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 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. Significance Reactive sulfur-containing compounds, such as l-cysteine hydropersulfide (CysSSH), reportedly form in mammals. However, the biological relevance of these reactive sulfur species remains unclear. We determined that CysSSH was synthesized from cystine by cystathionine β-synthase and cystathionine γ-lyase, which in turn may contribute to high levels of glutathione hydropersulfide (>100 μM) and other CysSSH derivatives of peptides/proteins formed in cells, tissues, and plasma from mice and humans. Compared with glutathione and hydrogen sulfide, CysSSH derivatives were superior nucleophiles and reductants and capable of regulating electrophilic cell signaling mediated by 8-nitroguanosine 3′,5′-cyclic monophosphate. Altogether, it is proposed that reactive Cys persulfides and S-polythiolation have critical regulatory functions in redox cell signaling.

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 inEscherichia coliand mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substratel-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.

The mammalian brain is highly vulnerable to oxygen deprivation, yet the mechanism underlying the brain’s sensitivity to hypoxia is incompletely understood. Hypoxia induces accumulation of hydrogen sulfide, a gas that inhibits mitochondrial respiration. Here, we show that, in mice, rats, and naturally hypoxia-tolerant ground squirrels, the sensitivity of the brain to hypoxia is inversely related to the levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize sulfide. Silencing SQOR increased the sensitivity of the brain to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological scavenging of sulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to hypoxia. These results illuminate the critical role of sulfide catabolism in energy homeostasis during hypoxia and identify a therapeutic target for ischemic brain injury.

For decades, reactive persulfide species including cysteine persulfide (CysSSH) have been known to exist endogenously in organisms. However, the physiological significance of endogenous persulfides remains poorly understood. That cystathionine β-synthase and cystathionine γ-lyase produced CysSSH from cystine was recently demonstrated. An endogenous sulfur transfer system involving CysSSH evidently generates glutathione persulfide (GSSH) that exists at concentrations greater than 100 μM in vivo. Because reactive persulfide species such as CysSSH and GSSH have higher nucleophilicity than parental cysteine (Cys) and glutathione do, these reactive species exhibit strong scavenging activities against oxidants, e.g., hydrogen peroxide, and electrophiles, which contributes to redox signaling regulation. Also, several papers indicated that various proteins and enzymes have Cys polysulfides including CysSSH at their specific Cys residues, which is called protein polysulfidation. Apart from the redox signaling regulatory mechanism, another plausible function of protein polysulfidation is providing protection for protein thiol residues against irreversible chemical modification caused by oxidants and electrophiles. Elucidation of the redox signaling regulatory mechanism of reactive persulfide species including small thiol molecules and thiol-containing proteins should lead to the development of new therapeutic strategies and drug discoveries for oxidative and electrophilic stress-related diseases.