Seven monoclonal antibodies, prepared to yeast holo- or apo-iso-1-cytochrome c by the method of Köhler and Milstein (Goding, J. W. (1983) Monoclonal Antibodies: Principles and Practice, Academic Press, Orlando, FL) were characterized by cross-reaction with a panel of evolutionarily related cytochromes c, apocytochromes c, fragments and homologous and hybrid fragment complexes, inhibition, competitive inhibition, and complementation and fluorescence titration. The results have permitted us to assign the specifically recognized amino acids as follows. IgG1 monoclonals: 4-74-6, Leu-63 and/or Asn-67 and/or Asn-68; 4-128-6, Glu-93; 4-145-10, Thr-74; 2-96-12, Asp-65; 2-34-19, Lys-59; and 10-28-86, trimethyl-lysine 77. IgM monoclonal 39-14, Pro-30 and His-31. With mAb 4-128-6 substitution of glutamic acid 93 with alanine, as it occurs in Candida cytochrome c, has resulted in a decrease in affinity by a factor of 10(4). A calculation appears to show that this value is too large to be accounted for solely by the sum of energy losses due to disruption of charge neutralization and changes of hydrophobic interaction including van der Waals interaction. This and similar results with mAb 10-28-86 have led us to the idea that some new extra interatomic interaction sensitive to differences in configuration of atomic groups may be present and perturbed in the substitution. Furthermore, the assumption of the presence of such interaction can explain the striking similarity between the antigen-antibody interaction (e.g. Geysen, H. M., Meloen, R. H., and Barteling, S. J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3998-4002) and a model system of horse cytochrome c three-fragment complex (Juillerat, M. A., and Taniuchi, H. (1986) J. Biol. Chem. 261, 2697-2711) with respect to the high specificity of interacting residues in the interface. Thus, by analogy to the hypothesis developed in the model system (Fisher, A., and Taniuchi, H. (1988) FASEB J. 2, A1338), we hypothesize that a closed interaction loop would be formed on the basis of contacting groups including glutamic acid 93 across or within the interface between the antigen and mAb 4-128-6 and mediate delocalized interaction to generate extra binding energy.