FIG. 7.

Autophosphorylation of PrrB, C-PrrB, and the Q93N mutant form of PrrB in the presence of ubiquinone. The autophosphorylation reactions were performed by using 13 μM purified proteins in the presence or absence of ubiquinone (coenzyme Q1). The reaction mixtures containing DTT (10 mM), ubiquinone (250 μM), or ubiquinol (250 μM) were incubated at 30°C for 20 min, and the reaction was started by the addition of ATP. To generate ubiquinol from ubiquinone and to perform autophosphorylation assays in the presence of ubiquinol, DTT was added to the ubiquinone stock solution as well as the reaction mixture to a final concentration of 10 mM. The autophosphorylation reaction in the presence of 10 mM DTT was performed as a negative control reaction for the ubiquinol-treated reaction. At the time points indicated, samples (10 μl) were removed and added to 3 μl of loading buffer to stop the reaction. Because the autophosphorylation rate of C-PrrB was lower than that of PrrB, the phosphorylation reaction with C-PrrB was performed for longer time. The amount of protein phosphorylation was quantified by SDS-PAGE.