Figure 6

Participation of Fe²⁺ in FlaR enzymatic activity. (a) Cysteine significantly stimulates rFlaR NADP-reductase activity (Spearman correlation, r = 1, **p = 0.0083). (b) Solutions containing either 10 µM Fe²⁺ or 200 µM NADP or 50 µM FAD were incubated with cysteine. The oxidative state of Fe²⁺ was assessed with ferrozine staining and monitored at A₅₆₂ nm. The oxidative state of FAD was monitored at A₄₅₀ nm and that of NADP monitored at A₃₄₀ nm. Fe²⁺, FAD and NADPH in aqueous solutions were allowed to oxidize for 2 hrs, and then cysteine was added for additional 2 hrs. Oxidation was observed only in Fe²⁺ solution which was significantly reduced upon cysteine addition. The results presented are the mean of three different experiments (one way ANOVA with Dunnett’s post hoc multiple comparisons test, n = 3 in each group, n = 3 in each group, ****p = 0.0001). (c) Increasing concentrations of Fe²⁺ significantly enhanced rFlaR NADP-reductase activity (Pearson correlation, r = 0.9947, ****p < 0.0001).