A hypothetical model of the cytochrome c peroxidase.cytochrome c complex (Poulos, T. L., and Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330) predicts charge interactions between aspartic acid residues of the peroxidase having a spatial distribution that is complementary to the distribution of essential and highly conserved residues of cytochrome c. In a first attempt to test this model, carboxyl groups of cytochrome c peroxidase have been modified with a water-soluble carbodiimide, either alone or in combination with a nucleophile. Modification led to the loss of up to 90% of the ferrocytochrome c peroxidase activity. At least 4-5 carboxyl groups out of a total of 48, but none of the heme carboxyls, were modified in a derivative with 14% residual activity. In the peroxidase.cytochrome c complex the rate of peroxidase inactivation is slowed and approximately 2 carboxyl groups are protected from chemical modification. In the presence of the carbodiimide, cytochrome c and peroxidase were cross-linked to form a covalent 1:1 complex and the linkage sites were preliminarily characterized. Cross-linking occurred to carboxyl groups of the NH2-terminal fragment 1-119 and of fragment 172-229. The four crucial aspartates of the hypothetical model are located in these same two sequence regions.