Reaction Of ONOO Research Articles

Aromatization of 9,10-Dihydroacridine Derivatives: Discovering a Highly Selective and Rapid-Responding Fluorescent Probe for Peroxynitrite.

As part of an effort to develop generally applicable strategies for creating probes suitable for detecting important molecular and ionic species, the oxidative aromatization of nonfluorescent 9,10-dihydroacridine derivatives triggered by peroxynitrite (ONOO-) led to the identification of compound 2H, 9-phenyl-9,10-dihydroacridine-4-carboxylic acid, as a rapid-responding fluorescent probe capable of detecting ONOO- with an extraordinary selectivity. Adding a little more than 1 equiv of ONOO- to a solution of 2H resulted in over 100-fold fluorescence enhancement. In sharp contrast, treating 2H with excessive amounts of other oxidants that often interfere with the detection of ONOO- failed to lead to noticeable fluorescence increase. The reaction of ONOO- with 2H shows a similar efficiency in the pH range of 2-8. Low cytotoxicity was observed for 2H and its aromatized product. Bioimaging experiments revealed the promising potential of 2H as a new fluorescent probe for the selective detection of intracellular ONOO-.

Catalytic Involvement of CO2 in the Mutagenesis Caused by Reactions of ONOO- with Guanine

The catalytic role of CO2 in reactions of ONOO- with guanine, leading to the formation of the mutagenic species 8-oxoguanine (8-oxoG) and 8-nitroguanine anion (8-nitroG-), was investigated by considering the reactions of nitrosoperoxycarbonate anion (ONOOCO2-), an adduct of ONOO- and CO2, with guanine at the B3LYP/6-31G** and B3LYP/AUG-cc-pVDZ levels of density functional theory in gas phase. In order to study bulk solvent effect, single-point energy calculations in aqueous media were carried out for all the species occurring in the reactions at the B3LYP/AUG-cc-pVDZ level of theory, by use of the polarizable continuum model (PCM). Vibrational frequency analysis was performed, and zero-point-energy (ZPE)-corrected total energies and Gibbs free energy changes at 298.15 K were obtained. The genuineness of the calculated transition states was confirmed by visually examining the vibrational modes and also by intrinsic reaction coordinate (IRC) calculations. The reaction between ONOOCO2- and guanine occurring through four different mechanisms leads to the formation of 8-oxoG or its anion, while the reaction between the same two species occurring through a different scheme leads to the formation of 8-nitroG-. It has been shown that the presence of a water molecule along with ONOOCO2- would not affect the reaction mechanisms significantly. Structures of the reactant complexes, product complexes and barrier energies involved in the reactions reveal that CO2 acts as a catalyst for the reaction between ONOO- and guanine. The cause of the catalytic action of CO2 is mainly due to intermediacy of the CO3 radical anion and NO2 radical into which ONOOCO2- is fragmented while reacting with guanine. The relative stabilities of the different product complexes suggest that the mutation caused by ONOO- in the presence of CO2 would mainly involve 8-oxoG.

Degradation of matrix glycosaminoglycans by peroxynitrite/peroxynitrous acid: Evidence for a hydroxyl-radical-like mechanism

The oxidant peroxynitrite/peroxynitrous acid (ONOO −/ONOOH) is generated at sites of inflammation via reaction of O 2 − with NO. Previous studies have shown that these species can oxidize cellular targets, but few data are available on damage to extracellular matrix and its components, despite evidence for matrix modification in a number of pathologies. In the current study we show that reaction of ONOO −/ONOOH with glycosaminoglycans results in extensive polymer fragmentation. Bolus authentic ONOO −/ONOOH modifies hyaluronan, heparin, and chondroitin, dermatan, and heparan sulfates, in a concentration-dependent, but O 2-independent, manner. The ONOO −/ONOOH generator 3-(4-morpholinyl)sydnoneimine produces similar time- and concentration-dependent damage. These reactions generate specific polymer fragments via cleavage at disaccharide intervals. Studies at different pH values, and in the presence of bicarbonate, are consistent with ONOOH, rather than the carbonate adduct, CO 3 − or ONOO −, being the source of damage. EPR spin trapping experiments have provided evidence for the formation of carbon-centered radicals on glycosaminoglycans and related monosaccharides; the similarity of these spectra to those obtained with authentic HO is consistent with fragmentation being induced by this oxidant. These data suggest that extracellular matrix fragmentation at sites of inflammation may be due, in part, to the formation and reactions of ONOOH.

Peroxynitrite: just an oxidative/nitrosative stressor or a physiological regulator as well?

British Journal of Pharmacology (2006) 148, 1–3. doi:10.1038/sj.bjp.0706693

Reactions of Peroxynitrite and Nitrite with Organic Molecules and Hemoglobin

In this work, the reactions of nitrite (NO2-) and peroxynitrite (ONOO-) with organic molecules as well as with hemoglobin (Hb) were examined and the potential interference with the detection of hydrogen peroxide and Hb was investigated. ONOO- at low concentrations (35-140 microM) induced a concentration-dependent oxidation of o-phenylenediamine and guaiacol, and this process can be improved by the addition of Hb in a concentration-dependent manner. This enhancing effect of Hb was possibly due to the formation of such highly reactive species as ferrylHb during the reaction of ONOO- and Hb. NO2- also oxidized the aromatic amine o-phenylenediamine, but its efficiency was much lower than that of ONOO-. A 300-fold excess of NO2- over hydrogen peroxide inhibited the oxidation of Pyrogallol Red mediated by hydrogen peroxide and Hb, which was due in part to the reaction of NO2- with Hb ferryl species compound I and compound II and the phenoxyl radical. These data suggest that ONOO- and NO2- can interfere with the detection of hydrogen peroxide. The overestimation or underestimation of the hydrogen peroxide detected is dependent upon the organic molecule utilized for detection and the relative rate of NO2-, superoxide, and ONOO- generation.

Metmyoglobin and methemoglobin catalyze the isomerization of peroxynitrite to nitrate.

Hemoproteins, in particular, myoglobin and hemoglobin, are among the major targets of peroxynitrite in vivo. The oxygenated forms of these proteins are oxidized by peroxynitrite to their corresponding iron(iii) forms (metMb and metHb). This reaction has previously been shown to proceed via the corresponding oxoiron(iv) forms of the proteins. In this paper, we have conclusively shown that metMb and metHb catalyze the isomerization of peroxynitrite to nitrate. The catalytic rate constants were determined by stopped-flow spectroscopy in the presence and absence of 1.2 mM CO(2) at 20 and 37 degrees C. The values obtained for metMb and metHb, with no added CO(2) at pH 7.0 and 20 degrees C, are (7.7 +/- 0.1) x 10(4) and (3.9 +/- 0.2) x 10(4) M(-1) s(-1), respectively. The pH-dependence of the catalytic rate constants indicates that HOONO is the species that reacts with the iron(iii) center of the proteins. In the presence of 1.2 mM CO(2), metMb and metHb also accelerate the decay of peroxynitrite in a concentration-dependent way. However, experiments carried out at pH 8.3 in the presence of 10 mM CO(2) suggest that ONOOCO(2)(-), the species generated from the reaction of ONOO(-) with CO(2), does not react with the iron(iii) center of Mb and Hb. Finally, we showed that different forms of Mb and Hb protect free tyrosine from peroxynitrite-mediated nitration. The order of efficiency is metMbCN < apoMb < metHb < metMb < ferrylMb < oxyHb < deoxyHb < oxyMb. Taken together, our data show that myoglobin is always a better scavenger than hemoglobin. Moreover, the globin offers very little protection, as the heme-free (apoMb) and heme-blocked (metMbCN) forms only partly prevent nitration of free tyrosine.

Loss of mitochondrial MnSOD in acute cardiac rejection: synergistic protection by low-dose cyclosporine plus vitamin C

Background: Tyrosine nitration (NO-Tyr) of proteins may occur via peroxynitrite (ONOO−), the product of nitric oxide (NO) and superoxide, or via secondary radicals formed after reaction of ONOO− and endogenous bicarbonate to form nitrosoperoxocarboxylate. Tyrosine nitration of purified manganese superoxide dismutase (MnSOD) by ONOO− leads to decreased enzyme activity, thereby increasing oxidative stress. Purpose: We examined the role of NO derived from inducible nitric oxide synthase (iNOS) on MnSOD protein and activity in acute cardiac rejection, and the benefits of treatment with vitamin C (VitC). Methods: Lewis (isograft) or Wistar-Furth (allograft) donor hearts were transplanted into Lewis recipient rats. Rats were untreated or treated daily with low-dose cyclosporine (CsA) 2.5 mg/kg i.m., VitC 120 mg/100 g gavage or VitC+CsA. We determined histological rejections scores, MnSOD activity, MnSOD and iNOS protein (Western blots), plasma NO metabolites and NO-Tyr (slot blot). Results: Rejection scores (untreated: 4.6 ± 0.2; VitC: 4.1 ± 0.4; CsA: 2.6 ± 0.3, P < 0.001 vs. untreated; VitC + CsA: 3.4 ± 0.4, P < 0.05 vs. untreated) as well as myocardial fractional shortening determined by sonomicrometry were improved by treatment (untreated: 0.77 ± 0.17; VitC: 1.45 ± 0.09; CsA: 2.24 ± 0.22, P < 0.01 vs. untreated; VitC + CsA: 1.77 ± 0.19, P < 0.05 vs. untreated). MnSOD activity was decreased on POD6 (5.8 ± 1.3 U/mg/min) but not POD4 (12.1 ± 2.1 U/mg/min) in untreated allografts vs. isografts (10.4 ± 2.5 U/mg/min). On POD6, MnSOD protein was decreased by 50% and was prevented by treatment with an iNOS dimerization inhibitor, L-N6-(1-iminoethyl) lysine (LNIL), (2.7 ± 0.3, 1.5 ± 0.4, 3.5 ± 0.4 arbitrary units for isografts, allografts and treated allografts, respectively). Loss of MnSOD protein was partially protected by CsA or VitC alone, but fully protected by VitC + CsA. VitC alone had no effect on iNOS protein while CsA alone or VitC + CsA decreased iNOS to a similar level. NO-Tyr was elevated by 3-fold in allografts at POD6. In contrast to effects of treatments on iNOS, VitC or CsA alone decreased NO-Tyr (untreated: 45 ± 9; VitC: 32 ± 1; CsA: 26 ± 6, ng NO-Tyr-BSA equivalents). Conclusions: Decreases in MnSOD protein occur via an iNOS-dependent pathway. As VitC alone did not change iNOS protein or NO production (i.e., NO metabolites) and the published rate constant of reaction of VitC with superoxide is modest, it is unlikely that VitC preserves MnSOD protein simply by limiting ONOO− formation. Rather, VitC may act to decrease NO-Tyr by quenching tyrosyl radicals produced secondary to ONOO−formation.

Interactions of Peroxynitrite, Tetrahydrobiopterin, Ascorbic Acid, and Thiols: IMPLICATIONS FOR UNCOUPLING ENDOTHELIAL NITRIC-OXIDE SYNTHASE

Tetrahydrobiopterin (BH4) serves as a critical co-factor for the endothelial nitric-oxide synthase (eNOS). A deficiency of BH4 results in eNOS uncoupling, which is associated with increased superoxide and decreased NO* production. BH4 has been suggested to be a target for oxidation by peroxynitrite (ONOO-), and ascorbate has been shown to preserve BH4 levels and enhance endothelial NO* production; however, the mechanisms underlying these processes remain poorly defined. To gain further insight into these interactions, the reaction of ONOO- with BH4 was studied using electron spin resonance and the spin probe 1-hydroxy-3-carboxy-2,2,5-tetramethyl-pyrrolidine. ONOO- reacted with BH4 6-10 times faster than with ascorbate or thiols. The immediate product of the reaction between ONOO- and BH4 was the trihydrobiopterin radical (BH3.), which was reduced back to BH4 by ascorbate, whereas thiols were not efficient in recycling of BH4. Uncoupling of eNOS caused by peroxynitrite was investigated in cultured bovine aortic endothelial cells (BAECs) by measuring superoxide and NO* using spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine and the NO*-spin trap iron-diethyldithiocarbamate. Bolus ONOO-, the ONOO- donor 3-morpholinosydnonimine, and an inhibitor of BH4 synthesis (2,4-diamino-6-hydroxypyrimidine) uncoupled eNOS, increasing superoxide and decreasing NO* production. Exogenous BH4 supplementation restored endothelial NO* production. Treatment of BAECs with both BH4 and ascorbate prior to ONOO- prevented uncoupling of eNOS by ONOO-. This study demonstrates that endothelial BH4 is a crucial target for oxidation by ONOO- and that the BH4 reaction rate constant exceeds those of thiols or ascorbate. We confirmed that ONOO- uncouples eNOS by oxidation of tetrahydrobiopterin and that ascorbate does not fully protect BH4 from oxidation but recycles BH3. radical back to BH4.

Carbon Dioxide Modulation of Peroxynitrite-Induced Mutagenesis of the supF Gene in pSP189

Peroxynitrite (ONOO(-)), a potent oxidant formed by the reaction of nitric oxide with superoxide anion, may play a significant role as an intermediate in NO(*)-related cytotoxicity and genotoxicity. In addition to causing other types of toxicity, peroxynitrite damages DNA and induces mutations in genetic targets following in vitro or in vivo exposure. It has recently been established that the reaction of CO(2) with ONOO(-) significantly changes its chemistry in biological media. The objective of this investigation was to characterize impacts of CO(2) on peroxynitrite-induced mutagenesis of the supF gene in the shuttle vector pSP189. The dose-response relationship between ONOO(-) concentration and mutation frequency was determined following bolus exposure of the plasmid suspended in 150 mM phosphate buffer at pH 7.4, with and without addition of 25 mM sodium bicarbonate. After treatment, plasmids were replicated in Escherichia coli MBL50 cells for mutant identification. CO(2) significantly reduced the mutagenic potency of peroxynitrite, in that increases in mutant fraction were reduced by 47-77% at ONOO(-) doses ranging from 0 to 4 mM. We also characterized the spectrum of mutations induced under these conditions and found mutational hotspots at positions 110, 113, 116, 141, 156, 168, and 172 in mutants induced in the presence of CO(2) and at positions 113, 124, 126, 141, and 156 in those induced in its absence. Among the 22 guanines present in the 84 nucleotide supF sequence, 18 were sites of mutation, including four mutational hotspots (G113, G116, G141, and G156), in plasmids exposed to ONOO(-) in the presence of bicarbonate. After exposure in the absence of bicarbonate, 14 of the 22 guanines were mutation sites, with hotspots located at five (G113, G124, G126, G141, and G156). Remaining mutations were located almost exclusively at cytosine residues. It is evident from these data that reactive intermediates formed through reaction of ONOO(-) with CO(2) play an important role in the mutagenicity of ONOO(-).

The reaction of ONOO– with carbonyls: Estimation of the half-lives of ONOOC(O)O– and O2NOOC(O)O–Based on the presentation given at Dalton Discussion No. 4, 10–13th January 2002, Kloster Banz, Germany.

The equilibrium constant for the formation of an adduct between a carbonyl and a hydroperoxide ion varies linearly with the acid dissociation constant of the hydroperoxide. Based on this relationship, the half-life of the adduct formed between ONOO- and CO2, ONOOC(O)O-, is estimated to be shorter than 100 ns. Consequently, this adduct should not play any role whatsoever in chemical or biological systems. O2NOO- and CO2 are believed to be involved in a fast equilibrium reaction forming an adduct, O2NOOC(O)O-. This adduct does not appear to homolyse either along the O-O or the N-O bond. Furthermore, at realistic CO2 concentrations, the equilibrium should be shifted far to the O2NOO- + CO2 side. Therefore, the rate of self-decomposition of O2NOO- into NO2- and O-2 is unaffected by the presence of bicarbonate.

The rate of homolysis of adducts of peroxynitrite to the C=O double bond.

Nucleophilic addition of the peroxynitrite anion, ONOO(-), to the two prototypical carbonyl compounds, acetaldehyde and acetone, was investigated in the pH interval 7.4-14. The process is initiated by fast equilibration between the reactants and the corresponding tetrahedral adduct anion, the equilibrium being strongly shifted to the reactant side. The adduct anion also undergoes fast protonation by water and added buffers. Consequently, the rate of the bimolecular reaction between ONOO(-) and the carbonyl is strongly dependent on the pH and on the concentration of the buffer. The pK(a) of the carbonyl-ONOO adduct was estimated to be approximately 11.8 and approximately 12.3 for acetone and acetaldehyde, respectively. It is shown that both the anionic and the neutral adducts suffer fast homolysis along the weak O-O bond to yield free alkoxyl and nitrogen dioxide radicals. The yield of free radicals was determined to be about 15% with both carbonyl compounds at low and high pH, while the remainder collapses to molecular products in the solvent cage. The rate constants for the homolysis of the adducts vary from ca. 3 x 10(5) to ca. 5 x 10(6) s(-1), suggesting that they cannot act as oxidants in biological systems. This small variation around a mean value of about 10(6) s(-1) suggests that the O-O bond in the adduct is rather insensitive to its protonation state and to the nature of its carbonyl precursor. An overall reaction scheme was proposed, and all the corresponding rate constants were evaluated. Finally, thermokinetic considerations were employed to argue that the formation of dioxirane as an intermediate in the reaction of ONOO(-) with acetone is an unlikely process.

Carbonate radical ion is the only observable intermediate in the reaction of peroxynitrite with CO(2).

The reaction of ONOO(-) with CO(2) at alkaline pH was recently reported to form a transient absorption with a maximum at 640 nm and a half-life of ca. 4 ms at 10 degrees C [Meli et al. (1999) Helv. Chim. Acta 82, 722-725]. This transient absorption was hardly affected by the presence of *NO, and therefore was attributed to the adduct ONOOC(O)O(-). This conclusion contradicts all current experimental results as it suggests that the decomposition of this adduct via homolysis of the O-O bond into CO(3)(*)(-) and *NO(2) is a minor pathway. In the present work the observations of Meli et al. will be shown to be artifacts resulting from light coming from the UV region. When these experiments are carried out in the presence of appropriate cutoff filters, the only observable intermediate formed in the reaction of ONOO(-) with CO(2) at alkaline pH is the carbonate radical ion with a maximum at 600 nm. This transient absorption is not observed in the presence of *NO or ferrocyanide. In the latter case ferricyanide is formed, and its yield was determined to be 66 +/- 2% of the initial concentration of peroxynitrite. The reaction of ONOO(-) with 16 mM CO(2) with and without ferrocyanide was also studied at pH 5.6-7.7 in the presence of 0.1 M phosphate, where both the initial pH and [CO(2)] remain constant. Under these conditions the rate constant of the decay of peroxynitrite was found to be identical to that of the formation of ferricyanide, indicating that ONOOC(O)(-) does not accumulate. These results confirm our earlier observations, i.e., the reaction of peroxynitrite with excess CO(2) takes place via the formation of about 33% CO(3)(*)(-) and *NO(2) radicals in the bulk of the solution.

Peptide nitration by peroxynitrite: characterisation of the nitration sites by liquid chromatography/tandem mass spectrometry

A number of recent studies have demonstrated that peroxynitrite (ONOO−) can react with a host of biomolecules, particularly those containing aromatic amino acid residues, resulting in a number of modifications which have been found in association with diverse pathological conditions. Electrospray ionisation tandem mass spectrometry with and without liquid chromatographic separation has been used to examine a series of model peptides following their treatment with peroxynitrite at physiological pH. The mass spectra of sequences containing two tyrosine residues showed the formation of both mononitrated and dinitrated species, while those with phenylalanine showed no detectable nitration. Tryptic digests of two of the investigated peptides were also examined by liquid chromatography/electrospray mass spectrometry, which yielded further information on the competition for NO2 by multiple nitration sites within a given sequence. In addition, tryptic digests of the mononitrated component of sequences containing tyrosine and tryptophan indicated that mononitration was limited to tyrosine. Furthermore, some of the presented data indicate that, as well as nitrotyrosine and nitrotryptophan formation, the reaction of ONOO− can result in oxidation as well as the formation of labile adducts with NO2. Copyright © 1999 John Wiley & Sons, Ltd.

31] Nitric oxide donor generation from reactions of peroxynitrite

Publisher Summary This chapter describes the methodology for the formation of nitric oxide (NO) donors via the reaction of peroxynitrite (ONOO - ) with glucose and other cellular metabolites. The formation of stable NO donors from reactions of ONOO - with glycerol, glucose, or thiol is not species specific because these compounds are physiologically active in arteries of different species. These NO donors produce a slowly developing and sustained decrease in tension consistent with the time required for the metabolic conversion of the NO donor to an NO - releasing metabolite. In vitro , NO donors are formed from the reaction of ONOO - with plasma and induce vascular relaxation and inhibit platelet aggregation, through a mechanism that is inhibited by the NO scavenger oxyhemoglobin (oxyHb). The pathophysiological consequences of the reactions of ONOO - with poly hydroxylated compounds are currently being investigated. In vivo , NO-dependent nitration and nitrosation reactions may be enhanced at sites of inflammation. Current thinking suggests that the increased production of O 2 - , in inflammatory cells, facilitates ONOO - formation and thus leads to the modification of NO reactivity in the blood vessel wall.

Formation of S-Nitrosothiols via Direct Nucleophilic Nitrosation of Thiols by Peroxynitrite with Elimination of Hydrogen Peroxide

Peroxynitrite (ONOO-), a potent oxidant formed by reaction of nitric oxide (NO.) with superoxide anion, can activate guanylyl cyclase and is able to induce vasodilation or inhibit platelet aggregation and leukocyte adhesion, via thiol-dependent formation of NO. Reaction of ONOO- with thiols is thought to proceed through formation of a S-nitrothiol (thionitrate; RSNO2) intermediate and yields low levels of S-nitrosothiols (thionitrites; RSNO), both of which are theoretical sources of NO. Kinetic analysis of NO. production after reaction of ONOO- with GSH established that NO. originates exclusively from the thionitrite GSNO. Further mechanistic investigations indicated that GSNO formation by ONOO- does not occur via one-electron oxidation mechanisms. Nitrosation of GSH could theoretically proceed via intermediate formation of the thionitrate GSNO2, which, after rearrangement to the corresponding sulfenyl nitrite (GSONO), can react with GSH to form GSNO and GSOH. However, no evidence for such a mechanism was found in experiments with NO2. or with the stable nitrothiol tert-butylthionitrate. Using high performance liquid chromatography with chemiluminescence detection, formation of H2O2 was observed after reaction of ONOO- with GSH under both aerobic and anaerobic conditions, at levels similar to the yield of GSNO, indicative of a direct nucleophilic nitrosation mechanism with elimination of HOO-. Our results indicate that ONOO- may contribute to S-nitrosation in vivo and that direct nitrosation of thiols or other nucleophilic substrates by ONOO- may represent an important and often overlooked component of NO. biochemistry.

S-Nitroglutathione, a Product of the Reaction between Peroxynitrite and Glutathione That Generates Nitric Oxide

Peroxynitrite (ONOO-) has been shown in studies on vascular relaxation and guanylate cyclase activation to react with glutathione (GSH), generating an intermediate product that promotes a time-dependent production of nitric oxide (NO). In this study, reactions of ONOO- with GSH produced a new substance, which was characterized by liquid chromatography, ultraviolet spectroscopy, and electrospray tandem mass spectrometry. The mass spectrometric data provided evidence that the product of this reaction was S-nitroglutathione (GSNO2) and that S-nitrosoglutathione (GSNO) was not a detectable product of this reaction. Further evidence was obtained by comparison of the spectral and chromatographic properties with synthetic standards prepared by reaction of GSH with nitrosonium or nitronium borofluorates. Both the synthetic and ONOO-/GSH-derived GSNO2 generated a protonated ion, GSNO2H+, at m/z 353, which was unusually resistant to decomposition under collision activation, and no fragmentation was observed at collision energy of 25 eV. In contrast, an ion at m/z 337 (GSNOH+), generated from the synthetic GSNO, readily fragmented with the abundant loss of NO at 9 eV. Reactions of ONOO- with GSH resulted in the generation of NO, which was detected by the head space/NO-chemiluminescence analyzer method. The generation of NO was inhibited by the presence of glucose and/or CO2 in the buffers employed. Synthetic GSNO2 spontaneously generated NO in a manner that was not significantly altered by glucose or CO2. Thus, ONOO- reacts with GSH to form GSNO2, and GSNO2 decomposes in a manner that generates NO.

Reactions of peroxynitrite with aldehydes as probes for the reactive intermediates responsible for biological nitration.

We have examined the reactions of peroxynitrite with short-chain aliphatic aldehydes to model the reaction of the peroxynitrite anion (ONOO-) with CO2. Aldehydes, like CO2, react rapidly with peroxynitrite and catalyze its decomposition. The pH dependence of the reaction is consistent with the addition of ONOO- (not ONOOH) to the carbonyl carbon atom of the free aldehyde forming a 1-hydroxyalkylperoxynitrite anion adduct (5), which structurally resembles the nitrosoperoxycarbonate adduct (1) formed from the reaction of ONOO- with CO2. Intermediate 5, or the secondary products derived from it, decays to give NO3- and regenerated aldehyde, with small but significant yields of H2O2, organic acids, and organic nitrates. In analogy with the peroxynitrite/CO2 system, it is suggested that 5 undergoes homolytic or heterolytic cleavage at the O-O bond, giving a caged radical pair [RCH(OH)O./ .NO2] (7) or intimate ion pair [RCH(OH)O -/+ NO2] (8). The radicals and ions in intermediates 7 and 8 can recombine within the solvent cage to form 1-hydroxyalkylnitrate [RCH(OH)ONO2] (6), which can then dissociate to give nitrate and regenerate the aldehyde. The aldehyde/ peroxynitrite adducts 5-8 mediate the oxidation of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) but not the nitration of 4-hydroxyphenylacetate. The significance of these findings is discussed in relation to the mechanism(s) of the CO2-catalyzed isomerization of peroxynitrite to nitrate and biological nitrations involving peroxynitrite/CO2 adducts.

The Effect of Bicarbonate on Oxidation by Peroxynitrite: Implication for Its Biological Activity

The effect of bicarbonate on the oxidation rates and yields of the reaction of peroxynitrite with NiII(cyclam), ferrocyanide, and ABTS was studied using the stopped-flow technique. In the absence of bicarbonate, NiII(cyclam) is oxidized directly by peroxynitrous acid whereas ferrocyanide and ABTS are oxidized by intermediates that are formed during the self-decomposition of peroxynitrous acid. In the presence of sufficient concentrations of bicarbonate, the rate-determining step of these oxidation processes is the reaction of ONOO- with CO2. The latter reaction results in the formation of an adduct ONOOCO2-, which is incapable of oxidizing directly these substrates. It is shown that about 33% of this adduct decomposes into highly oxidizing species, most probably into •NO2 and CO3•- via the homolysis of the peroxo O−O bond. Under physiological conditions, where the most abundant species is ONOO- and the concentration of CO2 exceeds 1 mM, the half-life of peroxynitrite is reduced, and consequently its diffusion distance is shortened. Therefore, indirect oxidation by peroxynitrite through intermediates that are formed during its self-decomposition will not take place in vivo, whereas direct oxidation by peroxynitrite may take place provided that the rate of this reaction competes efficiently with that of peroxynitrite with CO2. We conclude that the toxicity of peroxynitrite in vivo is most probably governed by ONOOCO2-, which has a reactivity different from that of peroxynitrite.

37] Reaction of peroxynitrite and hydrogen peroxide to produce singlet molecular oxygen (1Δg)

This chapter discusses the reaction of peroxynitrite and hydrogen peroxide to produce singlet molecular oxygen ( 1 Δ g ). Peroxynitrite (ONOO – ) is a biologically active species produced by the reaction of the superoxide anion radical (O 2 – ) with nitric oxide (NO . ). The decomposition of ONOO– generates the hydroxyl radical, a strong oxidant. Nitric oxide, identified as the endothelium-derived relaxing factor (EDRF), is formed by conversion of L -arginine to L -citrulline by NO synthase. The production of singlet molecular oxygen [O 2 ( 1 Δ g )] in the reaction of ONOO – with H 2 O 2 , uses (1) chemiluminescence (CL) measurement of dimol or monomol light emission in the visible and infrared spectral regions, and (2) water-soluble chemical trap, the sodium salt of anthracene 9,10-diyldiethyl disulfate (EAS), to provide evidence for the formation of O 2 ( l Δ g ) in this system. Peroxynitrite is synthesized in a quenched-flow reactor. The singlet molecular oxygen generation in the peroxynitrite-H 2 O 2 reaction is monitored by chemical trapping with EAS.

The role of the reactions of ·NO with superoxide and oxygen in biological systems: A kinetic approach

In this study we calculate the half-life of ·NO in its reactions with superoxide and with oxygen under various conditions using the known rate constants for these reactions. The measured half-life of ·NO in biological systems is 3–5 s, which agrees well with the calculated value for intracellular ·NO, but not for extracellular ·NO under normal physiological conditions. The autoxidation of ·NO to yield NO 2 as a final product cannot be responsible for such a short measured half-life under normal as well as pathologic conditions. Therefore, if there is direct evidence for the occurrence of the reaction of ·NO with O 2 in the medium, one has to assume that the steady state concentrations of free ·NO are much lower than those measured. The very low concentrations of free ·NO in biological systems may result from its reversible strong binding to biological molecules. Simulation of the mechanism of the autoxidation of ·NO indicates that the binding constants of ·NO to O 2 or to another ·NO are too small to account for the very low concentration of free ·NO in biological systems. Nevertheless, the reaction of ·NO with oxygen cannot be neglected in biological systems if the intermediate ONOO· reacts rapidly with a biological target. The biological damage caused by ONOO′ is expected to be due to the radical itself and to peroxynitrite, which is most probably formed via the reaction of ONOO· with the biological molecule.