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

Several ferryl states of the catalytic heme a₃-Cu_B center of the respiratory cytochrome c oxidases (CcOs) are observed during the reduction of O2 to H2O. One of the P-type ferryl forms, P_M, 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 P_M formation has not been experimentally established yet. Here, the generation of P_M 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 P_M 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 P_M production. The data indicate that during the first phase, the radical in P_M is quenched and spectrally similar second P-type ferryl form (P_R) 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).