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

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Ludmila Mikulova^1,a, Ivana Pechova^2,b, Daniel Jancura^2,c, Marek Stupak^3,d, and Marian Fabian^1e*

¹Center for Interdisciplinary Biosciences, Technology and Innovation Park, University of P. J. Safarik, 041 54 Košice, Slovak Republic

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

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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).

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KEY WORDS: cytochrome c oxidase, hydrogen peroxide, ferryl state, isothermal titration calorimetry

DOI: 10.1134/S0006297921010077

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