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 chemical biology of thiols (RSH, e.g., cysteine and cysteine‐containing proteins/peptides) has been a topic of extreme interest for many decades due to their reported roles in protein structure/folding, redox signaling, metal ligation, cellular protection, and enzymology. While many of the studies on thiol/sulfur biochemistry have focused on thiols, relatively ignored have been hydropersulfides (RSSH) and higher order polysulfur species (RSS_nH, RSS_nR, n > 1). Recent and provocative work has alluded to the prevalence and likely physiological importance of RSSH and related RSS_nH. RSSH of cysteine (Cys‐SSH) has been found to be prevalent in mammalian systems along with Cys‐SSH‐containing proteins. The RSSH functionality has not been examined to the extent of other biologically relevant sulfur derivatives (e.g., sulfenic acids, disulfides, etc.), whose roles in cell signaling are strongly indicated. The recent finding of Cys‐SSH biosynthesis and translational incorporation into proteins is an unequivocal indication of its fundamental importance and necessitates a more profound look into the physiology of RSSH. In this Review, we discuss the currently reported chemical biology of RSSH (and related species) as a prelude to discussing their possible physiological roles.

Irreversible oxidation of Cys residues to sulfinic/sulfonic forms typically impairs protein function. We found that persulfidation (CysSSH) protects Cys from irreversible oxidative loss of function by the formation of CysSSO_1-3H derivatives that can subsequently be reduced back to native thiols. Reductive reactivation of oxidized persulfides by the thioredoxin system was demonstrated in albumin, Prx2, and PTP1B. In cells, this mechanism protects and regulates key proteins of signaling pathways, including Prx2, PTEN, PTP1B, HSP90, and KEAP1. Using quantitative mass spectrometry, we show that (i) CysSSH and CysSSO₃H species are abundant in mouse liver and enzymatically regulated by the glutathione and thioredoxin systems and (ii) deletion of the thioredoxin-related protein TRP14 in mice altered CysSSH levels on a subset of proteins, predicting a role for TRP14 in persulfide signaling. Furthermore, selenium supplementation, polysulfide treatment, or knockdown of TRP14 mediated cellular responses to EGF, suggesting a role for TrxR1/TRP14-regulated oxidative persulfidation in growth factor responsiveness.

Nitric oxide (NO) is a ubiquitous signaling molecule involved in diverse physiological processes, including plant senescence and stomatal closure. The NO and cyclic GMP (cGMP) cascade is the main NO signaling pathway in animals, but whether this pathway operates in plant cells, and the mechanisms of its action, remain unclear. Here, we assessed the possibility that the nitrated cGMP derivative 8-nitro-cGMP functions in guard cell signaling. Mass spectrometry and immunocytochemical analyses showed that abscisic acid and NO induced the synthesis of 8-nitro-cGMP in guard cells in the presence of reactive oxygen species. 8-Nitro-cGMP triggered stomatal closure, but 8-bromoguanosine 3′,5′-cyclic monophosphate (8-bromo-cGMP), a membrane-permeating analog of cGMP, did not. However, in the dark, 8-bromo-cGMP induced stomatal opening but 8-nitro-cGMP did not. Thus, cGMP and its nitrated derivative play different roles in the signaling pathways that lead to stomatal opening and closure. Moreover, inhibitor and genetic studies showed that calcium, cyclic adenosine-5′-diphosphate-ribose, and SLOW ANION CHANNEL1 act downstream of 8-nitro-cGMP. This study therefore demonstrates that 8-nitro-cGMP acts as a guard cell signaling molecule and that a NO/8-nitro-cGMP signaling cascade operates in guard cells.