Specific interaction between protein and ubiquinone in succinate-ubiquinone reductase.

The lack of ubiquinone in the isolated succinate-ubiquinone reductase (Complex II) and in some preparations of ubiquinone-binding protein (QPs) has caused some concern about the nature of interaction between ubiquinone and QPs. When a low concentration of radioactively labeled ubiquinone derivative in dilute detergent solution was incubated with delipidated or intact succinate-ubiquinone reductase followed by high speed centrifugation, a stoichiometric amount of radioactive ubiquinone derivative was taken up by protein, indicating that the interaction between ubiquinone and protein is specific and strong. The specific interaction between ubiquinone and delipidated succinate-ubiquinone reductase was also confirmed by observation of a protein-dependent, pronase-sensitive spectral shift of ubiquinone. When ubiquinone was bound to succinateubiquinone reductase in aqueous solution, it showed an absorption maximum at 282 nm instead of 288 nm, the absorption peak when no protein is present. Delipidated succinate-ubiquinone reductase was reacted with a photoaffinity labeled ubiquinone derivative, followed by photolysis and electrophoresis in the presence of sodium dodecyl sulfate and 8-mercaptoethanol. The radioactivity was located mainly in the two low molecular weight subunits which contain QPs, indicating that ubiquinone is preferentially bound to the QPs. The specific interaction between ubiquinone and protein in succinate-ubiquinone reductase is stronger than that between the enzyme and Triton X-100. When succinatecytochrome c reductase was separated into succinateubiquinone and ubiquinol-cytochrome c reductases by treatment with Triton X-100 and calcium phosphate column chromatography, ubiquinone was bound to succinate-ubiquinone reductase. After a prolonged washing with 0.2% Emasolll30 solution, which removed all the bound Triton X-100, most of the ubiquinone remained bound to protein. The above results indicate that ubiquinone is specifically bound to protein.