Last year we showed that the cysteine residue in the active site of certain eukaryotic peroxiredoxin (Prx) enzymes undergoes reversible oxidation to sulfinic acid (Cys?SOOH) during catalysis. An enzyme named sulfiredoxin (Srx) has been identified as responsible for reversal of the resulting enzyme inactivation in yeast. We have now characterized mammalian orthologs of yeast Srx with an assay based on monitoring of the reduction of sulfinic Prx by immunoblot analysis with antibodies specific for the sulfinic state. Sulfinic reduction by mammalian Srx was found to be a slow process (kcat = 0.18/min) that requires ATP hydrolysis. ATP could be efficiently replaced by GTP, dATP, or dGTP but not by CTP, UTP, dCTP, or dTTP. Both glutathione and thioredoxin are potential physiological electron donors for the Srx reaction, given that their Km values (1.8 mM and 1.2 microM, respectively) are in the range of their intracellular concentrations and the Vmax values obtained with the two reductants were similar. Although its pKa is relatively low (~7.3), the active site cysteine of Srx remained reduced even when the active site cysteine of most Prx molecules became oxidized. Finally, depletion of human Srx by RNA interference suggested that Srx is largely responsible for reduction of the Cys?SOOH of Prx in A549 human cells. Various proapoptotic stimuli increase the production of superoxide and H2O2 by mitochondria. Whereas superoxide impairs mitochondrial function and is removed by Mn2+-dependent superoxide dismutase, the role and metabolism of mitochondrial H2O22 during apoptosis have remained unclear. The effects on apoptotic signaling of depletion of peroxiredoxin (Prx) III, a mitochondrion-specific H2O2-scavenging enzyme, have now been investigated by RNA interference in HeLa cells. Depletion of Prx III resulted in increased intracellular levels of H2O2 and sensitized cells to induction of apoptosis by staurosporine or TNF-alpha. The rates of mitochondrial membrane potential collapse, cytochrome c release, and caspase activation were increased in Prx III?depleted cells, and these effects were reversed by ectopic expression of Prx III or mitochondrion-targeted catalase. Depletion of Prx III also exacerbated damage to mitochondrial macromolecules induced by the proapoptotic stimuli. Our results suggest that Prx III is a critical regulator of the abundance of mitochondrial H2O2, which itself promotes apoptosis in cooperation with other mediators of apoptotic signaling.
