Thermodynamics of the transition from the ferryl (F) state to the oxidized form of the solubilized cytochrome c oxidase: implication for the proton pumping

Two ferryl intermediates have been identified in the membrane-bound respiratory heme-copper oxygen reductases (HCOs) during reduction of O₂ to water. Apparently, energy released by reduction of these two ferryl forms is utilized to build the transmembrane electrochemical proton gradient by two mechanisms. One of them, the proton pumping, is the key unresolved problem of the contemporary molecular bioenergetics. Even though the position of these ferryl forms in energy transformation is central, the direct and complete thermodynamic characterization of these intermediates is lacking. Here, thermodynamics of redox transition of one of these ferryl intermediates, the F state, was established by isothermal titration calorimetry (ITC) and density functional theory utilizing one representative of HCOs, bovine cytochrome c oxidase (CcO). In CcOs, the reduction of catalytic cytochrome a₃-Cu_B center is accomplished by electron transfer (ET) from ferrocytochrome c via copper Cu_A and cytochrome a center. The energy for the pumping is suggested to be released mainly during the transition of the catalytic center of F initiated by ET from cytochrome a. This transfer results in the conversion of Fe(IV)=O to Fe(III) state of heme a₃, yielding the oxidized CcO (O). Based on the enthalpy changes determined by ITC and available entropy values for this process, the estimated ΔG⁰ was found to be −24 kcal/mol, corresponding to the electrode potential of +1.3 V for the F/O couple (pH 8.0, 25 °C). Remarkably, the results indicate that major fraction of energy for the proton pumping is provided by the redox-dependent structural changes of cytochrome a.