Oxidation and Nitrosation of Thiols at Low Micromolar Exposure to Nitric Oxide

Abstract

Although the nitric oxide (⋅NO)-mediated nitrosation of thiol-containing molecules is increasingly recognized as an important post-translational modification in cell signaling and pathology, little is known about the factors that govern this processin vivo. In the present study, we examined the chemical pathways of nitrosothiol (RSNO) production at low micromolar concentrations of ⋅NO. Our results indicate that, in addition to nitrosation by the ⋅NO derivative dinitrogen trioxide (N2O3), RSNOs may be formed via intermediate one-electron oxidation of thiols, possibly mediated by nitrogen dioxide (⋅NO2), and the subsequent reaction of thiyl radicals with ⋅NO. In vitro, the formation ofS-nitrosoglutathione (GSNO) from ⋅NO and excess glutathione (GSH) was accompanied by the formation of glutathione disulfide, which could not be ascribed to the secondary reaction of GSH with GSNO. Superoxide dismutase significantly increased GSNO yields and the thiyl radical trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), inhibited by 45 and 98% the formation of GSNO and GSSG, respectively. Maximum nitrosation yields were obtained at an oxygen concentration of 3%, whereas higher oxygen tensions decreased GSNO and increased GSSG formation. When murine fibroblasts were exposed to exogenous ⋅NO, RSNO formation was sensitive to DMPO and oxygen tension in a manner similar to that observed with GSH alone. Our data indicate that RSNO formation is favored at oxygen concentrations that typically occur in tissues. Nitrosothiol formation in vivo depends not only on the availability of ⋅NO and O2 but also on the degree of oxidative stress by affecting the steady-state concentration of thiyl radicals. Although the nitric oxide (⋅NO)-mediated nitrosation of thiol-containing molecules is increasingly recognized as an important post-translational modification in cell signaling and pathology, little is known about the factors that govern this processin vivo. In the present study, we examined the chemical pathways of nitrosothiol (RSNO) production at low micromolar concentrations of ⋅NO. Our results indicate that, in addition to nitrosation by the ⋅NO derivative dinitrogen trioxide (N2O3), RSNOs may be formed via intermediate one-electron oxidation of thiols, possibly mediated by nitrogen dioxide (⋅NO2), and the subsequent reaction of thiyl radicals with ⋅NO. In vitro, the formation ofS-nitrosoglutathione (GSNO) from ⋅NO and excess glutathione (GSH) was accompanied by the formation of glutathione disulfide, which could not be ascribed to the secondary reaction of GSH with GSNO. Superoxide dismutase significantly increased GSNO yields and the thiyl radical trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), inhibited by 45 and 98% the formation of GSNO and GSSG, respectively. Maximum nitrosation yields were obtained at an oxygen concentration of 3%, whereas higher oxygen tensions decreased GSNO and increased GSSG formation. When murine fibroblasts were exposed to exogenous ⋅NO, RSNO formation was sensitive to DMPO and oxygen tension in a manner similar to that observed with GSH alone. Our data indicate that RSNO formation is favored at oxygen concentrations that typically occur in tissues. Nitrosothiol formation in vivo depends not only on the availability of ⋅NO and O2 but also on the degree of oxidative stress by affecting the steady-state concentration of thiyl radicals. nitrosothiol reactive nitrogen oxide species high performance liquid chromatography diethylenetriaminepentaacetic acid S-nitrosoglutathione superoxide dismutase spermine NONOate S-Nitrosothiols (RSNOs)1 are biological products formed from the interaction of nitric oxide (⋅NO) with thiol-containing molecules. In recent years, RSNOs have received increasing attention as intermediates in the transport, storage, and delivery of ⋅NO, as post-translational protein modifications in cell signaling and inflammatory processes, and as biochemical markers of reactive nitrogen oxide species (RNOS) (1Scharfstein J.S. Keaney J. Slivka A. Welch G.N. Vita J.A. Stamler J.S. J. Clin. Invest. 1994; 94: 1432-1439Crossref PubMed Scopus (258) Google Scholar, 2Stamler J.S. Lamas S. Fang F.C. Cell. 2001; 106: 675-683Abstract Full Text Full Text PDF PubMed Scopus (1133) Google Scholar). Although critical for understanding their biological roles, the mechanisms by which these⋅NO derivatives are formed in vivo are not clearly understood. Proposed pathways include the reaction of thiols with dinitrosyl-iron or nitrosylheme complexes (3Boese M. Mordvintcev P.I. Vanin A.F. Busse R. Mülsch A. J. Biol. Chem. 1995; 270: 29244-29249Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), direct reaction of thiols with ⋅NO in the presence of an electron acceptor such as NAD+ (4Gow A.J. Buerk D.G. Ischiropoulos H. J. Biol. Chem. 1997; 272: 2841-2845Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar), copper-catalyzed nitrosation (5Stubauer G. Giuffrè A. Sarti P. J. Biol. Chem. 1999; 274: 28128-28133Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and reaction of thiols with peroxynitrite (6Moro M.A. Darley-Usmar V.M. Goodwin D.A. Read N.G. Zamora-Pino R. Feelisch M. Radomski M.W. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6702-6706Crossref PubMed Scopus (337) Google Scholar, 7Van der Vliet A. 't Hoen P.A. Chr. Wong P.S.Y. Bast A. Cross C.E. J. Biol. Chem. 1998; 273: 30255-30262Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 8Balazy M. Kaminski P.M. Mao K. Tan J. Wolin M.S. J. Biol. Chem. 1998; 273: 32009-32015Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Prevalence of certain reaction pathways is likely to be attained within the context of specific proteins where the immediate environment may impact on the reactivity of thiols to RNOS. An increase in hydrophobicity within the core region of a protein, for example, may result in the accumulation of ⋅NO and RNOS with a consequential acceleration of nitrosation reactions within this region (9Rafikova O. Rafikov R. Nudler E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5913-5918Crossref PubMed Scopus (130) Google Scholar). A decrease in thiol pKa secondary to a change in the acid/base equilibrium of the surrounding amino acid residues renders thiols more susceptible to nucleophilic attack by RNOS (10Mannick J.B. Schonhoff C.M. Arch. Biochem. Biophys. 2002; 408: 1-6Crossref PubMed Scopus (96) Google Scholar). In cells, the spatial confinement of protein targets, the presence of hydrophobic domains, and the occurrence of multiple competing reactions that limit the availability of ⋅NO make it difficult to establish the quantitative contribution of the proposed chemical pathways (11Espey M.G. Miranda K.M. Thomas D.D. Wink D.A. J. Biol. Chem. 2001; 276: 30085-30091Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Regardless of their contribution to overall RSNO formation, it is evident that a better understanding of the chemical principles that dictate the outcome of nitrosation reactions within cells is required to interpret the possible significance of differences in RSNO content between different organs and tissues, in particular in view of a highly reducing environment containing millimolar concentrations of glutathione (GSH) and limiting micromolar concentrations of molecular oxygen (O2) (12Wardman P. von Sonntag C. Methods Enzymol. 1995; 251: 31-45Crossref PubMed Scopus (190) Google Scholar). Reactive nitrogen oxide species derived from the reaction of O2 with ⋅NO nitrosate thiols (13Wink D.A. Darbyshire J.F. Nims R.W. Saavedra J.E. Ford P.C. Chem. Res. Toxicol. 1993; 6: 23-27Crossref PubMed Scopus (479) Google Scholar, 14Kharitonov V. Sundquist A.R. Sharma V. J. Biol. Chem. 1995; 270: 28158-28164Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 15Keshive M. Singh S. Wishnok J.S. Tannenbaum S.R. Deen W.M. Chem. Res. Toxicol. 1996; 9: 988-993Crossref PubMed Scopus (114) Google Scholar, 16Goldstein S. Czapski G. J. Am. Chem. Soc. 1996; 118: 3419-3425Crossref Scopus (213) Google Scholar). The reaction is second order with respect to ⋅NO and first order in O2, consistent with the formation of dinitrogen trioxide (N2O3) as the nitrosating agent. 2⋅NO+O2→2⋅NO2Reaction 1 2⋅NO+2⋅NO2→2N2O3Reaction 2 N2O3+RSH→RSNO+NO2−+H+Reaction 3 The oxygen dependence of this process suggests that low O2 concentrations, which are in the range of 1 to 50 μm in most tissues (17Jones D.P. Am. J. Physiol. 1986; 86: C663-C675Crossref Google Scholar), will greatly limit the efficacy of this reaction. The second order of Reaction 1 with regard to⋅NO also dictates that the rate of RSNO formation via Reaction 3 is controlled by the local concentration of ⋅NO and that N2O3 would form quantitatively only when the concentration of ⋅NO rises to the micromolar range (18Beckman J.S. Koppenol W.H. Am. J. Physiol. 1996; 271: C1424-C1437Crossref PubMed Google Scholar).In vivo, conditions associated with the up-regulation of the inducible form of nitric oxide synthase (iNOS) are accompanied by the production of such levels. In addition, the reaction of ⋅NO with O2 is accelerated several hundredfold in the interior of lipid bilayers and proteins, suggesting that hydrophobic environments in cells and tissues may favor the formation of RSNOs via this pathway (9Rafikova O. Rafikov R. Nudler E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5913-5918Crossref PubMed Scopus (130) Google Scholar, 19Liu X. Miller M.J.S. Joshi M.S. Thomas D.D. Lancaster Jr., J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2175-2179Crossref PubMed Scopus (535) Google Scholar). Although the principles governing ⋅NO/O2-mediated nitrosation reactions have been delineated in earlier kinetic studies, the importance of nitrogen dioxide (⋅NO2) as an intermediate in the reaction of thiols with ⋅NO has been ignored. Nitrogen dioxide oxidizes thiols (Reaction 4) such as GSH with a rate constant that is approximately two orders of magnitude smaller than the rate constant for the reaction of ⋅NO2 with⋅NO (2 × 107m−1·s−1 compared with 1.1 × 109m−1·s−1 (20Huie R.E. Toxicology. 1994; 89: 193-216Crossref PubMed Scopus (116) Google Scholar,21Ford E. Hughes M.N. Wardman P. Free Radic. Biol. Med. 2002; 32: 1314-1323Crossref PubMed Scopus (213) Google Scholar)). RSH+⋅NO2→RS⋅+NO2−+H+Reaction 4 Accordingly, previous studies found negligible thiol oxidation by⋅NO and O2 because upon bolus addition of authentic⋅NO the initial concentrations of ⋅NO and⋅NO2 are very high, which favor Reaction 2 and result in high RSNO yields (13Wink D.A. Darbyshire J.F. Nims R.W. Saavedra J.E. Ford P.C. Chem. Res. Toxicol. 1993; 6: 23-27Crossref PubMed Scopus (479) Google Scholar, 15Keshive M. Singh S. Wishnok J.S. Tannenbaum S.R. Deen W.M. Chem. Res. Toxicol. 1996; 9: 988-993Crossref PubMed Scopus (114) Google Scholar). The reaction of excess ⋅NO with biological substrates is, however, of limited relevance for thein vivo situation. We reasoned that the slow in situ generation of ⋅NO involves rather low concentrations instead and that excess thiols can effectively compete with ⋅NO for ⋅NO2 (Reaction 4). As a consequence, thiol oxidation might be greatly increased and a fraction of RSNOs might be formed through the radical-radical combination reaction of ⋅NO with thiyl radicals (RS⋅). RS⋅+⋅NO→RSNOReaction 5 Under physiologically relevant conditions, the removal of thiyl radicals occurs through either geminate recombination or reaction with O2 and thiolates (12Wardman P. von Sonntag C. Methods Enzymol. 1995; 251: 31-45Crossref PubMed Scopus (190) Google Scholar). Consequently, Reaction 5 directly competes with Reactions 6–9. RS⋅+RS⋅→RSSRReaction 6 RS⋅+O2↔RSOO⋅Reaction 7 RS⋅+RS−↔RSSR⨪Reaction 8 RSSR⨪+O2→RSSR+O2⨪Reaction 9 In the presence of ambient O2 and excess thiol, Reaction 8 dominates because of the rapid removal of the disulfide radical anion (RSSR⨪) via Reaction 9 (12Wardman P. von Sonntag C. Methods Enzymol. 1995; 251: 31-45Crossref PubMed Scopus (190) Google Scholar). The effect of different O2 concentrations on RSNO yields at low⋅NO fluxes is unknown. It may be expected that lower, physiologically relevant O2 tensions will affect the relative amounts of oxidized and nitrosated products not only by reducing the amount of ⋅NO2 formed but also by limiting the impact of O2 on thiyl radical consumption. Here, we present evidence that the nitrosation of thiols by ⋅NO, both in vitro and in intact cells, occurs via the intermediate oxidation of thiols by ⋅NO2 and subsequent reaction of the thiyl radical with impact of O2NO. Under either condition, RSNO formation was maximal at physiologically relevant oxygen The between the present results and obtained in previous studies can be by the between ⋅NO and thiols NONOate was obtained from were from In a a reaction containing 1 μm and either ⋅NO or at the concentration was at in the the was with containing and in and by high performance liquid chromatography as of ⋅NO were an as M. J. Scopus Google Scholar, J.B. V. Methods Methods Enzymol. 1995; Scopus Google Scholar). the were with from of were with ⋅NO that was by through a 5 The ⋅NO were in concentration was typically an The ⋅NO were as in and on The initial rate of ⋅NO generation via of at and was In the rate of was directly by the decrease in at C.M. Wink D.A. Saavedra J.E. A. J.A. J. Med. Chem. PubMed Scopus Google Scholar). of GSH in and μm were with in of O2, by of with were to to oxygen tensions of and of a of was reaction a a at the were with and by as The products obtained from the reaction with ⋅NO in were by as K. J. A. 2001; PubMed Scopus Google Scholar). were a × at a rate of 1 with in The reaction products of GSH were at and the of was by with was a with a with were at in containing The cell was obtained from the were in in with and cells were in and to in with and with cell were with containing and the were for 2 in with different in O2 tensions of and The cells were with for 1 at was not by of the as by not cells were with containing μm The cells were with by and in 1 of 4 containing μm were a of were to a containing of The were on and in the for the addition of of and were for on RSNO formation was by the amount of in a as M. S. Singh M. J. 2002; PubMed Scopus Google Scholar). the of acid and of an containing and The was at via a and the was with nitrogen and The amounts of from the were by was and the results were to ⋅NO concentrations ⋅NO as a concentrations were of GSNO. of or the data were by of and when a was the was as a to two were the A of than was a The reaction of 1 GSH with ⋅NO in with ambient was by GSSG, and GSNO in this order as by with A and GSSG and GSNO were formed upon of 1 GSH with 1 the concentrations of GSSG, and formed concentrations of ⋅NO GSNO production was accompanied by GSSG formation. The could not be to the reaction of GSH with GSNO because the of GSNO with GSH not result in an increase in the GSSG concentration within the not The amount of GSNO formed was two to higher than the amount of GSSG formed at the ⋅NO concentration In the amount of GSNO was two to than that of GSSG at the concentration results indicate that the reaction of ⋅NO with O2 in the formation of an intermediate of GSH to GSSG and that high concentrations of ⋅NO could compete with GSH for this to limit GSSG formation and increase GSNO of GSSG, and formed upon of GSH with ⋅NO at ambient oxygen glutathione 1 was for with concentrations of ⋅NO in and formation was as in the for The concentrations are as the of in a glutathione 1 was for with concentrations of ⋅NO in and formation was as in the for The concentrations are as the of the and oxidative associated with the reaction of ⋅NO with O2, the was as a of ⋅NO because of rate of ⋅NO at C.M. Wink D.A. Saavedra J.E. A. J.A. J. Med. Chem. PubMed Scopus Google Scholar). In the presence of μm ⋅NO GSNO accumulation whereas GSSG concentration to increase the at which the concentration of GSSG that of GSNO by of GSNO with 1 GSH not result in a increase in GSNO within the not with the results obtained with ⋅NO, the amount of GSNO relative to GSSG increased with the rate of ⋅NO generation 2 was the generation of ⋅NO but increased in the presence of GSH The addition of superoxide dismutase to the reaction increased by the amount of GSNO formed and decreased formation by about The formation of GSSG, the increased O2 and the effect of were consistent with a pathway in which the thiyl formed from the one-electron oxidation of a The thiyl radical with the of GSH to form the glutathione disulfide radical anion which in O2 to the superoxide anion Reactions 6 to Under conditions, a fraction of ⋅NO to form peroxynitrite with a decrease in the of GSNO and an increase in the of formation of the radical has been to occur upon of with GSH S. J. Chem. Soc. 1998; Scopus Google Scholar). the of in the formation of GSSG and GSNO conditions, we the effect of the thiyl radical trap, 5,5-dimethyl-1-pyrroline N-oxide 4 We found that DMPO inhibited the formation of GSSG, and by and upon of μm with 1 GSH for 1 at The concentration of could not be because DMPO and results the formation of and the occurrence of a radical pathway for the oxidation and nitrosation of thiols at low micromolar of and DMPO on GSSG, and formation. GSH was for 2 with in with ambient containing μm and in are as compared with The the compared with GSH was with μm as in in the presence or of the thiyl radical DMPO in concentrations are as compared with The the compared with The availability of for reaction with ⋅NO might be by O2 not only because the formation is an process but also because O2 the of through reaction with the glutathione disulfide radical with formation of (Reaction 9 and P. von Sonntag C. Methods Enzymol. 1995; 251: 31-45Crossref PubMed Scopus (190) Google Scholar). we the effect of O2 tension on the reaction and O2 with We that at high O2 the of O2 by the disulfide radical the concentration of thiyl radicals for reaction with ⋅NO to form GSNO. an increased formation at high O2 tension might to a decrease in the of GSNO through peroxynitrite formation. We found that the amount of GSNO formed upon of GSH with a at O2 and that increasing O2 tension to inhibited GSNO formation by with GSSG formation increased in a manner with increasing O2 tension 5 In we that the rate of was not by in oxygen tension and that the of the not change upon with the different not the relevance of pathways for nitrosation we examined the of RSNO formation to the radical DMPO and to in oxygen fibroblasts cell were exposed to exogenous ⋅NO by with at The cells were for RSNO by as that ⋅NO reaction were thiols, were examined for their to and M. S. Singh M. J. 2002; PubMed Scopus Google Scholar). The in A and that more than of the obtained from cells exposed to ⋅NO but we also a and of A concentration of μm was to establish the of RSNO formation from the RSNO content was as as and at of 6 RSNO was found when the cells were with not The fibroblasts were exposed for to different concentrations of to initial from to RSNO formation was upon to and increased with increasing ⋅NO fluxes In the presence of μm of the cells with DMPO decreased RSNO content by Maximum nitrosation upon of the cells with O2 and was decreased by with the with oxygen results were consistent with obtained with GSH in that a radical pathway may a relevant process for RSNO formation in of DMPO and oxygen tension on RSNO content of fibroblasts exposed to exogenous ⋅NO. fibroblasts were for with the thiyl radical DMPO for 1 with μm RSNO content was as in the for The the compared with cells were for 2 in with different in a O2 tension of and The cells were with for 1 at and RSNO content was The the compared with The reaction of ⋅NO and O2 with thiols is to result in thiol nitrosation through the of dinitrogen trioxide (N2O3) formed from the combination of ⋅NO with 15Keshive M. Singh S. Wishnok J.S. Tannenbaum S.R. Deen W.M. Chem. Res. Toxicol. 1996; 9: 988-993Crossref PubMed Scopus (114) Google and M.G. Miranda K.M. Thomas D.D. S. Wink D.A. Acad. Sci. 2002; PubMed Scopus Google Scholar). In the context of the however, thiols are in excess relative to ⋅NO, and a fraction with thiols rather than ⋅NO (Reaction E. Hughes M.N. Wardman P. Free Radic. Biol. Med. 2002; 32: 1314-1323Crossref PubMed Scopus (213) Google Scholar). An important of the by thiols is the formation of oxidized through the intermediate formation of thiyl radicals studies found negligible or oxidation products suggesting differences between the and principles (13Wink D.A. Darbyshire J.F. Nims R.W. Saavedra J.E. Ford P.C. Chem. Res. Toxicol. 1993; 6: 23-27Crossref PubMed Scopus (479) Google Scholar, 14Kharitonov V. Sundquist A.R. Sharma V. J. Biol. Chem. 1995; 270: 28158-28164Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 15Keshive M. Singh S. Wishnok J.S. Tannenbaum S.R. Deen W.M. Chem. Res. Toxicol. 1996; 9: 988-993Crossref PubMed Scopus (114) Google Scholar). In the present study, we found that ⋅NO and O2 both oxidized and nitrosated The yields of GSNO and GSSG upon the relative concentrations of ⋅NO and consistent with a between ⋅NO and GSH for The formation of GSSG, the increased O2 upon increased GSH and the effect of were evidence for the occurrence of a radical in which O2 is to by the glutathione disulfide radical (Reaction a fraction of ⋅NO with in a manner to form peroxynitrite and the of GSNO and formation to superoxide of peroxynitrite formation, the occurrence of GSSG in amounts of GSNO that ⋅NO might oxidative reactions that were only ascribed to the reaction with the between the reactivity of ⋅NO intermediates and peroxynitrite may not be as as in as as both and reactions are accompanied by oxidation the most the importance of ⋅NO2 in reaction and the formation of the has been in previous Although and S. J. Chem. Soc. 1998; Scopus Google observed the formation of upon of GSH with in the presence of a trap, was to formation. The present that a fraction of RSNO formed conditions is derived from the combination of with⋅NO because we that the thiyl radical DMPO GSNO yields by We also that RSNO yields not increase with O2 concentration but were maximal at low O2 O2 the removal of thiyl radicals and the formation of the ⋅NO 8 and a decrease in results in a efficacy of reactions and a increase in the availability of ⋅NO and thiyl radicals for the formation is an but the concentration attained at O2 tension to is not limiting compared with the low fluxes of ⋅NO by As in the it would be very results obtained from studies in a understanding of the relevant chemical pathways that dictate of the of hydrophobic environments that and O2, spatial and in O2 availability and as as in and will affect nitrosation reactions (11Espey M.G. Miranda K.M. Thomas D.D. Wink D.A. J. Biol. Chem. 2001; 276: 30085-30091Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), the of in vitro results to the in vivo a the results of present a from which principles may be that are to in relevant An important of the present is that not limit RSNO formation. RSNO formation to be favored at oxygen concentrations that typically occur in tissues. because the rate of is second order with regard to oxidation and nitrosation reactions are by the availability of ⋅NO2 in a similar to the reactions mediated by of the as a the that RSNO formation at in through a radical for of the of the pathways that to RSNO formation in vivo and of to important of is that the concentration of RSNOs in not only be limited by the formation or availability of ⋅NO but also by the steady-state concentration of thiyl which may be by the local availability of RSNO formation was observed not only with GSH in vitro but also in intact cells with a in which GSH is by and for example, as as superoxide and It is to that conditions that are associated with an increased oxidative stress and a increase in one-electron oxidation of thiols may an important of thiyl radicals for reaction with ⋅NO. Under such conditions, at limiting the production and availability of thiyl radicals would be critical in the of cells and tissues to reactive nitrogen oxide We for