Metabolic Fate of Peroxynitrite in Aqueous Solution
1997; Elsevier BV; Volume: 272; Issue: 6 Linguagem: Inglês
10.1074/jbc.272.6.3465
ISSN1083-351X
AutoresSilvia Pfeiffer, Antonius C.F. Gorren, Kurt Schmidt, Ernst R. Werner, Bernhard Hansert, D. Scott Bohle, Bernd Mayer,
Tópico(s)Sulfur Compounds in Biology
ResumoPeroxynitrite, the reaction product of nitric oxide (NO) and superoxide (O2) is assumed to decompose upon protonation in a first order process via intramolecular rearrangement to NO3−. The present study was carried out to elucidate the origin of NO2− found in decomposed peroxynitrite solutions. As revealed by stopped-flow spectroscopy, the decay of peroxynitrite followed first-order kinetics and exhibited a pKa of 6.8 ± 0.1. The reaction of peroxynitrite with NO was considered as one possible source of NO2−, but the calculated second order rate constant of 9.1 × 104M−1 s−1 is probably too small to explain NO2− formation under physiological conditions. Moreover, pure peroxynitrite decomposed to NO2− without apparent release of NO. Determination of NO2− and NO3− in solutions of decomposed peroxynitrite showed that the relative amount of NO2− increased with increasing pH, with NO2− accounting for about 30% of decomposition products at pH 7.5 and NO3− being the sole metabolite at pH 3.0. Formation of NO2− was accompanied by release of stoichiometric amounts of O2 (0.495 mol/mol of NO2−). The two reactions yielding NO2− and NO3− showed distinct temperature dependences from which a difference in Eact of 26.2 ± 0.9 kJ mol−1 was calculated. The present results demonstrate that peroxynitrite decomposes with significant rates to NO2− plus O2 at physiological pH. Through formation of biologically active intermediates, this novel pathway of peroxynitrite decomposition may contribute to the physiology and/or cytotoxicity of NO and superoxide. Peroxynitrite, the reaction product of nitric oxide (NO) and superoxide (O2) is assumed to decompose upon protonation in a first order process via intramolecular rearrangement to NO3−. The present study was carried out to elucidate the origin of NO2− found in decomposed peroxynitrite solutions. As revealed by stopped-flow spectroscopy, the decay of peroxynitrite followed first-order kinetics and exhibited a pKa of 6.8 ± 0.1. The reaction of peroxynitrite with NO was considered as one possible source of NO2−, but the calculated second order rate constant of 9.1 × 104M−1 s−1 is probably too small to explain NO2− formation under physiological conditions. Moreover, pure peroxynitrite decomposed to NO2− without apparent release of NO. Determination of NO2− and NO3− in solutions of decomposed peroxynitrite showed that the relative amount of NO2− increased with increasing pH, with NO2− accounting for about 30% of decomposition products at pH 7.5 and NO3− being the sole metabolite at pH 3.0. Formation of NO2− was accompanied by release of stoichiometric amounts of O2 (0.495 mol/mol of NO2−). The two reactions yielding NO2− and NO3− showed distinct temperature dependences from which a difference in Eact of 26.2 ± 0.9 kJ mol−1 was calculated. The present results demonstrate that peroxynitrite decomposes with significant rates to NO2− plus O2 at physiological pH. Through formation of biologically active intermediates, this novel pathway of peroxynitrite decomposition may contribute to the physiology and/or cytotoxicity of NO and superoxide. INTRODUCTIONThe reaction between nitric oxide (NO) and superoxide anion (O2) yields peroxynitrite with a second order rate constant near the diffusion-controlled limit (k = 4.3-6.7 × 109M−1 s−1) (1Huie R.E. Padmaja S. Free Radical Res. Commun. 1993; 18: 195-199Crossref PubMed Scopus (2006) Google Scholar, 2Goldstein S. Czapski G. Free Radical Biol. & Med. 1995; 117: 12078-12084Google Scholar). The reaction constitutes an important sink for O2 because it is about twice as fast as the maximum velocity of SOD. 1The abbreviations used are:SODsuperoxide dismutaseSIN-13-(4-morpholinyl)-sydnoniminehydrochlorideHPLChigh performance liquid chromatographyDTPAdiethylenetriaminepentaacetic acid. Consequently, peroxynitrite has been implicated in many pathological conditions including stroke (3Dawson V.L. Dawson T.M. London E.D. Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6368-6371Crossref PubMed Scopus (2098) Google Scholar), heart disease (4Matheis G. Sherman M.P. Buckberg G.D. Haybron D.M. Young H.H. Ignarro L.J. Am. J. Physiol. 1992; 262PubMed Google Scholar), and atherosclerosis (5Hogg N. Darley-Usmar V.M. Graham A. Moncada S. Biochem. Soc. Trans. 1993; 21: 358-362Crossref PubMed Scopus (89) Google Scholar, 6White C.R. Brock T.A. Chang L.Y. Crapo J. Briscoe P. Ku D. Bradley W.A. Gianturco S.H. Gore J. Freeman B.A. Tarpey M.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1044-1048Crossref PubMed Scopus (655) Google Scholar). The potential cellular targets for peroxynitrite cytotoxicity include the antioxidants ascorbate, α-tocopherol, and uric acid (7Bartlett D. Church D.F. Bounds P.L. Koppenol W.H. Free Radical Biol. & Med. 1995; 18: 85-92Crossref PubMed Scopus (163) Google Scholar, 8Squadrito G.L. Jin X. Pryor W.A. Arch. Biochem. Biophys. 1995; 322: 53-59Crossref PubMed Scopus (121) Google Scholar, 9Hogg N. Joseph J. Kalyanaraman B. Arch. Biochem. Biophys. 1994; 314: 153-158Crossref PubMed Scopus (101) Google Scholar, 10Vasquez-Vivar J. Santos A.M. Junqueira B.C. Augusto O. Biochem. J. 1996; 314: 869-876Crossref PubMed Scopus (130) Google Scholar), protein and non-protein sulfhydryls (11Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar), DNA (12King P.A. Anderson V.E. Edwards J.O. Gustafson G. Plumb R.C. Suggs J.W. J. Am. Chem. Soc. 1992; 114: 5430-5432Crossref Scopus (244) Google Scholar), and membrane phospholipids (13Radi R. Beckman J.S. Bush K.M. Freeman B.A. Arch. Biochem. Biophys. 1991; 288: 481-487Crossref PubMed Scopus (2030) Google Scholar).Decomposition of peroxynitrite is complex (14Edwards J.O. Plumb R.C. Karlin K.D. Progress in Inorganic Chemistry. John Wiley & Sons, Inc., New York1994: 599-635Google Scholar, 15Koppenol W.H. Moreno J.J. Pryor W.A. Ischiropoulos H. Beckman J.S. Chem. Res. Toxicol. 1992; 5: 834-842Crossref PubMed Scopus (1274) Google Scholar). The anion is rather stable in alkaline solutions but decomposes rapidly (t1/2 = 1 s at pH 7.4, 37°C) upon protonation to peroxynitrous acid (ONOOH) (pKa = 6.8) (16Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6666) Google Scholar). Two pathways of ONOOH decomposition have been proposed. Some studies have argued that ONOOH is cleaved homolytically to generate hydroxyl and NO2 radicals. This hypothesis is based on the sensitivity to hydroxyl radical scavengers of certain peroxynitrite-induced reactions, including the formation of malondialdehyde from deoxyribose and the hydroxylation on the benzene ring of sodium benzoate, phenylalanine, and tyrosine (16Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6666) Google Scholar, 17van der Vliet A. O'Neill C.A. Halliwell B. Cross C.E. Kaur H. FEBS Lett. 1994; 339: 89-92Crossref PubMed Scopus (357) Google Scholar). Studies on decomposition of peroxynitrite by electron paramagnetic resonance spectroscopy with the spin traps 5,5-dimethyl-1-pyrroline N-oxide and 4-pyridyl-1-oxide-N-tert-butylnitrone also provided evidence for the formation of free hydroxyl radicals (18Augusto O. Gatti R.M. Radi R. Arch. Biochem. Biophys. 1994; 310: 118-125Crossref PubMed Scopus (167) Google Scholar, 19Pou S. Nguyen S.Y. Gladwell T. Rosen G.M. Biochim. Biophys. Acta. 1995; 1244: 62-68Crossref PubMed Scopus (83) Google Scholar). Against this, Koppenol et al. (15Koppenol W.H. Moreno J.J. Pryor W.A. Ischiropoulos H. Beckman J.S. Chem. Res. Toxicol. 1992; 5: 834-842Crossref PubMed Scopus (1274) Google Scholar) concluded from molecular dynamic calculations that homolytic cleavage of ONOOH is highly improbable. This was reinforced by the independence of the rate of ONOOH decomposition on solvent viscosity (20Pryor W.A. Jin X. Squadrito G.L. J. Am. Chem. Soc. 1996; 118: 3125-3128Crossref Scopus (52) Google Scholar). Based on these results, it was suggested that decomposition of ONOOH to NO3− involves formation of an activated intermediate (ONOOH*), which might account for the hydroxyl radical-like properties of peroxynitrite (15Koppenol W.H. Moreno J.J. Pryor W.A. Ischiropoulos H. Beckman J.S. Chem. Res. Toxicol. 1992; 5: 834-842Crossref PubMed Scopus (1274) Google Scholar, 21Pryor W.A. Jin X. Squadrito G.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11173-11177Crossref PubMed Scopus (360) Google Scholar).There are several methods for the detection of peroxynitrite in biological systems. Since ONOOH decomposition yields an intermediate that nitrates phenolic compounds (22Beckman J.S. Ischiropoulos H. Zhu L. van der Woerd M. Smith C. Chen J. Harrison J. Martin J.C. Tsai M. Arch. Biochem. Biophys. 1992; 298: 438-445Crossref PubMed Scopus (731) Google Scholar, 23Ischiropoulos H. Zhu L. Chen J. Tsai M. Martin J.C. Smith C.D. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 431-437Crossref PubMed Scopus (1420) Google Scholar), presence of nitrotyrosine in proteins was proposed to be evidence of peroxynitrite production in tissues (24Beckman J.S. Ye Y.Z. Anderson P.G. Chen J. Accavitti M.A. Tarpey M.M. White C.R. Biol. Chem. Hoppe-Seyler. 1994; 375: 81-88Crossref PubMed Scopus (1068) Google Scholar). However, using both a monoclonal antibody specifically recognizing peroxynitrite-modified proteins (24Beckman J.S. Ye Y.Z. Anderson P.G. Chen J. Accavitti M.A. Tarpey M.M. White C.R. Biol. Chem. Hoppe-Seyler. 1994; 375: 81-88Crossref PubMed Scopus (1068) Google Scholar) as well as a published HPLC method (17van der Vliet A. O'Neill C.A. Halliwell B. Cross C.E. Kaur H. FEBS Lett. 1994; 339: 89-92Crossref PubMed Scopus (357) Google Scholar), we failed to detect tyrosine nitration by authentic peroxynitrite at concentrations <0.1 mM. 2S. Pfeiffer, and B. Mayer, unpublished observations. Spectrophotometric determination of dihydrorhodamine 123 oxidation was described as another sensitive assay for the specific detection of peroxynitrite at submicromolar concentrations (25Kooy N.W. Royall J.A. Ischiropoulos H. Beckman J.S. Free Radical Biol. & Med. 1994; 16: 149-156Crossref PubMed Scopus (666) Google Scholar), but in our hands, interference of several redox-active compounds precluded application of this method in cell-free assay systems. 3P. Klatt, and B. Mayer, unpublished observations. Under certain experimental conditions, indirect evidence for peroxynitrite production can be obtained by comparing NO release in the absence and presence of SOD. The peroxynitrite donor compound SIN-1, for example, does not release detectable amounts of free NO unless SOD is present in amounts sufficient to outcompete the reaction with concomitantly produced O2 (26Schmidt K. Klatt P. Mayer B. Biochem. J. 1994; 301: 645-647Crossref PubMed Scopus (65) Google Scholar). Based on similar results obtained with purified neuronal NO synthase, we suggested that the enzyme generates NO and O2 simultaneously and hence functions as peroxynitrite synthase if incubated in vitro (27Mayer B. Klatt P. Werner E.R. Schmidt K. J. Biol. Chem. 1995; 270: 655-659Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). However, in contrast with the widely held view that peroxynitrite decomposes exclusively to NO3−, considerable amounts of NO2− were also found as a major stable product of SIN-1 or NO synthase under physiological conditions.2 Similarly, excess NO2− formation was observed in peroxynitrite producing cells (28Lewis R.S. Tamir S. Tannenbaum S.R. Deen W.M. J. Biol. Chem. 1995; 270: 29350-29355Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), suggesting that additional as yet unidentified reactions contribute to peroxynitrite decomposition.The present study was done to elucidate the fate of peroxynitrite in aqueous solution. Studies with the authentic compound, prepared in two different ways, identified a reaction leading to release of NO2− and O2 in a 2:1 stoichiometry as a route of peroxynitrite decomposition at pH ≥ 7.5.RESULTSDecomposition of peroxynitrite was monitored as decrease in absorbance at 302 nm at 20°C. As expected, decomposition at pH 3 was very fast and followed first order kinetics with a calculated rate constant (kcalc) of 0.86 ± 0.05 s−1 but slowed down at increasing pH. The kcalc values and corresponding Hill coefficients summarized in Table I demonstrate that peroxynitrite decay was first order under most conditions although Hill coefficients smaller than 1.0 were obtained at pH 8.0 (0.67 ± 0.02) and pH 11.0 (0.5 ± 0.1). Using the Hill equation for overall kinetic analysis of decomposition at pH 3-11, we calculated a pKa of 6.8 ± 0.1, which agrees well with published data (35Pryor W.A. Squadrito G.L. Am. J. Physiol. 1995; 12Google Scholar). The possible contribution of transition metals to peroxynitrite decomposition was studied with 0.6 mM peroxynitrite in 0.5 M phosphate buffer (pH 7.4) in the presence of Cu(NO3)2, Fe(NH4)(SO4)2, Fe(NH4)2(SO4)2, and the metal chelator DTPA. Rates of decomposition were affected neither by the metal salts (0.1 mM each) nor by DTPA (0.1 and 1 mM). At a concentration of 2.5 mM DTPA, the peroxynitrite decay rate was enhanced 10-fold.Table IApparent first-order rate constants of peroxynitrite decomposition as a function of pHpHkcalcav (s−1)Hill coefficient30.86 ± 0.050.91 ± 0.0740.82 ± 0.011.09 ± 0.0750.71 ± 0.021.16 ± 0.0560.61 ± 0.011.11 ± 0.0370.39 ± 0.020.83 ± 0.0380.08 ± 0.0090.67 ± 0.0290.0298 ± 0.0091.10 ± 0.03100.0033 ± 0.00011.16 ± 0.05110.00008 ± 0.000010.5 ± 0.1 Open table in a new tab Stopped-flow data showed that peroxynitrite decomposition was faster in the presence of ∼1 mM NO and that the increase in rate was dependent on the NO concentration. However, calculation of rate constants was difficult because the exact NO concentrations in these experiments were not known and the effect of NO was observed only as a relatively small increase of an already fast reaction. Therefore, we used an NO-sensitive electrode to measure the consumption of NO by known amounts of peroxynitrite. Fig. 1 shows a representative trace obtained by addition of 4 μl of a saturated NO solution to 1.8 ml of 0.1 M phosphate buffer, followed by two repetitive additions of peroxynitrite yielding concentrations of 0.75 μM each. Peroxynitrite induced a rapid consumption of NO with initial rates of 100 ± 9 nM s−1 and a stoichiometry close to 1:1 (0.75 μM peroxynitrite consumed 0.66 ± 0.06 μM NO). NO consumption (initial NO concentration 1-2 μM) was linear in the range of 0.25-1 μM peroxynitrite with initial rates ranging from 20 to 167 nM s−1 and a rate constant of 9.1 × 104M−1 s−1.We consistently observed that decomposition of peroxynitrite or [Me4N][ONOO] resulted in formation of about 70% NO3− and 30% NO2− at pH 7.4 and 37°C. As the NO2−/NO3− ratios were not affected by known metal chelators (Table II), our results do not support previous suggestions according to which formation of NO2− is due to contamination of peroxynitrite solutions with trace metals (36Plumb R.C. Edwards J.O. Herman M.A. Analyst. 1992; 117: 1639-1641Crossref Scopus (16) Google Scholar) but indicate that NO2− release results from an as yet unrecognized pathway of peroxynitrite decomposition. To address this issue, we measured NO2− and NO3− after peroxynitrite decomposition at pH 3-9 and found that the relative amount of NO2− increased with increasing pH (Fig. 2A). Assuming that these results were not due to a reaction of peroxynitrite with contaminants in the stock solutions, our findings led us to speculate that 2 mol of peroxynitrite decomposed to 2 mol of NO2− and 1 mol of O2. Indeed, using a Clark-type O2 sensor, we found that the pH-dependent formation of NO2− was accompanied by release of stoichiometric amounts of O2 (Fig. 2A). The replot of the data (Fig. 2B) revealed a correlation coefficient of 0.988 and a slope of 0.495, suggesting that NO2− and O2 were released in a 2:1 stoichiometry.Table IIFormation of NO2− and NO3− upon decomposition of peroxynitrite in the presence of metal chelatorsChelatorNO2−0.1 mM%Control32.4 ± 6.3EDTA27.7 ± 5.9Neocuproine29.0 ± 5.4Cuprizone32.8 ± 2.2Bathophenanthroline32.8 ± 7.3DTPA32.6 ± 2.5 Open table in a new tab Fig. 2Decomposition of peroxynitrite yields NO2− and oxygen. A, peroxynitrite (0.5 mM final initial concentration) was decomposed by incubation in 0.1 M phosphate buffer (pH 3.0-9.0) at 37°C for 1 h, followed by the determination of NO2−, NO3−, and O2 as described under "Experimental Procedures". NO2− and NO3− were determined after measurement of O2 release in the same vials. Data are means ± S.E. of six experiments. B, correlation between O2 and NO2− production (slope = 0.495, correlation coefficient = 0.988).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To corroborate these data and exclude possible artifacts, the experiments were repeated with [Me4N][ONOO]. Fig. 3A shows that formation of NO2− and O2 increased when [Me4N][ONOO] (0.25 mM) was decomposed at increasing pH. The linear correlation of NO2− versus O2 shown in Fig. 3B yielded a slope of 0.657 and a correlation coefficient of 0.983. Although these data nicely confirmed the results obtained with the conventional preparation, two interesting differences were observed. First, while release of NO2− and O2 was negligible when the Baeyer-Villinger preparation of peroxynitrite was decomposed at pH ≤6.5 (cf Fig. 2A), decomposition of [Me4N][ONOO] resulted in formation of significant amounts of NO2− and O2, even at low pH. Second, release of NO2− and O2 from [Me4N][ONOO] did not level off at high pH values and appeared to account for virtually 100% of the decomposition occurring at pH 9.0. In all experiments, the measured sum of NO2− plus NO3− was close to theoretical values.Fig. 3Decomposition of [Me4N][ONOO] yields NO2− and oxygen. A, [Me4N][ONOO] (0.25 mM final initial concentration) was decomposed by incubation in 0.5 M phosphate buffer (pH 3.0-9.0) at 37°C for 1 h, followed by the determination of NO2−, NO3−, and O2 as described under "Experimental Procedures." NO2− and NO3− were determined after measurement of O2 release in the same vials. Data are means ± S.E. of six experiments. B, correlation between O2 and NO2− production (slope = 0.657, correlation coefficient = 0.983).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The assumption that two different pathways of decomposition account for the formation of NO2− and NO3− was supported by a pronounced temperature sensitivity of the NO2−/NO3− ratio. As shown in Table III, decomposition of 0.1 mM [Me4N][ONOO] at pH 7.4 yielded 16.2 and 52.1% NO2− at 5 and 56°C, respectively. A similar increase of the NO2−/NO3− ratio was observed with three concentrations (0.1, 0.5, and 1 mM) of the Baeyer-Villinger preparation (not shown). We also determined the temperature dependence for the overall peroxynitrite decomposition rate between 5 and 50°C by stopped-flow spectroscopy. The Arrhenius plots showed a strictly linear relationship between ln kobs and T−1 at pH 5.0 and 7.4 (Fig. 4A). From the slope of the plots, values for Eact of 92.0 ± 2 kJ mol−1 and 90.0 ± 0.8 kJ mol−1 were calculated for decomposition at pH 5.0 and 7.4, respectively. Assuming that the NO2−/NO3− ratios reflect the kinetic partitioning of the two pathways leading to NO2− and NO3− formation, the difference in Eact of the reations (ΔEact) was estimated as 26.2 ± 0.9 kJ mol−1 (Fig. 4B).Table IIIEffect of temperature on formation of NO2− and NO3− from [Me4N][ONOO]TemperatureNO2−NO3−% NO2−°CμMμM515.0 ± 1.177.7 ± 0.116.22422.1 ± 1.668.9 ± 1.624.33732.8 ± 0.759.4 ± 1.435.65649.2 ± 1.945.4 ± 1.252.1 Open table in a new tab Fig. 4Arrhenius plots of peroxynitrite decomposition yielding NO2− and NO3−. A, peroxynitrite decomposition rates between 5 and 50°C were calculated from first-order fits. Peroxynitrite (final initial concentration 0.6 mM in 0.01 M NaOH) was mixed with 0.5 M acetate buffer (pH 3.0) or 0.5 M phosphate buffer (pH 7.4). Data are mean values of three experiments. B, the NO2−/NO3−data shown in Table I were replotted as ln((k1)/(k2)) with ((k1)/(k2)) = ((%NO2−)/(%NO3−)) versus (1/T). From the slope of the linear plot, the difference between the activation energies (ΔEact) of the two reactions yielding NO2− and NO3− was calculated. Data are the mean values of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The NO2−/NO3− ratio was independent of the initial peroxynitrite concentration. Decomposition of 0.01, 0.1, 0.5, and 1 mM peroxynitrite at pH 7.4 and 37°C resulted in formation of 29.6 ± 1.3, 25.5 ± 5.7, 27.3 ± 1.2, and 28.9 ± 1.5%, respectively, of NO2− (mean ± S.E.; n = 3 each).DISCUSSIONThe present study was carried out to identify the pathways of formation of NO2− in the course of peroxynitrite decomposition. Stopped-flow kinetic experiments confirmed that peroxynitrite decomposed rapidly upon protonation with a pKa of 6.8. The first order rate constants calculated for peroxynitrite decomposition at different pH values agreed well with previously published data (11Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar, 15Koppenol W.H. Moreno J.J. Pryor W.A. Ischiropoulos H. Beckman J.S. Chem. Res. Toxicol. 1992; 5: 834-842Crossref PubMed Scopus (1274) Google Scholar, 37Logager T. Sehested K. J. Phys. Chem. 1993; 97: 6664-6669Crossref Scopus (202) Google Scholar). Under physiological conditions (pH 7.4 and 37°C), decomposition consistently yielded about 30% NO2−, whereas NO3− was the sole product at pH <5.0. Some studies indicated that under certain experimental conditions, peroxynitrite does indeed decompose to NO2−, but this was attributed to minor side reactions catalyzed by contaminating trace metals (36Plumb R.C. Edwards J.O. Herman M.A. Analyst. 1992; 117: 1639-1641Crossref Scopus (16) Google Scholar, 38Hughes M.N. Nicklin H.G. J. Chem. Soc. 1970; : 925-1970Crossref Google Scholar). In our hands, NO2− release was not metal-catalyzed because it was affected neither by chelators nor by metal ions (0.1 mM).Previous studies showed that small amounts of NO are released upon peroxynitrite decomposition under certain conditions (39Zhu L. Gunn C. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 452-457Crossref PubMed Scopus (414) Google Scholar) and that peroxynitrite reacts with NO according to Equation 1 (40Crow J.P. Beckman J.S. Ignarro L. Murad F. Advances in Pharmacology. Academic Press, Inc., San Diego1995: 17-43Google Scholar, 41Miles A.M. Bohle D.S. Glassbrenner P.A. Hansert B. Wink D.A. Grisham M.B. J. Biol. Chem. 1996; 271: 40-47Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar).Lewis et al. (28Lewis R.S. Tamir S. Tannenbaum S.R. Deen W.M. J. Biol. Chem. 1995; 270: 29350-29355Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) observed that activated macrophages release more NO2− than expected and considered Equation 1 as a possible source for excess NO2−. To judge whether Equation 1 could account for NO2− formation under physiological conditions, we studied the reaction of peroxynitrite with NO using both stopped-flow kinetic spectroscopy and an NO-sensitive electrode. These experiments yielded an apparent second order rate constant of 9.1 × 104M−1 s−1, which is in the same order of magnitude as the rate constant determined for peroxynitrite reacting with CO2 (42Lymar S.V. Hurst J.K. J. Am. Chem. Soc. 1995; 117: 8867-8868Crossref Scopus (493) Google Scholar). However, the concentration of NO that would be required to render the reaction as fast as peroxynitrite decomposition (0.69 s−1 at pH 7.4 and 37°C) is so high (7.6 μM) that the reaction with NO is probably insignificant under most physiological conditions (43Laurent M. Lepoivre M. Tenu J.P. Biochem. J. 1996; 314: 109-113Crossref PubMed Scopus (85) Google Scholar).Since pure peroxynitrite decomposed to NO2− without detectable release of free NO, we considered additional pathways that could account for NO2− formation. Assuming that there were no other redox-active reaction partners of peroxynitrite present, we speculated that two peroxynitrite molecules might combine to release 2 molecules of NO2− and 1 molecule of O2 according to Equation 2.This hypothesis was corroborated by determination of NO2− and O2 formed upon decomposition of two different peroxynitrite preparations. With both products, we obtained linear correlations between NO2− and O2 release with slopes close to the theoretical value of 0.50. At pH 3.0-11.0, peroxynitrite decomposition was first order at most. Moreover, even though the temperature dependence of the NO2−/NO3− ratio clearly indicated that the two pathways have different activation energies (ΔEact = 26.2 ± 0.9 kJ mol−1), the Arrhenius plot for overall peroxynitrite decomposition at pH 7.4 (∼30% NO2−) was strictly linear and yielded an Eact value that was virtually identical to that observed at pH 5.0 (∼10% NO2−). The Eact of overall peroxynitrite decomposition was found to be rather high (92 ± 2 and 90.0 ± 0.8 kJ mol−1 at pH 5.0 and 7.4, respectively). A similar value (77.5 kJ mol−1 at pH 5.0) was reported by Koppenol et al. (15Koppenol W.H. Moreno J.J. Pryor W.A. Ischiropoulos H. Beckman J.S. Chem. Res. Toxicol. 1992; 5: 834-842Crossref PubMed Scopus (1274) Google Scholar).From these observations, we conclude that the rate-limiting step in both reactions is the same, a conformational change of ONOOH to an activated intermediate that either rearranges to HNO3 (15Koppenol W.H. Moreno J.J. Pryor W.A. Ischiropoulos H. Beckman J.S. Chem. Res. Toxicol. 1992; 5: 834-842Crossref PubMed Scopus (1274) Google Scholar, 35Pryor W.A. Squadrito G.L. Am. J. Physiol. 1995; 12Google Scholar) or undergoes a reaction with peroxynitrite anion to yield NO2− and O2 (this study). Any potential model must account for the thoroughly characterized kinetics of peroxynitrite decomposition as well as the stoichiometries of the end products. Further, a bimolecular rate law for either of the product determining steps is excluded because the partitioning of the two pathways does not depend on the concentration of peroxynitrite. Fig. 5 shows a hypothetical pathway of peroxynitrite decomposition that appears to be most consistent with the data presented both here and in the literature. According to this scheme, activated ONOOH can either isomerize to NO3− or decompose to HO and NO2 radicals. At alkaline pH, the OH radical may react with peroxynitrite anion yielding O2, NO, and OH−, and NO could react with NO2 radicals to yield N2O3 and finally nitrite.Fig. 5Hypothetical mechanism of peroxynitrite decomposition to nitrite and nitrate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The novel pathway of peroxynitrite decomposition described here could have important physiological consequences, as it possibly involves generation of intermediates with biological activities not attributed so far to peroxynitrite. In a recent paper, it was reported that peroxynitrite decomposition could lead to release of singlet O2 (44Khan A.U. J. Biolumin. Chemilumin. 1995; 10: 329-333Crossref PubMed Scopus (24) Google Scholar). If that observation were due to the novel reaction proposed here, peroxynitrite-dependent toxicity might be mediated by singlet O2 toxicity under certain pathophysiological conditions. Alternatively, decomposition to H NO2− and O2 may be responsible for the observed NO-like biological activity of peroxynitrite. At pH 7.4, peroxynitrite oxidizes hemoglobin to methemoglobin with an efficiency of about 20% (26Schmidt K. Klatt P. Mayer B. Biochem. J. 1994; 301: 645-647Crossref PubMed Scopus (65) Google Scholar), and it is tempting to speculate that this reaction represents scavenging by hemoglobin of the NO that is formed as intermediate during decomposition to NO2− and O2 (Fig. 5). Also, our working hypothesis involves intermediary formation of N2O3, a potent nitrosating agent that could account for the observed peroxynitrite-induced nitrosation of GSH, especially in light of our findings that the nitrosation reaction has a pronounced pH dependence and does not occur at significant rates below pH 7.5 (45Mayer B. Schrammel A. Klatt P. Koesling D. Schmidt K. J. Biol. Chem. 1995; 270: 17355-17360Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Accordingly, reactive intermediates formed in the course decomposition to NO2− and O2 could be responsible for stimulation of soluble guanylyl cyclase by peroxynitrite (45Mayer B. Schrammel A. Klatt P. Koesling D. Schmidt K. J. Biol. Chem. 1995; 270: 17355-17360Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), resulting in cyclic GMP-mediated biological effects such as vascular smooth muscle relaxation and inhibition of platelet aggregation (46Liu S. Beckman J.S. Ku D.D. J. Pharmacol. Exp. Ther. 1994; 268: 1114-1121PubMed Google Scholar, 47Moro M.A. Darley-Usmar V.M. Goodwin D.A. Read N.G. Zamorapino R. Feelisch M. Radomski M.W. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6702-6706Crossref PubMed Scopus (336) Google Scholar). INTRODUCTIONThe reaction between nitric oxide (NO) and superoxide anion (O2) yields peroxynitrite with a second order rate constant near the diffusion-controlled limit (k = 4.3-6.7 × 109M−1 s−1) (1Huie R.E. Padmaja S. Free Radical Res. Commun. 1993; 18: 195-199Crossref PubMed Scopus (2006) Google Scholar, 2Goldstein S. Czapski G. Free Radical Biol. & Med. 1995; 117: 12078-12084Google Scholar). The reaction constitutes an important sink for O2 because it is about twice as fast as the maximum velocity of SOD. 1The abbreviations used are:SODsuperoxide dismutaseSIN-13-(4-morpholinyl)-sydnoniminehydrochlorideHPLChigh performance liquid chromatographyDTPAdiethylenetriaminepentaacetic acid. Consequently, peroxynitrite has been implicated in many pathological conditions including stroke (3Dawson V.L. Dawson T.M. London E.D. Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6368-6371Crossref PubMed Scopus (2098) Google Scholar), heart disease (4Matheis G. Sherman M.P. Buckberg G.D. Haybron D.M. Young H.H. Ignarro L.J. Am. J. Physiol. 1992; 262PubMed Google Scholar), and atherosclerosis (5Hogg N. Darley-Usmar V.M. Graham A. Moncada S. Biochem. Soc. Trans. 1993; 21: 358-362Crossref PubMed Scopus (89) Google Scholar, 6White C.R. Brock T.A. Chang L.Y. Crapo J. Briscoe P. Ku D. Bradley W.A. Gianturco S.H. Gore J. Freeman B.A. Tarpey M.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1044-1048Crossref PubMed Scopus (655) Google Scholar). The potential cellular targets for peroxynitrite cytotoxicity include the antioxidants ascorbate, α-tocopherol, and uric acid (7Bartlett D. Church D.F. Bounds P.L. Koppenol W.H. Free Radical Biol. & Med. 1995; 18: 85-92Crossref PubMed Scopus (163) Google Scholar, 8Squadrito G.L. Jin X. Pryor W.A. Arch. Biochem. Biophys. 1995; 322: 53-59Crossref PubMed Scopus (121) Google Scholar, 9Hogg N. Joseph J. Kalyanaraman B. Arch. Biochem. Biophys. 1994; 314: 153-158Crossref PubMed Scopus (101) Google Scholar, 10Vasquez-Vivar J. Santos A.M. Junqueira B.C. Augusto O. Biochem. J. 1996; 314: 869-876Crossref PubMed Scopus (130) Google Scholar), protein and non-protein sulfhydryls (11Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar), DNA (12King P.A. Anderson V.E. Edwards J.O. Gustafson G. Plumb R.C. Suggs J.W. J. Am. Chem. Soc. 1992; 114: 5430-5432Crossref Scopus (244) Google Scholar), and membrane phospholipids (13Radi R. Beckman J.S. Bush K.M. Freeman B.A. Arch. Biochem. Biophys. 1991; 288: 481-487Crossref PubMed Scopus (2030) Google Scholar).Decomposition of peroxynitrite is complex (14Edwards J.O. Plumb R.C. Karlin K.D. Progress in Inorganic Chemistry. John Wiley & Sons, Inc., New York1994: 599-635Google Scholar, 15Koppenol W.H. Moreno J.J. Pryor W.A. Ischiropoulos H. Beckman J.S. Chem. Res. Toxicol. 1992; 5: 834-842Crossref PubMed Scopus (1274) Google Scholar). The anion is rather stable in alkaline solutions but decomposes rapidly (t1/2 = 1 s at pH 7.4, 37°C) upon protonation to peroxynitrous acid (ONOOH) (pKa = 6.8) (16Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6666) Google Scholar). Two pathways of ONOOH decomposition have been proposed. Some studies have argued that ONOOH is cleaved homolytically to generate hydroxyl and NO2 radicals. This hypothesis is based on the sensitivity to hydroxyl radical scavengers of certain peroxynitrite-induced reactions, including the formation of malondialdehyde from deoxyribose and the hydroxylation on the benzene ring of sodium benzoate, phenylalanine, and tyrosine (16Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6666) Google Scholar, 17van der Vliet A. O'Neill C.A. Halliwell B. Cross C.E. Kaur H. FEBS Lett. 1994; 339: 89-92Crossref PubMed Scopus (357) Google Scholar). Studies on decomposition of peroxynitrite by electron paramagnetic resonance spectroscopy with the spin traps 5,5-dimethyl-1-pyrroline N-oxide and 4-pyridyl-1-oxide-N-tert-butylnitrone also provided evidence for the formation of free hydroxyl radicals (18Augusto O. Gatti R.M. Radi R. Arch. Biochem. Biophys. 1994; 310: 118-125Crossref PubMed Scopus (167) Google Scholar, 19Pou S. Nguyen S.Y. Gladwell T. Rosen G.M. Biochim. 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A. 1994; 91: 11173-11177Crossref PubMed Scopus (360) Google Scholar).There are several methods for the detection of peroxynitrite in biological systems. Since ONOOH decomposition yields an intermediate that nitrates phenolic compounds (22Beckman J.S. Ischiropoulos H. Zhu L. van der Woerd M. Smith C. Chen J. Harrison J. Martin J.C. Tsai M. Arch. Biochem. Biophys. 1992; 298: 438-445Crossref PubMed Scopus (731) Google Scholar, 23Ischiropoulos H. Zhu L. Chen J. Tsai M. Martin J.C. Smith C.D. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 431-437Crossref PubMed Scopus (1420) Google Scholar), presence of nitrotyrosine in proteins was proposed to be evidence of peroxynitrite production in tissues (24Beckman J.S. Ye Y.Z. Anderson P.G. Chen J. Accavitti M.A. Tarpey M.M. White C.R. Biol. Chem. Hoppe-Seyler. 1994; 375: 81-88Crossref PubMed Scopus (1068) Google Scholar). However, using both a monoclonal antibody specifically recognizing peroxynitrite-modified proteins (24Beckman J.S. Ye Y.Z. Anderson P.G. Chen J. Accavitti M.A. Tarpey M.M. White C.R. Biol. Chem. Hoppe-Seyler. 1994; 375: 81-88Crossref PubMed Scopus (1068) Google Scholar) as well as a published HPLC method (17van der Vliet A. O'Neill C.A. Halliwell B. Cross C.E. Kaur H. FEBS Lett. 1994; 339: 89-92Crossref PubMed Scopus (357) Google Scholar), we failed to detect tyrosine nitration by authentic peroxynitrite at concentrations <0.1 mM. 2S. Pfeiffer, and B. Mayer, unpublished observations. Spectrophotometric determination of dihydrorhodamine 123 oxidation was described as another sensitive assay for the specific detection of peroxynitrite at submicromolar concentrations (25Kooy N.W. Royall J.A. Ischiropoulos H. Beckman J.S. Free Radical Biol. & Med. 1994; 16: 149-156Crossref PubMed Scopus (666) Google Scholar), but in our hands, interference of several redox-active compounds precluded application of this method in cell-free assay systems. 3P. Klatt, and B. Mayer, unpublished observations. Under certain experimental conditions, indirect evidence for peroxynitrite production can be obtained by comparing NO release in the absence and presence of SOD. The peroxynitrite donor compound SIN-1, for example, does not release detectable amounts of free NO unless SOD is present in amounts sufficient to outcompete the reaction with concomitantly produced O2 (26Schmidt K. Klatt P. Mayer B. Biochem. J. 1994; 301: 645-647Crossref PubMed Scopus (65) Google Scholar). Based on similar results obtained with purified neuronal NO synthase, we suggested that the enzyme generates NO and O2 simultaneously and hence functions as peroxynitrite synthase if incubated in vitro (27Mayer B. Klatt P. Werner E.R. Schmidt K. J. Biol. Chem. 1995; 270: 655-659Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). However, in contrast with the widely held view that peroxynitrite decomposes exclusively to NO3−, considerable amounts of NO2− were also found as a major stable product of SIN-1 or NO synthase under physiological conditions.2 Similarly, excess NO2− formation was observed in peroxynitrite producing cells (28Lewis R.S. Tamir S. Tannenbaum S.R. Deen W.M. J. Biol. Chem. 1995; 270: 29350-29355Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), suggesting that additional as yet unidentified reactions contribute to peroxynitrite decomposition.The present study was done to elucidate the fate of peroxynitrite in aqueous solution. Studies with the authentic compound, prepared in two different ways, identified a reaction leading to release of NO2− and O2 in a 2:1 stoichiometry as a route of peroxynitrite decomposition at pH ≥ 7.5.
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