| Abstract
| - The apparent equilibrium constant (Kapp) of the alkaline transition (AT) of beef heart cytochromec, obtained from pH titrations of the current intensities in cyclic voltammetry experiments, has beenmeasured as a function of the temperature from 5 to 65 °C, at different ionic strength (I = 0.01−0.2 M).The temperature profile of the pKapp values is biphasic and yields two distinct sets of ΔH°‘AT and ΔS°‘ATvalues below and above approximately 40 °C. In the low-temperature range, the process is endothermicand is accompanied by a small positive entropy change, while at higher temperatures it becomes lessendothermic and involves a pronounced entropy loss. The temperature dependence of the transitionthermodynamics is most likely the result of the thermal transition of native ferricytochrome c from alow-T to an high-T conformer which occurs at alkaline pH values at a temperature comparable withabove (Ikeshoji, T., Taniguchi, I., and Hawkridge, F. M. (1989) J. Electroanal. Chem. 270, 297−308;Battistuzzi, G., Borsari, M., Sola, M., and Francia, F. (1997) Biochemistry36, 16247−16258). Thus, it isapparent that the transitions of the two native conformers to the corresponding alkaline form(s) arethermodynamically distinct processes. It is suggested that this difference arises from either peculiartransition-induced changes in the hydration sphere of the protein or to the preferential binding of differentlysines to the heme iron in the two temperature ranges. Extrapolation of the Kapp values at null ionicstrength allowed the determination of the thermodynamic equilibrium constants (Ka) at each temperature,hence of the “true” standard thermodynamic parameters of the transition. The pKa value at 25 °C wasfound to be 8.0. A pKapp value of 14.4 was calculated for the alkaline transition of ferrocytochrome c at25 °C and I = 0.1 M. The much greater relative stabilization of the native state in the reduced as comparedto the oxidized form turns out to be almost entirely enthalpic in origin, and is most likely due to thegreater affinity of the methionine sulfur for the Fe(II) ion. Finally, it is found that the Debye−Hückeltheory fits the ionic strength dependence of the pKapp values, at least qualitatively, as observed previouslyfor the ionic strength dependence of the reduction potential of this protein class. It is apparent that theincrease in the pKapp values with increasing ionic strength is for the most part an entropic effect.
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