The cytochrome c/cytochrome c peroxidase system has been extensively investigated as a model for long-range electron transfer in biology. Two models for the structure of the one-to-one cytochrome c/cytochrome c peroxidase complex in solution exist: one is based upon computer docking of the two proteins and the second is based upon the structure of the complex in the crystalline state. Titration calorimetry is used to investigate the interaction of horse ferricytochrome c with baker's yeast cytochrome c peroxidase and with six cytochrome c peroxidase mutants. Five of the six peroxidase mutants eliminate a negative charge in the cytochrome c binding site by replacing a side-chain carboxylate with an amide. The sixth mutation replaces a surface alanine residue with phenylalanine. The binding affinity between cytochrome c and the cytochrome c peroxidase mutants varies from no significant change in comparison to the wild-type enzyme to a 4-fold decrease in the equilibrium association constant. The pattern of decreasing cytochrome c binding affinity for the cytochrome c peroxidase mutants is consistent with the cytochrome c binding domain defined by X-ray crystallography [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. For those mutants which have lower affinity for cytochrome c, the lower affinity is due to a decrease in the entropy change upon complex formation, consistent with the difference in hydration of carboxylate and amide groups.