SITE-SPECIFIC MUTAGENESIS STUDIES OF CYTOCHROMES-C

Cytochromes c are among the best characterized proteins, which consequently make them attractive candidates for study by mutagenesis. Site-specific mutagenesis studies have been reported for five species of cytochromes c, including those from Rhodobacter capsulatus, Saccharomyces cerevisiae, Drosophila melanogaster, rat and horse. The effect of mutations to highly conserved residues on redox potential indicates that substitutions of the M80 axial heme ligand result in the greatest effect (i.e., > 200 mV) while other mutations generally have a small negative effect (i.e., < 60 mV). Denaturation of the mutants suggests that conformational stability is generally decreased upon substitution of conserved residues, with the largest observed destabilization being approx. 4 kcal/mol. As judged by the pK for the alkaline transition of the mutants, the stability of the Fe-S bond can be increased or de-creased by approx. 1 kcal/mol with the largest effects occurring when the mutated group is proximal to the heme. Electron transfer reactions between cytochrome c mutants and various physiological partners are generally not affected to a large degree, an observation that is consistent with their function in vivo. Structural characterization of several mutants by X-ray crystallography suggests that site-specific substitutions generally do not disrupt the overall conformation but result in small local and remote structural perturbations. NMR characterization of several mutants supports the lack of large structural changes but suggests that changes in the dynamic properties of mutants often occur. Taken together, these observations suggest that comprehensive study of equivalent mutations in a number of species is necessary to understand the determinants of cytochrome c structure and function as well as the determinants of evolutionary conservation.