Ferrocytochrome c (Fe(II)cyt c) is similar to 10 kcal/mol (410 meV) more stable toward unfolding than Fe(III)cyt c, owing mainly to the stabilization of the ferroheme by hydrophobic encapsulation and enhanced iron-methionine bonding. To determine the magnitudes of these two components, we have measured the binding constants of N-acetylmethionine (AcMet) and imidazole to ferric and ferrous N-acetylmicroperoxidase-8 (AcMP8), a heme-containing proteolytic fragment of cyt c. Our results show that the AcMet affinity of the heme significantly increases upon reduction, as confirmed by electrochemical measurements, and this increase accounts for 130 meV of the 410-meV stability difference between Fe(II)- and Fe(III)cyt c. A 240-mV upshift in reduction potential in the folded protein is attributed mainly to water exclusion from the heme environment, based on an analysis of the potentials of eight structurally characterized c-type cytochromes. Our analysis shows that the potentials of heme proteins can be tuned by roughly 500 mV through variations in cofactor exposure to solvent.