Examination of ‘high-energy’ metastable state of the oxidized (OH) bovine cytochrome c oxidase: Proton uptake and reaction with H2O2

Reoxidized cytochrome c oxidase appears to be in a ‘high-energy’ metastable state (OH) in which part of the energy released in the redox reactions is stored. The OH is supposed to relax to the resting ‘as purified’ oxidized state (O) in a time exceeding 200 ms. The catalytic heme a3-CuB center of these two forms should differ in a protonation and ligation state and the transition of OH-to-O is suggested to be associated with a proton transfer into this center. Employing a stopped-flow and UV–Vis absorption spectroscopy we investigated a proton uptake during the predicted relaxation of OH. It is shown, using a pH indicator phenol red, that from the time when the oxidation of the fully reduced CcO is completed (∼25 ms) up to ∼10 min, there is no uptake of a proton from the external medium (pH 7.8). Moreover, interactions of the assumed OH, generated 100 ms after oxidation of the fully reduced CcO, and the O with H2O2 (1 mM), result in the formation of two ferryl intermediates of the catalytic center, P and F, with very similar kinetics and the amounts of the formed ferryl states in both cases. These results implicate that the relaxation time of the catalytic center during the OH-to-O transition is either shorter than 100 ms or there is no difference in the structure of heme a3-CuB center of these two forms. [Display omitted] •Metastable ‘high-energy’ (OH) and resting ‘as isolated’ oxidized cytochrome c oxidase (O).•Proton uptake during a predicted relaxation of the OH-to-O.•Proton uptake following the oxidation of fully reduced oxidase by O2.•Kinetics of peroxide stimulated oxidation at the catalytic center in both OH and O forms.