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Reaction between nitric oxide, glutathione, and oxygen in the presence and absence of protein: How are S-nitrosothiols formed? Free Radic Biol Med

Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Free Radical Biology and Medicine (Impact Factor: 5.71). 10/2009; 48(1):55-64. DOI: 10.1016/j.freeradbiomed.2009.10.026
Source: PubMed

ABSTRACT The reaction between NO, thiols, and oxygen has been studied in some detail in vitro due to its perceived importance in the mechanism of NO-dependent signal transduction. The formation of S-nitrosothiols and thiol disulfides from this chemistry has been suggested to be an important component of the biological chemistry of NO, and such subsequent thiol modifications may result in changes in cellular function and phenotype. In this study we have reinvestigated this reaction using both experiment and simulation and conclude that: (i) S-nitrosation through radical and nonradical pathways is occurring simultaneously, (ii) S-nitrosation through direct addition of NO to thiol does not occur to any meaningful extent, and (iii) protein hydrophobic environments do not catalyze or enhance S-nitrosation of either themselves or of glutathione. We conclude that S-nitrosation and disulfide formation in this system occur only after the initial reaction between NO and oxygen to form nitrogen dioxide, and that hydrophobic protein environments are unlikely to play any role in enhancing and targeting S-nitrosothiol formation.

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    • "(1)) is second order in [NO], autoxidation-mediated nitrosothiol formation is expected to be too slow to make an impact under physiological conditions [3–5,10,12,13,17,20,21]. At submicromolar NO concentrations a direct reaction between NO and thiols has been reported [22], although later studies (utilizing micromolar NO levels) could not confirm this [11] [23]. "
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    ABSTRACT: Although different routes for the S-nitrosation of cysteinyl residues have been proposed, the main in vivo pathway is unknown. We recently demonstrated that direct (as opposed to autoxidation-mediated) aerobic nitrosation of glutathione is surprisingly efficient, especially in the presence of Mg(2+). In the present study we investigated this reaction in greater detail. From the rates of NO decay and the yields of nitrosoglutathione (GSNO) we estimated values for the apparent rate constant of 8.9±0.4 and 0.55±0.06M(-1)•s(-1) in the presence and absence of Mg(2+). The maximum yield of GSNO was close to 100% in the presence of Mg(2+) but only about half as high in its absence. From the latter observation we conclude that, in the absence of Mg(2+), nitrosation starts by formation of a complex between NO and O2, which then reacts with the thiol. Omission of superoxide dismutase (SOD) reduced by half the GSNO yield in the absence of Mg(2+), demonstrating O2(-) formation. The reaction in the presence of Mg(2+) appears to involve formation of a Mg(2+)-GSH complex. SOD did not affect Mg(2+)-stimulated nitrosation, suggesting that no O2(-) is formed in that reaction. Replacing GSH by other thiols revealed that reaction rates increased with the pKa of the thiol, suggesting that the nucleophilicity of the thiol is crucial for the reaction, but that the thiol need not be deprotonated. We propose that in cells Mg(2+)-stimulated NO/O2-induced nitrosothiol formation may be a physiologically relevant reaction.
    Free Radical Biology and Medicine 09/2014; 76. DOI:10.1016/j.freeradbiomed.2014.08.024 · 5.71 Impact Factor
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    • "Cys are the most reactive protein residues to NO-derived reactive species at physiological pH, and they are known to be important for maintaining the conformation of proteins and essential residues at the active sites of enzymes. Some mechanisms have been proposed for SNO formation within the biological environment , as NO itself is a poor nitrosylating agent (Keszler, Zhang, & Hogg, 2010). Although the likely S-nitrosylating species in biological systems is dinitrogen trioxide (N 2 O 3 ), which is formed from O 2 and NO, a significant fraction of protein S-nitrosylation by NO may occur in the absence of O 2 (Foster et al., 2009) or through radical-based reaction mechanisms (Hess et al., 2005). "
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    ABSTRACT: Many of nitric oxide (NO) actions are mediated through the coupling of a nitroso moiety to a reactive cysteine leading to the formation of a S-nitrosothiol (SNO), a process known as S-nitrosylation or S-nitrosation. In many cases this reversible post-translational modification is accompanied by altered protein function and aberrant S-nitrosylation of proteins, caused by altered production of NO and/or impaired SNO homeostasis, has been repeatedly reported in a variety of pathophysiological settings. A growing number of studies are directed to the identification and characterization of those proteins that undergo S-nitrosylation and the analysis of S-nitrosoproteomes under pathological conditions is beginning to be reported. The study of these S-nitrosoproteomes has been fueled by advances in proteomic technologies that are providing researchers with improved tools for exploring this post-translational modification. Here we review novel refinements and improvements to these methods, and some recent studies of the S-nitrosoproteome in disease. © 2013 Wiley Periodicals, Inc. Mass Spec Rev.
    Mass Spectrometry Reviews 01/2014; 33(1). DOI:10.1002/mas.21373 · 8.05 Impact Factor
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    • "One-electron oxidation of · NO yields · NO 2 , which can oxidize thiols to thiyl radicals (Jourd'heuil et al., 2003; Keszler et al., 2010). In addition, ONOO − , the product of · NO and superoxide ( · O − 2 ), can form S-nitrosothiols either directly with thiolate anion (RS − ; van der Vliet et al., 1998) or through thiyl radicals (Goldstein et al., 1996; Keszler et al., 2010). So, any mechanism or cellular processes that could enhance RS · formation, such as increased superoxide formation or the action of peroxidases, has the potential to generate S-nitrosothiols, too. "
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    ABSTRACT: Nitric oxide (NO) is a reactive free radical with pleiotropic functions that participates in diverse biological processes in plants, such as germination, root development, stomatal closing, abiotic stress, and defense responses. It acts mainly through redox-based modification of cysteine residue(s) of target proteins, called protein S-nitrosylation.In this way NO regulates numerous cellular functions and signaling events in plants. Identification of S-nitrosylated substrates and their exact target cysteine residue(s) is very important to reveal the molecular mechanisms and regulatory roles of S-nitrosylation. In addition to the necessity of protein–protein interaction for trans-nitrosylation and denitrosylation reactions, the cellular redox environment and cysteine thiol micro-environment have been proposed important factors for the specificity of protein S-nitrosylation. Several methods have recently been developed for the proteomic identification of target proteins. However, the specificity of NO-based cysteine modification is still less defined. In this review, we discuss formation and specificity of S-nitrosylation. Special focus will be on potential S-nitrosylation motifs, site-specific proteomic analyses, computational predictions using different algorithms, and on structural analysis of cysteine S-nitrosylation.
    Frontiers in Plant Science 05/2013; 4:229. DOI:10.3389/fpls.2013.00229 · 3.95 Impact Factor
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