FIG 3 .

Effect of reduction and oxidation of bound ubiquinone on RegB kinase activity. (A) Effect of the reduction of bound ubiquinone on RegB C265S activity. Ten micromolar purified RegB C265S was kept at 22°C for 10 min in the absence (solid line with solid circles) or presence (dashed line with open circles) of NaBH₄ before ATP was added to start the kinase reaction. (B) Effect of oxidation of bound ubiquinone on RegB C265S activity. Purified RegB C265S was incubated in the absence (solid line with solid circles) or presence (dashed line with open circles) of 2 mM K₃Fe(CN)₆ before ATP was added to start the kinase reaction. (C) Effect of the oxidation of reduced bound ubiquinone on RegB C265S activity. Bound ubiquinone in purified RegB C265S was first reduced by NaBH₄ at room temperature (RT) for 10 min and then incubated at 22°C for 10 min in the absence (solid line with solid circles) or presence (dashed line with open circles) of 2 mM K₃Fe(CN)₆ before ATP was added to start the kinase reaction. (D) Effect of oxidation of bound ubiquinone on the activity of RegB″ C265S. RegB″ C265S without the transmembrane domain was treated as described in the legend to panel C as a control.