Redox chemistry and acid-base equilibria of mitochondrial plant cytochromes c

Mitochondrial cytochromes c from spinach, cucumber, and sweet potato have been investigated through direct electrochemical measurements and electronic and H-1 NMR spectroscopies, under conditions of varying temperature and pH. The solution behaviors of these plant cytochromes closely resemble, but do not fully reproduce, those of homologous eukaryotic species. The reduction potentials (E degrees') at pH 7 and 25 degrees C are +0.268 V (spinach), +0.271 V (cucumber), and +0.274 V (sweet potato) vs SHE. Three acid-base equilibria have been determined for the oxidized proteins with apparent pK(a) values of 2.5, 4.8, and 8.3-8.9, which are related to disruption of axial heme ligation, deprotonation of the solvent-exposed heme propionate-7 and replacement of the methionine axially bound to the heme iron with a stronger ligand, respectively. The most significant peculiarities with respect to the mammalian analogues include: (i) less negative reduction enthalpies and entropies (Delta S degrees'(rc) and Delta H degrees'(rc)) for the various protein conformers [low- and high-T native (N-1 and N-2) and alkaline (A)], whose effects at pH 7 and 25 degrees C largely compensate to produce E degrees' values very similar to those of the mammalian proteins; (ii) the N-1 - N-2 transition that occurs at a lower temperature (e.g., 30-35 degrees C vs 50 degrees C at pH 7.5) and at a lower pH (7 vs 7.5); and (iii) a more pronounced temperature-induced decrease in the pK(a) for the alkaline transition which allows observation of the alkaline conformer(s) at pH values as low as 7 upon increasing the temperature above 40 degrees C. Regarding the pH and the temperature ranges of existence of the various protein conformers, these plant cytochromes c are closer to bacterial cytochromes c(2).