Glutaredoxins rapidly reduce glutathione hydroper- and polysulfides

Hydropersulfides have gained attention in cell biology as excellent nucleophiles and membrane-protective radical scavengers. They form perthiyl radicals, which terminate radical chain reactions through self-recombination, leading to the formation of polysulfides. It is currently unknown how polysulfides are subsequently reduced again in non-enzymatic or enzymatic metabolic pathways. Here we used stopped-flow kinetic measurements in combination with mass spectrometry to show that the model class I glutaredoxin from the malaria parasite Plasmodium falciparum (PfGrx) rapidly reduces the polysulfides glutathione trisulfide (GS ₃ G) and glutathione tetrasulfide (GS ₄ G), yielding the glutathionylated enzyme and the corresponding glutathione hydropersulfide GSSH and hydrotrisulfide GS ₃ H. The second-order rate constants of these enzymatic reductions ≥10 ⁷ M ⁻¹ s ⁻¹ are even slightly higher than for glutathione disulfide (GSSG). In contrast, PfGrx was inactive or only moderately active using cystine or cysteine trisulfide as oxi-dants. GSSH and GS ₃ H are further reduced by PfGrx with second-order rate constants on the order of 10 ⁶ –10 ⁷ M ⁻¹ s ⁻¹ , yielding the glutathionylated enzyme as well as hydrogen sulfide (H ₂ S) and hydrogen disulfide (H ₂ S ₂ ), respectively. Thus, glutaredoxins specifically recognize the glutathione moiety of glutathione (hydro)polysulfides and glutathione hydropersulfide. Due to the rapid reduction of glutathionylated glutaredoxins by reduced glutathione (GSH), glutathione (hydro)per/polysulfides are efficiently converted to GSSG and H ₂ S or the corresponding hydrogen polysulfides. As a consequence, the steady-state concentration of glutathione (hydro)per/polysulfides should be tightly controlled in subcellular compartments containing active glutaredoxins and high GSH concentrations.