## 3 Results
### Quaternary Structures Characterization of Bacterial Thr-Prx and Ser-Prx  
Considering the higher abundance of Ser-Prx in Bacteria, we evaluated the role of Thr/Ser in AhpC enzymes. We selected to analyze recombinant AhpCs from _Pseudomonas aeruginosa_ (PaAhpC--a Thr-Prx) and AhpC from _Staphylococcus epidermidis_ (SeAhpC--a Ser-Prx), as they belong to the most abundant groups of Thr-Prx (Proteobacteria) and of Ser-Prx (Terrabacteria). As with yeast 2-Cys Prx, we performed reciprocal Ser-Thr mutations and we carried out SEC analysis of bacterial 2-Cys Prx. While PaAhpC was mostly decamer in the reduced form and dimer when oxidized (Figure 5A), PaAhpCT48 was predominantly decamer when oxidized or reduced (Figure 5B). Similarly, yeast Tsa1 and _Salmonella typhimurium_ AhpC (both Thr-Prx) are decamers when reduced and dimers when oxidized in disulfides, while the substitution of Thr to Ser confers higher stability to the decamer even when the 2-Cys Prx are oxidized [3; 49; 39]. Similarly to other Ser-Prx [3; 48], SeAhpC was presented mostly as decamer independently of the redox state although a small amount of dimers was present in the reduced form (Figure 5C). Additionally, most of SeAhpC8666 was presented as decamer and only a small fraction of the enzyme was detected as dimers, independently of the redox state (Figure 5D). It has been shown by SEC and TEM that the AhpC from _Enterococcus faecalis_, a naturally Ser-Prx, is a decamer independent of the redox state [43], similarly to yeast  
Figure 5: Size exclusion chromatography of PaAhpC and SeAhpC and Thr/Ser mutants. PaAhpC (**A**), PaAhpCT48 (**B**), SeAhpC (**C**), and SeAhpC866 (**D**), at a final concentration of 30 \(\upmu\)M, were treated with TCEP 5 mM (red lines) or H2O2 36 \(\upmu\)M (blue lines) for 30 min, at room temperature, before being submitted to SEC. The elution profile was monitored at a wavelength of 280 nm, at flow rate of 1 mL/min. Bovine thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa) were used as molecular standards. The experiments were performed at least three times. Tsa2 [3]. Thus, our results showed that the presence of Thr or Ser in the catalytic triad is a relevant factor related to the redox sensitive oligomerization, but additional features also interfere with the quaternary assembly of typical 2-Cys Prx [43; 45; 61]. Comparative Analysis of the Inactivation of Bacterial Thr-Prx and Ser-Prx by High hydroperoxide Concentrations and by Thermal Insult  
We also investigated if the presence of Thr or Ser in the catalytic triad could affect the susceptibility of bacterial 2-Cys Prx to hyperoxidation. In the experimental conditions tested, H\({}_{2}\)O\({}_{2}\) did not inhibit the NADPH consumption in the reaction mixtures containing Thr-Prx (Figure 6A,D) or Ser-Prx (Figure 6B,C) even at the highest doses (10 mM). However, when CHP was the substrate, a slight inhibition was observed to Thr-Prx (compare Figure 6E,F and Figure 6G,H). Again, we confirmed that the inhibition of peroxidase activity was a consequence of the C\({}_{\text{P}}\) hyperoxidation by western blot analysis performed using anti-SO\({}_{3}\) antibody (Supplementary Material, Figure S2). As the inhibition to bacterial Thr-Prx was less evident in relation to eukaryotic counterparts, we compared the NADPH consumption after 300 s from the data presented in Figure 2, Figure 3, and Figure 6 (Figure 7). For the yeast enzymes, we observed that NADPH consumption is quite similar between Thr-Prx and Ser-Prx at lower H\({}_{2}\)O\({}_{2}\) concentrations, but it is considerably higher in samples containing Ser-Prx when 5 or 10 mM H\({}_{2}\)O\({}_{2}\) were used (Figure 7A). When CHP was the substrate, the NADPH consumption was also more evident for Ser-Prx (Figure 7B). For the bacterial enzymes, there was no clear difference among the enzymes (Figure 7C).