Oxidation–Reduction Properties of the Regulatory Site of Spinach Phosphoribulokinase

The oxidation–reduction midpoint potential (Em) of the regulatory disulfide, formed between Cys16 and Cys55, of spinach chloroplast phosphoribulokinase has been determined both for the wild-type enzyme and for a C244S–C250S double mutant, using enzymatic activity to monitor the oxidation–reduction state of the regulatory disulfide. At pH 7.0,Emvalues for the two-electron reduction of the regulatory disulfide of −295 ± 10 and −290 ± 10 mV were measured for the wild-type and mutant, respectively. In contrast to the dependence of activity on ambient potential (Eh) observed for the wild-type enzyme and the double mutant, which both followed the Nernst equation for a two-electron process, high and constant activity was exhibited by a C16S–C244S–C250 triple mutant of the enzyme at allEhvalues tested.Emvalues for the wild-type enzyme were also measured at pH values of 6.7, 7.5, 7.7, and 8.2 and theEmvs pH data in this region give a good fit to a straight line with a slope of −60 mV/pH unit.