Association Constants for Metal Hexacyanide Binding to Cytochrome c

The binding ofCo(CN)₆]^3?, and that ofFe(CN)₆]^3? and Ru(CN)₆]^4? using a competitive method, to horse cytochrome c has been studied by ⁵⁹ Co NMR spectroscopy. At I = 0.07 M, without added salt and in ²H₂O at ph* 7.3 (measured in ²H₂O) and 25°C, there are at least two binding sites on ferricytochrome c and ferrocytochrome c for Co(CN)₆]^3?. Association constants were determined to be 2.0 ± 0.6 × 10³M^?1 and 1.5 ± 0.5 × 10²M^?1 respectively. with no effect of the oxidation state of the cytochrome. At higher ionic strength (I = 0.12 M adjusted with KCl the binding markedly decreased, and, although it was not possible to determine the precise binding stoichiometry and magnitude of association constants, it is clear that the association constants are ≤ 1.5 × 10^tM^?1 The binding of Ru(CN)₆]^4? at I = 0.07, without added salt and in ²H₂O at pH 1.3 and 23°C, was not precisely defined, but its binding strength relative to that of Fe(CN)₆]^3? was determined. Extrapolating this to I = 0.12 (KCl) suggests that under these conditions the association constant for Ru(CN)₆]^4? binding to ferricytochrome c is ≤ 3 × 10²M^?1.