The chemistry of hydropersulfides (RSSH) as related to possible physiological functions.

Hydropersulfides (RSSH) are oxidized thiol (RSH) derivatives that have been shown to be biologically prevalent with likely important functions (along with other polysulfur compounds). The functional utility of RSSH can be gleaned from their unique chemical properties. That is, RSSH possess chemical reactivity not present in other biologically relevant sulfur species that should allow them to be used in specific ways in biology as effector/signaling molecules. For example, compared to RSH, RSSH are considered to be superior nucleophiles, reductants and metal ligands. Moreover, unlike RSH, RSSH can be either reductants/nucleophiles or oxidants/electrophiles depending on the protonated state. It has also become clear that studies related to the chemical biology and physiology of hydrogen suflide (H2S) must also consider the effects of RSSH (and related polysulfur species) as they are biochemically linked. Herein is a discussion of the relevant chemistry of RSSH that can serve as a basis for understanding how RSSH can be used by cells to, for example, combat stresses and used in signaling. Also, discussed are some current experimental studies regarding the biological activity of RSSH that can be explained by their chemical properties.

A comparison of the chemical biology of hydropersulfides (RSSH) with other protective biological antioxidants and nucleophiles.

The hydropersulfide (RSSH) functional group has received significant recent interest due to its unique chemical properties that set it apart from other biological species. The chemistry of RSSH predicts that one possible biological role may be as a protectant against cellular oxidative and electrophilic stress. That is, RSSH has reducing and nucleophilic properties that may combat the potentially destructive biochemistry of toxicologically relevant oxidants and electrophiles. However, there are currently numerous other molecules that have established roles in this regard. For example, ascorbate and tocopherols are potent antioxidants that quench deleterious oxidative reactions and glutathione (GSH) is a well-established and highly prevalent biological protectant against electrophile toxicity. Thus, in order to begin to understand the possible role of RSSH species as protectants against oxidative/electrophilic stress, the inherent chemical properties of RSSH versus these other protectants will be discussed and contrasted.

Methods in sulfide and persulfide research.

Sulfides and persulfides/polysulfides (R-Sn-R', n > 2; R-Sn-H, n > 1) are endogenously produced metabolites that are abundant in mammalian and human cells and tissues. The most typical persulfides that are widely distributed among different organisms include various reactive persulfides-low-molecular-weight thiol compounds such as cysteine hydropersulfide, glutathione hydropersulfide, and glutathione trisulfide as well as protein-bound thiols. These species are generally more redox-active than are other simple thiols and disulfides. Although hydrogen sulfide (H2S) has been suggested for years to be a small signaling molecule, it is intimately linked biochemically to persulfides and may actually be more relevant as a marker of functionally active persulfides. Reactive persulfides can act as powerful antioxidants and redox signaling species and are involved in energy metabolism. Recent evidence revealed that cysteinyl-tRNA synthetases (CARSs) act as the principal cysteine persulfide synthases in mammals and contribute significantly to endogenous persulfide/polysulfide production, in addition to being associated with a battery of enzymes including cystathionine ß-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase, which have been described as H2S-producing enzymes. The reactive sulfur metabolites including persulfides/polysulfides derived from CARS2, a mitochondrial isoform of CARS, also mediate not only mitochondrial biogenesis and bioenergetics but also anti-inflammatory and immunomodulatory functions. The physiological roles of persulfides, their biosynthetic pathways, and their pathophysiology in various diseases are not fully understood, however. Developing basic and high precision techniques and methods for the detection, characterization, and quantitation of sulfides and persulfides is therefore of great importance so as to thoroughly understand and clarify the exact functions and roles of these species in cells and in vivo.

The reactions of hydropersulfides (RSSH) with myoglobin.

Hydropersulfides are reported to be good biological reductants, superior to thiols and akin to selenols. As such, they have been previously shown to reduce metalloproteins such as ferric myoglobin and ferric cytochrome c to their ferrous forms under conditions where little or no reduction from corresponding thiols is observed. Not surprisingly, the reduction of ferric myoglobin to ferrous myoglobin under aerobic conditions results in the generation of oxymyoglobin (dioxygen bound ferrous myoglobin). Previous studies have demonstrated that oxymyoglobin can also act as an oxidant with highly reducing species such as hydroxylamine and ascorbate. Considering the reducing properties of hydropersulfides, it is possible that they can also react with oxymyoglobin similarly to hydroxylamine or ascorbate. Herein, this reaction is examined and indeed hydropersulfides are found to react with oxymyoglobin similarly to other reducing species leading to a fleeting ferric myoglobin which is rapidly reduced to the ferrous form also by hydropersulfide.

Cysteine Trisulfide Protects E. coli from Electrophile-Induced Death through the Generation of Cysteine Hydropersulfide.

Hydropersulfide and polysulfide species have recently been shown to elicit a wide variety of biological and physiological responses. In this study, we examine the effects of cysteine trisulfide (Cys-SSS-Cys; also known as thiocystine) treatment on E. coli. Previous studies in mammalian cells have shown that Cys-SSS-Cys treatment results in protection from the electrophiles. Here, we show that the protective effect of Cys-SSS-Cys treatment against electrophile-induced cell death is conserved in E. coli. This protection correlates with the rapid generation of cysteine hydropersulfide (Cys-SSH) in the culture media. We go on to demonstrate that an exogenous phosphatase expressed in E. coli, containing only a single catalytic cysteine, is protected from electrophile-induced inactivation in the presence of hydropersulfides. These data together demonstrate that E. coli can utilize Cys-SSS-Cys to generate Cys-SSH and that the Cys-SSH can protect cellular thiols from reactivity with the electrophiles.

Interaction of Quinone-Related Electron Acceptors with Hydropersulfide Na2S2: Evidence for One-Electron Reduction Reaction.

We previously reported that 9,10-phenanthraquinone (9,10-PQ), an atmospheric electron acceptor, undergoes redox cycling with dithiols as electron donors, resulting in the formation of semiquinone radicals and monothiyl radicals; however, monothiols have little reactivity. Because persulfide and polysulfide species are highly reducing, we speculate that 9,10-PQ might undergo one-electron reduction with these reactive sulfides. In the present study, we explored the redox cycling capability of a variety of quinone-related electron acceptors, including 9,10-PQ, during interactions with the hydropersulfide Na2S2 and its related polysulfides. No reaction occurred when 9,10-PQ was incubated with Na2S; however, when 5 µM 9,10-PQ was incubated with either 250 µM Na2S2 or Na2S4, we detected extensive consumption of dissolved oxygen (84 µM). Under these conditions, both the semiquinone radicals of 9,10-PQ and their thiyl radical species were also detected using ESR, suggesting that a redox cycle reaction occurred utilizing one-electron reduction processes. Notably, the perthiyl radicals remained stable even under aerobic conditions. Similar phenomenon has also been observed with other electron acceptors, such as pyrroloquinoline quinone, vitamin K3, and coenzyme Q10. Our experiments with N-methoxycarbonyl penicillamine persulfide (MCPSSH), a precursor for endogenous cysteine persulfide, suggested the possibility of a redox coupling reaction with 9,10-PQ inside cells. Our study indicates that hydropersulfide and its related polysulfides are efficient electron donors that interact with quinones. Redox coupling reactions between quinoid electron acceptors and such highly reactive thiols might occur in biological systems.

The Uptake and Release of Polysulfur Cysteine Species by Cells: Physiological and Toxicological Implications.

Hydropersulfides and related polysulfides have recently become topics of significant interest due to their physiological prevalence and proposed biological functions. Currently, examination of the effects of hydropersulfide treatment on cells is difficult due to their lack of inherent stability with respect to disproportionation. Herein, it is reported that the treatment of a variety of cell types with cysteine trisulfide (also known as thiocystine; Cys-SSS-Cys), results in an increase in intracellular hydropersulfide levels (e.g., cysteine hydropersulfide; Cys-SSH, and glutathione hydropersulfide; GSSH). Thus, Cys-SSS-Cys represents a possible pharmacological agent for examining the effects of hydropersulfides on cell function/viability. It has also been found that cells with increased intracellular hydropersulfide levels can export Cys-SSH into the extracellular media. Interestingly, the Cys-SSH is the major hydropersulfide exported by cells, although GSSH is the predominant intracellular species. The possible implications of cellular export are discussed.

The reaction of alkyl hydropersulfides (RSSH, R = CH3 and tBu) with H2S in the gas phase and in aqueous solution.

The RSSH + H2S → RSH + HSSH reaction has been suggested by numerous labs to be important in H2S-mediated biological processes. Seven different mechanisms for this reaction (R = CH3, as a model) have been studied using the DFT methods (M06-2X and ωB97X-D) with the Dunning aug-cc-pV(T+d)Z basis sets. The reaction of CH3SSH with gas phase H2S has a very high energy barrier (>45 kcal mol-1), consistent with the available experimental observations. A series of substitution reactions R1-S-S-H + -S-R2 (R1 = Me, tBu, Ad, R2 = H, S-Me, S-tBu, S-Ad) have been studied. The regioselectivity is largely affected by the steric bulkiness of R1, but is much less sensitive to R2. Thus, when R1 is Me, all -S-R2 favorably attack the internal S atom, leading to R1-S-S-R2. While for R1 = tBu, Ad, all -S-R2 significantly prefer to attack the external S atom to form -S-S-R2. These results are in good agreement with the experimental observations.

Carbon disulfide. Just toxic or also bioregulatory and/or therapeutic?

The overview presented here has the goal of examining whether carbon disulfide (CS2) may play a role as an endogenously generated bioregulator and/or has therapeutic value. The neuro- and reproductive system toxicity of CS2 has been documented from its long-term use in the viscose rayon industry. CS2 is also used in the production of dithiocarbamates (DTCs), which are potent fungicides and pesticides, thus raising concern that CS2 may be an environmental toxin. However, DTCs also have recognized medicinal use in the treatment of heavy metal poisonings as well as having potency for reducing inflammation. Three known small molecule bioregulators (SMBs) nitric oxide, carbon monoxide, and hydrogen sulfide were initially viewed as environmental toxins. Yet each is now recognized as having intricate, though not fully elucidated, biological functions at concentration regimes far lower than the toxic doses. The literature also implies that the mammalian chemical biology of CS2 has broader implications from inflammatory states to the gut microbiome. On these bases, we suggest that the very nature of CS2 poisoning may be related to interrupting or overwhelming relevant regulatory or signaling process(es), much like other SMBs.

The chemical biology of HNO signaling.

Nitroxyl (HNO) is a simple molecule with significant potential as a pharmacological agent. For example, its use in the possible treatment of heart failure has received recent attention due to its unique therapeutic properties. Recent progress has been made on the elucidation of the mechanisms associated with its biological signaling. Importantly, the biochemical mechanisms described for HNO bioactivity are consistent with its unique and novel chemical properties/reactivity. To date, much of the biology of HNO can be associated with interactions and modification of important regulatory thiol proteins. Herein will be provided a description of HNO chemistry and how this chemistry translates to some of its reported biological effects.

Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling.

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 ß-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 H2O2 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 H2S can be generated from persulfide degradation, much of the reported biological activity associated with H2S may actually be that of persulfides. That is, H2S may act primarily as a marker for the biologically active of persulfide species.

The chemical biology of hydropersulfides (RSSH): Chemical stability, reactivity and redox roles.

Recent reports indicate the ubiquitous prevalence of hydropersulfides (RSSH) in mammalian systems. The biological utility of these and related species is currently a matter of significant speculation. The function, lifetime and fate of hydropersulfides will be assuredly based on their chemical properties and reactivity. Thus, to serve as the basis for further mechanistic studies regarding hydropersulfide biology, some of the basic chemical properties/reactivity of hydropersulfides was studied. The nucleophilicity, electrophilicity and redox properties of hydropersulfides were examined under biological conditions. These studies indicate that hydropersulfides can be nucleophilic or electrophilic, depending on the pH (i.e. the protonation state) and can act as good one- and two-electron reductants. These diverse chemical properties in a single species make hydropersulfides chemically distinct from other, well-known sulfur containing biological species, giving them unique and potentially important biological function.

Involvement of reactive persulfides in biological bismethylmercury sulfide formation.

Bismethylmercury sulfide (MeHg)2S has been found to be a detoxified metabolite of methylmercury (MeHg) that is produced by SH-SY5Y cells and in livers of rats exposed to MeHg. (MeHg)2S could be formed through the interactions between MeHg and sulfur species such as hydrogen sulfide (H2S or HS(-)), but the origin of its sulfur has not been fully identified. We herein examined the formation of (MeHg)2S through interactions between MeHg and persulfides, polysulfides, and protein preparations. Investigations using HPLC/atomic absorption spectrophotometry and EI-MS revealed that NaHS and Na2S4 react readily with MeHg to give (MeHg)2S, and similar results were found using GSH persulfide (GSSH) formed endogenously or generated enzymatically in vitro. (MeHg)2S was also formed by incubation of MeHg with liver and heart cytosolic fractions prepared from wild-type mice but not with those from mice lacking cystathionine γ-lyase (CSE) that catalyzes the formation of cysteine persulfide. Consistent with this, (MeHg)2S was detected in a variety of tissues taken from wild-type mice intraperitoneally injected with MeHg in vivo but not in those from MeHg-injected CSE knockout mice. By separating liver fractions by column chromatography, we found numerous proteins that contain persulfides: one of the proteins was identified as being glutathione S-transferase pi 1. These results indicate that the formation of (MeHg)2S can be attributed to interactions between MeHg and endogenous free persulfide species, as well as protein-bound cysteine persulfide.

Reaction Mechanisms of H2S Oxidation by Naphthoquinones.

1,4-naphthoquinones (NQs) catalytically oxidize H2S to per- and polysufides and sulfoxides, reduce oxygen to superoxide and hydrogen peroxide, and can form NQ-SH adducts through Michael addition. Here, we measured oxygen consumption and used sulfur-specific fluorophores, liquid chromatography tandem mass spectrometry (LC-MS/MS), and UV-Vis spectrometry to examine H2S oxidation by NQs with various substituent groups. In general, the order of H2S oxidization was DCNQ ~ juglone > 1,4-NQ > plumbagin >DMNQ ~ 2-MNQ > menadione, although this order varied somewhat depending on the experimental conditions. DMNQ does not form adducts with GSH or cysteine (Cys), yet it readily oxidizes H2S to polysulfides and sulfoxides. This suggests that H2S oxidation occurs at the carbonyl moiety and not at the quinoid 2 or 3 carbons, although the latter cannot be ruled out. We found little evidence from oxygen consumption studies or LC-MS/MS that NQs directly oxidize H2S2-4, and we propose that apparent reactions of NQs with inorganic polysulfides are due to H2S impurities in the polysulfides or an equilibrium between H2S and H2Sn. Collectively, NQ oxidation of H2S forms a variety of products that include hydropersulfides, hydropolysulfides, sulfenylpolysulfides, sulfite, and thiosulfate, and some of these reactions may proceed until an insoluble S8 colloid is formed.

The effects of nitroxyl (HNO) on H2O2 metabolism and possible mechanisms of HNO signaling.

Nitroxyl (HNO) possesses unique and potentially important biological/physiological activity that is currently mechanistically ill-defined. Previous work has shown that the likely biological targets for HNO are thiol proteins, oxidized metalloproteins (i.e. ferric heme proteins) and, most likely, selenoproteins. Interestingly, these are the same classes of proteins that interact with H2O2. In fact, these classes of proteins not only react with H2O2, and thus potentially responsible for the signaling actions of H2O2, but are also responsible for the degradation of H2O2. Therefore, it is not unreasonable to speculate that HNO can affect H2O2 degradation by interacting with H2O2-degrading proteins possibly leading to an increase in H2O2-mediated signaling. Moreover, considering the commonality between HNO and H2O2 biological targets, it also seems likely that HNO-mediated signaling can also be due to reactivity at otherwise H2O2-reactive sites. Herein, it is found that HNO does indeed inhibit H2O2 degradation via inhibition of H2O2-metaboilizing proteins. Also, it is found that in a system known to be regulated by H2O2 (T cell activation), HNO behaves similarly to H2O2, indicating that HNO- and H2O2-signaling may be similar and/or intimately related.

Cysteine hydropersulfide reduces lipid peroxidation and protects against myocardial ischaemia-reperfusion injury - Are endogenous persulfides mediators of ischaemic preconditioning?

Earlier studies revealed the presence of cysteine persulfide (CysSSH) and related polysulfide species in various mammalian tissues. CysSSH has both antioxidant and oxidant properties, modulates redox-dependent signal transduction and has been shown to mitigate oxidative stress. However, its functional relevance in the setting of myocardial ischaemia-reperfusion injury (IRI) remains unknown. The present study was undertaken to (1) study the dynamics of production and consumption of persulfides under normoxic and hypoxic conditions in the heart, and (2) determine whether exogenous administration of the CysSSH donor, cysteine trisulfide (Cys-SSS-Cys) at the onset of reperfusion rescues functional impairment and myocardial damage by interfering with lipid peroxidation. Utilising a well-established ex vivo Langendorff murine model, we here demonstrate that endogenous tissue concentrations of CysSSH are upregulated when oxygen supply is compromised (global myocardial ischaemia) and rapidly restored to baseline levels upon reperfusion, suggestive of active regulation. In a separate set of experiments, exogenous administration of Cys-SSS-Cys for 10 min at the onset of reperfusion was found to decrease malondialdehyde (MDA) concentrations, formation of 4-hydroxynonenal (4-HNE) protein adducts and rescue the heart from injury. Cys-SSS-Cys also restored post-ischaemic cardiac function, improving both coronary flow and left ventricular developed pressure (LVDP). Taken together, these results support the notion that endogenous CysSSH plays an important role as a "redox preconditioning" agent to combat the oxidative insult in myocardial IRI.

Small molecule signaling agents: the integrated chemistry and biochemistry of nitrogen oxides, oxides of carbon, dioxygen, hydrogen sulfide, and their derived species.

Several small molecule species formally known primarily as toxic gases have, over the past 20 years, been shown to be endogenously generated signaling molecules. The biological signaling associated with the small molecules NO, CO, H2S (and the nonendogenously generated O2), and their derived species have become a topic of extreme interest. It has become increasingly clear that these small molecule signaling agents form an integrated signaling web that affects/regulates numerous physiological processes. The chemical interactions between these species and each other or biological targets is an important factor in their roles as signaling agents. Thus, a fundamental understanding of the chemistry of these molecules is essential to understanding their biological/physiological utility. This review focuses on this chemistry and attempts to establish the chemical basis for their signaling functions.

The Biological/Physiological Utility of Hydropersulfides (RSSH) and Related Species: What Is Old Is New Again.

Significance: Hydrogen sulfide (H2S) is reported to be an important mediator involved in numerous physiological processes. H2S and hydropersulfides (RSSH) species are intimately linked biochemically. Therefore, interest in the mechanisms of the biological activity of H2S has led to investigations of the chemical biology of RSSH since they are likely to coexist in a biological system. Currently it is hypothesized that RSSH may be responsible for a least part of the observed H2S-mediated biology/physiology. Recent Advances: It has been recently touted that thiols (RSH) and RSSH have some important differences in terms of their chemical biology and that the generation of RSSH from RSH is purposeful to exploit these chemical differences as a response to a physiological or biological stress. This transformation may represent an unappreciated/unrecognized biological mechanism for dealing with cellular stresses. Critical Issues: Although recent studies indicate a diverse and potentially important chemical biology associated with RSSH species, these ideas have their foundations in early studies (some over 60 years old). It is vital to recognize the nature of this early work to fully appreciate the current ideas regarding RSSH biology. Importantly, these early studies were performed before the realization of purposeful H2S biosynthesis (before 1996). Future Directions: Taking clues from the past studies of RSSH chemistry and biology, progress in delineating the chemical biology of RSSH will continue. Determination of the possible relevance of RSSH chemical biology to signaling and cellular physiology will be a primary focus of many future studies. Antioxid. Redox Signal. 36, 244-255.