## 4 Discussion  
The roles of Thr or Ser in the catalytic triad of the typical 2-Cys Prx has been considered redundant, but recently, we (and others) showed that Ser-Prx are more stable in the decameric form [3; 49]. Here, we demonstrated that the presence of Ser or Thr in the active site affected also the C\({}_{\text{P}}\) hyperoxidation susceptibilities, especially for yeast 2-Cys Prx, besides being related to thermal resistance and the chaperone activities acquisition of AhpCs. Furthermore, we showed that the vast majority of multicellular organisms have Thr on catalytic triad. Ser-Prx is more prevalent in bacteria, which might be related with diverse ecological strategies in some groups of this domain. Although the typical 2-Cys Prx from bacteria are very resistant to hyperoxidation, we demonstrated that the Thr to Ser substitution slightly increased the resistance to inactivation of AhpC by CHP. Previously, eukaryotic 2-Cys Prx were described to be highly susceptible to oxidative inactivation in comparison with bacterial counterparts [40]. Accordingly, eukaryotic 2-Cys Prx were named as sensitive, while bacterial enzymes were referred as robust. Furthermore, authors identified the presence of GGLG and YF motifs exclusively in eukaryotic 2-Cys Prx, which were proposed to be positively selected during evolution [40]. The presence of the GGLG and YF motifs near the C\({}_{\text{P}}\) residue would represent a steric hindrance effect, decreasing the rate of intermolecular disulfide bond formation [37]. According to this model, C\({}_{\text{R}}\) and H\({}_{2}\)O\({}_{2}\) compete for C\({}_{\text{P}}\)-SOH. Therefore, enzymes with fast rates of condensation reaction between C\({}_{\text{R}}\) and C\({}_{\text{P}}\)-SOH would correspond to increased resistance to hyperoxidation. More recently, two other structural elements, named motifs A and B, were identified by conferring resistance to hyperoxidation in 2-Cys Prx [50]. Here, we analyzed the susceptibilities to hyperoxidation of sensitive (Tsa1, Tsa2) and robust (PaAhpC, SeAhpC) 2-Cys Prx carrying Thr or Ser in the catalytic triad. Remarkably, yeast Ser-Prx (Tsa1\({}^{\text{T}445}\) and Tsa2) are considerably more resistant to inactivation than Thr  
Figure 10: Comparative protection of PaAhpC and SeAhpC to citrate synthase (CS) aggregation by thermal stress. Chaperone activity of PaAhpC and SeAhpC was monitored by its ability to protect citrate synthase (CS) from thermally induced aggregation. The aggregation of 2 uM of CS in 40 mM HEPES-KOH pH 7.5 was monitored by light scattering (\(\lambda\) = 360 nm) for 90 min, either alone or in the presence of 40 uM PaAhpC (**A**) or SeAhpC (**B**). The assays were executed with the enzyme pre-heated at 48 \({}^{\circ}\)C for 1 h (red lines) or not (blue lines). The CS alone was used as negative control (black lines). The experiments were performed in triplicates under the same conditions. Prx (Tsa1 and Tsa2 54T) by hyperoxidation (Figure 2, Figure 3, and Figure 7) and this effect is only moderate in bacterial enzymes treated with CHP (Figures 6 and 7). Noteworthy, motifs A and B previously described as resistance factors to hyperoxidation [50] are also highly similar among the yeast and bacterial counterparts (Supplementary material, Table S1 and Figure S3). Therefore, the observed differences in inactivation between Tsa1 and Tsa2 or between PaAhpC and SeAhpC are probably not related to the compositions of motifs A and B in these enzymes. In fact, the natural substitution of catalytic triad Thr to Ser stands as a novel trait related to resistance or susceptibility to C\({}_{\text{P}}\) hyperoxidation. We also analyzed the crystallographic structures of Tsa1 (PDB = 3 SBC) and Tsa2 (PDB = 5 DVB) to gain insights on mechanisms underlying differences in the hyperoxidation susceptibilities. In our previous work [3], we observed that the side chain of catalytic Ser presents higher mobility than the Thr in Tsa1. The additional methyl group in Thr side chain interacts with an aromatic ring in the adjacent dimer.