The Reactivity of Ferricytochrome c with Ionic Ligands

The reactions of ferricytochrome c with anionic ligands involve the fission of one of the bonds to basic groups that hold the heme firmly within a crevice in the polypeptide structure. Denoting the group by X, the reactions thus take the form [see PDF for equation] where L is ligand and Pr is the protein liberated by the incoming ligand. With ferricytochrome c, the [H+] dependence of the equilibrium constant for complex formation is determined by the ionization equilibrium of the ligand species HL ⇌ H+ + L-, the coordinated water molecule -Fe+-H2O ⇌ FeOH + H+, and the nitrogenous base group which is liberrated -XH+ ⇌ X + H+. Studies of the equilibrium constants for the formation of the azide and cyanide complexes, over the pH range 4 to 8, show that there is a change in the [H+] dependence for both reactions at about pH 5. At 26° and ionic strength of 0.03, the following values were found: pKxH+ = 4.98 with ΔH = +9.2 kcal per mole and ΔS = +8 e.u. This indicates that the bond to the Fe, which is broken in complex formation, involves the glyoxalinium nitrogen atom of a histidine residue. The stability constants for the formation of the closed crevice form of ferricytochrome c at 26° and ionic strength of 0.03 are ΔF = -3.5 kcal per mole, ΔH = -18.0 kcal per mole, and ΔS = -48 e.u. At higher ionic strengths, deviations were observed from the pH dependence noted above. An extension of the mathematical analysis to include another group capable of accepting a proton leads to an expression for the equilibrium constant of the same form, and, on this basis, calculation shows that the pK would be about 4.4.