Js. Park et al., REGULATION OF THE REDOX ORDER OF 4 HEMES BY PH IN CYTOCHROME C(3) FROM DESULFOVIBRIO-VULGARIS MIYAZAKI F, Biochimica et biophysica acta. Protein structure and molecular enzymology, 1293(1), 1996, pp. 45-54
The assignment of H-1-NMR signals of the heme methyl and propionate gr
oups of cytochrome c(3) of D. vulgaris Miyazaki F was performed. The h
eme assignment was revised for hemes 2 and 3 (sequential heme numberin
g). Namely, heme 4 is mainly reduced at first with hemes 1, 2 and 3 fo
llowing it in this order, The p(2)H titration of heme methyl signals i
n four macroscopic oxidation states was performed in the p(2)H range o
f 5.2 to 9.0. While the heme methyl resonances in the fully oxidized s
tate showed just small changes with p(2)H, most resonances in the inte
rmediate oxidation states revealed clear p(2)H dependence. In particul
ar, the methyl resonances of heme 1 shifted significantly in the acidi
c region. Then, the chemical shifts of beta-CH2 (next to the carboxyl
group) of all propionate groups in the fully oxidized state were obser
ved at various p(2)H in the range of 4.5 to 9.0. Only the propionate g
roup 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 microsco
pic 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 r
emarkable p(2)H dependence, resulting in the change of the order of th
e redox potentials of four hemes. A significant change was also found
in the interacting potential between hemes 1 and 2. In the light of th
e p(2)H-titration experiments, the propionate at C-13 of heme 1 was id
entified as the most plausible ionizable group responsible for the p(2
)H dependence of microscopic redox potentials of heme 1 in the acidic
region.