Reactive sulfur species are inactivated and excreted as trimethylsulfonium ion by thiopurine S-methyltransferase

Reactive sulfur species (RSS) play essential roles in cellular signaling, redox regulation, and defense against electrophilic stress. However, excessive RSS are cytotoxic and must be strictly regulated. While RSS elimination has been canonically attributed to oxidative pathways, here we show that thiopurine S -methyltransferase (TPMT), a selenium-associated methyltransferase, promotes the detoxification and urinary excretion of RSS. In a mouse model, knockout of TPMT decreased urinary excretion of the trimethylsulfonium, suggesting that TPMT catalyzes a non-oxidative elimination of RSS via methylation. Cell-based assays demonstrated that TPMT exerts a protective effect against excess RSS. In vitro , TPMT methylated hydrosulfide, polysulfides (HSS − , HS 4 − ), and persulfides (GSS − ). ESI-MS analysis confirmed the formation of methylated GSS − . Kinetic analyses revealed substrate-dependent differences in methylation efficiency. In silico analyses suggested that steric hindrance of the substrate is a key determinant of the methylation efficiency. Collectively, these findings uncover an energy-dependent, non-oxidative pathway for the elimination of RSS beyond classical oxidative detoxification. Given that TPMT polymorphisms are clinically important determinants of thiopurine drug metabolism, variation in TPMT activity may likewise influence biological responses to RSS. Together, these results provide a foundation for understanding how endogenous methylation shapes RSS homeostasis.