Nitrite as a Substrate and Inhibitor of Myeloperoxidase
2000; Elsevier BV; Volume: 275; Issue: 16 Linguagem: Inglês
10.1074/jbc.275.16.11638
ISSN1083-351X
AutoresChristine J. van Dalen, Christine C. Winterbourn, Revathy Senthilmohan, Anthony J. Kettle,
Tópico(s)Vanadium and Halogenation Chemistry
ResumoMyeloperoxidase is a heme enzyme of neutrophils that uses hydrogen peroxide to oxidize chloride to hypochlorous acid. Recently, it has been shown to catalyze nitration of tyrosine. In this study we have investigated the mechanism by which it oxidizes nitrite and promotes nitration of tyrosyl residues. Nitrite was found to be a poor substrate for myeloperoxidase but an excellent inhibitor of its chlorination activity. Nitrite slowed chlorination by univalently reducing the enzyme to an inactive form and as a consequence was oxidized to nitrogen dioxide. In the presence of physiological concentrations of nitrite and chloride, myeloperoxidase catalyzed little nitration of tyrosyl residues in a heptapeptide. However, the efficiency of nitration was enhanced at least 4-fold by free tyrosine. Our data are consistent with a mechanism in which myeloperoxidase oxidizes free tyrosine to tyrosyl radicals that exchange with tyrosyl residues in peptides. These peptide radicals then couple with nitrogen dioxide to form 3-nitrotyrosyl residues. With neutrophils, myeloperoxidase-dependent nitration required a high concentration of nitrite (1 mm), was doubled by tyrosine, and increased 4-fold by superoxide dismutase. Superoxide is likely to inhibit nitration by reacting with nitrogen dioxide and/or tyrosyl radicals. We propose that at sites of inflammation myeloperoxidase will nitrate proteins, even though nitrite is a poor substrate, because the co-substrate tyrosine will be available to facilitate the reaction. Also, production of 3-nitrotyrosine will be most favorable when the concentration of superoxide is low. Myeloperoxidase is a heme enzyme of neutrophils that uses hydrogen peroxide to oxidize chloride to hypochlorous acid. Recently, it has been shown to catalyze nitration of tyrosine. In this study we have investigated the mechanism by which it oxidizes nitrite and promotes nitration of tyrosyl residues. Nitrite was found to be a poor substrate for myeloperoxidase but an excellent inhibitor of its chlorination activity. Nitrite slowed chlorination by univalently reducing the enzyme to an inactive form and as a consequence was oxidized to nitrogen dioxide. In the presence of physiological concentrations of nitrite and chloride, myeloperoxidase catalyzed little nitration of tyrosyl residues in a heptapeptide. However, the efficiency of nitration was enhanced at least 4-fold by free tyrosine. Our data are consistent with a mechanism in which myeloperoxidase oxidizes free tyrosine to tyrosyl radicals that exchange with tyrosyl residues in peptides. These peptide radicals then couple with nitrogen dioxide to form 3-nitrotyrosyl residues. With neutrophils, myeloperoxidase-dependent nitration required a high concentration of nitrite (1 mm), was doubled by tyrosine, and increased 4-fold by superoxide dismutase. Superoxide is likely to inhibit nitration by reacting with nitrogen dioxide and/or tyrosyl radicals. We propose that at sites of inflammation myeloperoxidase will nitrate proteins, even though nitrite is a poor substrate, because the co-substrate tyrosine will be available to facilitate the reaction. Also, production of 3-nitrotyrosine will be most favorable when the concentration of superoxide is low. diethylenetriaminepentaacetic acid 4β-phorbol 12-myristate 13-acetate phosphate-buffered saline high-performance liquid chromatography The presence of 3-nitrotyrosine at sites of inflammation has been used to implicate peroxynitrite in inflammatory tissue damage (1.Haddad I.Y. Ischiropoulos H. Holm B.A. Beckman J.S. Baker J.R. Matalon S. Am. J. Physiol. 1993; 265: L555-L564PubMed Google Scholar, 2.Haddad I.Y. Pataki G. Hu P. Galliani C. Beckman J.S. Matalon S. J. Clin. Invest. 1994; 94: 2407-2413Crossref PubMed Scopus (577) Google Scholar). Peroxynitrite is formed from the rapid reaction of nitric oxide with superoxide (k ∼ 5 × 109m−1 s−1) (3.Huie R.E. Padmaja S. Free Radic. Res. Commun. 1993; 18: 195-199Crossref PubMed Scopus (2043) Google Scholar, 4.Goldstein S. Czapski G. Free Radic. Biol. Med. 1995; 19: 505-510Crossref PubMed Scopus (383) Google Scholar, 5.Kobayashi K. Miki M. Tagawa S. J. Chem. Soc. Dalton Trans. 1995; 1995: 2885-2889Crossref Google Scholar). It nitrates free tyrosine and tyrosyl residues in proteins (6.Beckman 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 (1084) Google Scholar). However, it was recently found that peroxynitrite is extremely inefficient at nitrating tyrosine when it is formed at physiological fluxes of superoxide and nitric oxide (7.Pfeiffer S. Mayer B. J. Biol. Chem. 1998; 273: 27280-27285Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). This result suggests that other mechanisms are likely to contribute to production of 3-nitrotyrosine in vivo. One route may involve the neutrophil protein myeloperoxidase. This heme enzyme has been shown to catalyze nitration of tyrosine and tyrosyl residues in peptides and proteins (8.Eiserich J.P. Hristova M. Cross C.E. Jones A.D. Freeman B.A. Halliwell B. van der Vliet A. Nature. 1998; 391: 393-397Crossref PubMed Scopus (1379) Google Scholar). To assess the potential of myeloperoxidase to contribute to formation of 3-nitrotyrosine at sites of inflammation, it is essential to appreciate how this enzyme catalyzes nitration of proteins. Myeloperoxidase is the most abundant protein in neutrophils and is also present in monocytes (9.Kettle A.J. Winterbourn C.C. Redox Rep. 1997; 3: 3-15Crossref PubMed Scopus (602) Google Scholar, 10.Klebanoff S.J. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. Lippincott Williams & Wilkins, Philadelphia1999: 721-768Google Scholar). Ferric myeloperoxidase reacts with hydrogen peroxide, which is produced by stimulated neutrophils, to form the redox intermediate compound I (Reaction 1). Compound I is strongly oxidizing and reacts with a variety of substrates. Its main physiological substrate is assumed to be chloride, which undergoes a two-electron oxidation to form hypochlorous acid (Reaction 2). This is the most powerful oxidant produced by neutrophils in appreciable amounts. Under physiological conditions, thiocyanate is an equally preferred substrate and is oxidized to hypothiocyanite (11.Van Dalen C.J. Whitehouse M. Winterbourn C.C. Kettle A.J. Biochem. J. 1997; 327: 487-492Crossref PubMed Scopus (352) Google Scholar). In addition to this halogenation activity, myeloperoxidase acts as a classical peroxidase. Compound I is reduced by organic substrates (RH) in a one-electron reaction to form compound II and a free radical product (Reaction 3). Compound II then reacts with a second molecule of RH to regenerate the native enzyme (Reaction 4). MP3++H2O2→compound I REACTION 1 Compound I+Cl−→MP3++HOCl REACTION 2 Compound I+RH→compound II+R· REACTION 3 Compound II+RH→MP3++R· REACTION 4 NO2−+HOCl→NO2Cl+HO− REACTION 5Eiserich and co-workers (12.Eiserich J.P. Cross C.E. Jones A.D. Halliwell B. van der Vliet A. J. Biol. Chem. 1996; 271: 19199-19208Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar) have proposed that myeloperoxidase catalyzes nitration by two distinct mechanisms. They showed that hypochlorous acid reacts with nitrite to form nitryl chloride (Reaction 5) (12.Eiserich J.P. Cross C.E. Jones A.D. Halliwell B. van der Vliet A. J. Biol. Chem. 1996; 271: 19199-19208Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). This species both nitrates and chlorinates tyrosine to give 3-nitrotyrosine and 3-chlorotyrosine. Given that Reaction 5 is relatively slow compared with the reaction of hypochlorous acid with thiols and amines (13.Panasenko O.M. Brivida K. Klotz L.-O. Sies H. Arch. Biochem. Biophys. 1997; 343: 254-259Crossref PubMed Scopus (90) Google Scholar, 14.Folkes L.K. Candeias L.P. Wardman P. Arch. Biochem. Biophys. 1995; 323: 120-126Crossref PubMed Scopus (302) Google Scholar), it is unlikely that nitryl chloride is a significant nitrating agent in vivo. They also demonstrated that nitrite is oxidized directly by myeloperoxidase to a species that is capable of nitrating tyrosine (15.van der Vliet A. Eiserich J.P. Halliwell B. Cross C.E. J. Biol. Chem. 1997; 272: 7617-7625Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar). A two-electron oxidation would result in the production of the nitronium cation, whereas removal of one electron would give nitrogen dioxide. The rate of oxidation of substrates by peroxidases is strongly dependent on their one-electron potentials. Although substrates with reduction potentials of 1.05–1.2 V are readily oxidized by compound I of myeloperoxidase, they are poor peroxidase substrates because they are unable to reduce compound II (16.Kettle A.J. Winterbourn C.C. Biochem. Pharmacol. 1991; 41: 1485-1492Crossref PubMed Scopus (166) Google Scholar). Nitrite has a one-electron reduction potential of 0.99 V (17.Koppenol W.H. Free Radic. Biol. Med. 1998; 25: 385-391Crossref PubMed Scopus (308) Google Scholar). Thus, on thermodynamic grounds nitrite should be a poor substrate for myeloperoxidase and inhibit its chlorination activity. To reconcile this argument with the experimental findings that myeloperoxidase catalyzes nitration of tyrosine and proteins, we have investigated the mechanism of oxidation of nitrite by this enzyme. In previous studies, tyrosine or small peptides containing tyrosyl residues have been used to investigate myeloperoxidase-catalyzed nitration. These compounds are substrates for myeloperoxidase and will be oxidized by the enzyme to produce tyrosyl radicals (18.Heinecke J.W. Li W. Francis G.A. Goldstein J.A. J. Clin. Invest. 1993; 91: 2866-2872Crossref PubMed Scopus (304) Google Scholar, 19.Tien M. Arch. Biochem. Biophys. 1999; 367: 61-66Crossref PubMed Scopus (51) Google Scholar), which may influence nitration. To more realistically mimic protein nitration, we have used peptides containing tyrosyl residues that are not oxidized directly by myeloperoxidase. We show that nitrite is indeed a poor substrate for myeloperoxidase but free tyrosine facilitates nitration of tyrosyl residues by acting as a co-substrate in the reaction. Myeloperoxidase was purified from human leukocytes as described previously (20.Kettle A.J. Winterbourn C.C. Biochem. J. 1988; 252: 529-536Crossref PubMed Scopus (157) Google Scholar). Its purity index (A 430/A 280) was at least 0.80, and its concentration was determined using ε43091,000 m−1·cm−1/heme (21.Odajima T. Yamazaki I. Biochim. Biophys. Acta. 1970; 206: 71-77Crossref PubMed Scopus (131) Google Scholar). Solutions of sodium nitrite (BDH Chemicals) were prepared daily and kept on ice. 5,5′-Dithiobis-2-nitrobenzoic acid, acetyl-Ser-Gln-Asn-Tyr-Pro-Val-Val-amide (peptide I), Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser (peptide II), acetyl-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln (peptide III), diethylenetriaminepentaacetic acid (DTPA),1l-tyrosine, superoxide dismutase, and 4β-phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma. 3-Nitro-l-tyrosine was from Fluka Chemica (Buchs, Switzerland). dl-Methionine was from BDH Chemicals. COMPLETE™ protease inhibitor mixture tablets, which are inhibitory to a large spectrum of serine proteases, were from Roche Molecular Biochemicals. 5-Thio-2-nitrobenzoic acid was prepared from 5,5′-dithiobis-2-nitrobenzoic acid as described previously (22.Kettle A.J. Winterbourn C.C. Methods Enzymol. 1994; 233: 502-512Crossref PubMed Scopus (225) Google Scholar). Hydrogen peroxide solutions were prepared daily by diluting a 30% stock, and the concentration was calculated by measuring its absorbance at 240 nm (ε240 43.6m−1·cm−1) (23.Beers R.J. Sizer I.W. J. Biol. Chem. 1952; 195: 133-140Abstract Full Text PDF PubMed Google Scholar). Inactive superoxide dismutase was prepared by treating 4.5 mg/ml enzyme with 88 mm hydrogen peroxide in 10 mm carbonate buffer, pH 10.9, for 1 h. It was then dialyzed against 10 mmphosphate buffer, pH 7.4, containing 140 mm sodium chloride and 10 μg/ml catalase (24.Hodgson E.K. Fridovich I. Biochemistry. 1975; 14: 5294-5299Crossref Scopus (693) Google Scholar). The activity of purified myeloperoxidase was measured by continuously monitoring hydrogen peroxide concentration with a YSI 2510 oxidase probe fitted to a YSI model 25 oxidase meter (Yellow Springs Instrument Co., Yellow Springs, OH). The electrode was covered with a single layer of dialysis tubing, except when using tyrosine. In these experiments an exclusion membrane (22.Kettle A.J. Winterbourn C.C. Methods Enzymol. 1994; 233: 502-512Crossref PubMed Scopus (225) Google Scholar) was placed directly on the electrode and then covered with dialysis tubing. The electrode was calibrated against known concentrations of hydrogen peroxide. All reactions were started with the addition of myeloperoxidase. For experiments containing nitrite, the electrode was equilibrated at each concentration of nitrite. The visible absorption spectrum of myeloperoxidase was recorded during the oxidation of chloride with and without nitrite using a Beckman 7500 diode array spectrophotometer. Each spectrum was recorded over 2 s and is an average of 20 spectra. Hypochlorous acid production by purified myeloperoxidase was determined by measuring accumulation of taurine chloramine (22.Kettle A.J. Winterbourn C.C. Methods Enzymol. 1994; 233: 502-512Crossref PubMed Scopus (225) Google Scholar). Myeloperoxidase (10 nm) was incubated with 10 mm taurine in 10 mm sodium phosphate buffer, pH 7.4, plus 140 mm sodium chloride (PBS) at 21 °C for 5 min. The reaction was started by addition of 30 μmhydrogen peroxide and stopped with 20 μg/ml catalase. The amount of taurine chloramine formed was assayed with 5-thio-2-nitrobenzoic acid. Neutrophils were isolated from normal individuals according to established procedures (25.Boyum A. Scand. J. Clin. Lab. Invest. 1968; 21: 77-89Crossref PubMed Scopus (991) Google Scholar). Neutrophils (1 × 106) were stimulated with PMA (100 ng/ml) and incubated at 37 °C in 1 ml of PBS containing 5 mm glucose, 1 mmCaCl2, and 0.5 mm MgCl2, 20 mm taurine, and varying concentrations of nitrite as indicated. Reactions were stopped after 30 min with catalase, and neutrophils were pelleted at 10,000 × g for 2 min. The supernatant was assayed for taurine chloramine as described previously (22.Kettle A.J. Winterbourn C.C. Methods Enzymol. 1994; 233: 502-512Crossref PubMed Scopus (225) Google Scholar). The NADPH-oxidase activity of neutrophils was determined using the hydrogen peroxide electrode (22.Kettle A.J. Winterbourn C.C. Methods Enzymol. 1994; 233: 502-512Crossref PubMed Scopus (225) Google Scholar). Neutrophils were stimulated with PMA under the conditions described above for production of hypochlorous acid and the accumulation of hydrogen peroxide was monitored for 20 min in the absence or presence of 200 μmnitrite. Nitration of the peptides by purified myeloperoxidase was carried out at 21 °C in 50 mm sodium phosphate buffer, pH 7.4, containing 100 μm nitrite, 20 μm DTPA, and 100 μm peptide. Reactions were started by the addition of 100 μm hydrogen peroxide, stopped after 1 h with catalase (20 μg/ml), then analyzed by HPLC (see below). Neutrophils (2 × 106/ml) were incubated at 37 °C in PBS with 100 μm peptide I, 5 mm glucose, 1 mm CaCl2, 0.5 mm MgCl2, 1 mm nitrite, and additions of 100 μm tyrosine, 10 μg/ml superoxide dismutase, 1 mm methionine, 100 μm azide, or 20 μg/ml catalase. The cells were stimulated with PMA (100 ng/ml). After 1 h, reactions were stopped by adding 20 μg/ml catalase and placing cells on ice. Neutrophils were pelleted by centrifugation at 10,000 × g for 5 min. COMPLETE™ protease inhibitors (0.8 mg/ml) were then added, and the supernatant fraction was stored at −20 °C until analyzed by HPLC. Nitrite concentrations were monitored using the Griess reagent (26.Tsikas D. Gutzki F.M. Rossa S. Bauer H. Neumann C. Dockendorff K. Sandmann J. Frolich J.C. Anal. Biochem. 1997; 244: 208-220Crossref PubMed Scopus (137) Google Scholar). Products of nitration reactions were analyzed by HPLC using a 5-μmSpherisorb ODS-2 RP-18 column with a gradient of 50 mmpotassium dihydrogen phosphate, pH 3.0/methanol, 80/20 (v/v) for 5 min, changing to a ratio of 50/50 (v/v) over 10 min, remaining at this ratio for a further 10 min, and subsequently returning to the original ratio. Tyrosine, 3-nitrotyrosine, peptide, and nitrated peptide were monitored by UV detection (Philips PU 4120 diode array detector) at 274 nm, and identification of products was made by the comparative time of elution with standards and by spectral matching. As no standard for nitrated peptides was available, quantification of products was made by comparison of the area under the curve for each peak at 274 nm relative to a standard curve for 3-nitrotyrosine. After nitration, peptide I was injected into the HPLC, and fractions were collected every minute from just before the parent peak eluted until just after the major modified peptide eluted. Fractions were dried down, dissolved in 0.1% trifluoroacetic acid, then desalted by solid-phase extraction on a reverse phase C18 column (Alltech extract-clean C18 column, 500 mg/2.8 ml) equilibrated with 0.1% trifluoroacetic acid. Bound peptides were washed off the column with 80% methanol. They were then dried down and hydrolyzed under nitrogen for at least 18 h at 110 °C using 6n HBr supplemented with 1% phenol. The content of 3-nitrotyrosine, 3-chlorotyrosine, and tyrosine in the parent and nitrated peptides was assayed using stable isotopic dilution gas chromatography mass spectroscopy essentially as described by Heinecke and co-workers (27.Heinecke J.W. Hsu F.F. Crowley J.R. Hazen S.L. Leeuwenburgh C. Mueller D.M. Rasmussen J.E. Turk J. Methods Enzymol. 1999; 300: 124-144Crossref PubMed Scopus (83) Google Scholar), except that amino acids were derivatized with trifluoroacetic anhydride. For amino acid analysis, hydrolyzed samples were derivatized with o-phthalaldehyde (28.Jarrett H.W. Cooksy K.D. Ellis B. Anderson J.M. Anal. Biochem. 1986; 153: 189-198Crossref PubMed Scopus (187) Google Scholar). To determine how effective nitrite is as a substrate for myeloperoxidase, we measured enzyme activity by monitoring the loss of hydrogen peroxide (Fig. 1). Steady state loss of hydrogen peroxide was minimal at concentrations of nitrite measured in biological fluids (0.5–210 μm) (29.Green L.C. Wagner D.A. Glogowski J. Skipper P.L. Wishnok J.S. Tannenbaum S.R. Anal. Biochem. 1982; 126: 131-138Crossref PubMed Scopus (11073) Google Scholar, 30.Gaston B. Reilly J. Drazen J.M. Fackler J. Ramdev P. Arnelle D. Mullins M.E. Sugarbaker D.J. Chee C. Singel D.J. Loscalzo J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10957-10961Crossref PubMed Scopus (585) Google Scholar, 31.Leone A.M. Francis P.L. Rhodes P. Moncada S. Biochem. Biophys. Res. Commun. 1994; 200: 951-957Crossref PubMed Scopus (197) Google Scholar), but increased with increasing concentration of nitrite. The activity of myeloperoxidase could not be determined above 10 mm nitrite because the hydrogen peroxide electrode could no longer be equilibrated. Therefore, it was not possible to determine the specificity constant for nitrite. An alternative approach to gauging how readily nitrite will be oxidized by myeloperoxidase in vivo is to compare the turnover numbers of myeloperoxidase at physiological concentrations of competing substrates. Chloride and thiocyanate concentrations in plasma range between 100–140 mm and 20–120 μm, respectively (10.Klebanoff S.J. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. Lippincott Williams & Wilkins, Philadelphia1999: 721-768Google Scholar). Therefore, we determined the turnover numbers of myeloperoxidase at 100 μm nitrite, 100 μm thiocyanate, or 100 mm chloride. The concentration of hydrogen peroxide was 30 μm. The turnover number for nitrite was calculated from the steady state rate of hydrogen peroxide loss to be 0.7 s−1. For chloride and thiocyanate, the turnover numbers were 28.5 and 30.5 s−1, respectively. Thus, nitrite is a comparatively poor substrate for myeloperoxidase. The effect of nitrite on the chlorination activity of myeloperoxidase was determined by continuously monitoring the loss of hydrogen peroxide in the presence of chloride (Fig.2 A). Under the conditions of this assay essentially all the hydrogen peroxide consumed by myeloperoxidase is converted to hypochlorous acid (22.Kettle A.J. Winterbourn C.C. Methods Enzymol. 1994; 233: 502-512Crossref PubMed Scopus (225) Google Scholar). As shown in Fig. 2 A, on addition of myeloperoxidase, there was an initial rapid loss of hydrogen peroxide that slowed over time. When nitrite was present, the loss of hydrogen peroxide was markedly inhibited. There was progressive inhibition of enzyme activity with increasing concentration of nitrite up to a maximum of 80% (Fig.2 B). The concentration of nitrite that resulted in 50% maximal inhibition of myeloperoxidase activity (IC50) was 1.3 μm. Taurine chloramine formation, which is a measure of production of hypochlorous acid, was also assayed under the conditions described in Fig. 2 A. With 10 μmnitrite, production of taurine chloramine was inhibited by 79 ± 8% (n = 3). This confirms that nitrite was inhibiting the conversion of hydrogen peroxide to hypochlorous acid. To check whether myeloperoxidase was reversibly or irreversibly inactivated by nitrite, we added tyrosine to the reaction system subsequent to inhibition of the enzyme. Since tyrosine is a good peroxidase substrate it should reverse inhibition that is due to accumulation of compound II. Indeed, upon addition of 100 μm tyrosine, enzyme activity was completely restored (Fig. 2 A). We confirmed that nitrite converts the enzyme to compound II by recording the absorption spectra of myeloperoxidase during its reaction with hydrogen peroxide and chloride. In the absence of nitrite, addition of hydrogen peroxide to myeloperoxidase and chloride caused a minimal change in the absorption spectrum of the enzyme. However, when 10 μm nitrite was present, there was a shift in the Soret maximum to about 450 nm and a new peak appeared at 627 nm (Fig. 3). Based on the extinction coefficients for the native enzyme and its redox intermediates (32.Hoogland H. Van Kuilengurg A. van Reil C. Muijers A.O. Wever R. Biochim. Biophys. Acta. 1987; 916: 76-82Crossref PubMed Scopus (45) Google Scholar), these changes indicate that about 80% of ferric myeloperoxidase was converted to compound II. Compound II was unstable and decayed back to the native enzyme within 5 min. We also determined the effect of nitrite on production of hypochlorous acid by human neutrophils. Hypochlorous acid was trapped with taurine, and accumulated taurine chloramine was measured (22.Kettle A.J. Winterbourn C.C. Methods Enzymol. 1994; 233: 502-512Crossref PubMed Scopus (225) Google Scholar). Under the conditions of our experiments, scavenging of hypochlorous acid by nitrite would have been negligible, as taurine was present in a 50-fold excess and has a rate constant for its reaction with hypochlorous acid 100 times that for nitrite (14.Folkes L.K. Candeias L.P. Wardman P. Arch. Biochem. Biophys. 1995; 323: 120-126Crossref PubMed Scopus (302) Google Scholar). In the absence of superoxide dismutase, nitrite caused modest inhibition of hypochlorous acid formation (Fig. 4). Adding superoxide dismutase to cells caused an approximately 50% increase in the production of hypochlorous acid, as has been reported previously (33.Kettle A.J. Gedye C.A. Winterbourn C.C. Biochem. Pharmacol. 1993; 45: 2003-2010Crossref PubMed Scopus (80) Google Scholar). This occurs because superoxide is prevented from reacting with myeloperoxidase and ensures that the enzyme is present predominantly in its active form (20.Kettle A.J. Winterbourn C.C. Biochem. J. 1988; 252: 529-536Crossref PubMed Scopus (157) Google Scholar). With superoxide dismutase present, nitrite was considerably more effective at inhibiting production of hypochlorous acid. The IC50 was 12 μm. These results demonstrate that superoxide limits the ability of nitrite to inhibit myeloperoxidase-dependent production of hypochlorous acid by neutrophils. Nitrite was not acting by inhibiting the NADPH-oxidase because, at 200 μm, it had no effect on hydrogen peroxide production by neutrophils (result not shown). In view of our result that nitrite is a poor substrate and inhibits chlorination activity, it is surprising that effective nitration of tyrosine, peptides, and proteins by both isolated myeloperoxidase and neutrophils has been demonstrated (8.Eiserich J.P. Hristova M. Cross C.E. Jones A.D. Freeman B.A. Halliwell B. van der Vliet A. Nature. 1998; 391: 393-397Crossref PubMed Scopus (1379) Google Scholar, 15.van der Vliet A. Eiserich J.P. Halliwell B. Cross C.E. J. Biol. Chem. 1997; 272: 7617-7625Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar, 34.Sampson J.B. Ye Y.Z. Rosen H. Beckman J.S. Arch. Biochem. Biophys. 1998; 356: 207-213Crossref PubMed Scopus (301) Google Scholar). To reconcile these findings, we investigated nitration of the peptide acetyl-Ser-Gln-Asn-Tyr-Pro-Val-Val-amide (peptide I) at physiological concentrations of nitrite, chloride, and tyrosine. We chose this heptapeptide because its tyrosyl residue should not be oxidized directly by myeloperoxidase (18.Heinecke J.W. Li W. Francis G.A. Goldstein J.A. J. Clin. Invest. 1993; 91: 2866-2872Crossref PubMed Scopus (304) Google Scholar, 19.Tien M. Arch. Biochem. Biophys. 1999; 367: 61-66Crossref PubMed Scopus (51) Google Scholar). This was confirmed using the hydrogen peroxide electrode (results not shown). Consequently, it should be a good model for investigating how myeloperoxidase nitrates proteins. Also, it should be poorly chlorinated because it does not contain an amino group (35.Domigan N.M. Charlton T.S. Duncan M.W. Winterbourn C.C. Kettle A.J. J. Biol. Chem. 1995; 270: 16542-16548Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Therefore nitration of the tyrosyl residue should not be overwhelmed by formation of 3-chlorotyrosine. To establish that the peptide could undergo nitration, it was incubated with a high concentration of nitrite plus tyrosine, myeloperoxidase, and hydrogen peroxide. Nitration was then monitored using HPLC with UV detection at 274 nm (Fig. 5 A). With the complete reaction system, two new peaks in addition to tyrosine and peptide appeared in the chromatogram (Fig. 5 A,trace a). The peak eluting immediately after tyrosine was identified as 3-nitrotyrosine based on its retention time and absorption spectrum (not shown). The late eluting peak was attributed to the peptide containing a 3-nitrotyrosyl residue based on the following results. When one of either the peptide, nitrite, myeloperoxidase, or hydrogen peroxide were omitted from the reaction system, the late eluting peak was not formed (Fig. 5 A,trace b), indicating that it was derived from the peptide and an oxidation product of nitrite. This peak was also absent when the peptide was incubated with myeloperoxidase and hydrogen peroxide plus tyrosine or chloride (not shown). Thus it could not be attributed to tyrosylated or chlorinated peptide. The absorption spectrum of the peak had maxima at 280 and 350 nm, which is characteristic of a nitrated tyrosyl residue (Fig. 5 B). To further characterize the modified peptide, fractions of eluant were collected from the HPLC corresponding to the parent peptide and the late eluting peak. The fractions were then analyzed by gas chromatography with mass spectrometry after the hydrolyzed amino acids were derivatized with trifluoroacetic acid anhydride. In this assay, 3-nitrotyrosine is reduced and detected as 3-aminotyrosine. Tyrosine and 3-chlorotyrosine are also measured in this assay. In the fractions corresponding to the parent peptide only tyrosine was present. In fractions corresponding to the late eluting peak, a component had a mass spectrum with major ions having m/z of 526 and 429 mass units (Fig. 6). This mass spectrum is expected for derivatized 3-aminotyrosine. The relative amount of 3-nitrotyrosine to tyrosine was 300:1. These results demonstrate that the late eluting peak is due to a peptide that contains 3-nitrotyrosine. Hydrolyzed fractions from the HPLC were also assayed for their amino acid content. The late eluting peak contained all the amino acids of the parent peptide except tyrosine (result not shown). 3-Nitrotyrosine was not amenable to detection by amino acid analysis but these results confirmed that the tyrosyl residue was modified upon nitration. Under the reaction conditions given in the legend to Fig. 5 A, 35% of the hydrogen peroxide was used to nitrate the peptide. When 100 mm chloride was added to the reaction system, 3-chlorotyrosine was detected by gas chromatography with mass spectrometry in fractions that eluted from the HPLC between the parent peptide and the nitrated peptide. In the presence of a physiological concentration of nitrite (100 μm), myeloperoxidase catalyzed nitration of peptide I (Table I). The yield was about 1% of the hydrogen peroxide used by the enzyme. Nitration was minimally affected by 100 mm chloride, but in the presence of chloride and methionine, it was negligible. This result is best explained by a change in the nitrating species to nitryl chloride when chloride was present in the reaction system. Nitryl chloride is produced when nitrite reacts with hypochlorous acid (12.Eiserich J.P. Cross C.E. Jones A.D. Halliwell B. van der Vliet A. J. Biol. Chem. 1996; 271: 19199-19208Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). Methionine would prevent its formation by scavenging hypochlorous acid and consequently block nitration. It is therefore apparent that in the presence of chloride, there was insufficient direct oxidation of nitrite by myeloperoxidase to promote nitration of the peptide. Addition of 10 μmtyrosine (Table I) or 4-hydroxyphenylacetic acid (result not shown) to the reaction system promoted a 3-fold increase in nitration, which was not inhibited by chloride even in the presence of methionine. This result demonstrates that tyrosine or tyrosyl radicals played a crucial role in nitration. Furthermore, nitryl chloride was not responsible for nitrating the peptide. In the presence of chloride and methionine, a threshold concentration of about 50 μm nitrite was required for myeloperoxidase to nitrate the peptide (Fig.7). Adding tyrosine eliminated this threshold and, at 100 μm nitrite or less, it enhanced nitration by at least 4-fold. Tyrosine had no effect on the extent of nitration above 250 μm nitrite (Fig. 7,inset). Nitration of two other unrelated peptides that contained tyrosyl residues was also enhanced by free tyrosine (TableI), which indicates that the effect of tyrosine is not unique to peptide I.Table IEffects tyrosine and chloride on nitration of peptides by myeloperoxidaseCondition[Nitrated peptide]μmPeptide I alone0.00 ± 0.00 +MPO, H2O2, NO2−0.93 ± 0.18 +Cl−0.92 ± 0.26 +Cl−, methionine0.05 ± 0.00 +Tyrosine2.80 ± 0.22 +Tyrosine, Cl−3.55 ± 0.48 +Tyrosine, Cl−, methionine3.87 ± 0.68Peptide II alone0.00 ± 0.00 +MPO, H2O2, NO2−2.54 ± 0.18 +Tyrosine4.65 ± 0.54Peptide III alone0.00 ± 0.00 +MPO, H2O2, NO2−2.36 ± 0.40 +Tyrosine5.05 ± 0.30Peptides (100 μm) were nitrated by adding 100 μm hydrogen peroxide to 50 nm myeloperoxidase (MPO) and 100 μm nitrite in 50 mm phosphate buffer, pH 7.4, at 21 °C. Reactions were stopped after 1 h by adding 20 μg/ml of catalase. Where indicated, 10 μmtyrosine, 100 mm chloride, or 1 mm methionine were included. Results are means and ranges of at least duplicate experiments. Open table in a new tab Peptides (100 μm) were nitrated by adding 100 μm hydrogen peroxide to 50 nm myeloperoxidase (MPO) and 100 μm nitrite in 50 mm phosphate buffer, pH 7.4, at 21 °C. Reactions were stopped after 1 h by adding 20 μg/ml of catalase. Where indicated, 10 μmtyrosine, 100 mm chloride, or 1 mm methionine were included. Results are means and ranges of at least duplicate experiments. Gas chromatography with mass spectrometry analysis of the reaction system containing 100 μm each of peptide I, nitrite, and hydrogen peroxide, plus 100 mm chloride and 10 μm tyrosine under the conditions given in Table I, revealed that 3% of the hydrogen peroxide was used to produce 3-chlorotyrosine. This result confirms that peptide I, which lacks a free amine group, is a poor substrate for chlorination. When neutrophils were stimulated with PMA in the presence of 100 μm nitrite there was no detectable nitration of peptide I. There was also none when the concentration of peptide I was increased from 100 to 500 μm. The lack of nitration was not due to consumption of the nitrite because there was no appreciable loss of nitrite (not shown). With 1 mm nitrite, less than 1% of the peptide was nitrated (Fig. 8). The extent of nitration increased dramatically in the presence of superoxide dismutase (Fig. 8), whereas inactive enzyme had no effect (not shown). These results indicate that superoxide suppresses formation of 3-nitrotyrosine on the peptide. Tyrosine almost doubled the efficiency of the reaction. Methionine, which scavenges hypochlorous acid, enhanced nitration of the peptide, which excludes the involvement of nitryl chloride. No nitrated peptide was detected with unstimulated cells or when azide and catalase were added to the reaction system. These results confirm that nitration was dependent on myeloperoxidase and an active respiratory burst. In this investigation we have demonstrated that at concentrations less than 100 μm, nitrite is a poor substrate for myeloperoxidase, and nitration of tyrosyl residues is extremely inefficient. However, free tyrosine acts as a co-substrate of myeloperoxidase and enhances the nitration reaction. We obtained these results using peptides that contained tyrosyl residues that could not be oxidized directly by myeloperoxidase and were therefore good models for protein nitration. We propose that at sites of inflammation myeloperoxidase will nitrate proteins, even though nitrite is a poor substrate, because tyrosine will be available to facilitate the reaction. We found that above 250 μm nitrite, tyrosine was no longer required to promote nitration. Such high concentrations of nitrite have not been detected in vivo, but they may be feasible at localized sites where there are high fluxes of nitric oxide. In accord with our findings, high concentrations of nitrite are required for nitration of bovine serum albumin (34.Sampson J.B. Ye Y.Z. Rosen H. Beckman J.S. Arch. Biochem. Biophys. 1998; 356: 207-213Crossref PubMed Scopus (301) Google Scholar) and low density lipoproteins (36.Podrez E.A. Schmitt D. Hoff H.F. Hazen S.L. J. Clin. Invest. 1999; 103: 1547-1560Crossref PubMed Scopus (427) Google Scholar). Constraints on myeloperoxidase-dependent nitration identified in our study are that with neutrophils it is inefficient and largely attenuated by superoxide. Thus, the concentration of superoxide at sites of inflammation will have a significant impact on formation of 3-nitrotyrosine via this route. Within phagosomes, where the flux of superoxide is high, nitration would not be expected to occur. In contrast, when there is extracellular release of myeloperoxidase and when superoxide is trapped within phagosomes (37.Thomas M.J. Hedrick C.C. Smith S. Pang J. Jerome W.G. Willard A.S. Shirley P.S. J. Leukoc. Biol. 1992; 51: 591-596Crossref PubMed Scopus (10) Google Scholar), nitration should proceed. Consistent with this conclusion, Jiang and Hurst (38.Jiang Q. Hurst J.K. J. Biol. Chem. 1997; 272: 32767-32772Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) found that fluorescein-coated particles were readily nitrated in extracellular reactions of neutrophils, but no nitrated products were detected within their phagosomes. Our investigation into how nitrite affects the chlorination activity of myeloperoxidase has provided insights into how it will influence production of hypochlorous acid in vivo. It has also revealed how this anion is oxidized by the enzyme and demonstrated that nitrogen dioxide must be the initial product. We found that nitrite is a potent inhibitor of the purified enzyme because it converts myeloperoxidase to compound II. However, it was a poor inhibitor of the production of hypochlorous acid by neutrophils. The discrepancies between the results with the purified enzyme and neutrophils are largely explained by the effect superoxide has on the activity of myeloperoxidase. Superoxide would prevent inhibition of chlorination by reducing compound II back to the native enzyme and thereby maintain enzyme turnover (20.Kettle A.J. Winterbourn C.C. Biochem. J. 1988; 252: 529-536Crossref PubMed Scopus (157) Google Scholar). In support of our findings, nitrite has been shown to have little effect on intraphagosomal chlorination until its concentration exceeds 1 mm (38.Jiang Q. Hurst J.K. J. Biol. Chem. 1997; 272: 32767-32772Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Therefore, nitrite will not attenuate the generation of hypochlorous acid at sites of inflammation. The reduction of compound I to compound II is a one electron reaction. Therefore, when nitrite converts myeloperoxidase to compound II, it must be oxidized to nitrogen dioxide. Compound II will also oxidize nitrite to nitrogen dioxide and complete the classical peroxidase cycle (Reactions 1, 3, and 4). However, because nitrite is so effective at inhibiting the enzyme's chlorination activity, reduction of compound II by nitrite must be slow. Thus, at physiological concentrations of nitrite, its oxidation and the nitration of proteins are inefficient because the enzyme lingers at compound II and is turned over slowly. The nitrogen dioxide produced by myeloperoxidase will nitrate tyrosyl residues. It reacts with tyrosyl residues (p-TyrH) with a rate constant of 3.2 × 105m−1 s−1 to form tyrosyl radicals (Reaction 6) (39.Prutz W.A. Monig H. Butler J. Land E.J. Arch. Biochem. Biophys. 1985; 243: 125-134Crossref PubMed Scopus (333) Google Scholar). Tyrosyl radicals also react with nitrogen dioxide and at a rate that is almost diffusion controlled (k = 3 × 109m−1s−1) to give 3-nitrotyrosine (Reaction 7) (39.Prutz W.A. Monig H. Butler J. Land E.J. Arch. Biochem. Biophys. 1985; 243: 125-134Crossref PubMed Scopus (333) Google Scholar). NO2·+pTyrH→NO2−+pTyr⋅+H+ REACTION 6 NO2·+pTyr⋅→pTyrNO2 REACTION 7Adding tyrosine would eliminate the need for Reaction 6 because tyrosyl radicals would be efficiently generated in Reactions 3 and 4 (40.McCormick M.L. Gaut J.P. Lin T.S. Britigan B.E. Buettner G.R. Heinecke J.W. J. Biol. Chem. 1998; 273: 32030-32037Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and then rapidly exchange with tyrosyl residues in the peptide (18.Heinecke J.W. Li W. Francis G.A. Goldstein J.A. J. Clin. Invest. 1993; 91: 2866-2872Crossref PubMed Scopus (304) Google Scholar). As a consequence, the dominant reaction for nitrogen dioxide would be coupling with tyrosyl radicals (Reaction 7). Thus, at low nitrite concentrations, where production of nitrogen dioxide is limiting, tyrosine will enhance nitration by ensuring that most of the nitrogen dioxide formed is trapped by tyrosyl radicals. In previous studies (8.Eiserich J.P. Hristova M. Cross C.E. Jones A.D. Freeman B.A. Halliwell B. van der Vliet A. Nature. 1998; 391: 393-397Crossref PubMed Scopus (1379) Google Scholar, 15.van der Vliet A. Eiserich J.P. Halliwell B. Cross C.E. J. Biol. Chem. 1997; 272: 7617-7625Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar) the effect of tyrosine would not have been apparent because the targets for nitration were either free tyrosine or tripeptides containing tyrosyl residues that are directly oxidized by myeloperoxidase to tyrosyl radicals. We found that neutrophils did not promote detectable nitration of the peptide I unless nitrite was present at 1 mm. It is not apparent why nitration by the cells was less efficient than with the isolated enzyme. Nitrite could not have had a substantial effect on release of myeloperoxidase from cells because its inhibition of hypochlorous acid product was only modest. Nitration was largely suppressed by superoxide. The most plausible explanation for this inhibition is that superoxide reacted with both nitrogen dioxide and tyrosyl radical. The reaction with tyrosyl radical is fast, having a second order rate constant of 1.5 × 109m−1 s−1 (41.Jin, F., Leitich, J., and von Sonntag, C. (1993) J. Chem. Soc. Perkin Trans. II 1583–1588Google Scholar). Reaction of superoxide with nitrogen dioxide is thermodynamically favorable and should also be fast (17.Koppenol W.H. Free Radic. Biol. Med. 1998; 25: 385-391Crossref PubMed Scopus (308) Google Scholar). In conclusion, we have shown that at physiological concentrations of nitrite, the propensity of myeloperoxidase to catalyze nitration of tyrosyl residues is greatly enhanced by tyrosine because it acts as a co-substrate in the reaction. Although stimulated neutrophils can promote nitration, it will be not be favored due to inhibition by superoxide. Therefore, before production of 3-nitrotyrosine at sites of inflammation is attributed to myeloperoxidase, it will be necessary to show that other specific footprints of this enzyme, such as 3-chlorotyrosine (35.Domigan N.M. Charlton T.S. Duncan M.W. Winterbourn C.C. Kettle A.J. J. Biol. Chem. 1995; 270: 16542-16548Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 42.Kettle A.J. FEBS Lett. 1996; 379: 103-106Crossref PubMed Scopus (163) Google Scholar) and 3,5-dichlorotyrosine (43.Kettle A.J. Methods Enzymol. 1999; 300: 111-120Crossref PubMed Scopus (28) Google Scholar), are also present.
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