenhouse gas nitrous oxide. As ammonia-oxidizing bacteria are the most dominant ammonia-oxidizing microbes in fertilized agricultural soils, understanding their responses to a variety of environmental conditions is essential for curbing the negative environmental effects of nitrification. Notably, oxygen limitation has been reported to significantly increase nitric oxide and nitrous oxide production during nitrification. Here, we investigate the physiology of the best-characterized ammonia-oxidizing bacterium, Nitrosomonas europaea, growing under oxygen-limited conditions.

Project description:Cytochrome c(m552) (cyt c(m552)) from the ammonia-oxidizing Nitrosomonas europaea is encoded by the cycB gene, which is preceded in a gene cluster by three genes encoding proteins involved in the oxidation of hydroxylamine: hao, hydroxylamine oxidoreductase; orf2, a putative membrane protein; cycA, cyt c(554). By amino acid sequence alignment of the core tetraheme domain, cyt c(m552) belongs to the NapC/TorC family of tetra- or pentaheme cytochrome c species involved in electron transport from membrane quinols to a variety of periplasmic electron shuttles leading to terminal reductases. However, cyt c(m552) is thought to reduce quinone with electrons originating from HAO. In this work, the tetrahemic 27 kDa cyt c(m552) from N. europaea was purified after extraction from membranes using Triton X-100 with subsequent exchange into n-dodecyl beta-d-maltoside. The cytochrome had a propensity to form strong SDS-resistant dimers likely mediated by a conserved GXXXG motif present in the putative transmembrane segment. Optical spectra of the ferric protein contained a broad ligand-metal charge transfer band at approximately 625 nm indicative of a high-spin heme. Mossbauer spectroscopy of the reduced (57)Fe-enriched protein revealed the presence of high-spin and low-spin hemes in a 1:3 ratio. Multimode EPR spectroscopy of the native state showed signals from an electronically interacting high-spin/low-spin pair of hemes. Upon partial reduction, a typical high-spin heme EPR signal was observed. No EPR signals were observed from the other two low-spin hemes, indicating an electronic interaction between these hemes as well. UV-vis absorption data indicate that CO (ferrous enzyme) or CN(-) (ferric or ferrous enzyme) bound to more than one and possibly all hemes. Other anionic ligands did not bind. The four ferrous hemes of the cytochrome were rapidly oxidized in the presence of oxygen. Comparative modeling, based on the crystal structure and conserved residues of the homologous NrfH protein from Desulfovibrio of cyt c(m552), predicted some structural elements, including a Met-ligated high-spin heme in a quinone-binding pocket, and likely axial ligands to all four hemes.

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STUDIES ON THE OXIDATION OF AMMONIA BY NITROSOMONAS.

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