The interaction of arylazido ubiquinone derivative with mitochondrial ubiquinol-cytochrome c reductase.

An arylazido ubiquinone derivative, 2,tdimethoxy5-methyl-6{10-[4-(azid0-2-nitroanilinopropionoxy)]decyl} -1,4-benzoquinone, restores about 60% of the electron transfer activity to ubiquinoneand phospholipid-depleted succinateor ubiquinol-cytochrome c reductases, compared to that restored by 2,3-dimethoxy5-methyl-6-(1O-bromodecyl)-1,4-benzoquinone. The restored activity is fully sensitive to illumination with long wavelength W light, suggesting that this azido ubiquinone derivative is bound to the ubiquinone-binding protein at the ubiquinone binding site. Inhibition upon illumination paralleled the amount of azido ubiquinone derivative incorporated into protein. The enzymatic activity of the photolyzed reductase could be restored to a maximum of 40% upon treatment with excess 2,3-dimethoxy-5-methy1-6-(10-bro1nodecy1)-1,4benzoquinone. When the ubiquinone present in the reductase was not removed before addition of the azido ubiquinone derivative, very little inhibition was observed upon illumination under the above conditions, indicating that no exchange between the azido ubiquinone derivative and the intrinsic ubiquinone takes place. If the azido ubiquinone derivative was added to depleted reductase together with 2,3-dimethoxy-5methy1-6-(10-bromodecy1)-1,4-benzoquinone, the maximal activity (that observed when 2,3-dimethoxy-5methy1-6-(10-bromodecy1)-1,4-benzoquinone alone was used) was obtained. By reacting the 3H-labeled azido ubiquinone derivative with depleted reductase, and performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photolysis, the distribution of radioactive ubiquinone derivative among the subunits of ubiquinol-cytochrome c reductase can be obtained. The most heavily labeled subunits were found to be the subunits with mobilities relative to cytochrome c of 0.475 and 0.841 in the Weber and Osborn system. These were previously identified as cytochrome b proteins. Addition of phospholipids before photolysis had little effect on the distribution pattern of radioactivity. Treatment of the system with antimycin A, which inhibits all the restored activity, also produced no significant effect on the distribution of radioactivity among the subunits, suggesting that antimycin A is not bound to same site as ubiquinone. An iron sulfur protein inhibitor, 5-nundecyl-6-hydroxy-4,7-dioxobenzothiazole, also showed little effect on the binding pattern, although it did decrease the total radioactivity incorporated by 258, suggesting that the inhibitor does not compete directly €or the ubiquinone binding site.