Sulfhydration by a hydrogen sulfide anion and electrophile thiolation by reactive sulfur species (RSS) such as persulfides/polysulfides (e.g., R-S-SH/R-S-S<inf>n</inf>-H(R)) are unique reactions in electrophilic signaling. Using 1,2-dihydroxynaphthalene-4-thioacetate (1,2-NQH<inf>2</inf>-SAc) as a precursor to 1,2-dihydroxynaphthalene-4-thiol (1,2-NQH<inf>2</inf>-SH) and a generator of reactive oxygen species (ROS), we demonstrate that protein thiols can be modified by a reactive sulfenic acid to form disulfide adducts that undergo rapid cleavage in the presence of glutathione (GSH). As expected, 1,2-NQH<inf>2</inf>-SAc is rapidly hydrolyzed and partially oxidized to yield 1,2-NQ-SH, resulting in a redox cycling reaction that produces ROS through a chemical disproportionation reaction. The sulfenic acid forms of 1,2-NQ-SH and 1,2-NQH<inf>2</inf>-SH were detected by derivatization experiments with dimedone. 1,2-NQH<inf>2</inf>-SOH modified Keap1 at Cys171 to produce a Keap1-S-S-1,2-NQH<inf>2</inf> adduct. Subsequent exposure of A431 cells to 1,2-NQ or 1,2-NQH<inf>2</inf>-SAc caused an extensive chemical modification of cellular proteins in both cases. Protein adduction by 1,2-NQ through a thio ether (C-S-C) bond slowly declined through a GSH-dependent S-transarylation reaction, whereas that originating from 1,2-NQH<inf>2</inf>-SAc through a disulfide (C-S-S-C) bond was rapidly restored to the free protein thiol in the cells. Under these conditions, 1,2-NQH<inf>2</inf>-SAc activated Nrf2 and upregulated its target genes, which were enhanced by pretreatment with buthionine sulfoximine (BSO), to deplete cellular GSH. Pretreatment of catalase conjugated with poly(ethylene glycol) suppressed Nrf2 activation by 1,2-NQH<inf>2</inf>-SAc. These results suggest that RSS-mediated reversible electrophilic signaling takes place through sulfenic acids formation under oxidative stress.
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