THE EFFECTS OF SURFACE-CHARGES ON THE REDOX POTENTIAL OF CYTOCHROME-C2 FROM THE PURPLE PHOTOTROPHIC BACTERIUM RHODOBACTER-CAPSULATUS

Four site-directed mutants of Rhodobacter capsulatus cytochrome c2, which substitute lysines at three positions with aspartate or glutamate, have been prepared. Mutations included the single charge substitutions K12D, K14E, and K32E and a double charge substitution K14E K32E. Characterization of the ionic strength dependence of the wild-type and mutant redox potentials in the "nonbinding" buffer Tris-cacodylate suggests that (i) at zero ionic strength introduction of negatively charged groups stabilizes the oxidized state by 11-14 mV per charge and (ii) at high ionic strengths where the charged groups are masked, the effects of single charge substitutions are overcome; however, the redox potential of the double charge substitution is still affected. These results indicate that at physiological ionic strengths charge distribution only affects redox potential when the heme environment has been perturbed by a structural perturbation and that the determinants of redox potential in c-type cytochromes is primarily due to the local heme environment. © 1991.