Isotope effects and intermediates in the reduction of NO by P450NOR

Daiber, Andreas; Nauser, Thomas; Takaya, Naoki; Kudo, Takashi; Weber, P.; Hultschig, C.; Shoun, Hirofumi; Ullrich, Volker

doi:10.1016/s0162-0134(01)00386-5

Cited by 86 publications

(118 citation statements)

References 29 publications

Supporting

Mentioning

103

Contrasting

Order By: Relevance

“…The EPR spectrum of this sample shows signals for Fe III -NH 2 OH and {FeNO} 7 (Fig. 4D) (35,36) and Fe II -HNO, which has been characterized in only one biological system, the myoglobin-HNO complex (37,38). Finally, because N 2 O is the product of NH 2 OH oxidation by cyt P460 under our experimental conditions, an Fe-N 2 O complex is also possible.…”

Section: Resultsmentioning

confidence: 82%

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.

159

212

View full text Add to dashboard Cite

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 ...

show abstract

Section: Resultsmentioning

confidence: 82%

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.

159

212

View full text Add to dashboard Cite

show abstract

“…N 2 O þ H 2 O). The chemical entity of I was proposed to be ferric-hydroxylamine radical complex, as shown in figure 5 [35]. The reaction mechanism (figure 5) is also supported by a quantum-chemical calculation [36].…”

mentioning

confidence: 70%

“…(b) Reaction scheme of P450nor [34]. The structure of I (444 nm species) as an Fe 3þ -hydroxylamine radical complex was proposed by Daiber et al [35]. dinucleotide; NAAD) [43].…”

mentioning

confidence: 99%

See 1 more Smart Citation

Fungal denitrification and nitric oxide reductase cytochrome P450nor

Shoun

Fushinobu

Jiang

et al. 2012

Phil. Trans. R. Soc. B

211

189

View full text Add to dashboard Cite

We have shown that many fungi (eukaryotes) exhibit distinct denitrifying activities, although occurrence of denitrification was previously thought to be restricted to bacteria (prokaryotes), and have characterized the fungal denitrification system. It comprises NirK (copper-containing nitrite reductase) and P450nor (a cytochrome P450 nitric oxide (NO) reductase (Nor)) to reduce nitrite to nitrous oxide (N 2 O). The system is localized in mitochondria functioning during anaerobic respiration. Some fungal systems further contain and use dissimilatory and assimilatory nitrate reductases to denitrify nitrate. Phylogenetic analysis of nirK genes showed that the fungal-denitrifying system has the same ancestor as the bacterial counterpart and suggested a possibility of its proto-mitochondrial origin. By contrast, fungi that have acquired a P450 from bacteria by horizontal transfer of the gene, modulated its function to give a Nor activity replacing the original Nor with P450nor. P450nor receives electrons directly from nicotinamide adenine dinucleotide to reduce NO to N 2 O. The mechanism of this unprecedented electron transfer has been extensively studied and thoroughly elucidated. Fungal denitrification is often accompanied by a unique phenomenon, co-denitrification, in which a hybrid N 2 or N 2 O species is formed upon the combination of nitrogen atoms of nitrite with a nitrogen donor (amines and imines). Possible involvement of NirK and P450nor is suggested.

show abstract

“…The unique reactivity of the Mn-QDO suggests a metalmediated electron transfer mechanism rather than metal-activation of the substrate's inherent base-catalyzed reactivity. There has been much discussion over the electronic nature of bonding in transition metal HNO complexes (50), for instance in the putative catalytic intermediate in nitric oxide reductase P450nor (51). An HNO-Mn II species with a d 5 electronic configuration is unprecedented, as all isolable HNO-metal complexes are diamagnetic with low-spin d 6 metal ions.…”

Section: Discussionmentioning

confidence: 99%

Nitrosyl hydride (HNO) replaces dioxygen in nitroxygenase activity of manganese quercetin dioxygenase

Kumar

Zapata

Ramirez

et al. 2011

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

View full text Add to dashboard Cite

Quercetin dioxygenase (QDO) catalyzes the oxidation of the flavonol quercetin with dioxygen, cleaving the central heterocyclic ring and releasing CO. The QDO from Bacillus subtilis is unusual in that it has been shown to be active with several divalent metal cofactors such as Fe, Mn, and Co. Previous comparison of the catalytic activities suggest that Mn(II) is the preferred cofactor for this enzyme. We herein report the unprecedented substitution of nitrosyl hydride (HNO) for dioxygen in the activity of Mn-QDO, resulting in the incorporation of both N and O atoms into the product. Turnover is demonstrated by consumption of quercetin and other related substrates under anaerobic conditions in the presence of HNO-releasing compounds and the enzyme. As with dioxygenase activity, a nonenzymatic base-catalyzed reaction of quercetin with HNO is observed above pH 7, but no enhancement of this basal reactivity is found upon addition of divalent metal salts. Unique and regioselective N-containing products ( 14 N∕ 15 N) have been characterized by MS analysis for both the enzymatic and nonenzymatic reactions. Of the several metallo-QDO enzymes examined for nitroxygenase activity under anaerobic condition, only the Mn(II) is active; the Fe(II) and Co(II) substituted enzymes show little or no activity. This result represents an enzymatic catalysis which we denote nitroxygenase activity; the unique reactivity of the Mn-QDO suggests a metalmediated electron transfer mechanism rather than metal activation of the substrate's inherent base-catalyzed reactivity.nitroxyl | quercetinase D ioxygenases are enzymes that incorporate both atoms of dioxygen into a targeted substrate, e.g., the enzyme catechol 1,2-dioxygenase cleaves the C-C bond between catechol's two hydroxyl groups forming the dicarboxylate cis,cis-muconic acid (1). Although highly divergent, there are two broadly defined classes of nonheme dioxygenases: those that directly bind dioxygen at the metal center or those in which dioxygen binds to the substrate that has been activated by interaction with the metal center (2). Such questions also apply to quercetin dioxygenase (QDO), which catalyzes the oxidation of the flavonol quercetin (1) with dioxygen ( Fig. 1). During turnover, QDO cleaves the central heterocyclic ring of quercetin, releasing CO, and yielding the corresponding depside. The QDO from Bacillus subtilis is unusual in that it has been shown to be active with several divalent metal cofactors such as Cu, Fe, Mn, and Co. Comparison of the catalytic activities suggest that Mn(II) is the preferred cofactor for this enzyme (3).The simple molecule nitrosyl hydride (HNO) is the singly reduced and protonated form of nitric oxide, NO. HNO has garnered much interest because it demonstrates a physiological reactivity distinctly different from its congener nitric oxide (4, 5). In a series of papers, we have reported the preparation and characterization of the HNO adduct of myoglobin (HNO-Mb) by reduction of 7) and by trapping of free HNO by deoxymyoglobin (Mb-Fe II ) (8)...

show abstract

Isotope effects and intermediates in the reduction of NO by P450NOR

Cited by 86 publications

References 29 publications

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

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

Fungal denitrification and nitric oxide reductase cytochrome P450nor

Nitrosyl hydride (HNO) replaces dioxygen in nitroxygenase activity of manganese quercetin dioxygenase

Contact Info

Product

Resources

About