Different oligomerization dynamics of reduced and oxidized human peroxiredoxin 1 and 2

Human peroxiredoxin 1 and peroxiredoxin 2 (HsPrx1 and HsPrx2), both cytosolic antioxidant enzymes share more than 90% sequence similitude, peroxide substrate specificity, reactivity, and an oligomeric ensemble of five homodimers forming a decamer. However, it is suggested that they serve different purposes in the cell. The question, whether the decamer-dimer equilibrium is relevant to the peroxidase activity and signaling functions has a long-standing history within the field, yet assessing its significance is still a challenge. We have studied the oligomerization dynamics of HsPrx1 and HsPrx2 in their dithiol and disulfide forms to find differences that could provide an explanation for their distinct functions. In this study, we performed analytic size exclusion chromatography (SEC) and fluorescence emission lifetime phasor analysis (FELPA) at different protein concentrations and quantified the relative fraction of the decamer species. We observed that reduced HsPrx2 forms stable decamers that do not fully dissociate, while HsPrx1 exhibits a highly cooperative transition from dimers to decamers with increasing concentration. Disulfide formation at the active site has a larger disruptive effect on the oligomerization equilibrium of HsPrx2 than that of HsPrx1. By performing kinetic measurements using FELPA, we observed that HsPrx2 goes from oxidized dimers to reduced decamers almost 20 times faster than HsPrx1 upon addition of DTT. Lastly, both SEC and FELPA results revealed that the mixture of reduced HsPrx1 and HsPrx2 yields hybrid decamers, that have not been looked for in vivo yet.