INFLUENCE OF PH ON THE EPR AND REDOX PROPERTIES OF CYTOCHROME-C OXIDASE IN DETERGENT SOLUTION AND IN PHOSPHOLIPID VESICLES

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 (aa3) 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 a3 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 H2O 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. © 1979.