2Department of Biophysics, Faculty of Science, University of P. J. Safarik, 041 54 Košice, Slovak Republic
3Department of Medical and Clinical Biochemistry, Faculty of Medicine, University of P. J. Safarik, 040 11 Košice, Slovak Republic
* To whom correspondence should be addressed.
Received August 28, 2020; Revised November 25, 2020; Accepted November 25, 2020
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).
KEY WORDS: cytochrome c oxidase, hydrogen peroxide, ferryl state, isothermal titration calorimetry