The SOD1 Inhibitor, LCS-1, Oxidizes H2S to Reactive Sulfur Species, Directly and Indirectly, through Conversion of SOD1 to an Oxidase.

LCS-1, a putative selective inhibitor of SOD1, is a substituted pyridazinone with rudimentary similarity to quinones and naphthoquinones. As quinones catalytically oxidize H2S to biologically active reactive sulfur species (RSS), we hypothesized LCS-1 might have similar attributes. Here, we examine LCS-1 reactions with H2S and SOD1 using thiol-specific fluorophores, liquid chromatography-mass spectrometry, electron paramagnetic resonance (EPR), UV-vis spectrometry, and oxygen consumption. We show that LCS-1 catalytically oxidizes H2S in buffer solutions to form RSS, namely per- and polyhydrosulfides (H2Sn, n = 2-6). These reactions consume oxygen and produce hydrogen peroxide, but they do not have an EPR signature, nor do they affect the UV-vis spectrum. Surprisingly, LCS-1 synergizes with SOD1, but not SOD2, to oxidize H2S to H2S3-6. LCS-1 forms monothiol adducts with H2S, glutathione (GSH), and cysteine (Cys), but not with oxidized glutathione or cystine; both thiol adducts inhibit LCS-1-SOD1 synergism. We propose that LCS-1 forms an adduct with SOD1 that disrupts the intramolecular Cys57-Cys146 disulfide bond and transforms SOD1 from a dismutase to an oxidase. This would increase cellular ROS and polysulfides, the latter potentially affecting cellular signaling and/or cytoprotection.

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 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.

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.

The antioxidant and oxidant properties of hydropersulfides (RSSH) and polysulfide species.

It has become apparent that hydrogen sulfide (H2S), hydropersulfides (RSSH) and other polysulfide species are all intimately linked biochemically. Indeed, at least some of the biological activity attributed to hydrogen sulfide (H2S) may actually be due to its conversion to RSSH and derived polysulfur species (and vice-versa). The unique chemistry associated with the hydropersulfide functional group (-SSH) predicts that it possesses possible protective properties that can help a cell contend with oxidative and/or electrophilic stress. However, since RSSH and polysulfides possess chemical properties akin to disulfides (RSSR), they can also be sources of oxidative/electrophilic stress/signaling as well. Herein are discussed the unique chemistry, possible biochemistry and the physiological implications of RSSH (and polysulfides), especially as it pertains to their putative cellular protection properties against a variety of stresses and/or as possible stressors/signaling agents themselves.

The reaction of hydropersulfides (RSSH) with S-nitrosothiols (RS-NO) and the biological/physiological implications.

S-Nitrosothiol (RS-NO) generation/levels have been implicated as being important to numerous physiological and pathophysiological processes. As such, the mechanism(s) of their generation and degradation are important factors in determining their biological activity. Along with the effects on the activity of thiol proteins, RS-NOs have also been reported to be reservoirs or storage forms of nitric oxide (NO). That is, it is hypothesized that NO can be released from RS-NO at opportune times to, for example, regulate vascular tone. However, to date there are few established mechanisms that can account for facile NO release from RS-NO. Recent discovery of the biological formation and prevalence of hydropersulfides (RSSH) and their subsequent reaction with RS-NO species provides a possible route for NO release from RS-NO. Herein, it is found that RSSH is capable of reacting with RS-NO to liberate NO and that the analogous reaction using RSH is not nearly as proficient in generating NO. Moreover, computational results support the prevalence of this reaction over other possible competing processes. Finally, results of biological studies of NO-mediated vasorelaxation are consistent with the idea that RS-NO species can be degraded by RSSH to release NO.

Hydropersulfides (RSSH) and Nitric Oxide (NO) Signaling: Possible Effects on S-Nitrosothiols (RS-NO).

S-Nitrosothiol (RS-NO) formation in proteins and peptides have been implicated as factors in the etiology of many diseases and as possible regulators of thiol protein function. They have also been proposed as possible storage forms of nitric oxide (NO). However, despite their proposed functions/roles, there appears to be little consensus regarding the physiological mechanisms of RS-NO formation and degradation. Hydropersulfides (RSSH) have recently been discovered as endogenously generated species with unique reactivity. One important reaction of RSSH is with RS-NO, which leads to the degradation of RS-NO as well as the release of NO. Thus, it can be speculated that RSSH can be a factor in the regulation of steady-state RS-NO levels, and therefore may be important in RS-NO (patho)physiology. Moreover, RSSH-mediated NO release from RS-NO may be a possible mechanism allowing RS-NO to serve as a storage form of NO.

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.

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.

Comment on "Evidence that the ProPerDP method is inadequate for protein persulfidation detection due to lack of specificity".

The recent report by Fan et al alleged that the ProPerDP method is inadequate for the detection of protein persulfidation. Upon careful evaluation of their work, we conclude that the claim made by Fan et al is not supported by their data, rather founded in methodological shortcomings. It is understood that the ProPerDP method generates a mixture of cysteine-containing and non-cysteine-containing peptides. Instead, Fan et al suggested that the detection of non-cysteine-containing peptides indicates nonspecific alkylation at noncysteine residues. However, if true, then such peptides would not be released by reduction and therefore not appear as products in the reported workflow. Moreover, the authors' biological assessment of ProPerDP using Escherichia coli mutants was based on assumptions that have not been confirmed by other methods. We conclude that Fan et al did not rigorously assess the method and that ProPerDP remains a reliable approach for analyses of protein per/polysulfidation.

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.

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.

Predicting the Possible Physiological/Biological Utility of the Hydropersulfide Functional Group Based on Its Chemistry: Similarities Between Hydropersulfides and Selenols.

Significance: Hydropersulfides (RSSH) and related polysulfide species (RSnR, n > 2, R = alkyl, H) are highly biologically prevalent with likely important physiological functions. Due to their prevalence, many labs have begun to investigate their possible roles, especially with regards to their protective, redox, and signaling properties. Recent Advances: A significant amount of work has been performed while delineating the chemical reactivity/chemical properties of hydropersulfides, and it is clear that their overall chemistry is distinct from all other biologically relevant sulfur species (e.g., thiols, disulfides, sulfenic acids, etc.). Critical Issues: One way to predict and ultimately understand the biological functions of hydropersulfides is to focus on their unique chemistry, which should provide the rationale for why this unique functionality is present. Interestingly, some of the chemical properties of RSSH are strikingly similar to those of selenols (RSeH). Therefore, it may be important to consider the known functions of selenoproteins when speculating about the possible functions of RSSH species. Future Directions: Currently, many of the inherent chemical differences between hydropersulfides and other biological sulfur species have been established. It remains to be determined, however, whether and how these differences are utilized to accomplish specific biochemical/physiological goals. A significant aspect of elucidating the biological utility of hydropersulfides will be to determine the mechanisms of regulation of their formation and/or biosynthesis, that is, based on whether it can be determined under what cellular conditions hydropersulfides are made, more meaningful speculation regarding their functions/roles can be developed.

The chemical biology of hydrogen sulfide and related hydropersulfides: interactions with biologically relevant metals and metalloproteins.

Hydrogen sulfide and related/derived persulfides (RSnH, RSSnR, n > 1) have been the subject of recent research interest because of their reported physiological signaling roles. In spite of their described actions, the chemical/biochemical mechanisms of activity have not been established. From a chemical perspective, it is likely that metals and metalloproteins are possible biological targets for the actions of these species. Thus, the chemical biology of hydrogen sulfide and persulfides with metals and metalloproteins will be discussed as a prelude to future speculation regarding their physiological function and utility.

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.

Manganese Porphyrin-Based SOD Mimetics Produce Polysulfides from Hydrogen Sulfide.

Manganese-centered porphyrins (MnPs), MnTE-2-PyP5+ (MnTE), MnTnHex-2-PyP5+ (MnTnHex), and MnTnBuOE-2-PyP5+ (MnTnBuOE) have received considerable attention because of their ability to serve as superoxide dismutase (SOD) mimetics thereby producing hydrogen peroxide (H2O2), and oxidants of ascorbate and simple aminothiols or protein thiols. MnTE-2-PyP5+ and MnTnBuOE-2-PyP5+ are now in five Phase II clinical trials warranting further exploration of their rich redox-based biology. Previously, we reported that SOD is also a sulfide oxidase catalyzing the oxidation of hydrogen sulfide (H2S) to hydrogen persulfide (H2S2) and longer-chain polysulfides (H2Sn, n = 3-7). We hypothesized that MnPs may have similar actions on sulfide metabolism. H2S and polysulfides were monitored in fluorimetric assays with 7-azido-4-methylcoumarin (AzMC) and 3',6'-di(O-thiosalicyl)fluorescein (SSP4), respectively, and specific polysulfides were further identified by mass spectrometry. MnPs concentration-dependently consumed H2S and produced H2S2 and subsequently longer-chain polysulfides. This reaction appeared to be O2-dependent. MnP absorbance spectra exhibited wavelength shifts in the Soret and Q bands characteristic of sulfide-mediated reduction of Mn. Taken together, our results suggest that MnPs can become efficacious activators of a variety of cytoprotective processes by acting as sulfide oxidation catalysts generating per/polysulfides.

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.

Polysulfide stabilization by tyrosine and hydroxyphenyl-containing derivatives that is important for a reactive sulfur metabolomics analysis.

The physiological importance of reactive sulfur species (RSS) such as cysteine hydropersulfide (CysSSH) has been increasingly recognized in recent years. We have established a reactive sulfur metabolomics analysis by using RSS metabolic profiling, which revealed appreciable amounts of RSS generated endogenously and ubiquitously in both prokaryotic and eukaryotic organisms. The chemical nature of these polysulfides is not fully understood, however, because of their reactive or complicated redox-active properties. In our study here, we determined that tyrosine and a hydroxyphenyl-containing derivative, ß-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM), had potent stabilizing effects on diverse polysulfide residues formed in CysSSH-related low-molecular-weight species, e.g., glutathione polysulfides (oxidized glutathione trisulfide and oxidized glutathione tetrasulfide). The protective effect against degradation was likely caused by the inhibitory activity of hydroxyphenyl residues of tyrosine and HPE-IAM against alkaline hydrolysis of polysulfides. This hydrolysis occurred via heterolytic scission triggered by the hydroxyl anion acting on polysulfides that are cleaved into thiolates and sulfenic acids, with the hydrolysis being enhanced by alkylating reagents (e.g. IAM) and dimedone. Moreover, tyrosine prevented electrophilic degradation occurring in alkaline pH. The polysulfide stabilization induced by tyrosine or the hydroxyphenyl moiety of HPE-IAM will greatly improve our understanding of the chemical properties of polysulfides and may benefit the sulfur metabolomics analysis if it can be applied successfully to any kind of biological samples, including clinical specimens.

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.