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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- 3 H] 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 C II-3 and CI I-4 from Complex II. Specific binding of 3′-azidocarboxin and ubiquinone reductase activity were missing when the enzyme was recombined with peptide C II-4 devoid of C II-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 C II-3 + C II-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.

Publisher Summary This chapter discusses the four aspects of mammalian succinate dehydrogenase: (1) a critical comparison of assay methods, (2) activation–deactivation of the enzyme, (3) the active site of the enzyme, and (4) the comparison of the properties of various purified preparations including recent improvements of procedures for isolating the reconstitutively active form in high yield and with a high turnover number. Because the catalytic turnover of succinate dehydrogenase is faster than the rate-limiting step in the respiratory chain, artificial electron acceptors are usually used for assays of the enzyme in order to ensure that full activity is being measured. Of these, phenazine methosulfate (PMS) with either DCIP or cytochrome c as the terminal oxidant, may be used with the particulate or soluble preparations. Ferricyanide has been widely used for the assay of succinate dehydrogenase. Polarographic or manometric measurements of the succinate–PMS reaction are not recommended, as they are less sensitive and the rate is limited by the oxygen concentration.

Publisher Summary This chapter focuses on covalently bound flavin active center of succinate dehydrogenase. It has been known since the early 1950s that membrane fragments of heart mitochondria contain a form of flavin which is not released by denaturation but is extracted in soluble form only after extensive proteolysis. Kearney isolated the first pure flavin peptide from a proteolytic digest of highly purified succinate dehydrogenase by a series of ion exchange chromatographic procedures. The flavin peptide and its acid hydrolysis product differed from free flavins in three important respects: they were much more water soluble, they showed a considerable hypsochromic shift of the absorption spectrum in the neutral, oxidized form, with the 375-mμ band shifted to 345–350 mμ, and they showed a characteristic pH fluorescence curve, not seen with free flavins.

Publisher Summary This chapter discusses the flavokinase. In the course of the assay method, the FMN formed during the enzymatic reaction is measured in a protein-free filtrate. The method is based on the different distributions of FMN and riboflavin between benzyl alcohol and water. After extraction of the protein-free filtrate with benzyl alcohol, the flavin content of the water phase may be estimated spectrophotometrically at 450 mμ or fluorometrically, and the FMN content is calculated from a formula derived from the distribution coefficients of the flavins between benzyl alcohol and the aqueous solution. Preparations of flavokinase from brewer's yeast and intestinal mucosa are described. The steps described in the purification procedure are: (1) autolysis, (2) ammonium sulfate precipitation, (3) precipitation at pH 5.0 and adsorption of impurities with alumina, (4) fractionation with ammonium sulfate at pH 6.0 and 7.2, (5) differential denaturation. The purified enzyme acts on riboflavin, dichloroflavin [6,7-dichloro-9-(D-l'-ribityl)isoalloxazine], and arabitylflavin [6,7-dimethyl-9-(D-l'-arabityl)isoalloxazine], but not on isoriboflavin [5,6-dimethyl-9-(D-l'-ribityl)isoalloxazine], 6-carbon alcohol derivatives of isoalloxazinesuch as galactoflavin, sorbitylflavin, and dulcitylflavin, alloxazine, or ring compounds of other than the isoalloxazine configuration.