Regulation of the redox order of four hemes by pH in cytochrome C3 from D. vulgaris Miyazaki F

The assignment of 1 H-NMR signals of the heme methyl and propionate groups of cytochrome c 3 of D. vulgaris Miyazaki F was performed. The heme assignment was revised for hemes 2 and 3 (sequential heme numbering). Namely, heme 4 is mainly reduced at first with hemes 1, 2 and 3 following it in this order. The p 2 H titration of heme methyl signals in four macroscopic oxidation states was performed in the p 2 H range of 5.2 to 9.0. While the heme methyl resonances in the fully oxidized state showed just small changes with p 2 H most resonances in the intermediate oxidation states revealed clear p 2 H dependence. In particular, the methyl resonances of heme 1 shifted significantly in the acidic region. Then, the chemical shifts of β-CH 2 (next to the carboxyl group) of all propionate groups in the fully oxidized state were observed at various p 2 H in the range of 4.5 to 9.0. Only the propionate group at C-13 (IUPAC-IUB nomenclature) of heme 1 showed a clear change in this p 2 H range, its titration curve being similar to those of the methyl resonances of heme 1 in the intermediate oxidation states. pK a of the propionate group was 5.95 ± 0.05. Analysis of the microscopic formal redox potentials was carried out for the observations at p 2 H 5.2, 7.1 and 9.0. The redox potentials of heme 1 showed the most remarkable p 2 H dependence, resulting in the change of the order of the redox potentials of four berries. A significant change was also found in the interacting potential between hemes 1 and 2. In the light of the p 2 H-titration experiments, the propionate at C-13 of heme 1 was identified as the most plausible ionizable group responsible for the p 2 H dependence of microscopic redox potentials of heme 1 in the acidic region.