Structural Basis of Biological NO Generation by Octaheme Oxidoreductases

Maalcke, Wouter J.; Dietl, Andreas; Marritt, Sophie J.; Butt, Julea N.; Jetten, Mike S. M.; Keltjens, Jan T.; Barends, Thomas R. M.; Kartal, Boran

doi:10.1074/jbc.m113.525147

Cited by 87 publications

(175 citation statements)

References 61 publications

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“…The revised N. europaea HAO product is consistent with that of the K. stuttgartiensis kustc1061 protein that Kartal and coworkers (20) purified and characterized; in their paper, the authors proposed that a tyrosine above the active site pocket (Y358) present in N. europaea HAO but absent in K. stuttgartiensis kustc1061 facilitates the {FeNO} 6 reaction with H 2 O to form NO 2 -. However, our results indicate that the N. europaea HAO and kustc1061 NH 2 OH oxidation products are identical and, therefore, Y358 does not promote NO 2 -formation.…”

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

confidence: 54%

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: 54%

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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“…In the case of HOX, the covalently bound trimer (Ͼ200 kDa), and, to a lesser extent, also the dimeric (ϳ130 kDa) and monomeric forms (ϳ55 kDa) of the protein complex were detected after incubation with the antiserum (Fig. 3C) (5). Three specific bands were visible when incubating blotted crude extract with the antibody against NXR, representing the subunits NxrA (ϳ130 kDa), NxrB (ϳ50 kDa), and NxrC (ϳ35 kDa) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…All enzymes were purified from CE using liquid chromatography. Purification of HZS, HDH, and HOX was performed as described previously (1,5). The HAO-like protein kustc0458, together with its partner kustc0457 and NXR, were purified as follows.…”

Section: Methodsmentioning

confidence: 99%

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Immunogold Localization of Key Metabolic Enzymes in the Anammoxosome and on the Tubule-Like Structures of Kuenenia stuttgartiensis

et al. 2015

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Anaerobic ammonium-oxidizing (anammox) bacteria oxidize ammonium with nitrite as the terminal electron acceptor to form dinitrogen gas in the absence of oxygen. Anammox bacteria have a compartmentalized cell plan with a central membrane-bound "prokaryotic organelle" called the anammoxosome. The anammoxosome occupies most of the cell volume, has a curved membrane, and contains conspicuous tubule-like structures of unknown identity and function. It was suggested previously that the catalytic reactions of the anammox pathway occur in the anammoxosome, and that proton motive force was established across its membrane. Here, we used antibodies raised against five key enzymes of the anammox catabolism to determine their cellular location. The antibodies were raised against purified native hydroxylamine oxidoreductase-like protein kustc0458 with its redox partner kustc0457, hydrazine dehydrogenase (HDH; kustc0694), hydroxylamine oxidase (HOX; kustc1061), nitrite oxidoreductase (NXR; kustd1700/03/04), and hydrazine synthase (HZS; kuste2859-61) of the anammox bacterium Kuenenia stuttgartiensis. We determined that all five protein complexes were exclusively located inside the anammoxosome matrix. Four of the protein complexes did not appear to form higher-order protein organizations. However, the present data indicated for the first time that NXR is part of the tubule-like structures, which may stretch the whole length of the anammoxosome. These findings support the anammoxosome as the locus of catabolic reactions of the anammox pathway. IMPORTANCEAnammox bacteria are environmentally relevant microorganisms that contribute significantly to the release of fixed nitrogen in nature. Furthermore, the anammox process is applied for nitrogen removal from wastewater as an environment-friendly and cost-effective technology. These microorganisms feature a unique cellular organelle, the anammoxosome, which was proposed to contain the energy metabolism of the cell and tubule-like structures with hitherto unknown function. Here, we purified five native enzymes catalyzing key reactions in the anammox metabolism and raised antibodies against these in order to localize them within the cell. We showed that all enzymes were located within the anammoxosome, and nitrite oxidoreductase was located exclusively at the tubule-like structures, providing the first insights into the function of these subcellular structures.

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“…However, an octaheme HAO-like protein from the anaerobic NH 3 -oxidizing microorganism Kuenenia stuttgartiensis was shown to oxidize NH 2 OH to NO instead of NO − 2 (27). An X-ray crystal structure of the K. stuttgartiensis HAO-like enzyme features a tyrosine cross-linked active site and heme distribution that was identical to N. europaea HAO.…”

Section: Significancementioning

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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Structural Basis of Biological NO Generation by Octaheme Oxidoreductases

Cited by 87 publications

References 61 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

Immunogold Localization of Key Metabolic Enzymes in the Anammoxosome and on the Tubule-Like Structures of Kuenenia stuttgartiensis

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

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