Abstract
The EPR signals of oxidized and partially reduced cytochrome oxidase have been studied at pH 6.4, 7.4, and 8.4. Isolated cytochrome oxidase in both non-ionic detergent solution and in phospholipid vesicles has been used in reductive titrations with ferrocytochrome c. The g values of the low- and high-field parts of the low-spin heme signal in oxidized cytochrome oxidase are shown to be pH dependent. In reductive titrations, low-spin heme signals at g 2.6 as well as rhombic and nearly axial high-spin heme signals are found at pH 8.4, while the only heme signals appearing at pH 6.4 are two nearly axial g 6 signals. This pH dependence is shifted in the vesicles. The g 2.6 signals formed in titrations with ferrocytochrome c at pH 8.4 correspond maximally to 0.25–0.35 heme per functional unit ( aa 3) of cytochrome oxidase in detergent solution and to 0.22 heme in vesicle oxidase. The total amount of high-spin heme signals at g 6 found in partially reduced enzyme is 0.45–0.6 at pH 6.4 and 0.1–0.2 at pH 8.4. In titrations of cytochrome oxidase in detergent solution the g 1.45 and g 2 signals disappear with fewer equivalents of ferrocytochrome c added at pH 8.4 compared to pH 6.4. The results indicate that the environment of the hemes varies with the pH. One change is interpreted as cytochrome a 3 being converted from a high-spin to a low-spin form when the pH is increased. Possibly this transition is related to a change of a liganded H 2O to OH − with a concomitant decrease of the redox potential. Oxidase in phosphatidylcholine vesicles is found to behave as if it experiences a pH, one unit lower than that of the medium.
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