In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper

Ensign, Scott A.; Hyman, Michael R.; Arp, Daniel J.

doi:10.1128/jb.175.7.1971-1980.1993

Cited by 167 publications

(113 citation statements)

References 59 publications

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“…N. europaea (ATCC 19178) was cultured as described, with minor modifications (Ensign et al, 1993;Stein & Arp, 1998). The standard (Fe-replete) medium contained 10 mM Fe 3+ (FeCl 3 ) complexed with EDTA to prevent Fe from precipitation.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the ferrioxamine uptake system of Nitrosomonas europaea

2007

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Section: Methodsmentioning

confidence: 99%

Characterization of the ferrioxamine uptake system of Nitrosomonas europaea

2007

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“…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).…”

mentioning

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.

173

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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“…Therefore, it is assumed that their key enzyme, the ammonia monooxygenase (AMO), is highly conserved. However, little is known about the structure and function of AMO since the enzyme has not been purified [14,44,45]. Inactivation experiments suggested that the proteins AmoA and AmoB were possible subunits of AMO [33].…”

Section: Discussionmentioning

confidence: 99%

Genera-Specific Immunofluorescence Labeling of Ammonia Oxidizers with Polyclonal Antibodies Recognizing Both Subunits of the Ammonia Monooxygenase

Fiencke

Bock

2004

Microb Ecol

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Polyclonal antibodies that recognize the two subunits AmoA and AmoB of the ammonia monooxygenase (AMO) were applied to identify ammonia-oxidizing bacteria by immunofluorescence (IF) labeling in pure, mixed, and enriched cultures. The antibodies against the AmoA were produced using a synthetic peptide of the AmoA of Nitrosomonas eutropha, whereas the antibodies against the AmoB had been developed previously is against the whole B-subunit of the AMO [Pinck et al. (2001) Appl Environ Microbiol 67:118-124]. Using IF labeling, the AmoA antibodies were specific for the detection of all species of the genus Nitrosomonas. In contrast, the antiserum against AmoB labeled all genera of ammonia oxidizers of the b-subclass of Proteobacteria (Nitrosomonas, Nitrosospira, Nitrosolobus, and Nitrosovibrio). The fluorescence signals of the AmoA antibodies were spread all over the cells, whereas the signals of the AmoB antibodies were associated with the cytoplasmic membranes. The specificity of the reactions of the antisera with ammonia oxidizers were proven in pure and mixed cultures, and the characteristic IF labeling and the morphology of the cells enabled their identification at the genus level. The genus-specific IF labeling could be used to identify ammonia oxidizers enriched from various habitats. In enrichment cultures of natural sandstone, cells of the genera Nitrosomonas, Nitrosovibrio, and Nitrosospira were detected. Members of the genus Nitrosovibrio and Nitrosolobus were most prominent in enriched garden soil samples, whereas members of the genus Nitrosomonas dominated in enriched activated sludge. The antibodies caused only slight background fluorescence on sandstone and soil particles compared to oligonucleotide probes, which could not be used to detect ammonia oxidizers on these materials because of strong nonspecific fluorescence.

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In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper

Cited by 167 publications

References 59 publications

Characterization of the ferrioxamine uptake system of Nitrosomonas europaea

Characterization of the ferrioxamine uptake system of Nitrosomonas europaea

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

Genera-Specific Immunofluorescence Labeling of Ammonia Oxidizers with Polyclonal Antibodies Recognizing Both Subunits of the Ammonia Monooxygenase

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