Thermodynamics of the alkaline transition of cytochrome c

The apparent equilibrium constant (K-app) Of the alkaline transition (AT) o f beef heart cytochrome c, obtained from pH titrations of the current inten sities in cyclic voltammetry experiments, has been measured as a function o f the temperature from 5 to 65 degrees C, at different ionic strength (I = 0.01-0.2 M). The temperature profile of the pK(app) values is biphasic and yields two distinct sets of Delta H-o'(AT) and Delta S-o'(AT) values below and above approximately 40 degrees C. In the low-temperature range, the pro cess is endothermic and is accompanied by a small positive entropy change, while at higher temperatures it becomes less endothermic and involves a pro nounced entropy loss; The temperature dependence of the transition thermody namics is most likely the result of the thermal transition of native ferric ytochrome c from a low-T to an high-T conformer which occurs at alkaline pH values at a temperature comparable with above (Ikeshoji, T., Taniguchi, I. , and Hawkridge, F. M. (1989) J. Electroanal. Chem. 270, 297-308; Battistuz zi, G., Borsari, M, Sola, M., and Francia, F. (1997) Biochemistry 36, 16247 -16258). Thus, it is apparent that the transitions of the two native confor mers to the corresponding alkaline form(s) are thermodynamically distinct p rocesses. It is suggested that this difference arises from either peculiar transition-induced changes in the hydration sphere of the protein or to the preferential binding of different lysines to the heme iron in the two temp erature ranges. Extrapolation of the K-app values at null ionic strength al lowed the determination of the thermodynamic equilibrium constants (K-a) at each temperature, hence of the "true" standard thermodynamic parameters of the transition. The pK(a) value at 25 degrees C was found to be 8.0, A pK( app) value of 14.4 was calculated for the alkaline transition of ferrocytoc hrome c at 25 degrees C and I = 0.1 M. The much greater relative stabilizat ion of the native state in the reduced as compared to the oxidized form tur ns out to be almost entirely enthalpic in origin, and is most likely due to the greater affinity of the methionine sulfur for the Fe(II) ion. Finally, it is found that the Debye-Huckel theory fits the ionic strength dependenc e of the pK(app) values, at least qualitatively, as observed previously for the ionic strength dependence of the reduction potential. of this protein class. It is apparent that the increase in the pK(app) values with increasi ng ionic strength is for the most part: an entropic effect.