Understanding the molecular mechanisms responsible for regulation of the redox potentials (Em) of proteins is a problem of immense fundamental and practical importance. Cytochromes c (cyts c), in which heme Fe is coordinated to His side chain imidazole N and Met side chain S atoms as axial ligands at the redox center, are some of the best characterized redox active proteins. Homologous cyts c, thermophilic Hydrogenobacter thermophilus cytochrome c552 (HT) and mesophilic Pseudomonas aeruginosa cytochrome c551 (PA), exhibit a unique thermodynamic property such that, despite their structural similarity, HT is significantly more stable than PA. Site-directed mutants of PA, for which amino acid substitutions were selected with reference to the corresponding residues in HT, exhibited thermostabilities between those of PA and HT. Furthermore, we found that cyt c with higher stability in its oxidized form exhibits a lower Em value. This finding provides novel insights into the functional regulation of cyts c, which could be utilized for tuning the Em value of the proteins by means of protein engineering.