Thermodynamics of the P-type Ferryl Form of Bovine Cytochrome c Oxidase.

Several ferryl states of the catalytic heme a3-CuB center of the respiratory cytochrome c oxidases (CcOs) are observed during the reduction of O2 to H2O. One of the P-type ferryl forms, PM, is produced by the reaction of the two-electron reduced CcO with O2. In this state, the heme a3 iron is in the ferryl state and a free radical should be also present at the catalytic center. However, the energetics of the PM formation has not been experimentally established yet. Here, the generation of PM by the reaction of oxidized bovine CcO (O) with one molecule of H2O2 was investigated by the isothermal titration calorimetry and UV-Vis absorption spectroscopy. Two kinetic phases, corresponding to the formation of PM and its endogenous conversion back to O, were resolved by both methods. The ΔH of the entire process (-66 kcal/mol H2O2) was larger than the heat (-50.8 kcal/mol O2) liberated during O2 reduction by ferrocytochrome c (pH 8, 25°C). Interestingly, ΔH of the first phase (-32 kcal/mol ferryl state) far exceeds the enthalpy of the PM production. The data indicate that during the first phase, the radical in PM is quenched and spectrally similar second P-type ferryl form (PR) is produced. Additionally, it was shown that the entropy contribution to the Gibbs energy change (ΔG = -46 kcal/mol O2) during the catalytic reduction of O2 by ferrocytochrome c is negligible (-0.7 cal·mol-1·K-1).

Modulation of the electron-proton coupling at cytochrome a by the ligation of the oxidized catalytic center in bovine cytochrome c oxidase.

Cytochrome a was suggested as the key redox center in the proton pumping process of bovine cytochrome c oxidase (CcO). Recent studies showed that both the structure of heme a and its immediate vicinity are sensitive to the ligation and the redox state of the distant catalytic center composed of iron of cytochrome a3 (Fea3) and copper (CuB). Here, the influence of the ligation at the oxidized Fea33+-CuB2+ center on the electron-proton coupling at heme a was examined in the wide pH range (6.5-11). The strength of the coupling was evaluated by the determination of pH dependence of the midpoint potential of heme a (Em(a)) for the cyanide (the low-spin Fea33+) and the formate-ligated CcO (the high-spin Fea33+). The measurements were performed under experimental conditions when other three redox centers of CcO are oxidized. Two slightly differing linear pH dependencies of Em(a) were found for the CN- and the formate-ligated CcO with slopes of -13 mV/pH unit and -23 mV/pH unit, respectively. These linear dependencies indicate only a weak and unspecific electron-proton coupling at cytochrome a in both forms of CcO. The lack of the strong electron-proton coupling at the physiological pH values is also substantiated by the UV-Vis absorption and electron-paramagnetic resonance spectroscopy investigations of the cyanide-ligated oxidized CcO. It is shown that the ligand exchange at Fea3+ between His-Fea3+-His and His-Fea3+-OH- occurs only at pH above 9.5 with the estimated pK >11.0.