Direct Evidence for Complex Formation Between 1‐Cys Peroxiredoxin and Glutathione S‐Transferase pi

Abstract

A heterodimer complex forms between 1-Cys peroxiredoxin (1-Cys Prx) with a C-terminal His-tag and glutathione S-transferase pi (GST pi) upon incubation at pH 8.0 in buffer containing 20% 1,6-hexanediol, followed by dialysis against buffer containing 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 shows the two proteins are present in equimolar amounts. In the heterodimer, 1-Cys Prx is fully active, while the GST pi activity is ~25% of that of the GST pi homodimer. In contrast, the 1-Cys Prx homodimer is inactive in the presence of free GSH. The yield of heterodimer is decreased in the absence of 1,6-hexanediol or of 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/heterodimer. Based on mutagenesis, for Prx, neither Cys-47 nor Ser-32 is required for heterodimer formation, but Cys-47 is essential for Prx activation. For GST pi, Cys-47 and Tyr-7 (at the GSH-binding site) are needed for heterodimer formation. We conclude that reactivation of oxidized 1-Cys Prx by GST pi occurs by heterodimerization of 1-Cys Prx and GST pi harboring bound GSH, followed by glutathionylation of 1-Cys Prx and then formation of an inter-subunit disulfide. Finally, the GSH-mediated reduction of the disulfide regenerates the active site sulfhydryl of 1-Cys Prx. (Supp. By NIH R01-CA66561, F31-GM75387 & P01-HL79063.)