Isomerization and denaturation of homologous cytochromes c: correlation between local and gross conformational changes.

To elucidate the relationship between local and gross conformational changes, three types of conformational changes, i.e., alkaline isomerization, ligand replacement, and guanidine hydrochloride (GuHCl) denaturation, of a set of homologous and modified cytochromes c were investigated by spectroscopic methods. Cytochromes c examined include the horse, tuna, Candida, monomeric Saccharomyces, and disulfide-linked dimeric Saccharomyces proteins. Correlations were found between the apparent pK (pKa) for the isomerization and the equilibrium constants for the binding of imidazole and azide to the heme iron: the lower the pKa value, the higher the binding constant. This is explained by the fact that the isomerization and ligand replacement involve similar conformational changes localized around the sixth coordination position of the heme. A good correlation was also observed between the susceptibilities to local and gross conformational changes: the lower the pKa value for the isomerization, the lower the GuHCl concentration for the midpoint of the denaturation. Thermodynamic analysis suggests that this correlation is not due to the involvement of similar conformational changes in the two processes. Cooperative stabilization of the tertiary structure is proposed to interpret this correlation.