## 3 Results
### Quaternary Structures Characterization of Bacterial Thr-Prx and Ser-Prx
However, we could detect a lower NADPH consumption for Thr-Prx (PaAhpC and SeAhpC\({}^{\text{S46T}}\)) compared to the reciprocal Ser-Prx (PaAhpC\({}^{\text{T43S}}\) and SeAhpC) (Figure 7D). These results indicate that bacterial Ser-Prx present a subtle resistance to hyperoxidation to CHP, but not to H\({}_{2}\)O\({}_{2}\) compared to the bacterial Thr-Prx. We also evaluated the inactivation of Thr-Prx and Ser-Prx by thermal stress. The differential scanning fluorimetry (DSF) curve for PaAhpC presented a lag phase and the determined \(T_{\text{m}}\) was 58.5 \(\pm\) 1.4 \({}^{\circ}\)C, while PaAhpC\({}^{\text{T44S}}\) displayed a sudden increase in the fluorescence and the \(T_{\text{m}}\) of 66.1 \(\pm\) 2.3 \({}^{\circ}\)C (Figure 8A). Therefore, replacement of the catalytic triad Thr by Ser resulted in an increase about 8 \({}^{\circ}\)C in the \(T_{\text{m}}\) (Figure 8A). For SeAhpC and SeAhpC\({}^{\text{S46T}}\), the shapes of the thermal sigmoidal profiles were very similar, although the Ser to Thr substitution resulted in a decrease of \(\approx\)9 \({}^{\circ}\)C of the \(T_{\text{m}}\) (61.3 \(\pm\) 0.5 and 52.7 \(\pm\) 0.6 \({}^{\circ}\)C, respectively) (Figure 8B). Therefore, the presence of Ser in the catalytic triad increased the thermal stabilities of the AhpCs. Since the thermal stability increased in Ser-Prx, we tested if the proteins retained their peroxidase activities after the thermal pre-treatments. The Thr-Prx (PaAhpC and SeAhpC\({}^{\text{S46T}}\)) were partially inactivated after being heated to 55 \({}^{\circ}\)C (Figure 9A,D). However, the effect of temperature was much more noticeable for SeAhpC\({}^{\text{S46T}}\), where the pre-treatment at 55 \({}^{\circ}\)C abolished the peroxidase activity of the enzyme (Figure 9D). This result agreed with the determined \(T_{\text{m}}\) values, as the enzyme SeAhpC\({}^{\text{S46T}}\) displayed the lowest \(T_{\text{m}}\) value. On the other hand, the Ser-Prx (PaAhpC\({}^{\text{T43S}}\) and SeAhpC) retained the activity in all temperatures tested (Figure 9B,C). Figure 6: NADPH oxidation assay for evaluation of AhpC hyperoxidation of _P. aeruginosa_ and _S. epidermidis_ using the heterologous _E. coli_ Trx system. Reactions were executed containing PaAhpC (**A**,**E**), PaAhpC\({}^{\text{T43S}}\) (**B**,**F**), SeAhpC (**C**,**G**), or SeAhpC\({}^{\text{S46T}}\) (**D**,**H**) (3 \(\upmu\)M), EcTrx (6 \(\upmu\)M), EcTrxR (0.9 \(\upmu\)M), NADPH (150 \(\upmu\)M), 50 mM HEPES pH 7.4, 1 mM sodium azide, and 100 \(\upmu\)M DTPA. The experiments were started adding growing concentrations of H\({}_{2}\)O\({}_{2}\) (**A**–**D**) or CHP (**E**–**H**) (0 \(\upmu\)M = \(\upmu\) black, 500 \(\upmu\)M = \(\upmu\) blue, 1 mM = \(\times\) orange, 5 mM = \(\upmu\) green and 10 mM = \(\upmu\) red). The reactions were incubated for 5 min/37 \({}^{\circ}\)C. The oxidation of NADPH was monitored spectrophotometrically at 340 nm. The error bars represent the standard deviations. All experiments were performed in triplicate and repeated at least three times, yielding consistent results. Figure 8: AhpC thermal unfolding monitored by DSF. Differential scanning fluorimetry of PaAhpC (**A**, blue line), PaAhpC\({}^{\text{TM3S}}\) (**A**, red line), SeAhpC (**B**, red line), and SeAhpC\({}^{\text{S46T}}\) (**B**, blue line). Proteins were previously reduced with DTT (20 mM), desalted (PD10, GE Healthcare), and incubated with SYBR Orange dye. The fluorescence scanning was carried out using a temperature gradient from 25 \({}^{\circ}\)C to 95 \({}^{\circ}\)C. The melting temperature (\(T_{\text{m}}\)) was determined by the Boltzmann equation to determine the midpoint of thermal denaturation. The experiments were performed in triplicate and repeated at least three times with similar results. Figure 7: Evaluation of Thr-Prx and Ser-Prx inhibition.