The alkaline conformation (state IV) of yeast iso-1-ferricytochrome c and variants in which selected lysyl residues were replaced with alanyl residues has been studied by H-1 NMR spectroscopy, electronic spectroscopy, EPR spectroscopy, direct electrochemistry, pH-jump kinetics, and temperature-dependent circular dichroism spectroscopy. On the basis of the NMR studies, Lys73 and Lys79 are shown to replace Met80 as the axial ligand in the two conformers of state IV that were detected in previous studies (Hong, X. L.; Dixon, D. W. FEES Lett. 1989, 246, 105-108; Ferrer, J. C.; Guillemette, J. G.; Bogumil, R.; Inglis, S. C.; Smith, M.; Mauk, A. G. J. Am. Chem. Sec. 1993, 115, 7507-7508). The pK(a) for the conformational equilibrium between state III (native conformation) and state N of the wild-type protein (8.70(2)) is found to be intermediate between that of the Lys73 bound conformer (8.44(1)) and that of the Lys79 bound conformer (8.82(2)) (0.1 M NaCl, 25 degrees C) as are the kinetic parameters for the conversion of native protein to each of the two alkaline conformers and the midpoint reduction potentials of the two alkaline forms. The EPR spectra of the Lys73Ala and Lys79Ala variants permit interpretation of the corresponding spectrum of the wild-type protein as the sum of two component conformers. The Lys79Ala variant is slightly more susceptible to thermal denaturation at pH 6.15, but the Lys73Ala variant is less thermally stable than the wild-type cytochrome or the Lys79Ala variant at alkaline pH. The Lys73Ala/Lys79Ala double variant retains the spectroscopic characteristics of the native cytochrome at moderately high pH and appears to undergo a change of axial ligation only under more alkaline conditions (pK(a) similar to 10.5). This observation suggests that the coordination of the amine ligands is a significant contribution toward the driving force for formation of the state IV conformers. These results establish the axial ligation of yeast iso-1-ferricytochrome c state IV, characterize the kinetics with which state III converts to state IV, and establish the electrochemical properties and thermal stabilities of the two conformers that constitute state TV. The results of this work are discussed with reference to pH-dependent structural behavior of other proteins, the mechanism by which these conformers of the ferricytochrome are formed, and the relationship of the present results to those reported previously for the formation of state IV from state III.