Analysis of the ionization constants and heats of ionization of reduced and oxidized horse heart cytochrome c

Simultaneous curve fitting for the ionization parameters of oxidized and reduced horse heart cytochrome c in 0.15 M KCl and 20°C yields values for the ionization constants (as p K ′) and the heats of ionization (Δ H i ) which can reconstruct either the potentiometric or thermal titration curves. Reduced cytochrome c requires 8 sets of groups, whereas oxidized cytochrome c requires 10 sets of groups. The additional groups in the oxidized preparation appear to involve the ferriheme (p K ′, 9.25; Δ H i , 13.7 kcal/mol) and a tyrosine (p K ′ ≃ 10.24) that is not present in the reduced form. The potentiometric and thermal difference curves (reduced – oxidized) involve the appearance of 17 kcal/mol centered at pH 9.7 and 5.8 kcal/mol centered at pH 4.9. The carboxyl groups in both species appear to be normal for the hydrogen‐bonded form. Only one histidine has normal ionization properties (p K ′, 6.7; Δ H i , 7.5 kcal/mol), as do 17 of the lysine residues (p K ′, 10.8; Δ H i , 11.5 kcal/mol).