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 (GS3G) and glutathione tetrasulfide (GS4G), yielding the glutathionylated enzyme and the corresponding glutathione hydropersulfide GSSH and hydrotrisulfide GS3H. The second-order rate constants of these enzymatic reductions [≥]107 M-1s-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 oxidants. GSSH and GS3H are further reduced by PfGrx with second-order rate constants on the order of 106-107 M-1s-1, yielding the glutathionylated enzyme as well as hydrogen sulfide (H2S) and hydrogen disulfide (H2S2), 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 (hy-dro)per/polysulfides are efficiently converted to GSSG and H2S 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.