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• W2077386186 abstract "We investigated the effects of a cysteine residue on tyrosine nitration in several model peptides treated with myeloperoxidase (MPO), H2O2, and nitrite anion (NO2-) and with horseradish peroxidase and H2O2. Sequences of model peptides were acetyl-Tyr-Cys-amide (YC), acetyl-Tyr-Ala-Cys-amide (YAC), acetyl-Tyr-Ala-Ala-Cys-amide (YAAC), and acetyl-Tyr-Ala-Ala-Ala-Ala-Cys-amide (YAAAAC). Results indicate that nitration and oxidation products of tyrosyl residue in YC and other model peptides were barely detectable. A major product detected was the corresponding disulfide (e.g. YCysCysY). Spin trapping experiments with 5,5′-dimethyl-1-pyrroline N-oxide (DMPO) revealed thiyl adduct (e.g. DMPO-SCys-Tyr) formation from peptides (e.g. YC) treated with MPO/H2O2 and MPO/H2O2/NO2-. The steady-state concentrations of DMPO-thiyl adducts decreased with increasing chain length of model peptides. Blocking the sulfydryl group in YC with methylmethanethiosulfonate (that formed YCSSCH3) totally inhibited thiyl radical formation as did substitution of Tyr with Phe (i.e. FC) in the presence of MPO/H2O2/NO2-. However, increased tyrosine nitration, tyrosine dimerization, and tyrosyl radical formation were detected in the MPO/H2O2/NO2-/YCSSCH3 system. Increased formation of S-nitrosated YC (YCysNO) was detected in the MPO/H2O2/·NO system. We conclude that a rapid intramolecular electron transfer reaction between the tyrosyl radical and the Cys residue impedes tyrosine nitration and induces corresponding thiyl radical and nitrosocysteine product. Implications of this novel intramolecular electron transfer mechanism in protein nitration and nitrosation are discussed. We investigated the effects of a cysteine residue on tyrosine nitration in several model peptides treated with myeloperoxidase (MPO), H2O2, and nitrite anion (NO2-) and with horseradish peroxidase and H2O2. Sequences of model peptides were acetyl-Tyr-Cys-amide (YC), acetyl-Tyr-Ala-Cys-amide (YAC), acetyl-Tyr-Ala-Ala-Cys-amide (YAAC), and acetyl-Tyr-Ala-Ala-Ala-Ala-Cys-amide (YAAAAC). Results indicate that nitration and oxidation products of tyrosyl residue in YC and other model peptides were barely detectable. A major product detected was the corresponding disulfide (e.g. YCysCysY). Spin trapping experiments with 5,5′-dimethyl-1-pyrroline N-oxide (DMPO) revealed thiyl adduct (e.g. DMPO-SCys-Tyr) formation from peptides (e.g. YC) treated with MPO/H2O2 and MPO/H2O2/NO2-. The steady-state concentrations of DMPO-thiyl adducts decreased with increasing chain length of model peptides. Blocking the sulfydryl group in YC with methylmethanethiosulfonate (that formed YCSSCH3) totally inhibited thiyl radical formation as did substitution of Tyr with Phe (i.e. FC) in the presence of MPO/H2O2/NO2-. However, increased tyrosine nitration, tyrosine dimerization, and tyrosyl radical formation were detected in the MPO/H2O2/NO2-/YCSSCH3 system. Increased formation of S-nitrosated YC (YCysNO) was detected in the MPO/H2O2/·NO system. We conclude that a rapid intramolecular electron transfer reaction between the tyrosyl radical and the Cys residue impedes tyrosine nitration and induces corresponding thiyl radical and nitrosocysteine product. Implications of this novel intramolecular electron transfer mechanism in protein nitration and nitrosation are discussed. There is increasing evidence for generation of inflammatory oxidants including the reactive oxygen/nitrogen species in the progression and pathogenesis of cardiovascular, pulmonary, and neurodegenerative diseases (1Kooy K.W. Lewis S.J. Royall J.A. Ye Y.Z. Kelly D.R. Beckman J.S. Crit. Care Med. 1997; 25: 812-819Crossref PubMed Scopus (219) Google Scholar, 2Turko I.V. Murad F. Pharmacol. 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Neurosci. 2001; 21: 8053-8061Crossref PubMed Google Scholar, 9Schopfer F.J. Baker P.R. Freeman B.A. Trends Biochem. Sci. 2003; 28: 646-654Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). Supporting evidence came from the identification of the post-translational modification of protein and lipid oxidation/nitration marker products (10Rubbo H. Radi R. Trujillo M. Telleri R. Kalyanaraman B. Barnes S. Kirk M. Freeman B.A. J. Biol. Chem. 1994; 269: 26066-26075Abstract Full Text PDF PubMed Google Scholar, 11Lanone S. Manivet P. Callebert J. Launay J.-M. Payen D. Aubier M. Boczkowski J. Mebazaa A. Biochem. J. 2002; 366: 399-404Crossref PubMed Scopus (51) Google Scholar, 12Greenacre S.A. Ischiropoulos H. Free Radic. Res. 2001; 34: 541-581Crossref PubMed Scopus (478) Google Scholar). Prominent nitrative, nitrosative, and oxidative reactions in tissues include tyrosine nitration, cysteine and tryptophan nitrosation, tyrosine, tryptophan, histidine, and methionine oxidation and lipid oxidation/nitration (12Greenacre S.A. Ischiropoulos H. Free Radic. Res. 2001; 34: 541-581Crossref PubMed Scopus (478) Google Scholar, 13Rassaf T. Bryan N.S. Kelm M. Feelisch M. Free Radic. Biol. Med. 2002; 33: 1590-1596Crossref PubMed Scopus (167) Google Scholar, 14Zhang Y.Y. Xu A.M. Nomen M. Walsh M. Keaney Jr., J.F. Loscalzo J. J. Biol. Chem. 1996; 271: 14271-14279Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 15White 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 (662) Google Scholar). The goal of the present study was to monitor the influence of a cysteine residue on tyrosine nitration. Several studies have shown that tyrosine nitration is a selective process that is controlled by various microenvironmental factors (hydrophobicity, CO2 levels, membrane oxygen concentration, acidic environment, and amino acid sequence) (16Khairutdinov R.F. Coddington J.W. Hurst J.K. Biochemistry. 2000; 39: 14238-14249Crossref PubMed Scopus (71) Google Scholar, 17Liu X. Miller M.J. Joshi M.S. Thomas D.D. Lancaster Jr., J.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2175-2179Crossref PubMed Scopus (533) Google Scholar, 18Marla S.S. Lee J. Groves J.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14243-14248Crossref PubMed Scopus (282) Google Scholar, 19Denicola A. Batthyany C. Lissi E. Freeman B.A. Rubbo H. Radi R. J. Biol. Chem. 2002; 277: 932-936Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 20Denicola A. Souza J.M. Radi R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3566-3571Crossref PubMed Scopus (370) Google Scholar, 21Ischiropoulos H. Arch. Biochem. Biophys. 1998; 356: 1-11Crossref PubMed Scopus (924) Google Scholar). Previously, we have shown, using membrane-incorporated tyrosine analogs and tyrosyl peptides, that dityrosine formation is not a significant reaction process for tyrosyl radicals in membranes due to hindrance of free diffusion of tyrosyl radicals (22Zhang H. Joseph J. Feix J. Hogg N. Kalyanaraman B. Biochemistry. 2001; 40: 7675-7686Crossref PubMed Scopus (76) Google Scholar). In addition, the location of the tyrosyl probe in the membrane determines the transmembrane nitration profile (23Zhang H. Bhargava K. Keszler A. Feix J. Hogg N. Joseph J. Kalyanaraman B. J. Biol. Chem. 2003; 278: 8969-8978Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Literature data show that the CO2 levels greatly influence the kinetics of tyrosyl nitration in proteins (24Gow A. Duran D. Thom S.R. Ischiropoulos H. Arch. Biochem. Biophys. 1996; 333: 42-48Crossref PubMed Scopus (280) Google Scholar, 25Radi R. Denicola A. Freeman B.A. Methods Enzymol. 1999; 301: 353-367Crossref PubMed Scopus (103) Google Scholar). Factors influencing nitration of tyrosyl residues in protein are not fully known. As discussed in previous reviews (12Greenacre S.A. Ischiropoulos H. Free Radic. Res. 2001; 34: 541-581Crossref PubMed Scopus (478) Google Scholar, 21Ischiropoulos H. Arch. Biochem. Biophys. 1998; 356: 1-11Crossref PubMed Scopus (924) Google Scholar, 26Souza J.M. Daikhin E. Yudkoff M. Raman C.S. Ischiropoulos H. Arch. Biochem. Biophys. 1999; 371: 169-178Crossref PubMed Scopus (290) Google Scholar), the local environment of tyrosine residues within the secondary and tertiary structure of the protein will probably influence the site of tyrosine nitration. Although no specific amino acid sequence criteria exist for predicting tyrosine nitration or lack thereof, it has been shown, as originally suggested, that protein tyrosyl nitration is (i) enhanced when tyrosine is situated closer to a negatively charged amino acid (i.e. glutamate or aspartate) and (ii) decreased when tyrosine residue is present in the vicinity of a cysteinyl or methionine residue (12Greenacre S.A. Ischiropoulos H. Free Radic. Res. 2001; 34: 541-581Crossref PubMed Scopus (478) Google Scholar, 21Ischiropoulos H. Arch. Biochem. Biophys. 1998; 356: 1-11Crossref PubMed Scopus (924) Google Scholar, 26Souza J.M. Daikhin E. Yudkoff M. Raman C.S. Ischiropoulos H. Arch. Biochem. Biophys. 1999; 371: 169-178Crossref PubMed Scopus (290) Google Scholar). However, detailed quantitative analysis of these effects on tyrosyl nitration is lacking, although a more recent report focused on the effect of lysine residues on tyrosyl nitration (27Shao B. Bergt C. Fu X. Green P. Voss J.C. Oda M.N. Oram J.F. Heinecke J.W. J. Biol. Chem. 2005; 280: 5983-5993Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Understanding the biophysical/biochemical mechanisms that determine the motif for nitration-sensitive tyrosine residues has physiological and pathophysiological relevance (28Gow A.J. Farkouh C.R. Munson D.A. Posencheg M.A. Ischiropoulos H. Am. J. Physiol. 2004; 287: L262-L268Crossref PubMed Scopus (337) Google Scholar). Two major pathways were proposed to be responsible for tyrosine nitration in vivo (29Brennan M.L. Wu W. Fu X. Shen Z. Song W. Frost H. Vadseth C. Narine L. Lenkiewicz E. Borchers M.T. Lusis A.J. Lee J.J. Lee N.A. Abu-Soud H.M. Ischiropoulos H. Hazen S.L. J. Biol. Chem. 2002; 277: 17415-17427Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar, 30Beckman J.S. Chem. Res. Toxicol. 1996; 9: 836-844Crossref PubMed Scopus (916) Google Scholar, 31Ischiropoulos 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 (1434) Google Scholar, 32Pfeiffer S. Lass A. Schmidt K. Mayer B. FASEB J. 2001; 15: 2355-2364Crossref PubMed Scopus (97) Google Scholar, 33Jourd'heuil D. Jourd'heuil F.L. Kutchukian P.S. Musah R.A. Wink D.A. Grisham M.B. J. Biol. Chem. 2001; 276: 28799-28805Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 34MacPherson J.C. Comhair S.A. Erzurum S.C. Klein D.F. Lipscomb M.F. Kavuru M.S. Samoszuk M.K. Hazen S.L. J. Immunol. 2001; 166: 5763-5772Crossref PubMed Scopus (254) Google Scholar, 35Pryor W.A. Squadrito G.L. Am. J. Physiol. 1995; 68: L699-L722Google Scholar). These involve either the catalytic action of heme peroxidases (e.g. myeloperoxidase, eosinophil peroxidase) using nitrite and H2O2 as substrates and/or the nitrative chemistry of peroxynitrite. In this study, we investigated the effects of cysteine on tyrosyl nitration (or tyrosine on cysteine oxidation) in model peptides subjected to oxidation by myeloperoxidase (MPO) 2The abbreviations used are: MPOmyeloperoxidaseDBNBS3,5-dibromo-4-nitrosobenzenesulfonic acidDMPO5,5′-dimethyl-1-pyrroline N-oxideDTPAdiethylenetriaminepentaacetic acidEPRelectron paramagnetic resonanceFCN-acetyl phenylalaninylcysteine amideMMTSmethylmethanethiosulfonate·NO2nitrogen dioxide radicalYCN-acetyl tyrosylcysteine amideYACN-acetyl tyrosylalaninylcysteine amideYAACN-acetyl tyrosylalaninylalaninylcysteine amideYCysCysYdisulfide formed from YCY(NO2)CN-acetyl nitrotyrosylcysteine amideFmocN-(9-fluorenyl)-methoxycarbonylDICdiisopropylcarbodiimideHOBt1-hydroxybezotriazoleESI-MSelectrospray ionization-mass spectrometryHRPhorseradish peroxidaseHPLChigh pressure liquid chromatographyYCysNOS-nitrosated YCMSmass spectrometryDEA-NO2-(N,N-diethylamino)-diazenolate-2-oxide sodium salt./H2O2 and NO2-. Results indicate that a rapid intramolecular electron transfer between the tyrosyl radical and the cysteine residue controls the extent of tyrosine nitration and cysteine oxidation by MPO/H2O2/NO2-. The influence of intramolecular electron transfer reactions in protein nitration and nitrosation reactions is discussed. myeloperoxidase 3,5-dibromo-4-nitrosobenzenesulfonic acid 5,5′-dimethyl-1-pyrroline N-oxide diethylenetriaminepentaacetic acid electron paramagnetic resonance N-acetyl phenylalaninylcysteine amide methylmethanethiosulfonate nitrogen dioxide radical N-acetyl tyrosylcysteine amide N-acetyl tyrosylalaninylcysteine amide N-acetyl tyrosylalaninylalaninylcysteine amide disulfide formed from YC N-acetyl nitrotyrosylcysteine amide N-(9-fluorenyl)-methoxycarbonyl diisopropylcarbodiimide 1-hydroxybezotriazole electrospray ionization-mass spectrometry horseradish peroxidase high pressure liquid chromatography S-nitrosated YC mass spectrometry 2-(N,N-diethylamino)-diazenolate-2-oxide sodium salt. The following chemicals and enzymes were purchased from various sources as indicated: tyrosine, hydrogen peroxide, sodium nitrite, sodium bicarbonate, 3-nitrotyrosine, N-ethylmaleimide, cysteine, and GSH were from Sigma; myeloperoxidase was from Calbiochem. Rink amide methylbenzhydrylamine resin and all Fmoc-protected amino acids were from Calbiochem. Diisopropylcarbodiimide (DIC), 1-hydroxybezotriazole (HOBt), triisopropylsilane, methylmethanethiosulfonate (MMTS), N-methylpyrrolidione (NMP), and piperidine were purchased from Fisher. The sequences of the peptides used in this study are given in Fig. 2S. The peptides were chemically synthesized using the standard Fmoc solid phase peptide-based synthetic procedure on an Advanced Chemtech model 90 synthesizer (Louisville, KY) (23Zhang H. Bhargava K. Keszler A. Feix J. Hogg N. Joseph J. Kalyanaraman B. J. Biol. Chem. 2003; 278: 8969-8978Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Rink amide methylbenzhydrylamine resin (loading 0.72 mmol/g) was used as a solid support. Fmoc-protected amino acids were coupled as HOBt-esters. All amino acids were double coupled using HOBt/DIC. The following steps were performed in the reaction vessel for each double coupling: deprotection of the Fmoc group with 20% piperidine in NMP for 30 min (twice), three NMP washes, two dichloromethane washes, first coupling for 1 h with a 5-fold excess of Fmoc amino acid in 0.5 m HOBt and 0.5 m DIC, second coupling using a fresh addition of the same reagent for 1 h, followed by three NMP washes, and two dichloromethane washes. Final acetylation was performed using an acetic anhydride/HOBt/DIC mixture for 30 min (twice). The resin was washed twice with dichloromethane and three times with methanol and then dried under vacuum prior to cleavage. The peptide was deprotected and cleaved from the resin with 90% trifluoroacetic acid containing triisopropylsilane for 3 h at room temperature. The resin was removed by filtration and washed with trifluoroacetic acid, and the combined trifluoroacetic acid filtrates were evaporated to dryness under a steam of dry N2 gas. The oily residue was washed three times with cold ether to remove the scavengers, and the dry crude peptide was dissolved in acetonitrile/H2O (1:1) and lyophilized. The crude peptides were purified by a semipreparative reverse phase HPLC on a RP-C18 (10 × 250 mm) column using a CH3CN/water gradient (5-25% CH3CN over 60 min) containing 0.1% trifluoroacetic acid at a flow rate of 3 ml/min with detection at 280 nm. The disulfide of YC peptide (YCysCysY) was synthesized as follows. YC peptide (30 mm) in a phosphate buffer (100 mm, pH 7.4) containing 1 mm DTPA was incubated with 30 mm hydrogen peroxide at room temperature for 1 h. The reaction mixture was injected into a RP-C18 semipreparatory column, and YC peptide disulfide was eluted at 26 min using a CH3CN/water gradient (5-25% CH3CN over 60 min) containing 0.1% trifluoroacetic acid at a flow rate of 3 ml/min. Detection was at 280 nm. The structure of the disulfide product was confirmed by ESI-MS analysis (M + H+, 649.3). The YC peptide dityrosine (bis-N-acetyl tyrosylcysteine amide) was prepared as follows. The YC disulfide (15 mm) was incubated with 10 mm H2O2 and 100 μg of horseradish peroxidase (HRP) in a phosphate buffer (100 mm, pH 7.4) containing 100 μm DTPA for 20 min. The reaction mixture was then mixed with β-mercaptoethanol (500 mm), and HRP was removed by ultracentrifugation (Mr 3000 cut-off). The product (YC dityrosine) was purified by a preparative HPLC (C-18, 250 × 10 mm) using a fluorescence detector (excitation 294 nm; emission, 410 nm). The YC dityrosine was eluted by a linear CH3CN gradient as above. The product was further confirmed by ESI-MS. Nitrated YC peptide (Y(NO2)C) was prepared as follows. The YCysCysY peptide (1 mm) was mixed with peroxynitrite (10 mm) in a phosphate buffer (100 mm, pH 7.4) containing DTPA (100 μm) for 20 min. The reaction mixture was then mixed with 500 mm β-mercaptoethanol and incubated for 20 min. The resulting Y(NO2)C peptide was purified using a preparative HPLC. Nitro peptides show a characteristic UV-visible spectrum. Upon adding NaOH, the 350 nm absorption peak in MeOH was shifted to 430 nm with an extinction coefficient of 4100 m-1 m-1. The structure of Y(NO2)C was verified by liquid chromatography/mass spectrometry on an Agilent 1100 series liquid chromatograph/mass spectrometer. 20 mm YC was mixed with 20 mm sodium nitrite in 0.03 m HCl for 10 min at room temperature. The solution was then neutralized by 100 mm phosphate buffer, pH 7.4 containing 100 μm DTPA. YCysNO (yield >95%) was verified by ESI-MS (M + H+; 355), UV absorption (λmax = 335 nm) and by Hg2+-induced cleavage. Typically, peptides (0.3 mm) were incubated with NaNO2 (0.5 mm), H2O2 (0.1 mm), and MPO (30 nm) in a phosphate buffer (100 mm, pH 7.4) containing DTPA (100 μm) at room temperature for 30 min. Reactions were stopped by adding catalase (200 units) and analyzed by HPLC. Repeat injections in 24 h showed no significant oxidation of YC under these experimental conditions. All reagents were purged with argon gas for 60 min before experiments. YC (150 μm) was incubated with H2O2 (50 μm), MPO (30 nm), and DEA-NO (10-50 μm) in a phosphate buffer (50 mm, pH 7.4) containing DTPA (100 μm) at room temperature for 10 min. Reactions were stopped by adding iodoacetamide (20 mm) and analyzed by HPLC. Repeat injections within 24 h showed no significant loss of YCysNO under these experimental conditions. Typically, 20 μl of sample was injected into an HPLC system (HP1100) with a C-18 column (250 × 4.6 mm) equilibrated with 5% CH3CN in 0.1% trifluoroacetic acid. The peptide and its product were separated by a linear increase of CH3CN concentration to 25% in 60 min at a flow rate of 1 ml/min. The elution was monitored using the on-line UV-visible and fluorescence detectors. YC and nitrated YC were eluted at 12 and 17.5 min, respectively. Nitration of tyrosine with or without cysteine by MPO/H2O2/NaNO2 was performed under the same conditions, and the products were analyzed as reported previously (36Goss S.P. Hogg N. Kalyanaraman B. Arch. Biochem. Biophys. 1999; 363: 333-340Crossref PubMed Scopus (64) Google Scholar). Thiyl Radical Trapping—A typical incubation mixture consisted of a peptide or tyrosine (1 mm) and cysteine (1 mm), MPO (50 nm), H2O2 (1 mm), and 150 mm DMPO in a phosphate buffer (100 mm, pH 7.4) containing DTPA (100 μm). The reaction was initiated by adding H2O2. Samples were subsequently transferred to a 100-μl capillary tube, and EPR spectra were recorded within 30 s after starting the reaction. EPR spectra were recorded at room temperature on a Bruker ER 200 D-SRC spectrometer operating at 9.8 GHz and a cavity equipped with a Bruker Aquax liquid sample cell. Typical spectrometer parameters were as follows: scan range, 100 G; field set, 3505 G; time constant, 0.64 ms; scan time, 10 s; modulation amplitude, 1.0 G; modulation frequency, 100 kHz; receiver gain, 5 × 104; and microwave power, 10 milliwatts. The spectra shown were the average of 10 scans. Tyrosyl Radical Trapping—Incubations consisting of a peptide or tyrosine (1 mm) and cysteine (1 mm), MPO (100 nm), and DBNBS (20 mm) in a phosphate buffer (0.1 m, pH 7.4) containing DTPA (0.1 mm) were rapidly mixed with H2O2 (1 mm). Samples were subsequently transferred to a 100-μl capillary tube, and ESR spectra were taken within 30 s after starting the reaction. Typical spectrometer parameters were as follows: scan range, 100 G; field set, 3505 G; time constant, 0.64 ms; scan time, 20 s; modulation amplitude, 2.0 G; modulation frequency, 100 kHz; receiver gain, 5 × 105; and microwave power, 20 milliwatts. The spectra shown were the average of 30 scans. The minimum energy conformations of the peptides and their global minima in the presence of water molecules were computed by the Metropolis Monte Carlo approach using a random variation of the randomly selected torsional angles (37von Freyberg B. Braun W.J. Comput. Chem. 1991; 12: 1065Crossref Scopus (105) Google Scholar). The starting configurations of the peptides were linear, corresponding to an all-trans backbone configuration with an N-terminal acetyl and C-terminal amide group. The molecular mechanics calculations were performed using an Amber 99 force field methodology. The structures calculated by Metropolis Monte Carlo were reminimized by the conjugate gradient method. Energy minimization was terminated when the gradient root mean square was below 0.01 mol/kcal. The selected lower energy conformers of each peptide were solvated by TIP3P water molecules and reminimized again. Characterization of Tyrosylcysteine Peptides—TABLE ONE lists the amino acid sequences of model peptides and oxidation/nitration products along with the mass spectral data. The intramolecular distances between the tyrosyl oxygen atom and the cysteinyl sulfur atom calculated from the lowest energy conformations in an aqueous environment using the HyperChem 7.1 Package Program (Hypercube Inc.) are shown (see supplemental Fig. 1S).TABLE ONECharacterization of peptidesPeptidesM + H+Distance between Oy and Sc atomsÅYC326.14.45YAC397.24.42YAAC468.24.75YAAAAC610.24.54YCysCysY649.2CYYC649.2Y(NO2)C371.2 Open table in a new tab MPO/H2O2/NaNO2-dependent Oxidation and Nitration of Peptides Containing Tyrosine and Cysteine Residues—The aim of these experiments was to determine the effect of the cysteine residue on nitration and oxidation of the tyrosine group present in model peptides (see supplemental Fig. 2S). MPO/H2O2/NaNO2-dependent nitration/oxidation of YC peptide was monitored by HPLC with UV-visible detection at 280 nm for tyrosyl residue and at 350 nm for nitro tyrosyl residue. The HPLC/fluorescence (excitation, 290 nm; emission, 410 nm) was used for detecting the dityrosyl product of YC (Fig. 1). The authentic YC peptide (300 μm), YC nitration product (Y(NO2)C; 0.1 μm), dityrosyl product (bis-N-acetyl tyrosylcysteine amide; 0.1 μm), and the disulfide product (YCysCysY; 150 μm) were detected at 16, 18, 29, and 32 min, respectively (Fig. 1, A-C). Incubation of YC peptide with MPO, H2O2, and NaNO2 in a phosphate buffer (100 mm, pH 7.4) containing DTPA (100 μm) for 30 min at room temperature failed to yield detectable levels of nitrated tyrosine or the dityrosyl product (Fig. 1, A and C). However, upon UV-visible analysis at 280 nm, a new product eluting at 32 min was detected (Fig. 1A, a-d). By comparison with the HPLC profile of the authentic YC disulfide, the peak detected at 32 min was attributed to YCysCysY, a disulfide formed from YC. Incubations of YC with MPO/H2O2 also yielded YCysCysY but no dityrosine peptide (bis-N-acetyl tyrosylcysteine amide) (Fig. 1C, a-e). In the presence of H2O2 alone, a slight increase in disulfide YCysCysY was detected (Fig. 1A, a) due to H2O2-dependent oxidation of cysteine to the corresponding disulfide. These data are in contrast to the results obtained with tyrosine alone in the MPO/H2O2/NO2- system, where a substantial but significant increase in nitrotyrosine and dityrosine formation was noted (see TABLE TWO).TABLE TWONitration and oxidation products of YC dipeptide and other model peptidesExperimental conditionsaAll reactions were incubated at room temperature for 0.5 h, and concentrations were determined by HPLCProductYCMPONaNO2H2O2YCYCysCysYY (NO2)CCYYCμmnmμmμmμm300305005055 ± 20115 ± 12NDbND, not detectedND30030050182 ± 761 ± 10NDND3000050271 ± 1117 ± 4NDND3003050010041 ± 12120 ± 10NDND300300100129 ± 1871 ± 4NDND30000100252 ± 1130 ± 6NDNDYC-SSCH3 (300 μm)30500100ND22 ± 71.4 ± 0.1Tyr (300 μm)3050010023 ± 25.3 ± 0.1YAC (300 μm)3050050117 ± 193 ± 8NDNDYAC (300 μm)30050216 ± 339 ± 1NDNDYAC (300 μm)0050277 ± 718 ± 1NDNDYAAC (300 μm)3050050215 ± 242 ± 2NDNDYAAC (300 μm)30050248 ± 225 ± 1NDNDYAAC (300 μm)0050278 ± 312 ± 2NDNDYAAAAC (300 μm)3050050254 ± 322 ± 2NDNDYAAAAC (300 μm)30050273 ± 712 ± 3NDNDYAAAAC (300 μm)0050283 ± 38 ± 2NDNDa All reactions were incubated at room temperature for 0.5 h, and concentrations were determined by HPLCb ND, not detected Open table in a new tab Additional confirmation of disulfide formation in this system (i.e. YC/MPO/H2O2/NO2-) was obtained by incubating the reaction mixture with β-mercaptoethanol. As shown in Fig. 1A, the disulfide YCysCysY was reduced back to the parent YC peptide. To further demonstrate that YC disulfide is the only oxidation product formed in this system, an additional aliquot of fresh reagents (MPO/H2O2/NaNO2) was added to the original reaction mixture after 30 min and incubated further for another 30 min. HPLC analysis did not reveal any other products other than the disulfide YCysCysY (Fig. 1A). Further confirmation of the product analysis was obtained by mass spectrometry. Fig. 1A (trace d, inset) shows the HPLC/ESI-MS analysis of the incubation mixture obtained 30 min after oxidation of YC by MPO/H2O2/NaNO2. The peak eluting at 32 min in Fig. 1A was analyzed by HPLC/ESI-MS. The mass spectral analysis of this peak is identical to the m/z pattern of the authentic YC disulfide (M + H+: 649; M + 2H+: 325). Incubation of other model peptides (YAC, YAAC, and YAAAAC) with MPO/H2O2/NaNO2 also yielded exclusively the corresponding disulfide products (i.e. YACysCysAY, YAACysCysAAY, and YAAAACysCysAAAAY) with little or no formation of nitrated and dityrosyl products; as shown in TABLE TWO, the yields of disulfide decreased with increasing peptide chain lengths. These results indicate that MPO/H2O2/NaNO2-dependent oxidation of YC and related homologs yields the corresponding disulfide as the only major product and that the extent of formation of nitrated tyrosine and dityrosine oxidation products was negligible (TABLE TWO). Intermediacy of Thiyl Radicals Formed during MPO/H2O2/NO2--dependent Oxidation of Tyrosylcysteine Model Peptides—To investigate whether MPO/H2O2/NaNO2 induces formation of disulfide from YC, YAC, YAAC, or YAAAAC peptides via a radical mechanism, we used the DMPO spin trap to detect the corresponding thiyl radicals (38Saez G. Thornalley P.J. Hill H.A. Hems R. Bannister J.V. Biochim. Biophys. Acta. 1982; 719: 24-31Crossref PubMed Scopus (245) Google Scholar, 39Harman L.S. Mottley C. Mason R.P. J. Biol. Chem. 1984; 259: 5606-5611Abstract Full Text PDF PubMed Google Scholar). DMPO-thiyl radical adducts exhibit a distinct EPR spectral pattern (38Saez G. Thornalley P.J. Hill H.A. Hems R. Bannister J.V. Biochim. Biophys. Acta. 1982; 719: 24-31Crossref PubMed Scopus (245) Google Scholar, 39Harman L.S. Mottley C. Mason R.P. J. Biol. Chem. 1984; 259: 5606-5611Abstract Full Text PDF PubMed Google Scholar, 40Kalyanaraman B. Karoui H. Singh R.J. Felix C.C. Anal. Biochem. 1996; 241: 75-81Crossref PubMed Scopus (53) Google Scholar, 41Karoui H. Hogg N. Frejaville C. Tordo P. Kalyanaraman B. J. Biol. Chem. 1996; 271: 6000-6009Abstract Full Text PDF PubMed Scopus (216) Google Scholar). Incubation of YC with MPO, H2O2, and NaNO2 in a phosphate buffer containing DTPA (100 μm) yielded a four-line EPR spectrum (Fig. 2A) with the hyperfine coupling constants, αN = 15.2 G and αH = 16.2 G, that are similar to the values reported for the DMPO-glutathionyl adduct (αN = 15.2 G and αH = 16.4 G). Pretreatment of the YC peptide with N-ethylmaleimide, a thiol-blocking reagent, inhibited the DMPO-thiyl adduct formation. To verify that tyrosyl residue is required for thiyl radical formation in YC peptide, we performed the oxidation of a dipeptide, FC (tyrosine replaced by a phenylalanine) in the MPO/H2O2/NO2- system. Oxidation of FC in MPO/H2O2 and DMPO did not yield a detectable ESR signal (Figs. 2A and 3C). A small amount of DMPO-SCysF adduct (αN = 15.1 G and αH = 16.3 G) detected in the presence of added NO2- was presumably from ·NO2-mediated oxidation of the" @default.
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• W2077386186 title "Intramolecular Electron Transfer between Tyrosyl Radical and Cysteine Residue Inhibits Tyrosine Nitration and Induces Thiyl Radical Formation in Model Peptides Treated with Myeloperoxidase, H2O2, and NO2-" @default.
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