Association constants for metal hexacyanide binding to cytochrome c.

The binding of [Co(CN)6]3-, and that of [Fe(CN)6]3- and [Ru(CN)6]4- using a competitive method, to horse cytochrome c has been studied by 59Co NMR spectroscopy. At I = 0.07 M, without added salt and in 2H2O at pH* 7.3 (measured in 2H2O) and 25 degrees 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 X 10(3) M-1 and 1.5 +/- 0.5 X 10(2) M-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 less than or equal to 1.5 X 10(2) M-1. The binding of [Ru(CN)6]4- at I = 0.07, without added salt and in 2H2O at pH* 7.3 and 23 degrees 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 less than or equal to 3 X 10(2) M-1.