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• W1977448081 abstract "The objective of this study was to identify a potential mechanism for S-nitrosation of proteins. Therefore, we assessed S-nitrosation of bovine serum albumin by dinitrosyl-iron-di-L-cysteine complex [(NO)2Fe(L-cysteine)2], a compound similar to naturally occurring iron-nitrosyls. Within 5-10 min, (NO)2Fe(L-cysteine)2 generated paramagnetic albumin-bound dinitrosyl-iron complex and S-nitrosoalbumin in a ratio of 4:1. Although S-nitroso-L-cysteine was concomitantly formed in low amounts, its concentration was not sufficient to account for formation of S-nitrosoalbumin via a trans-S-nitrosation reaction. Low oxygen tension did not affect S-nitrosation by the dinitrosyl-iron complex thus excluding the involvement of oxygenated NOx-species in the nitrosation reaction. Blockade of albumin histidine residues by pyrocarbonate, which prevented formation of dinitrosyl-iron-albumin complex, did not inhibit S-nitrosation of albumin. Thus, S-nitrosation of albumin by (NO)2Fe(L-cysteine)2 can proceed by direct attack of a nitrosyl moiety on the protein thiolate, without previous binding of the iron. We conclude that protein-bound dinitrosyl-iron complexes detected in high concentrations in certain tissues provide a reservoir of S-nitrosating species, e.g. low molecular dinitrosyl iron complexes. The objective of this study was to identify a potential mechanism for S-nitrosation of proteins. Therefore, we assessed S-nitrosation of bovine serum albumin by dinitrosyl-iron-di-L-cysteine complex [(NO)2Fe(L-cysteine)2], a compound similar to naturally occurring iron-nitrosyls. Within 5-10 min, (NO)2Fe(L-cysteine)2 generated paramagnetic albumin-bound dinitrosyl-iron complex and S-nitrosoalbumin in a ratio of 4:1. Although S-nitroso-L-cysteine was concomitantly formed in low amounts, its concentration was not sufficient to account for formation of S-nitrosoalbumin via a trans-S-nitrosation reaction. Low oxygen tension did not affect S-nitrosation by the dinitrosyl-iron complex thus excluding the involvement of oxygenated NOx-species in the nitrosation reaction. Blockade of albumin histidine residues by pyrocarbonate, which prevented formation of dinitrosyl-iron-albumin complex, did not inhibit S-nitrosation of albumin. Thus, S-nitrosation of albumin by (NO)2Fe(L-cysteine)2 can proceed by direct attack of a nitrosyl moiety on the protein thiolate, without previous binding of the iron. We conclude that protein-bound dinitrosyl-iron complexes detected in high concentrations in certain tissues provide a reservoir of S-nitrosating species, e.g. low molecular dinitrosyl iron complexes. S-Nitrosation of protein thiols by L-arginine- and nitrovasodilator-derived NO and/or subsequent further reaction of the S-nitrosothiol has been proposed to initiate widespread biological signal and effector pathways(1Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Google Scholar, 2Stamler J.S. Cell. 1994; 78: 931-936Google Scholar, 3Brüne B. Dimmler S. Molina y Vedia L. Lapetina E.G. Life Sci. 1994; 54: 61-70Google Scholar). Direct evidence has been provided that a small fraction of circulating serum albumin in human and dogs is S-nitrosated under physiological conditions(4Stamler J.S. Jaraki O. Osborne J. Simon D.I. Keaney J. Vita J. Singel D. Valeri C.R. Loscalzo J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7674-7677Google Scholar). Furthermore, indirect evidence suggests that S-nitrosation modulates the function of proteases, cytoskeletal proteins, membrane receptors(5Lipton S.A. Choi Y.-B. Pan Z.-H. Lei S.Z. Chen H.-S.V. Sucher N.J. Loscalzo J. Singel D. Stamler J.S. Nature. 1993; 364: 626-632Google Scholar), membrane ion channels(6Bolotina V.M. Najibi S. Palacino J.J. Pagano P.J. Cohen R.A. Nature. 1994; 368: 850-853Google Scholar), GTP-binding proteins(7Lander H.M. Sehajpal P.K. Novogrodsky A. J. Immunol. 1993; 151: 7182-7187Google Scholar), protein kinases(8Gopalakrishna R. Chen Z.H. Gundimeda U. J. Biol. Chem. 1993; 268: 27180-27185Google Scholar), phosphotyrosine protein phosphatases(9Caselli A. Camici G. Manao G. Moneti G. Pazzagli L. Cappugi G. Ramponi G. J. Biol. Chem. 1994; 269: 24878-24882Google Scholar), transcription factors(10Lander H.M. Sehajpal P.K. Levine D.M. Novogrodsky A. J. Immunol. 1993; 150: 1509-1515Google Scholar, 11Henderson S.A. Lee P.H. Aeberhard E.E. Adams J.W. Ignarro L.J. Murphy W.J. Sherman M.P. J. Biol. Chem. 1994; 269: 25239-25242Google Scholar), and glutathione reductase(12Becker K. Gui M. Schirmer R.H. Eur. J. Biochem. 1995; (in press)Google Scholar). However, the mechanism by which proteins are S-nitrosated in vivo is unknown. It is likely that nitrosation proceedes by electrophilic attack of a nitrosyl-cation (NO+) or a partially positively charged NO (NOδ+) on a sufficiently reactive nucleophilic thiol group within target proteins(1Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Google Scholar). It has been proposed that naturally occurring metabolites of NO, such as peroxynitrite (ONOO-), nitrogen dioxide (NO2), or dinitrogentrioxide (N2O3), account for the S-nitrosation(1Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Google Scholar, 2Stamler J.S. Cell. 1994; 78: 931-936Google Scholar). However, ONOO- was shown to oxidize thiols primarily by a mechanism not involving an S-nitrosothiol as an intermediate(13Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Google Scholar, 14Wink D. Nims R.W. Darbyshire J.F. Christodoulou D. Hanbauer I. Cox G.W. Laval F. Laval J. Cook J.A. Krishna M.C. DeGraff W.G. Mitchell J.B. Chem. Res. Toxicol. 1994; 7: 519-525Google Scholar). Furthermore, it is not known whether the steady state levels of NO2 and N2O3 attained in NO generating tissues are sufficiently high to account for biological S-nitrosation. It is therefore possible that other endogenous nitrosating species are operative. Since artificial metal-nitrosyls, like sodium nitroprusside, perform nitrosation of nucleophils (15Bottomley F. Accts. Chem. Res. 1978; 11: 158-163Google Scholar) we speculated that endogenous iron-nitrosyl complexes might do so as well(16Mülsch A. Drug Res. 1994; 44 (408–411.): 3aGoogle Scholar). These comprise so called dinitrosyl-iron complexes, which are generated by various cells and tissues concomitantly with NO(17Hibbs J.B. Taintor R.R. Vavrin Z. Granger D.L. Drapier J. Amber I.J. Lancaster J.R. Nitric Oxide from L -Arginine: A Bioregulatory System. Elsevier Science, Amsterdam1990: 189-224Google Scholar, 18Drapier J. Pellat C. Henry Y. J. Biol. Chem. 1991; 266: 10162-10167Google Scholar, 19Lancaster J.R. Hibbs J.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1223-1227Google Scholar, 20Stadler J. Bergonia H.A. Di Silvio M. Sweetland M.A. Billiar T.R. Simmons R.L. Lancaster J.R. Arch. Biochem. Biophys. 1993; 302: 4-11Google Scholar, 21Mülsch A. Mordvintcev P. Vanin A. Busse R. Biochem. Biophys. Res. Commun. 1993; 196: 1303-1308Google Scholar, 22Vanin A.F. Mordvintcev P.I. Hauschildt S. Mülsch A. Biochim. Biophys. Acta. 1993; 1177: 37-42Google Scholar, 23Lancaster J.R. Werner-Felmayer G. Wachter H. Free Rad. Biol. Med. 1994; 16: 869-870Google Scholar, 24Bastian N.R. Yim C.Y. Hibbs J.B. Samlowski W.E. J. Biol. Chem. 1994; 269: 5127-5131Google Scholar). The paramagnetic dinitrosyl-iron moiety is attached to as yet unidentified proteins(25Woolum J.C. Commoner B. Biochim. Biophys. Acta. 1970; 201: 131-140Google Scholar, 26Vanin A.F. Osipov A.N. Kubrina L.N. Burbaev D.S. Nalbandyan R.M. Studia Biophys. 1975; 49: 13-25Google Scholar), primarily via protein thiols and histidines, as judged from the characteristics of the EPR signals(21Mülsch A. Mordvintcev P. Vanin A. Busse R. Biochem. Biophys. Res. Commun. 1993; 196: 1303-1308Google Scholar, 22Vanin A.F. Mordvintcev P.I. Hauschildt S. Mülsch A. Biochim. Biophys. Acta. 1993; 1177: 37-42Google Scholar, 27McDonald C.C. Philips W.D. Mower H.F. J. Am. Chem. Soc. 1965; 87: 3319-3326Google Scholar, 28Woolum J.C. Tiezzi E. Commoner B. Biochim. Biophys. Acta. 1968; 160: 311-320Google Scholar, 29Burbaev D.S. Vanin A.F. Blumenfeld L.A. Zhurnal Strukt. Chimii (USSR). 1971; 12: 252-258Google Scholar). With regard to a ligand-exchange equilibrium between low mass thiol and protein thiol ligands(13Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Google Scholar, 21Mülsch A. Mordvintcev P. Vanin A. Busse R. Biochem. Biophys. Res. Commun. 1993; 196: 1303-1308Google Scholar, 22Vanin A.F. Mordvintcev P.I. Hauschildt S. Mülsch A. Biochim. Biophys. Acta. 1993; 1177: 37-42Google Scholar, 30Vanin A.F. Kiladze S.V. Kubrina L.N. Biofizika (USSR). 1975; 20: 1068-1073Google Scholar), it is conceivable that low mass dinitrosyl-iron complexes exist biologically, albeit at low steady state levels. For instance, the addition of cell membrane-permeable low mass thiols effects intracellular mobilization and transmembraneous release of the dinitrosyl-iron moiety from intracellular proteins of NO-generating cells(21Mülsch A. Mordvintcev P. Vanin A. Busse R. Biochem. Biophys. Res. Commun. 1993; 196: 1303-1308Google Scholar, 22Vanin A.F. Mordvintcev P.I. Hauschildt S. Mülsch A. Biochim. Biophys. Acta. 1993; 1177: 37-42Google Scholar, 30Vanin A.F. Kiladze S.V. Kubrina L.N. Biofizika (USSR). 1975; 20: 1068-1073Google Scholar, 31Mülsch A. Mordvintcev P.I. Vanin A.F. Busse R. FEBS Lett. 1991; 294: 252-256Google Scholar). In oxygenated aqueous media the biological half-life of the low molecular forms is inversely proportional to their concentration, amounting to a few seconds below 1 μM, and to about 10 min at 100 μM, for instance(31Mülsch A. Mordvintcev P.I. Vanin A.F. Busse R. FEBS Lett. 1991; 294: 252-256Google Scholar). In contrast, proteinacious dinitrosyl-iron complexes are stable for hours in the absence of low mass thiols. According to theoretical considerations, the dinitrosyl-iron complex and related di- and tetranucleated structures bear a positive charge on the NO(29Burbaev D.S. Vanin A.F. Blumenfeld L.A. Zhurnal Strukt. Chimii (USSR). 1971; 12: 252-258Google Scholar, 32Butler A.R. Glidewell C. Li M.-H. Adv. Inorg. Chem. 1988; 32: 335-393Google Scholar). The overall charge of the complex with two thiolate ligands is -1(32Butler A.R. Glidewell C. Li M.-H. Adv. Inorg. Chem. 1988; 32: 335-393Google Scholar), if the charge of the thiolate residues is zero or neutralized (at isosbestic pH of L-cysteine, for instance). Thus, its paramagnetism is best explained by assuming a d7 electron configuration at the central iron ion (effective spin system S = 1/2). However, the formal oxidation state of the iron will be -1 if the NO ligands take pure nitrosyl character (NO+). In reality, some back donation of electron density to NO might decrease its electrophilicity, which then carries only a partial positive charge (δ+), similar to the situation with mononitrosyl-metal complexes (33Bottomley F. Accts. Chem. Res. 1978; 11: 158-163Google Scholar). Thus, this complex can be regarded as a natural source of NO+ or NOδ+, capable of S-nitrosation reactions according to Fig. Z1(RS = low mass thiol; R′SH = low mass or protein thiol). To test this hypothesis we assessed S-nitrosation of the free cysteine residue of bovine serum albumin (BSA) 1The abbreviations used are: BSAbovine serum albuminDEPCdiethylpyrocarbonateDNICdinitrosyl-iron-di-L-cysteine. by the dinitrosyl-iron-di-L-cysteine complex. We observed that this complex S-nitrosates albumin, in support of our hypothesis that dinitrosyl-iron complexes are intermediates in the biological NO:S-nitrosothiol pathway. bovine serum albumin diethylpyrocarbonate dinitrosyl-iron-di-L-cysteine. Bromophenol blue, fatty acid-free BSA (96-99% pure), L-cysteine, diethylpyrocarbonate (DEPC), EDTA sodium salt, glutathione, N-ethylmaleimide, Sephadex G-25 were supplied by Sigma, Deisenhofen, Germany. Xylene cyanole was from Roth, Karlsruhe, Germany. NO gas was obtained by reaction of FeSO4× 7H2O (Fluka, Buchs, Switzerland) with NaNO2 in 5 N HCl and was purified by low temperature high vacuum (p = 0.01 mm Hg) distillation (31Mülsch A. Mordvintcev P.I. Vanin A.F. Busse R. FEBS Lett. 1991; 294: 252-256Google Scholar). Dinitrosyl-iron complex was prepared by mixing evacuated (10 min high vacuum) solutions of FeSO4 (5 mg/ml) and neutralized L-cysteine (72 mM) in a Thunberg-type reaction vessel under an atmosphere of pure NO gas (PNO 500 mm Hg; the NO was added 3 min before mixing). Upon mixing the solution immediately turned green. After 1 min the solution was evacuated for 2 min by high vacuum to remove excessive NO, immediately frozen, and stored in liquid nitrogen. The molar ratio of Fe2+ to L-cysteine was 1:20. S-Nitroso-L-cysteine and S-nitroso-BSA were synthetized by mixing at 20°C either L-cysteine (100 mM) or fatty acid-free BSA (2 mM) with an equimolar amount of sodium nitrite in 0.5 M H2SO4 for 5 min. The S-nitrosothiols were frozen and stored at -70°C until use. The yields of S-nitroso-L-cysteine and S-nitroso-BSA were 90 and 100% with respect to free thiol added (BSA-thiol/BSA = 0.4 ± 0.04 mol/mol), as calculated from the typical S-nitrosothiol absorbance spectra, using the reported molar absorption coefficients: S-nitroso-L-cysteine, ɛ338 = 940 and ɛ545 = 22 M-1 cm-1 (MeOH; 34); and S-nitroso-BSA, ɛ334 = 870 (H2O; 35). BSA-thiols were titrated by 5,5′-dithiobis(2-nitrobenzoic acid)(36Riddles P.W. Blakeley R.L. Zerner B. Methods Enzymol. 1983; 91: 49-60Google Scholar), using the absorbance of the thionitrobenzoate-anion at 412 nm (ɛ = 1.36 × 104M-1 cm-1) as a quantitative measure of thiol concentration. Protein was determined by the Bio-Rad method, using BSA as a standard. UV visible spectra were recorded on a Kontron 941 Plus spectrometer. S-Nitrosothiols were assessed by diazotization of β-naphthol and azo-coupling with N,N-ethylenediamine in the presence and absence of Hg2+ ions (3 mM) according to Saville(37Saville B. Analyst. 1958; 83: 670-672Google Scholar). Hg2+ releases NO+ from S-nitrosothiols(37Saville B. Analyst. 1958; 83: 670-672Google Scholar), which under acid conditions gives a positive Griess reaction, similar to acidified nitrite. The concentration of the red azo-compound was determined after 15 min by measuring the absorption at 570 nm on a microplate reader (MR 600, Dynatech, Alexandria, VA). In each experiment S-nitrosothiol/nitrite were determined in quadruplicate. The content of S-nitrosothiol accounted for the mean difference of absorption readings of Hg2+-containing versus Hg2+-free samples. A calibration curve was established in each experiment with freshly synthesized S-nitroso-L-cysteine and sodium nitrite as standards (1-100 μM). The detection limit was 1 μM. In some experiments the mixture of BSA, DNICs and the Griess reagent was frozen at specific time points and analyzed by EPR spectroscopy. Nitrate was determined after its reduction to nitrite by means of a cadmium reductor (38Green L.C. Wagner D.A. Glogowski J. Skipper P.L. Wishnok J.S. Tannenbaum S.R. Anal. Biochem. 1982; 126: 131-138Google Scholar) and then assessed by the Griess reaction as described above. Electrophoresis of DNIC was performed at 5°C in a horizontal 1% (w/v) agarose slab gel (20 mM KH2PO4 buffer, pH 7.5 or 6.5). 70 mML-cysteine was included in the gel buffer to prevent decomposition of DNIC. Freshly thawed stock solutions of the complex (40 μl) were loaded in slots at equidistant positions from the electrodes in the middle of the gel. The anionic dyes bromphenol blue and xylene cyanole were loaded in separate slots. Electrophoresis was started immediately at 50 V. Electrophoretic mobility relative to the anionic dyes was estimated from the migration of the green band of DNIC after 1 h. Thereafter the band corresponding to DNIC was excised, frozen, and analyzed by EPR spectroscopy. EPR spectra were recorded on a Bruker EPR 300E spectrometer at about 80 K with frozen aqueous solutions (0.6 ml) introduced into a quartz Dewar (5-mm inner diameter) chilled with liquid nitrogen. Some samples (25 μl of aqueous solution) were also recorded at 293 K in a quartz capillary tube (1 mm inner diameter). The EPR instrument was operated at a microwave frequency of 9.60 GHz, microwave power 20 mW, modulation frequency 100 kHz, modulation amplitude 5 Gauss, time constant 0.1-1.3 s. The concentration of DNIC was calculated by comparison with the EPR signal of a DNIC-L-cysteine standard based on double integration of the first derivative EPR signals(31Mülsch A. Mordvintcev P.I. Vanin A.F. Busse R. FEBS Lett. 1991; 294: 252-256Google Scholar). The single thiol group accessible on BSA was blocked by incubation of the protein (2 mM in 0.1 M potassium Pi buffer, pH 7.4) for 5 min at 20°C with either 20 mMN-ethylmaleimide, or for 30 min with 6 mM HgCl2(13Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Google Scholar). Modification of histidine residues was performed by adding a 10-fold molar excess of DEPC and subsequent incubation for 2 h at 20°C(39Lee M. Arosio P. Cozzi A. Chasteen N.D. Biochemistry. 1994; 33: 3679-3687Google Scholar). In control experiments it was verified that DEPC did not alter the EPR spectrum of low mass DNIC, indicating that both compounds did not directly interact. In frozen state at 77 K, DNIC exhibited an anisotropic EPR signal of axial symmetry with g factors g⊥ = 2.04 and g‖ = 2.01 (Fig. 1a)(28Woolum J.C. Tiezzi E. Commoner B. Biochim. Biophys. Acta. 1968; 160: 311-320Google Scholar, 29Burbaev D.S. Vanin A.F. Blumenfeld L.A. Zhurnal Strukt. Chimii (USSR). 1971; 12: 252-258Google Scholar, 30Vanin A.F. Kiladze S.V. Kubrina L.N. Biofizika (USSR). 1975; 20: 1068-1073Google Scholar, 31Mülsch A. Mordvintcev P.I. Vanin A.F. Busse R. FEBS Lett. 1991; 294: 252-256Google Scholar). In liquid phase at 20°C this spectrum was transformed into a narrow isotropic signal at gav = 2.03 with a 13 line hyperfine structure (Fig. 1e). The isotropy results from the high tumbling rate of the small paramagnetic molecule in liquid phase, which averages out any anisotropy of g values. The small coupling constant (A = 0.07 millitesla) and the absence of further fine structures points to a low spin configuration (S = 1/2). The unpaired electron is mainly localized on the iron atom(27McDonald C.C. Philips W.D. Mower H.F. J. Am. Chem. Soc. 1965; 87: 3319-3326Google Scholar, 29Burbaev D.S. Vanin A.F. Blumenfeld L.A. Zhurnal Strukt. Chimii (USSR). 1971; 12: 252-258Google Scholar, 32Butler A.R. Glidewell C. Li M.-H. Adv. Inorg. Chem. 1988; 32: 335-393Google Scholar). The hyperfine splitting results from the interaction of the unpaired electron with four protons of the methylene groups of both L-cysteines and two nitrogen nuclei of the NO groups. Similar EPR spectra were obtained if DNIC was synthesized with glutathione or N-acetyl-L-cysteine instead of L-cysteine (data not shown). In agarose gel electrophoresis conducted at pH 6.5 (the isoelectric pH of L-cysteine) DNIC moved toward the anode, indicating that DNIC carries a net negative charge. The electrophoretic mobility of the complex was 0.38 times that of the anionic dyes bromphenol blue and 0.85 times that of xylene cyanol, which migrated 2.9 and 1.3 cm/h, respectively. The electrophoretic mobility of DNIC increased at pH 7.5, presumably due to increased negative charge on the L-cysteine-carboxylates. EPR spectroscopic analysis of the gel band bearing DNIC revealed the pure EPR spectrum of DNIC. Therefore, DNIC seems to migrate in the electric field as an intact complex anion. To assess its decomposition kinetics, DNIC was incubated at 37°C for defined periods and was then quickly frozen for cryogenic EPR analysis. The rate of decomposition of DNIC, as estimated from the decrease in the EPR signal intensity, was inversely correlated to the initial concentration of DNIC (Fig. 2). DNIC reacted immediately with BSA to form a paramagnetic protein-bound dinitrosyl-iron complex, amounting to about 0.4 mol of DNIC bound per mol of BSA. In the frozen state this BSA-DNIC exhibited an EPR spectrum identical to that of low mass DNIC (Fig. 1a). The macromolecular nature of the paramagnetic species was confirmed by showing that, in contrast to the EPR signal of low mass DNIC (Fig. 1e), the anisotropy of the BSA-DNIC EPR signal did not change in liquid phase at room temperature (data not shown). This preservation of anisotropy in liquid media is due to the slow rotation rate of paramagnetic macromolecules. According to its characteristic EPR features the dinitrosyl-iron moiety must be attached to two nuclear equivalent ligands(25Woolum J.C. Commoner B. Biochim. Biophys. Acta. 1970; 201: 131-140Google Scholar, 26Vanin A.F. Osipov A.N. Kubrina L.N. Burbaev D.S. Nalbandyan R.M. Studia Biophys. 1975; 49: 13-25Google Scholar, 27McDonald C.C. Philips W.D. Mower H.F. J. Am. Chem. Soc. 1965; 87: 3319-3326Google Scholar, 28Woolum J.C. Tiezzi E. Commoner B. Biochim. Biophys. Acta. 1968; 160: 311-320Google Scholar, 29Burbaev D.S. Vanin A.F. Blumenfeld L.A. Zhurnal Strukt. Chimii (USSR). 1971; 12: 252-258Google Scholar, 30Vanin A.F. Kiladze S.V. Kubrina L.N. Biofizika (USSR). 1975; 20: 1068-1073Google Scholar). In the case of BSA-DNIC, these ligands are L-cysteines, one of which is the single reduced cysteinyl-thiol available on native BSA (cysteine 34), the other is provided by free L-cysteine. The apparently subequimolar recovery of BSA-DNIC (0.4 mol/mol BSA) resulted from the limited availability of cysteine 34, which is masked by disulfide formation with glutathione, L-cysteine, and BSA dimer formation in commercial BSA preparations(40Jocelyn P.C. Biochemistry of the SH Group. Academic Press, London1972Google Scholar). This was confirmed by titration of BSA with 5,5′-dithiobis(2-nitrobenzoic acid). When BSA-DNIC was passed over a desalting column (Sephadex G-25) to remove excessive L-cysteine (DNIC was synthesized with 20 mol of L-cysteine per mol of iron, although only two L-cysteines are directly included into the complex), the EPR signal of BSA-DNIC changed to rhombic symmetry with g factors g1 = 2.05, g2- = 2.04, and g3 = 2.01 (Fig. 1b). These EPR features were reminiscent of dinitrosyl-iron complexes with one non-thiol ligand, presumably a histidine-imidazole, as described previously(28Woolum J.C. Tiezzi E. Commoner B. Biochim. Biophys. Acta. 1968; 160: 311-320Google Scholar, 29Burbaev D.S. Vanin A.F. Blumenfeld L.A. Zhurnal Strukt. Chimii (USSR). 1971; 12: 252-258Google Scholar, 30Vanin A.F. Kiladze S.V. Kubrina L.N. Biofizika (USSR). 1975; 20: 1068-1073Google Scholar). If the free thiol group of BSA was blocked by N-ethylmaleimide, or the preformed BSA-DNIC was treated with HgCl2, another rhombic EPR signal was generated, with g factor values g1 = 2.055, g2 = 2.035, and g3 = 2.01 (Fig. 1c). These EPR features were similar to that of imidazole-ligated DNIC(28Woolum J.C. Tiezzi E. Commoner B. Biochim. Biophys. Acta. 1968; 160: 311-320Google Scholar, 29Burbaev D.S. Vanin A.F. Blumenfeld L.A. Zhurnal Strukt. Chimii (USSR). 1971; 12: 252-258Google Scholar, 30Vanin A.F. Kiladze S.V. Kubrina L.N. Biofizika (USSR). 1975; 20: 1068-1073Google Scholar). Thus, it can be inferred that the dinitrosyl-iron moiety was attached to the thiol-blocked BSA via histidine nitrogen atoms. If, on the other hand, the histidine-imidazole residues of BSA were carboxylated by DEPC, formation of BSA-DNIC was completely abolished (data not shown). This finding suggests that the carboxylated histidine-imidazole restricts access of DNIC to the free thiol group of BSA and is compatible with structures of the diverse BSA-DNIC isoforms as shown in Fig. Z2. In the presence of acid (0.5 N HCl), DNIC and BSA-DNIC rapidly transformed into a paramagnetic complex of rhombic symmetry with g factor values g1 = 2.06, g2 = 2.05, and g3 = 2.02 (Fig. 1d). Formation of this complex was not affected by the Griess reagent. The “acid” complex formed by 40 μM DNIC decayed by apparent first order kinetics with a half-life of 20.4 ± 1.3 min (n = 8). HgCl2 did not affect the formation, stability, and EPR features of this complex (data not shown). To identify NOx from decomposed DNIC the latter was incubated at 37°C in 100 mM potassium Pi (pH 7.4) for defined periods and then assessed for nitrate, nitrite, and S-nitrosothiol (see “Experimental Procedures”). DNIC (20 μM in iron) generated 30 ± 2 μM NO2 after 3 min and 40 ± 1 μM after 30 min of incubation (n = 3). A freshly thawed stock solution of DNIC (3.6 mM) contained less than 0.5% S-nitrosothiol positive material, which waned within 3 min. Nitrate was not detectable at any time. These findings demonstrate that DNIC generates exclusively NO2 as a stable NOx metabolite. DNIC (0.75 mM) and BSA (1 mM) at 37°C rapidly and transiently generated an S-nitrosothiol, with a maximal concentration (85 ± 12 μM; n = 5) achieved at 5-8 min after mixing both reactants (Fig. 3). A similar transient S-nitrosation (maximally 40 ± 8 μMS-nitrosothiol) was observed after mixing DNIC (0.75 mM) with glutathione (20 mM). To study the influence of L-cysteine introduced with the DNIC stock solution on S-nitrosation of BSA, BSA-DNIC was generated as described before and then desalted by means of a Sephadex G-25 column (1.6 × 8 cm; 0.1 M potassium Pi buffer, pH 7.0). All manipulations were conducted at 4°C to decelerate the S-nitrosation reaction, although some S-nitrosation during the procedure could not be avoided. The protein fraction was quickly aliquoted, one aliquot was taken for EPR analysis to confirm the presence of BSA-DNIC, the other aliquot was halved and incubated in the presence and absence of 20 mML-cysteine. A low amount of S-nitrosoprotein co-eluted from the column (t = 0 min value) with BSA-DNIC, but did not further increase upon incubation at 37°C in the absence of L-cysteine (open symbols; Fig. 4). In contrast, the subsequent addition of L-cysteine largely enhanced formation of S-nitrosothiol by a time course similar to that shown in Fig. 3(closed symbols; Fig. 4). To ascertain that BSA and not a low mass thiol was S-nitrosated by DNIC, a preincubated (5 min, 37°C) mixture (0.8 ml) of BSA (1 mM) and DNIC (0.4 mM) was fractionated at 4°C into protein and low mass constituents by the aforementioned desalting column technique. Individual fractions (1 ml) were immediately assessed for S-nitrosothiol and protein. As shown in Fig. 5, the S-nitrosothiol eluted mainly with the protein fraction (27 ± 3% recovery with respect to DNIC), and only a minor part was associated with the low mass (salt) fraction (5 ± 2% recovery). Low mass S-nitrosothiols are principally capable of transnitrosation reactions according to the following equilibrium(35Meyer D.J. Kramer H. Özer N. Coles B. Ketterer B. FEBS Lett. 1994; 345: 177-180Google Scholar, 41Barnett D.J. McAninly J. Williams D.L.H. J. Chem. Soc. Perkin. Trans. 1994; 2: 1131-1133Google Scholar, 42Arnelle D.R. Stamler J.S. Arch. Biochem. Biophys. 1995; 318: 279-285Google Scholar) : R1SNO+R2S−⇔R1S−+R2SNO(Eq. 1) To assess how efficiently low mass S-nitrosothiols could accomplish trans-S-nitrosation, BSA (0.5 mM) was incubated for 5 min with S-nitroso-L-cysteine and S-nitrosoglutathione (0.5 mM). This mixture (0.8 ml) was passed through the Sephadex column to analyze the protein and salt fraction for S-nitrosoprotein and low mass S-nitrosothiol, respectively. Significant trans-S-nitrosation of BSA (54 ± 4 nmol; n = 3) was detected with S-nitroso-L-cysteine, accounting for about 13% of the S-nitroso-L-cysteine added. About 26% of the S-nitroso-L-cysteine was recovered in the salt fraction (Fig. 6), and the remainder (about 60%) of S-nitroso-L-cysteine had decomposed to nitrite. In contrast, S-nitrosoglutathione added was recovered entirely in the salt fraction and did not generate measurable amounts of S-nitroso-BSA (data not shown). Addition of 8 mML-cysteine markedly decreased (5 ± 0.6-fold; n = 3) the extent of trans-S-nitrosation of BSA by S-nitroso-L-cysteine. The following experiments aimed to clarify whether or not carboxylation of BSA histidine with DEPC, which leaves the single BSA thiol in a reduced state, affected S-nitrosation of BSA by DNIC. Although formation of BSA-DNIC was reduced by 90%, S-nitrosation of BSA was altered (Fig. 7). These findings show that S-nitrosation of BSA by DNIC does not necessarily depend on previous formation of BSA-DNIC. To exclude the possibility that an oxygenated NOx species (14Wink D. Nims R.W. Darbyshire J.F. Christodoulou D. Hanbauer I. Cox G.W. Laval F. Laval J. Cook J.A. Krishna M.C. DeGraff W.G. Mitchell J.B. Chem. Res. Toxicol. 1994; 7: 519-525Google Scholar) generated by DNIC-derived NO and molecular oxygen accounted for S-nitrosation of BSA, the influence of reduced ambient oxygen tension on this reaction was analyzed. Buffered (100 mM potassium Pi, pH 7.4) solutions of BSA (1 mM) and DNIC (0.4 mM) were evacuated (p < 0.1 mm Hg) separately in a Thunberg flask until bubbling stopped and then mixed for 5 min. The mixture was removed by means of an airtight syringe and loaded on a desalting column, avoiding contact with air. The column was eluted with deoxygenated and nitrogen-saturated buffer and S-nitrosothiol was determined immediately in the eluting protein and salt fractions. The concentration of S-nitroso-BSA achieved in the anaerobic reaction mixture (70.5 ± 17.6 μM) was not significantly different from that obtained in parallel experiments conducted in the presence of ambient oxygen (63.6 ± 7.6 μM; n = 3). The objective of this investigation was to delineate a molecular mechanism accounting for biological S-nitrosation of protein thiols by NO(1Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Google Scholar, 2Stamler J.S. Cell. 1994; 78: 931-936Google Scholar, 3Brüne B. Dimmler S. Molina y Vedia L. Lapetina E.G. Life Sci. 1994; 54: 61-70Google Scholar). NO per se is not capable of this reaction and re" @default.
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