Ferroptosis is a type of cell death caused by radical-driven lipid peroxidation, leading to membrane damage and rupture. Here we show that enzymatically produced sulfane sulfur (S 0 ) species, specifically hydropersulfides, scavenge endogenously generated free radicals and, thereby, suppress lipid peroxidation and ferroptosis. By providing sulfur for S 0 biosynthesis, cysteine can support ferroptosis resistance independently of the canonical GPX4 pathway. Our results further suggest that hydropersulfides terminate radical chain reactions through the formation and self-recombination of perthiyl radicals. The autocatalytic regeneration of hydropersulfides may explain why low micromolar concentrations of persulfides suffice to produce potent cytoprotective effects on a background of millimolar concentrations of glutathione. We propose that increased S 0 biosynthesis is an adaptive cellular response to radical-driven lipid peroxidation, potentially representing a primordial radical protection system. NATURE CHEMiCAL BioLoGy | www.nature.com/naturechemicalbiology Articles NaTurE CHEmICal BIOlOGyprotect against ferroptosis independently of GPX4 by supplying sulfur to S 0 biosynthetic pathways. (3) The elevation of endogenous S 0 levels makes cells more resistant to LPO and ferroptosis, whereas the lowering of S 0 levels sensitizes cells to LPO and ferroptosis. (4) An increase of endogenous S 0 levels lowers endogenous radical load, and a decrease of endogenous S 0 levels increases endogenous radical load. (5) Exogenously supplied S 0 donors suppress ferroptosis in various cellular models. ( 6) Persulfides catalyze GSH-dependent radical scavenging in vitro, as they are regenerated via an autocatalytic cycle that couples radical reduction to glutathione oxidation through the formation and recombination of perthiyl radicals. Together, these results establish hydropersulfides as important radical scavengers and modulators of ferroptotic cell death. ResultsCystine uptake can inhibit ferroptosis independently of GPX4. It is well-understood that cellular cystine (Cys 2 ) uptake contributes to ferroptosis resistance. Inside the cell, Cys 2 is quickly reduced to Cys. Cys is needed for the synthesis of GSH, which, in turn, allows GPX4 to reduce lipid peroxides. However, it has been shown more recently that Cys is also a source of sulfur for S 0 species, including glutathione hydropersulfide and hydropolysulfides (GSS x H) 14,21 . Analyzing the cells used in this study, we found that about 1% of the total GSH pool is persulfidated (Extended Data Fig. 1a). We, therefore, asked if S 0 could contribute to ferroptosis resistance by suppressing LPO in a GPX4-independent manner (Fig. 1a). To address this question, we first assessed how endogenous S 0 levels change when cells are primed to undergo ferroptosis. Treatment of cells with the GPX4 inhibitor RSL3 or the lipophilic oxidant cumene hydroperoxide (CHP) increased endogenous S 0 levels (Fig. 1b and Extended Data Fig. 1b). Similarly, genetic deletion of floxed Gpx4 in Pfa1 cells 22 ...

Protein S-persulfidation (P-SSH) is recognized as a common posttranslational modification. It occurs under basal conditions and is often observed to be elevated under stress conditions. However, the mechanism(s) by which proteins are persulfidated inside cells have remained unclear. Here we report that 3-mercaptopyruvate sulfur transferase (MPST) engages in direct protein-to-protein transpersulfidation reactions beyond its previously known protein substrates thioredoxin and MOCS3/Uba4, associated with H2S generation and transfer RNA thiolation, respectively. We observe that depletion of MPST in human cells lowers overall intracellular protein persulfidation levels and identify a subset of proteins whose persulfidation depends on MPST. The predicted involvement of these proteins in the adaptation to stress responses supports the notion that MPST-dependent protein persulfidation promotes cytoprotective functions. The observation of MPST-independent protein persulfidation suggests that other protein persulfidases remain to be identified.