Analogues of the 39-residue CNBr fragment of horse cytochrome c (66-104) have been prepared by total chemical synthesis. Conformationally assisted ligation of these peptides with the native cytochrome c fragment 1-65 (homoserine lactone form) occurred in high yield. Semisynthetic protein molecules of the expected molecular weight were obtained that had folded structures similar to the native molecule as shown by spectral properties and by cross-reactivity with a panel of monoclonal antibodies sensitive to the three-dimensional integrity of cytochrome c. Point mutations were introduced into the horse sequence at three strongly conserved sites: Tyr67, Thr78, and Ala83. The contributions of these 3 residues to the stability of the heme crevice were estimated by titration of the 695 nm absorption due to coordination of ferric iron by the sixth ligand methionine sulfur. The roles of these residues in catalysis of electron transfer and in establishing the value of the redox potential of cytochrome c were also investigated. The hydroxyl group of Tyr67 modulates the spectral properties of the heme and has a profound influence on its redox properties, but hydrogen bonding involving this phenolic hydroxyl does not stabilize the heme crevice. In contrast, we find that Thr78 is strongly stabilizing and that asparagine is not an adequate substitute for this residue because of the greater entropic cost of burying its side chain. The low biological activity of analogues modified at this position, despite normal redox potentials, imply a role for Thr78 in the electron transfer mechanism. The replacement of Ala83 by proline induces a similar phenomenon. An involvement of this residue in the catalysis of electron transfer provides an explanation of the low reactivity of plant mitochondrial cytochromes c in mammalian redox systems.