We highlight methodological caveats potentially arising from these pitfalls and conclude that current derivatization strategies often fail to adequately capture physiological speciation of sulfur species.

In aqueous systems, the reaction between H S and RSSR results in the formation of equilibria whereby H S, RSH, RSSR, RSSH and RSSSR are present. In a biological system, any of these species can be responsible for the observed biological activity. These equilibrium species can also be generated via the reaction of RSH with RSSSR. Due to these equilibria, Cys-SSS-Cys can be a method for generating any of the other species. Importantly, HEK293T cells treated with Cys-SSS-Cys results in increased levels of hydropersulfides, allowing examination of the biological effects of RSSH.

The reversible oxidation of protein cysteine residues (Cys-SH) is a key reaction in cellular redox signaling involving initial formation of sulfenic acids (Cys-SOH), which are commonly detected using selective dimedone-based probes. Here, we report that significant portions of dimedone-tagged proteins are susceptible to cleavage by DTT reflecting the presence of perthiosulfenic acid species (Cys-SSOH) due to similar oxidation of hydropersulfides (Cys-SSH), since Cys-S-dimedone adducts are stable toward DTT. Combined studies using molecular modeling, mass spectrometry, and cell-based experiments indicate that Cys-SSH are readily oxidized to Cys-SSOH, which forms stable adducts with dimedone-based probes. We additionally confirm the presence of Cys-SSH within protein tyrosine kinases such as EGFR, and their apparent oxidation to Cys-SSOH in response NADPH oxidase activation, suggesting that such Cys-SSH oxidation may represent a novel, as yet uncharacterized, event in redox-based signaling.

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Aims:The aim of the present study was to investigate the biochemical properties of nitrosopersulfide (SSNO -), a key intermediate of the nitric oxide (NO)/sulfide cross talk. Results: We obtained corroborating evidence that SSNOis indeed a major product of the reaction of S-nitrosothiols with hydrogen sulfide (H 2 S). It was found to be relatively stable (t 1/2 *1 h at room temperature) in aqueous solution of physiological pH, stabilized by the presence of excess sulfide and resistant toward reduction by other thiols. Furthermore, we here show that SSNOescapes the reducing power of the NADPHdriven biological reducing machineries, the thioredoxin and glutathione reductase systems. The slow decomposition of SSNOproduces inorganic polysulfide species, which effectively induce per/polysulfidation on glutathione or protein cysteine (Cys) residues. Our data also demonstrate that, in contrast to the transient activation by inorganic polysulfides, SSNOinduces long-term potentiation of TRPA1 (transient receptor potential ankyrin 1) channels, which may be due to its propensity to generate a slow flux of polysulfide in situ. Innovation: The characterized properties of SSNOwould seem to represent unique features in cell signaling by enabling sulfur and nitrogen trafficking within the reducing environment of the cytosol, with targeted release of both NO and polysulfide equivalents. Conclusion: SSNOis a surprisingly stable bioactive product of the chemical interaction of S-nitrosothiol species and H 2 S that is resistant to reduction by the thioredoxin and glutathione systems. As well as generating NO, it releases inorganic polysulfides, enabling transfer of sulfane sulfur species to peptide/protein Cys residues. The sustained activation of TRPA1 channels by SSNOis most likely linked to all these properties.