| Abstract
| - Ferrocytochrome c (Fe(II)cyt c) is ∼10 kcal/mol (410 meV) more stable toward unfolding thanFe(III)cyt c, owing mainly to the stabilization of the ferroheme by hydrophobic encapsulation and enhancediron−methionine bonding. To determine the magnitudes of these two components, we have measured thebinding 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 theheme significantly increases upon reduction, as confirmed by electrochemical measurements, and this increaseaccounts for 130 meV of the 410-meV stability difference between Fe(II)- and Fe(III)cyt c. A 240-mV upshiftin 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 analysisshows that the potentials of heme proteins can be tuned by roughly 500 mV through variations in cofactorexposure to solvent.
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