Using entropies of reaction to predict changes in protein stability: tyrosine-67-phenylalanine variants of rat cytochrome c and yeast Iso-1 cytochromes c

Abstract

Using the voltammetric method of square-wave voltammetry, a direct electrochemical examination was made of the wild type and Tyr67Phe mutant of both rat cytochrome c and yeast iso-1-cytochrome c. In addition to determining the equilibrium reduction potential ( E 0′) for each cytochrome, the entropy of reaction, Δ S 0′ Rxn(Δ S 0′ Rxn= S 0′ Red− S 0′ Ox), for the reduction process was determined via the non-isothermal method. Having determined Δ S 0′ Rxn and E 0′, Δ H 0′ was calculated. For rat cytochrome c, it was found that Δ S 0′ Rxn=−43 J mol −1 K −1 for the wild type and −53 J mol −1 K −1 for the Tyr67Phe variant, with the Δ H 0′ for both the wild type and variant nearly identical, indicating that the changes in reduction potential and probably stability are due to changes in Δ S 0′ Rxn. In contrast the measured Δ S 0′ Rxn for yeast iso-1-cytochrome c demonstrated significant changes in both entropic and enthalpic contributions in going from wild type to mutant cytochrome c. The entropy of reaction provides information regarding the relative degree of solvation, and very likely the degree of compactness, of the oxidized state versus the reduced state of the redox protein. A thermodynamic scheme and stability derivation are presented that show how the entropies of reaction of wild type versus variant cytochromes contribute to and predict changes in stability in going from oxidized to reduced protein. For yeast iso-1-cytochrome c, the thermodynamically predicted change in stability was very close to the experimentally observed value, based on previous differential scanning calorimetric stability measurements. While such data is not available for rat cytochrome c, consideration of the enormously increased local stability of the rat oxidized cytochrome c variant predicts that the reduced rat variant will be even more stable than the already stabilized oxidized variant.