Direct evidence for the formation of a complex between 1-cysteine peroxiredoxin and glutathione S-transferase pi with activity changes in both enzymes.
Glutathione S-transferase pi (GST pi) 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 pi 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 pi activity is approximately 25% of that of the GST pi 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 pi, 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 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 intersubunit disulfide. Finally, the GSH-mediated reduction of the disulfide regenerates the reduced active-site sulfhydryl of 1-Cys Prx.
Ralat
Luis A
LA
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Manevich
Yefim
Y
Fisher
Aron B
AB
Colman
Roberta F
RF
D003545:Cysteine / Q000737:chemistry*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004793:Enzyme Reactivators / Q000737:chemistry / Q000378:metabolism; D051549:Glutathione S-Transferase pi / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism*; D054465:Peroxiredoxin VI; D054464:Peroxiredoxins; D017434:Protein Structure, Tertiary; D013438:Sulfhydryl Compounds / Q000737:chemistry
D004793:Enzyme Reactivators; D009097:Multienzyme Complexes; D013438:Sulfhydryl Compounds; D010544:Peroxidases; C515940:PRDX1 protein, human; C515977:PRDX6 protein, human; D054465:Peroxiredoxin VI; D054464:Peroxiredoxins; D051549:Glutathione S-Transferase pi; D003545:Cysteine