Properties of Bovine Heart Mitochondrial Cytochrome

A large-scale preparation of the two-subunit protein complex (QPs) that converts succinate dehydrogenase into succinate-ubiquinone reductase from cytochrome 6-c, particles is achieved by a procedure involving Triton X100 solubilization and calcium phosphate column chromatography at different pH values. The isolated two-subunit QPs contains 25 nmol of cytochrome basofmg of protein and is able to reconstitute with soluble succinate dehydrogenase to form a TTFA-sensitive succinate-ubiquinone reductase. The maximum reconstitutive activity is 100 @mol of succinate oxidized per min per mg of QPs protein at 23 “C. Although cytochrome beso in isolated BPS is not succinate reducible and its dithionite reduced form is reactive to carbon monoxide, cytochrome beao is shown to be physically associated with succinate dehydrogenase by the following observations. 1) The dithionite reduced form of cytochrome bas0 in isolated QPs has a symmetrical a-absorption peak, which upon reconstitution with succinate dehydrogenase becomes slightly broadened and shows a shoulder at around 553 nm, identical to that of cytochrome b6eo in succinate-ubiquinone reductase. 2) Upon addition of succinate dehydrogenase to QPs, about 50% of the reduced form of cytochrome bas0 in the QPs becomes insensitive to carbon monoxide treatment. 3) The redox potential of cytochrome baa0 in QPs is -144 mV which is higher than that of cytochrome bsao in succinate-ubiquinone reductase (-185 mV). Upon addition of succinate dehydrogenase, the redox potential of about 46% of the cytochrome baeo in QPs preparation becomes identical to that of cytochrome bas0 in succinate-ubiquinone reductase. 4) Cytochrome base in the QPs preparation shows two epr signals, g = 3.07 and g = 2.92, whereas cytochrome bseo in succinate-ubiquinone reductase exhibits only one epr signal at g = 3.46. When QPs is reconstituted with succinate dehydrogenase to form succinate-ubiquinone reductase, the g = 3.46 epr signal reappears at the expense of the g = 3.07 signal. Based on epr measurement at liquid helium temperature, about 18% of the total cytochrome b in the isolated active succinate-cytochrome c reductase is cytochrome bsa0, indicating that cytochrome bas0 is indeed a unique cytochrome b and not a denatured product of cytochrome bsez or bees.