Studies on the binding of carboxin analogs to succinate dehydrogenase.

Carboxin (5,6-dihydro-2-methyl-l,4-oxathiin-3-carboxanilide) and a number of other carboxanilides are known to be potent inhibitors of succinate oxidation in membrane preparations from animal tissues, fungi, and bacteria, acting at the succinate dehydrogenase-coenzyme Q junction. The kinetics and the maximal extent of inhibition of succinate oxidation by carboxanilides and by thenoyltrifluoroacetone are the same in a variety of enzyme preparations from various organisms. The two types of inhibitors are thought to act, therefore, at the same site. In the present study the binding properties of a labeled carboxanilide, [G-3H]2,4,5-trimethyl-3-carboxanilinofuran, were studied in Complex II and in inner membrane preparations from beef heart mitochondria. The furan was found to inhibit succinate oxidation at the same site as other carboxanilides with a Ki of about 0.1 PM. On titration of Complex II or of inner membrane samples with the furan it was found to combine both at the site(s) responsible for inhibition and other sites, unrelated to the inhibition, since binding to the particles occurred linearly long after saturation of the inhibition site(s). These observations paralleled the behavior of rotenone and piericidin A, which are bound both at specific sites responsible for the inhibition of NADH oxidation and at unspecific sites. In contrast to rotenone and piericidin A, however, the furan was readily dissociated from the site(s) associated with the inhibition of succinate oxidation by dilution or by washing of the particles, with full recovery of the activity, without dissociating it from the unspecific sites. In order to determine the binding constant of the furan at the specific (inhibition) site(s), binding to Complex II was determined at a series of concentrations of the labeled inhibitor in the presence and absence of unlabeled thenoyltrifluoroacetone, which prevents binding only at the specific site(s). The resulting KD agreed well with the Ki calculated from the inhibition of the succinate-2,6dichlorophenolindophenol reductase activity. On progressive extraction of succinate dehydrogenase from Complex II with perchlorate the specific binding site gradually disappeared, suggesting the involvement of this protein in the specific binding. Calculations from Scatchard plots, however, indicate that the amount of furan bound at the specific site is