Figure 2. NADPH-dependent peroxidase activity of EcAhpC and EcAhpC_1-186-YFSKHN.

Sensitivity of (A) EcAhpC and (B) EcAhpC_1-186-YFSKHN to over-oxidation was determined by the measurement of NADPH oxidation at 1 mM (red), 10 mM (green), 20 mM (blue), and 30 mM H₂O₂ (magenta). The background oxidation without Prxs is shown as a control in black. The decrease in NADPH-oxidation with increasing concentrations of H₂O₂ is visible for both EcAhpC and EcAhpC_1-186-YFSKHN. (C) Percentage of enzyme activity at each H₂O₂ concentration was calculated for WT EcAhpC and its mutants (Mean ± 1 SD) using the rate of NADPH oxidation at each concentration of H₂O₂. The rate of activity at the lowest concentration of H₂O₂ was taken as 100%. With increasing H₂O₂ concentrations, the percentage of enzyme activity decreased. (D) NAPDH oxidation of EcAhpC and EcAhpC_1-186-YFSKHN measured at 30 μM H₂O₂ is shown as a representative for all other measurements done at various H₂O₂ concentrations to establish the enzyme kinetic parameters. Michaelis-Menten plot of (E) EcAhpC and (F) EcAhpC_1-186-YFSKHN was done by fitting data of at least ten concentrations of H₂O₂.