NADPH oxidation assay of PaAhpC and SeAhpC after treatment with compounds C1 and C2.

To perform the assays PaAhpC and SeAhpC samples were previously reduced with DTT (5 mM/1h). The DTT excess was removed, and the enzymes were treated with 40 molar equivalents of C1 (A and C, red square) or C2 (B and D, red square) for 1hour at room temperature. The compounds excess was removed, and the peroxidase activity was accessed by the NADPH oxidation in reactions containing: 3 μM of PaAhpC or SeAhpC, 6 μM EcTrx, 0.9 μM EcTrxR and 150 μM NADPH in buffer 50 mM HEPES (pH = 7.4), 100 μM DTPA and 1 mM sodium azide. Reactions were incubated at 37°C for 5 minutes before being initiated by the addition of H2O2 (500 μM) and monitored spectrophotometrically at 340 nm, 37°C, for 5 minutes. Reactions containing PaAhpC or SeAhpC without natural compounds treatment (green square) were used as positive controls for peroxidase activity, and reactions without AhpC (black square) were used as negative controls. PaAhpC or SeAhpC samples previously treated with NEM were used as control of protein inhibition (blue square). All experiments were performed at least three times in triplicate.