H2O2 addition to the oxidized cytochrome c oxidase reconstituted in liposomes brings about a red shift of the Soret band of the enzyme and an increased absorption in the visible region with two distinct peaks at ∼570 and 605 nm. Throughout pH range 6–8.5, the spectral changes at 570 nm and in the Soret band titrate with very similar pH-independent K d values of 2–3,μM. At the same time, K d of the peroxide complex measured at 605 nm increases markedly with increased H+ activity reaching the value of 18 ± 2 μM at pH 6.0. This finding may indicate the presence of two different H2O2-binding sites in the enzyme with different affinity for the ligand at acid pH. The Soret and 570 nm band effects are suggested to report H2O2 coordination to heme iron of a 3, whereas the maximum at 605 nm could arise from H2O2 binding to Cu a3 followed by the enzyme transition into the ‘pulsed’ (or ‘420/605’) conformation. Possible implication of the two H2O2-binding sites for the cytochrome oxidase redox and proton-pumping mechanisms are discussed.
