The kinetic and spectroscopic changes accompanying the binding of two monoclonal antibodies to the oxidized form of horse heart cytochrome c have been investigated. The two epitopes recognized by the antibodies are distinct and noninteracting: antibody 2B5 binds to native cytochrome c near a type II turn (residue 44) while antibody 5F8 binds on the opposite face of the protein near the amino terminus of an a-helical segment (residue 60). Antibody-cytochrome c binding obeys a simple bimolecular reaction mechanism with second-order rate constants approaching those expected for diffusion-limited proteinprotein interactions. The association rate constants have small activation enthalpies and are inversely dependent on solvent viscosity, as expected for diffusion-controlled reactions. There is a moderate ionic strength dependence of the rate of association between the 2BS antibody and cytochrome c, with the rate constant increasing about 4-fold as the ionic strength is varied between 0.14 and 0 M. Comparison of the rates for antibody-cytochrome c complex formation for binding to the reduced-native, oxidized-native, and alkaline conformations shows that for MAb 2BS the forward rate constant depends slightly on cytochrome c conformation. Investigation of the pH-induced transition between the native and alkaline conformational states for free cytochrome c and for antibody-cytochrome c complexes shows that antibody binding stabilizes the native form of the protein. The rate of the native to alkaline conformational change is decreased for cytochrome c in complex with antibody 2B5 but is unchanged in complexes with antibody 5F8, suggesting a structural change in the epitope near residue 44 but no change in the epitope near residue 60 between the alkaline conformation and the alkaline-native transition state. A combination of electronic absorption, fluorescence, circular dichroism, and magnetic circular dichroism spectroscopies has been used to probe the geometric and electronic structure of the heme in free cytochrome c and antibody-complexed cytochrome c. Antibody binding to cytochrome c causes no change in the magnetic circular dichroism spectrum, suggesting that the coordination sphere of the metal ion remains unchanged. Changes in heme absorption and circular dichroism spectra accompanying binding of the 2B5 antibody to cytochrome c show that binding alters the heme environment, possibly by inducing a structural change within the protein antigen. This antibody is known to bind in the vicinity of the heme crevice. The antibody 5F8 which binds on the opposite surface does not elicit these effects. © 1992, American Chemical Society. All rights reserved.