Studies with ubiquinone-depleted submitochondrial particles. Essentiality of ubiquinone for the interaction of succinate dehydrogenase, NADH dehydrogenase, and cytochrome b.

Abstract

Submitochondrial particles were prepared from beef‐heart mitochondria by sonication in the presence of EDTA. The particles were lyophilized and repeatedly extracted with pentane until no ubiquinone was found in the extract. Treatment of the ubiquinone‐depleted particles with pentane containing a suitable concentration of ubiquinone (ubiquinone‐50) and subsequent quick washing with ubiquinone‐free pentane resulted in a “re‐incorporation” of ubiquinone in an amount similar to that present in the original particles (3–6 nmoles/mg protein). The ubiquinone‐depleted particles exhibited very low or no succinate and NADH oxidase activities, which were restored upon the re‐incorporation of ubiquinone to the levels found in the lyophilized particles before extraction with pentane. Partial (about 50%) extraction of ubiquinone resulted in markedly decreased succinate and NADH oxidase activities. Added cytochrome c did not replace ubiquinone in restoring the succinate or NADH oxidase activity of ubiquinone‐depleted particles. It stimulated the NADH oxidase, but not the succinate oxidase, activity of the “ubiquinone‐incorporated” particles, but the same stimulation occurred with the lyophilized particles before ubiquinone extraction. The normal, lyophilized, and “ubiquinone‐incorporated” particles contained equal amounts of both total and enzymatically reducible cytochromes. In the presence of KCN, NADH reduced the cytochromes, including cytochrome b, only at insignificant rates in the ubiquinone‐depleted particles as compared to the normal and lyophilized preparations, and these rates were greatly stimulated upon the re‐incorporation of ubiquinone. Succinate caused a rapid partial (about 25%) reduction of cytochrome b, but not of the rest of the cytochromes, in the ubiquinone‐depleted particles. This reduction occurred also in the absence of KCN, and the fraction of cytochrome b, so reduced was not reoxidized when succinate oxidation was inhibited by malonate. Evidence for the occurrence of such an enzymatically non‐oxidizable form of cytochrome b was also obtained in the normal, lyophilized and “ubiquinone‐incorporated” particles, but, in those cases, this cytochrome b was reduced by both succinate and NADH. In the presence of antimycin A, all cytochrome b in the ubiquinone‐depleted particles was rapidly reduced by succinate but not by NADH. The normal and lyophilized particles catalyzed a rotenone‐sensitive oxidation of NADH by fumarate. This reaction was completely absent from the ubiquinone‐depleted particles and was restored upon the re‐incorporation of ubiquinone. N,N,N′,N′‐Tetramethyl‐p‐phenylenediamine catalyzed an NADH and succinate oxidase activity in antimycin A‐inhibited particles. This NADH oxidase activity was partially sensitive to rotenone in the normal, lyophilized and “ubiquinone‐incorporated” particles, but completely rotenone‐insensitive in the ubiquinone‐depleted particles. All four types of particles were active in catalyzing the antimycin A‐sensitive oxidation of menadiol. It is concluded that uniquinone is essential for the interaction of succinate dehydrogenase, NADH dehydrogenase and cytochrome b, and that this interaction is a requisite for the normal function of the respiratory chain. Functionally modified forms of cytochrome b, arising as a consequence of structural damage or antimycin A treatment, are discussed in relation to existing information and proposals concerning the role of cytochrome b and ubiquinone in electron transport.