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

Using the voltammetric method of square-wave voltammetry, a direct electroc hemical examination was made of the wild type and Tyr67Phe mutant of both r at cytochrome c and yeast iso-1-cytochrome c. In addition to determining th e equilibrium reduction potential (E-0') for each cytochrome, the entropy o f reaction, Delta S-Rxn(0)'(Delta S-Rxn(0)' = S-Red(0)' - S-Ox(0)'), for th e reduction process was determined via the non-isothermal method. Having de termined Delta S-Rxn(0)' and E-0', Delta H-0' was calculated. For rat cytoc hrome c, it was found that Delta S-Rxn(0)' = -43 J mol(-1) K-1 for the wild type and -53 J mol(-1) K-1 for the Tyr67Phe variant, with the Delta H-0' f or both the wild type and variant nearly identical, indicating that the cha nges in reduction potential and probably stability are due to changes in De lta S-Rxn(0)'. In contrast the measured Delta S-Rxn(0)' for yeast iso-1-cyt ochrome c demonstrated significant changes in both entropic and enthalpic c ontributions 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 d erivation are presented that show how the entropies of reaction of wild typ e versus variant cytochromes contribute to and predict changes in stability in going from oxidized to reduced protein. For yeast iso-l-cytochrome c, t he thermodynamically predicted change in stability was very close to the ex perimentally observed value, based on previous differential scanning calori metric stability measurements. While such data is not available for rat cyt ochrome c, consideration of the enormously increased local stability of the rat oxidized cytochrome c variant predicts that the reduced rat variant wi ll be even more stable than the already stabilized oxidized variant. (C) 19 99 Elsevier Science B.V. All rights reserved.