CYCLIC VOLTAMMETRY AND H-1-NMR OF RHODOPSEUDOMONAS-PALUSTRIS CYTOCHROME C(2) - PROBING SURFACE-CHARGES THROUGH ANION-BINDING STUDIES

The effects of increasing concentrations of Cl-, C10(4)(-), and HCO3- on the redox potential of Rhodopseuclonzorzns palustris cytochrome c(2 ) indicate that the two polyatomic anions bind specifically to the pro tein at one site, while chloride simply exerts an ionic atmosphere eff ect. The change in E(o) upon specific anion binding allows us to probe for the influence of surface charges on the redox potential of cytoch romes c. The decrease in redox potential at null ionic strength (Delta E(1=0)(o)) due to anion neutralization of one positive surface charge was found to be 23 mV with perchlorate and 33 mV with bicarbonate. Th ese values compare reasonably well with previous theoretical predictio ns and estimates of the effect of charge alteration on the E(o) values in cytochromes c chemically modified or mutated at surface lysines. T hese Delta E(o) values, determined on the unmodified protein, are unpr ecedented for c-type cytochromes. The anion-induced chemical shift cha nges of the hyperfine-shifted heme H-1-NMR resonances of the oxidized protein yield lower limit values of 53 M(-1) and 18 M(-1) for the affi nity constant for specific HCO3- and ClO4- binding, respectively.