## 4 Discussion
Thus, the B-factor of active site regions in the 10 chains of Tsa2 structure are higher than observed to Tsa1. Now, we analyzed the interactions of catalytic Thr and Ser in an attempt to explain the differences in hyperoxidation resistance. We observed that the Ser44 in some chains of Tsa2 are in position quite similar to Thr44 in Tsa1 (Figure 11A). However, in three chains, the Ser44 of Tsa2 are found in an alternative position compared to that one observed to Thr44. Consequently, the motif GGLG is moved away from the C-terminal \(\alpha\)-helix where YF motif is found (Figure 11B) and a helix upstream to the GGLG motif is also deformed. We propose that the high mobility of Ser side chain makes the interaction between GGLG and C-terminal motif YF more unstable and, therefore, facilitate the approximation between C\({}_{\text{P}}\) and C\({}_{\text{R}}\) and disulfide formation. In fact, our data indicated that, in yeast enzymes (displaying the GGLG/YF motifs), the differences in hyperoxidation are more pronounced between Ser-Prx and Thr-Prx. Accordingly, [PERSON] and colleagues (2021) [61] showed that substitution of human Prx2 C\({}_{\text{R}}\) by amino acids that disrupts the C-terminal structure is able to affect the enzyme activity, indicating an interconnection between the C-terminal region and the active site microenvironment in eukaryotes. These data indicate that even regions located far from the active site can exert effect in the 2-Cys Prx activity. Figure 11: Active site, GGLG and YF motif regions of Tsa1 and Tsa2. (**A**) Superposition of Tsa1C47S (3 SBC = pink) and Tsa2C47S (5 DVB = light blue) show similar position of Thr44 (Tsa1) and Ser44 (Tsa2). Motif GGLG (red in Tsa1 and blue in Tsa2) and CT \(\alpha\)-helix (dark red in Tsa1 and dark blue in Tsa2) are also in quite similar position. (**B**) When Ser44 is found in an alternative conformation, GGLG motif is displaced to a further position, while CT \(\alpha\)-helix do not appear in the crystallographic structure probably due to being more mobile and, consequently, with low resolution. Crystallographic structures are presented in cartoon and highlighted amino acids are presented as balls and sticks. Graphical models were generated by PyMOL. In contrast, Thr/Ser substitutions in PaAhpC and SeAhpC produce less pronounced effects on the susceptibilities to hyperoxidation, possibly due to GGLG and YF motifs are absent at these enzymes. We compared the crystallographic structure of a Thr-Prx (_Salmonella typhimurium_ AhpC-PDB entry 1 YEP) with the only AhpC containing Ser in the PDB database (_Enterococcus faecalis_--PDB entry 5Y63), but we did not observe any major structural differences. However, it is worth to mention that disulfide formation in bacterial AhpC belonging to Thr-Prx group is considerable faster (75 s\({}^{-1}\)) compared to human enzymes such as Prx2 and Prx3 (1.7 and 22 s\({}^{-1}\), respectively) [38; 64]. We also observed that 2-Cys Prx are more susceptible to hyperoxidation by organic hydroperoxides than H\({}_{2}\)O\({}_{2}\) (Figures 2, 3 and 6), as previously described [34], which may be related with the environment close to the active site pocket. Crystallographic observations revealed the presence of large hydrophobic portions around this region [40; 43; 48]. In fact, _alpc_ gene inactivation frequently render bacteria with increased sensitivity to organic peroxide and AhpC over-expression invariably leads to a gain of resistance against these oxidants [65; 66; 67; 68; 69; 70; 71]. Moreover, _Mycobacterium tuberculosis_ and _Helicobacter pylori_ mutants for _alpc_ gene accumulated higher levels of lipid hydroperoxides during aerobic growth, when compared to wild type strains [72]. Considering that presence of Ser in catalytic triad makes 2-Cys Prx resistant to C\({}_{\text{P}}\) hyperoxidation, more stable as a decamer and increase its chaperone activity, we speculate that these enzymes may present complementary functions, protecting cells from severe stressful conditions.