Alternative pathway of H2S and polysulfides production from sulfurated catalytic-cysteine of reaction intermediates of 3-mercaptopyruvate sulfurtransferase

Abstract

It has been known that hydrogen sulfide and/or polysulfides are produced from a (poly)sulfurated sulfur-acceptor substrate of 3-mercaptopyruvate sulfurtransferase (MST) via thioredoxin (Trx) reduction in vitro. In this study, we used thiosulfate as the donor substrate and the catalytic reaction was terminated on the formation of a persulfide or polysulfides. We can present alternative pathway of production of hydrogen sulfide and/or polysulfides from (poly)sulfurated catalytic-site cysteine of reaction intermediates of MST via Trx reduction. Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometric analysis revealed that after prolonged incubation of MST with thiosulfate, a trisulfide adduct becomes predominant at the sulfurated catalytic-site cysteine. When these adducts were reduced by Trx with reducing system (MST:Escherichia coli Trx:E. coli Trx reductase:NADPH = 1:5:0.02:12.5 molar ratio), liquid chromatography with tandem mass spectrometric analysis for monobromobimane-derivatized H₂S_n revealed that H₂S₂ first appeared, and then H₂S and H₂S₃ did later. The results were confirmed by high-performance liquid chromatography-fluorescence analysis.

Keywords: 3-Mercaptopyruvate sulfurtransferase; Hydrogen sulfide; Monobromobimane; Polysulfide; Reaction intermediate; Thiosulfate.