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

Several ferryl states of the catalytic heme a(3)-Cu-B center of the respiratory cytochrome c oxidases (CcOs) are observed during the reduction of O-2 to H2O. One of the P-type ferryl forms, P-M, is produced by the reaction of the two-electron reduced CcO with O-2. In this state, the heme a(3) 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 Delta H of the entire process (-66 kcal/mol H2O2) was larger than the heat (-50.8 kcal/mol O-2) liberated during O-2 reduction by ferrocytochrome c (pH 8, 25 degrees C). Interestingly, Delta H 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 (Delta G = -46 kcal/mol O-2) during the catalytic reduction of O-2 by ferrocytochrome c is negligible (-0.7 cal.mol(-1).K-1).