Purification and characterization of the peroxiredoxin 5-thioredoxin 2 complex: unraveling the redox regeneration of peroxiredoxin 5 by thioredoxin 2 within the peroxiredoxin catalytic cycle.
Peroxiredoxin (Prx) is a critical cysteine-based peroxidase that detoxifies reactive oxygen species to maintain cellular peroxide homeostasis and prevent oxidative stress. Peroxiredoxin 5 (Prx5 or PRDX5) is the unique atypical 2-cysteine Prx in humans, which is able to scavenge the peroxides through redox-active cysteines. However, the precise regeneration mechanism of Prx5 by thioredoxin (Trx) within the catalytic cycle of Prx is not yet well understood. Here, we developed a design strategy through disulfide bond crosslinking to demonstrate that both Cys151 and Cys47 of Prx5 are able to form intermolecular disulfide bonds with thioredoxin 2 (Trx2). Gel filtration was further performed to validate the formation of Prx5-Trx2 complex in solution. We presented a scheme for obtaining the purified Prx5-Trx2 complex, and elucidated that this complex is a heterotetramer with a 1:1 molar ratio of both proteins. Predicted structure model of Prx5-Trx2 complex revealed that each Trx2 protein engages exclusively with a single subunit of the Prx5 dimer, which is distinctly different from the interaction mode observed in yeast Ahp1-Trx2 complex. Collectively, these results provide novel mechanistic insights into the transient Prx5-Trx2 complex formation and the electron transfer from electron donor Trx2 to Prx5 during the catalytic redox regeneration step.