Nitrosopersulfide (SSNO^-) is a unique cysteine polysulfidating agent with reduction-resistant bioactivity

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 SSNO - is indeed a major product of the reaction of S-nitrosothiols with hydrogen sulfide (H 2S). 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 SSNO - escapes the reducing power of the NADPH-driven biological reducing machineries, the thioredoxin and glutathione reductase systems. The slow decomposition of SSNO - produces 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, SSNO - induces 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 SSNO - would 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: SSNO - is a surprisingly stable bioactive product of the chemical interaction of S-nitrosothiol species and H 2S 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 SSNO - is most likely linked to all these properties. Antioxid. Redox Signal. 33, 1277–1294. </p

Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell?

BACKGROUND AND PURPOSE: Posttranslational modifications of cysteine (Cys) residues represent a major aspect of redox biology, and their reliable detection is key in providing mechanistic insights. The metastable character of these modifications and cell lysis-induced artifactual oxidation render current state-of-the-art protocols to rely on alkylation-based stabilization of labile Cys derivatives before cell/tissue rupture. An untested assumption in these procedures is that for all Cys derivatives alkylation rates are faster than their dynamic interchange. However, when the interconversion of Cys derivatives is not rate-limiting, then electrophilic labeling is under Curtin-Hammett control and hence the final alkylated mixture may not represent the speciation that prevailed before alkylation.KEY RESULTS: We here present evidence that in the majority of cases, the speciation of alkylated polysulfide/thiol derivatives indeed depends on the experimental conditions. Our results reveal that alkylation perturbs sulfur speciation in both a concentration- and time-dependent manner, and that strong alkylating agents can cleave polysulfur chains. Moreover, we show that labeling of sulfenic acids with dimedone also affects Cys speciation, suggesting that part of the endogenous pool of products previously believed to represent sulfenic acid species may in fact represent polysulfides.EXPERIMENTAL APPROACH: These observations were obtained using buffered aqueous solutions of inorganic-, organic-, cysteine-, glutathione- and GAPDH-polysulfide species. Additional experiments in human plasma and serum revealed that monobromobimane can extract sulfide from the endogenous sulfur pool by shifting speciation equilibria, suggesting caution should be exercised when interpreting experimental results using this tool.CONCLUSION AND IMPLICATION: We highlight methodological caveats potentially arising from these pitfalls and conclude that current derivatization strategies often fail to adequately capture physiologic speciation of sulfur species.</p