Using methodology developed herein, it is found that reactive persulfides and polysulfides are formed endogenously from both small molecule species and proteins in high amounts in mammalian cells and tissues. These reactive sulfur species were biosynthesized by two major sulfurtransferases: cystathionine β-synthase and cystathionine γ-lyase. Quantitation of these species indicates that high concentrations of glutathione persulfide (perhydropersulfide >100 μM) and other cysteine persulfide and polysulfide derivatives in peptides/proteins were endogenously produced and maintained in the plasma, cells, and tissues of mammals (rodent and human). It is expected that persulfides are especially nucleophilic and reducing. This view was found to be the case, because they quickly react with H 2 O 2 and a recently described biologically generated electrophile 8-nitroguanosine 3′,5′-cyclic monophosphate. These results indicate that persulfides are potentially important signaling/effector species, and because H 2 S can be generated from persulfide degradation, much of the reported biological activity associated with H 2 S may actually be that of persulfides. That is, H 2 S may act primarily as a marker for the biologically active of persulfide species.thiol redox | hydrogen sulfide | electrophilic signaling | polysulfidomics H ydrogen sulfide (H 2 S) has been suggested to be an endogenous small molecule signaling species (1) by unknown mechanisms. Our laboratory recently showed that the presence of hydrogen sulfide anion (HS − ) may be responsible for the regulation and metabolism of various important electrophilic species [e.g., 8-nitroguanosine 3′,5′-cyclic GMP (8-nitro-cGMP)] (2). However, these studies also indicated that reactive intermediates other than HS − likely react with the electrophiles of interest. These previous studies alluded to the generation of a more reactive sulfur species capable of reacting with electrophiles, such as 8-nitro-cGMP. As reported herein, it was determined that reactive sulfur intermediates, such as hydropersulfides (RSSH) and polysulfides [RS(S) n H and RS(S) n SR], are formed in appreciable amounts during sulfur amino acid metabolism and possess important chemical and biological properties. Some of these sulfide species have long been known as sulfane sulfur compounds, which were suggested to exist endogenously in mammalian systems (1,(3)(4)(5). Reports also indicated that a hydropersulfide moiety with the general molecular formula RSSH may be formed on specific protein cysteine (Cys) residues, most typically of sulfur-transferring enzymes (i.e., sulfurtransferases) during enzymatic reactions (1, 5). Although such persulfide chemical reactivity is thought to be involved in the catalytic activity of particular enzymes (e.g., rhodanese, Cys desulfurases, and sulfide:quinone oxidoreductase) (6, 7), the more general physiological function and occurrence of Cys persulfides (CysSSH) and related species in cells and tissues, especially mammals, were unclear. Moreover, the exact chemical nature ...

Nitric oxide (NO) in oxygen-containing aqueous solution has a short half-life that is often attributed to a rapid oxidation to both NOj-and NOT. The chemical fate ofNO in aqueous solution is often assumed to be the same as that in air, where NO is oxidized to NO2 followed by dimerization to N204. Water then reacts with N204 to form both NO-and NO-. We report here that NO in aqueous solution containing oxygen is oxidized primarily to NO -with little or no formation of NO3. In the presence of oxyhemoglobin or oxymyoglobin, however, NO and NO-were oxidized completely to N03. Methemoglobin was inactive in this regard. The unpurified cytosoLic fraction from rat cerebellum, which contains constitutive NO synthase activity, catalyzed the conversion of L-arginine primarily to NO3-(NOiT/NOiT ratio = 0.25). After chromatography on DEAE-Sephacel or affinity chromatography using 2',5'-ADP-Sepharose 4B, active fractions containing NO synthase activity catalyzed the conversion of L-arginine primarily to NO-(NOj-/NOj ratio = 5.6) or only to NOT, Sufficient evidence has been amassed to indicate a wide biological role for endogenous nitric oxide (NO) in modulating physiological and pathophysiological processes (1). NO is synthesized in various cell types by a family ofisoforms ofNO synthase (2). Some isoforms are constitutive and activated by calcium, whereas other isoforms are inducible and regulated by transcriptional mechanisms. Both isoforms catalyze the same complex oxidation of L-arginine to NO plus L-citrulhne (3-6). The mechanism of catalysis of NO synthase is similar to that for the cytochrome P450 monooxygenases in that molecular oxygen is incorporated into the substrate by reactions involving NADPH, flavins, and heme (7).To appreciate the diverse biological actions of NO, it is essential to understand not only the biosynthesis but also the metabolism of NO and the chemistry of NO in aqueous solution. NO pure aqueous solution, however, display half-lives of 500 sec or longer (10). This means that, in the presence of biological tissues, NO is rapidly converted to a less-active or inactive product. The chemical lability of NO in cells and tissues has been attributed to a rapid oxidation to both NOj and NO3 (11)(12)(13)(14). The common belief that NO is oxidatively metabolized to both NOj and NO3 derives largely from experiments with intact cells, tissues, and whole animals rather than pure aqueous systems. For example, macrophages that have been activated in culture to induce NO synthase activity generate both . Moreover, endogenous NO-production in whole animals cannot be observed by assaying plasma or urine because of the nearly complete oxidation of NO or NO -to NO-(12). NO gas reacts with oxygen to form NO2 gas, which dimerizes to N204. N204 dismutates spontaneously in water to form NO-(as HNO2) and NO-(as HNO3) (16). The assumption is commonly made that NO in an aqueous solution containing oxygen generates NO-and NO-. This assumption is inconsistent with chemical studies showing that pure aqueous solutions...

Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substrate l-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction.

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The redox siblings nitroxyl (HNO) and nitric oxide (NO) have often been assumed to undergo casual redox reactions in biological systems. However, several recent studies have demonstrated distinct pharmacological effects for donors of these two species. Here, infusion of the HNO donor Angeli's salt into normal dogs resulted in elevated plasma levels of calcitonin gene-related peptide, whereas neither the NO donor diethylamine͞NONOate nor the nitrovasodilator nitroglycerin had an appreciable effect on basal levels. Conversely, plasma cGMP was increased by infusion of diethylamine͞NONOate or nitroglycerin but was unaffected by Angeli's salt. These results suggest the existence of two mutually exclusive response pathways that involve stimulated release of discrete signaling agents from HNO and NO. In light of both the observed dichotomy of HNO and NO and the recent determination that, in contrast to the O2͞O 2 ؊ couple, HNO is a weak reductant, the relative reactivity of HNO with common biomolecules was determined. This analysis suggests that under biological conditions, the lifetime of HNO with respect to oxidation to NO, dimerization, or reaction with O2 is much longer than previously assumed. Rather, HNO is predicted to principally undergo addition reactions with thiols and ferric proteins. Calcitonin gene-related peptide release is suggested to occur via altered calcium channel function through binding of HNO to a ferric or thiol site. The orthogonality of HNO and NO may be due to differential reactivity toward metals and thiols and in the cardiovascular system, may ultimately be driven by respective alteration of cAMP and cGMP levels.Angeli's salt ͉ superoxide dismutase ͉ heme protein ͉ cGMP ͉ calcitonin gene-related peptide D uring the last two decades, discussion of the chemistry of nitric oxide (NO) in biological systems has primarily focused on the nitrosylation of heme proteins such as soluble guanylyl cyclase and the production of reactive nitrogen oxide species (RNOS) (1-3). The RNOS literature has largely been concerned with nitrogen dioxide (NO 2 ), dinitrogen trioxide (N 2 O 3 ), and peroxynitrite (ONOO Ϫ ), which are formed through reaction with molecular oxygen or superoxide (O 2 Ϫ ) (4-6). Recently, however, there has been increased interest in the one-electron reduction product of NO, nitroxyl (HNO͞NO Ϫ ; nitrosyl hydride͞nitroxyl anion). Of particular note are studies suggesting that oxidation of L-arginine by NO synthase (NOS) leads to production of nitroxyl rather than NO under certain conditions (7-10). In this light, elucidation of the chemical biology of nitroxyl has acquired new importance.Comparisons of the toxicological and pharmacological properties of nitrogen oxide donor compounds have revealed that NO and HNO in general elicit distinct responses under a variety of biological conditions. In vitro, HNO reacts with O 2 to generate potent oxidizing species capable of cleaving DNA, thereby augmenting oxidative damage (3, 11). The RNOS formed by NO autoxidation do not cause these cellular a...

. Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers. Am J Physiol Cell Physiol 287: C246 -C256, 2004; 10.1152/ ajpcell.00516.2003.-Except for the role of NO in the activation of guanylate cyclase, which is well established, the involvement of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in signal transduction remains controversial, despite a large body of evidence suggestive of their participation in a variety of signaling pathways. Several problems have limited their acceptance as signaling molecules, with the major one being the difficulty in identifying the specific targets for each pathway and the chemical reactions supporting reversible oxidation of these signaling components, consistent with a second messenger role for ROS and RNS. Nevertheless, it has become clear that cysteine residues in the thiolate (i.e., ionized) form that are found in some proteins can be specific targets for reaction with H 2O2 and RNS. This review focuses on the chemistry of the reversible oxidation of those thiolates, with a particular emphasis on the critical thiolate found in protein tyrosine phosphatases as an example. hydrogen peroxide; thiolate; nitrosothiol; nitric oxide; signal transduction ALTHOUGH THE INVOLVEMENT OF FREE RADICALS in biology was assumed for many years to be restricted to damaging reactions, the discovery of the endogenous generation of NO in mammalian systems and the finding that this small, freely diffusing, chemically unique species participates in specific signal transduction pathways represented an important new paradigm and expanded views of the possible nature of cell communication and/or signaling processes. This novel role in signal transduction for ⅐NO and other reactive nitrogen species (RNS) now extends to reactive oxygen species (ROS) such as H 2 O 2 and is gaining greater acceptance. The skepticism that still exists about these molecules acting as second messengers in various signaling pathways may vanish with better understanding of their chemistry, particularly regarding the differences in reactivity at high concentrations (mainly associated with pathology and toxicology) from those at low concentrations generated under physiological conditions in response to stimuli. Several excellent reviews have been published regarding evidence supporting a role for ROS and RNS in signaling (26,32,75,82,87,117,119), and we (35, 36) previously described the general properties that define a second messenger and showed how ROS and RNS fit into this role. In this review, the main focus is on the chemistry that may provide specificity, a necessary property of second messengers that has remained the most elusive in the study of ROS and RNS. REACTIVE SPECIES AS SECOND MESSENGERSSecond messengers are generated at the time of receptor activation, are short-lived, and act specifically on effectors to transiently alter their activity. Indeed, ROS and RNS can be generated at the time of receptor activation and are short-lived, as a...

We review gases that can affect oxidative stress and that themselves may be radicals. We discuss O(2) toxicity, invoking superoxide, hydrogen peroxide, and the hydroxyl radical. We also discuss superoxide dismutase (SOD) and both ground-state, triplet oxygen ((3)O(2)), and the more energetic, reactive singlet oxygen ((1)O(2)). Nitric oxide ((*)NO) is a free radical with cell signaling functions. Besides its role as a vasorelaxant, (*)NO and related species have other functions. Other endogenously produced gases include carbon monoxide (CO), carbon dioxide (CO(2)), and hydrogen sulfide (H(2)S). Like (*)NO, these species impact free radical biochemistry. The coordinated regulation of these species suggests that they all are used in cell signaling. Nitric oxide, nitrogen dioxide, and the carbonate radical (CO(3)(*-)) react selectively at moderate rates with nonradicals, but react fast with a second radical. These reactions establish "cross talk" between reactive oxygen (ROS) and reactive nitrogen species (RNS). Some of these species can react to produce nitrated proteins and nitrolipids. It has been suggested that ozone is formed in vivo. However, the biomarkers that were used to probe for ozone reactions may be formed by non-ozone-dependent reactions. We discuss this fascinating problem in the section on ozone. Very low levels of ROS or RNS may be mitogenic, but very high levels cause an oxidative stress that can result in growth arrest (transient or permanent), apoptosis, or necrosis. Between these extremes, many of the gasses discussed in this review will induce transient adaptive responses in gene expression that enable cells and tissues to survive. Such adaptive mechanisms are thought to be of evolutionary importance.

Hydrogen sulfide (H2S) is an endogenously generated and putative signaling/effector molecule. In spite of its numerous reported functions, the chemistry by which it elicits its functions is not understood. Moreover, recent studies allude to the existence of other sulfur species besides H2S that may play critical physiological roles. Herein, the basic chemical biology of H2S as well as other related or derived species is discussed and reviewed. A particular focus of this review are the per- and poly-sulfides which are likely in equilibrium with free H2S and which may be important biological effectors themselves.