Direct Evidence for the Formation of a Complex between 1-Cysteine Peroxiredoxin and Glutathione S-Transferase π with Activity Changes in Both Enzymes

Glutathione S-tranferase π (GST π) has been shown to reactivate oxidized 1-cysteine peroxiredoxin (1-Cys Prx, Prx VI, Prdx6, and AOP2). We now demonstrate that a heterodimer complex is formed between 1-Cys Prx with a C-terminal His6 tag and GST π upon incubation of the two proteins at pH 8.0 in buffer containing 20% 1,6-hexanediol to dissociate the homodimers, followed by dialysis against buffer containing 2.5 mM glutathione (GSH) but lacking 1,6-hexanediol. The heterodimer can be purified by chromatography on nickel-nitriloacetic acid agarose in the presence of GSH. N-Terminal sequencing showed that equimolar amounts of the two proteins are present in the isolated complex. In the heterodimer, 1-Cys Prx is fully active toward either H2O2 or phospholipid hydroperoxide, while the GST π activity is ∼25% of that of the GST π homodimer. In contrast, the 1-Cys Prx homodimer lacks peroxidase activity even in the presence of free GSH. The heterodimer is also formed in the presence of S-methylglutathione, but no 1-Cys Prx activity is found under these conditions. The yield of heterodimer is decreased in the absence of 1,6-hexanediol or GSH. Rapid glutathionylation of 1-Cys Prx in the heterodimer is detected by immunoblotting. Subsequently, a disulfide-linked dimer is observed on SDS−PAGE, and the free cysteine content is decreased by 2 per heterodimer. The involvement of particular binding sites in heterodimer formation was tested by site-directed mutagenesis of the two proteins. For 1-Cys Prx, neither Cys47 nor Ser32 is required for heterodimer formation but Cys47 is essential for 1-Cys Prx activation. For GST π, Cys47 and Tyr7 (at or near the GSH-binding site) are needed for heterodimer formation but three other cysteines are not. We conclude that reactivation of oxidized 1-Cys Prx by GST π occurs by heterodimerization of 1-Cys Prx and GST π harboring bound GSH, followed by glutathionylation of 1-Cys Prx and then formation of an intersubunit disulfide. Finally, the GSH-mediated reduction of the disulfide regenerates the reduced active-site sulfhydryl of 1-Cys Prx.