The apparent equilibrium constant (K-app) Of the alkaline transition (AT) of beef heart cytochrome c, obtained from pH titrations of the current intensities in cyclic voltammetry experiments, has been measured as a function of 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 process is endothermic and is accompanied by a small positive entropy change, while at higher temperatures it becomes less endothermic and involves a pronounced entropy loss; The temperature dependence of the transition thermodynamics is most likely the result of the thermal transition of native ferricytochrome 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; Battistuzzi, G., Borsari, M, Sola, M., and Francia, F. (1997) Biochemistry 36, 16247-16258). Thus, it is apparent that the transitions of the two native conformers to the corresponding alkaline form(s) are thermodynamically distinct processes. 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 temperature ranges. Extrapolation of the K-app values at null ionic strength allowed 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 ferrocytochrome c at 25 degrees C and I = 0.1 M. The much greater relative stabilization of the native state in the reduced as compared to the oxidized form turns 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 dependence 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 increasing ionic strength is for the most part: an entropic effect.

Thermodynamics of the alkaline transition of cytochrome c / Battistuzzi, Gianantonio; Borsari, Marco; Loschi, Lodovica; A., Martinelli; Sola, Marco. - In: BIOCHEMISTRY. - ISSN 0006-2960. - STAMPA. - 38:(1999), pp. 7900-7907.

Thermodynamics of the alkaline transition of cytochrome c

BATTISTUZZI, Gianantonio;BORSARI, Marco;LOSCHI, Lodovica;SOLA, Marco
1999

Abstract

The apparent equilibrium constant (K-app) Of the alkaline transition (AT) of beef heart cytochrome c, obtained from pH titrations of the current intensities in cyclic voltammetry experiments, has been measured as a function of 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 process is endothermic and is accompanied by a small positive entropy change, while at higher temperatures it becomes less endothermic and involves a pronounced entropy loss; The temperature dependence of the transition thermodynamics is most likely the result of the thermal transition of native ferricytochrome 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; Battistuzzi, G., Borsari, M, Sola, M., and Francia, F. (1997) Biochemistry 36, 16247-16258). Thus, it is apparent that the transitions of the two native conformers to the corresponding alkaline form(s) are thermodynamically distinct processes. 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 temperature ranges. Extrapolation of the K-app values at null ionic strength allowed 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 ferrocytochrome c at 25 degrees C and I = 0.1 M. The much greater relative stabilization of the native state in the reduced as compared to the oxidized form turns 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 dependence 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 increasing ionic strength is for the most part: an entropic effect.
38
7900
7907
Thermodynamics of the alkaline transition of cytochrome c / Battistuzzi, Gianantonio; Borsari, Marco; Loschi, Lodovica; A., Martinelli; Sola, Marco. - In: BIOCHEMISTRY. - ISSN 0006-2960. - STAMPA. - 38:(1999), pp. 7900-7907.
Battistuzzi, Gianantonio; Borsari, Marco; Loschi, Lodovica; A., Martinelli; Sola, Marco
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/305640
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