Examination of 'high-energy' metastable state of the oxidized (O) bovine cytochrome c oxidase: Proton uptake and reaction with HO.

Reoxidized cytochrome c oxidase appears to be in a 'high-energy' metastable state (O) in which part of the energy released in the redox reactions is stored. The O is supposed to relax to the resting 'as purified' oxidized state (O) in a time exceeding 200 ms. The catalytic heme a-Cu center of these two forms should differ in a protonation and ligation state and the transition of O-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 O. 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 O, generated 100 ms after oxidation of the fully reduced CcO, and the O with HO (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 O-to-O transition is either shorter than 100 ms or there is no difference in the structure of heme a-Cu center of these two forms.