SITE OF ACTION OF CARBOXAMIDES IN MITOCHONDRIAL COMPLEX II

Abstract

The systemic fungicide carboxin, 5, 6-dihydro-2-methyl 1,4-oxathiin-3-carboxanilide, and structurally related carboxamides are potent and highly specific inhibitors of succinate oxidation in the mitochondrial respiratory chain of fungi and beef heart muscle, acting in Complex II (succinate-ubiquinone reductase) at the same site and manner as the classical inhibitor thenoyltrifluoroacetone (TTF). Electron spin resonance data suggests that inhibition of Complex II by carboxanilides and TTF occurs between the high potential iron-sulfur center S-3 of succinate dehydrogenase and ubiquinone. The exact site of action of carboxin in Complex II has been probed by means of equilibrium dialysis (dark) and photoaffinity-labelling experiments using [G-3H] 3′-azidocarboxin in the presence or absence of unlabeled TTF to displace bound carboxanilide and thereby distinguish specific from non-specific binding sites. The equilibrium dialysis experiments with 3′-azidocarboxin showed that the TTF-displaceable carboxanilide binds to Complex II and to a reconstituted system of succinate dehydrogenase plus peptides CII-3 and CII-4 from Complex II. Specific binding of 3′-azidocarboxin and ubiquinone reductase activity were missing when the enzyme was recombined with peptide CII-4 devoid of CII-3 by chymotrypsin digestion. Soluble succinate dehydrogenase and peptides alone did not exhibit specific binding thus, the binding site is available only when the 70s and 30s subunits of the dehydrogenase are in proper positioning with the small peptides and phospholipid components. Photoaffinity-labelling experiments showed that the specific binding of 3′-azidocarboxin in Complex II was on peptides CII-3 + CII-3 and phospholipids. The subunits of succinate dehydrogenase were not labeled by specifically-bound 3′-azidocarboxin although the presence of the enzyme in the complex was essential for inhibitor binding.