The effect of binding ions on the oxidation of horse heart ferrocytochrome c

The effect of various specific binding ions on the rate of oxidation by potassium ferricyanide of electrodialyzed horse heart ferroeytochrome c was studied. The ionic strength was kept constant using Tris–cacodylate buffer, pH 7.0. Either the Tris or cacodylate ion was replaced by the binding ion studied. At an ionic strength of 0.194 M (24 °C), replacing cacodylate by chloride decreased the bimolecular oxidation rate constant from about 13.3 × 106 (Tris–cacodylate) to about 8.8 × 106 M−1 s−1 (Tris–chloride). Comparable decreases were found when cacodylate was replaced by phosphate or when Tris was replaced by potassium. When picrate replaced cacodylate (ionic strength 0.0485 M) a larger decrease was found, from about 5.2 × 107 to about 0.8 × 107 M−1 s−1. Data at intermediate ion concentrations were consistent with a simple cooperative binding model. The calculated association constants for chloride, potassium, and phosphate were in the range of 2–20 M−1, while for picrate it was 500 M−1. The data were consistent with one bound ion per ferroeytochrome c molecule, except for picrate, for which two binding sites were suggested. The results were interpreted by the hypothesis that the picrate ion binds near the solvent-exposed heme edge. The electron transfer reaction of ferricyanide was also presumed to take place through this region. The other ions probably bind at some distance from the heme edge and were suggested to exert their effect by perturbing the proteins' solvent shell. Consistent with this was the effect of replacing H2O by D2O which decreased the oxidation rate constant by about 50%.