## 3 Results  
### Presence of a Ser in the Catalytic Triad Increased Resistance of Yeast 2-Cys Prx to Hyperoxidation  
Previously, we observed that the presence of a Ser residue in the catalytic triad stabilized yeast 2-Cys Prx in the decameric state even in the disulfide form [3]. Here, we investigated if the presence of a Ser or Thr in the catalytic triad might also affect the hyperoxidation susceptibilities of these enzymes.  
The decrease of NADPH consumption at increasing hydroperoxides concentrations (H\({}_{2}\)O\({}_{2}\) in Figure 2 and CHP in Figure 3) is consistent with 2-Cys Prx inhibition by C\({}_{\text{P}}\) hyperoxidation. Tsa1 (a Thr-Prx) was remarkably more susceptible to inactivation (Figure 2A) when compared to Tsa2 (a Ser-Prx) (Figure 2C). At concentrations higher than 1 mM, H\({}_{2}\)O\({}_{2}\) significantly inhibited Tsa1 (Figure 2A), while high doses of H\({}_{2}\)O\({}_{2}\) (10 mM) did not inactivate Tsa2 (Figure 2C). Then, NADPH assays with mutant proteins (Tsa1T44S and Tsa254T)were performed. Tsa1T44S (Figure 2B) was more resistant to H\({}_{2}\)O\({}_{2}\) inactivation than wild type Tsa1 (Figure 2A). In contrast, Tsa2544T (Figure 2D) was more susceptible to inactivation than wild type Tsa2 (Figure 2C). Therefore, these results indicated that Thr-Prx are more susceptible to hyperoxidation than Ser-Prx.  
We then verified the susceptibilities of these yeast 2-Cys Prx to hyperoxidation by an organic hydroperoxide (CHP). The peroxidase activities of Thr-Prx were almost abolished in all concentrations tested for Tsa1 (Figure 3A) and Tsa2544T (Figure 3D). In contrast, Ser-Prx were considerably more resistant to inactivation (Figure 3B,C).  
We confirmed that 2-Cys Prx inactivation occurred due to C\({}_{\text{P}}\) hyperoxidation, by western blot assays using anti-SO\({}_{3}\)H antibody (Supplementary Material, Figure S1) from aliquots of samples described in Figures 2 and 3. Hyperoxidized 2-Cys Prx migrated as a dimer (-46 kDa) in non-reducing SDS-PAGE, when one of the active sites was in the disulfide form and the other with the C\({}_{\text{P}}\) hyperoxidized (C\({}_{\text{P}}\)-SO\({}_{3}\)H) or as monomer, when all C\({}_{\text{Ps}}\) were hyperoxidized.  
Two aspects deserve to be highlighted from these results. As already observed [58], Tsa1 is more sensitive to hyperoxidation by CHP than to H\({}_{2}\)O\({}_{2}\) (Figures 2A and 3A). We now observed that the same characteristic for Tsa2 (Figures 2C and 3C). Additionally, we verified that Ser-Prx (Tsa2 and Tsa1T44S) are more resistant to hyperoxidation than Thr-Prx (Tsa1 and Tsa2544T) (Figures 2 and 3). Accordingly, \(\Delta\)_tsa2_ yeast strains are more sensitive to organic hydroperoxides than \(\Delta\)_tsa1_[59].  
Figure 2: Tsa1, Tsa2, and mutant inhibition by H\({}_{2}\)O\({}_{2}\). _S. cerevisiae_ Tsa1 (**A**), Tsa1T44S (**B**), Tsa2 (**C**), or Tsa2544T (**D**) were exposed to increasing amounts of H\({}_{2}\)O\({}_{2}\). The assays were performed with 1 \(\upmu\)M Prx, 2 \(\upmu\)M Trx1, 0.3 \(\upmu\)M TrxR1, and increased amounts of H\({}_{2}\)O\({}_{2}\) (0 \(\upmu\)M = \(\bullet\) black, 500 \(\upmu\)M = \(\blacksquare\) blue, 1 mM = \(\times\) orange, 5 mM = \(\blacktriangledown\) green and 10 mM = \(\blacklozenge\) red) in buffer containing 50 mM HEPES pH 7.4, 100 \(\upmu\)M DTPA, 1 mM sodium azide and 150 \(\upmu\)M NADPH. The decrease in NADPH absorbance was followed at 340 nm. The error bars represent the standard deviation. All the experiments were performed in triplicate and repeated at least three times with similar results.