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 H 2 O. 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 H 2 O 2 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 H 2 O 2 ) was larger than the heat (–50.8 kcal/mol O 2 ) liberated during O 2 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 O 2 ) during the catalytic reduction of O 2 by ferrocytochrome c is negligible (–0.7 cal·mol –1 ·K –1 ).