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Summary Reductive titrations and rapid kinetic studies are reported on extensively or completely activated, particulate and solubilized succinate dehydrogenase (SD) preparations. There is one iron-sulfur (Fe−S) center of the ferredoxin type present per flavin which is reduced by succinate, but even in activated preparations at most 60% of these centers were reduced within the turnover time of the enzymes. Flavin semiquinone formation does not precede or significantly lag behind the reduction of the Fe−S centers. On reduction of the soluble enzymes an accumulation of semiquinone is observed. No qualitative difference between the behavior of preparations containing 4 Fe or 8 Fe per flavin was found. Succinate-ubiquinone reductase (Complex II) contains an Fe−S component with properties of high-potential Fe−S proteins (See Ruzicka and Beinert, Biochem. Biophys. Res. Communs. this issue). It occurs at a concentration close to that of the bound flavin and has been observed to be reduced by succinate at approximately the same rate as the ferredoxin type (g=1.94) component. With dithionite, reduction of additional Fe−S groups (0.2 to 0.5 per flavin) is observed but the significance of this is uncertain.

Summary Succinate dehydrogenase (SD) in intact, respiring mitochondria undergoes activation and deactivation in response to the metabolic state of the mitochondria. Highest SD activity is observed in state 4 and lowest in state 2 or in the presence of uncouplers, while in state 3 the level of activation is intermediate and varies with the nature of the substrate. Transition from the active to inactive (unactivated) state of SD occurs in mitochondria with a lower energy of activation (10 Kcal/mole) than in soluble or membrane preparations (33 to 36 Kcal/mole). In addition to activation by succinate and CoQ 10 H 2 , the enzyme in mitochondria is uniquely activated by ATP (or a compound in equilibrium with it), a process which has not been previously observed in submitochondrial particles and is oligomycin-insensitive. In tightly coupled mitochondria reversible activation by all these agents may occur concurrently, but experimental conditions are described to study the action of each type of activator independently.

The amino acid sequence of the coenzyme binding site of beef heart succinate dehydrogenase is serine-histidine-threonine-valine-alanine. Flavin adenine dinucleotide is covalently bound to a ring nitrogen of histidine through the 8α position of the isoalloxazine ring system.

It is well known that succinate dehydrogenase (SD) is transformed from an inactive to an active state on combination with substrates or competitive inhibitors and that SD becomes deactivated on removing the activator. Activation has a high temperature coefficient and is thought to be due to a conformational change in the enzyme. In the present paper it is shown that in well washed membrane preparations, in which SD occurs largely in the deactivated form, NADH induces activation of SD. The activation progresses as long as NADH is present; once NADH is removed by oxidation, SD reverts rapidly to the deactivated form. NADH-induced activation has the same temperature coefficient as that produced by succinate and the kinetic properties of SD activated by either agent are the same. Activation is not due to a combination of NADH with SD, since rhein and piericidin, inhibitors of NADH oxidation, interfere with the activation of SD by NADH. Extraction of CoQ10 with pentane prevents the activation by NADH nearly completely, while readdition of CoQ10 restores activability by NADH. It is suggested that the agent responsible for this type of activation is reduced CoQ and that NADH serves only to maintain CoQ10 in the reduced state. Since thenoyltrifluoroacetone prevents succinate oxidation but not activation of SD by NADH, it appears that CoQH2 can interact with and induce changes in SD in the absence of electron transport. The possibility that this type of rapid activation-deactivation may be of regulatory significance in the Krebs cycle is discussed.

Previous studies in this laboratory have shown that the active center of hepatic monoamine oxidase contains flavin dinucleotide covalently linked to the peptide chain via the 8α position of the flavin but that, unlike in succinate dehydrogenase, the linkage is not to histidine but to another amino acid. A pure flavin pentapeptide has now been isolated from monoamine oxidase which yields on acid hydrolysis or digestion with aminopeptidase M 1 mole each of serine and tyrosine and 2 of glycine and gives a positive test for sulfur. Oxidation of the peptide with performic acid yields, in addition to the amino acids mentioned, cysteic acid. The physical and chemical properties of the peptide are in accord with the conclusion that the amino acid substituted on the 8α group of the flavin is cysteine in thioether linkage. Edman degradation followed by dansylation revealed the sequence

Flavin peptides derived from monoamine oxidase, free from succinate dehydrogenase flavin, were obtained by digestion of outer membranes of beef liver mitochondria with trypsin and chymotrypsin and purification by various chromatographic methods. The flavin peptides show the same hypsochromic shift of the optical absorption spectrum as flavin peptides from succinate dehydrogenase: the 370 mμ band of the neutral oxidized flavin is shifted to 340 mμ, whereas the cation shows a peak at 370 mμ. The ESR spectrum of the monoamine oxidase flavin cation radical also resembles that of succinate dehydrogenase flavin in that the total width is reduced from 49 G (in riboflavin) to at least 45 G, and the line width from 3.8 G (in riboflavin) to 2.3 G. The covalently bound flavins of monoamine oxidase and succinate dehydrogenase differ, however, in that the former shows the same fluorescence intensity between pH 3.4 and 8, while the latter is quenched with a pK of 4.5 ± 0.1. These observations indicate that the FAD of monoamine oxidase is covalently linked to the peptide chain through the 8α-CH 3 group of riboflavin but histidine is not the immediate substituent, as in succinate dehydrogenase. Hydrolysis of flavin peptides from monoamine oxidase in 6 N HCl at 95° yields a derivative chromatographically distinct from free flavins which is ninhydrin-positive and thus contains an amino acid bound to the 8α-position.

Soluble succinate dehydrogenase prepared from acetone powders of submitochondrial particles is almost entirely in the deactivated state and contains 0.5 mole of oxalacetate (OAA) per mole of histidyl flavin. OAA is dissociated by succinate, malonate, IDP, ITP, and high concentrations of anions at elevated temperatures, but not significantly in the cold, with concurrent activation of the enzyme; the high energy of activation observed for OAA release and for activation suggests that a conformation change in the protein is involved. On removal of OAA, a reversible activation-deactivation cycle dependent on the pH is demonstrable. Submitochondrial particles behave similarly but appear to contain 1 mole of tightly bound OAA per histidyl flavin in the deactivated state.

Summary Previous studies established that succinate dehydrogenase (SD) is reversibly activated by three types of compounds: substrates, reduced CoQ10, and ATP. Each of these mechanisms have been shown to operate in intact mitochondria in regulation of SD activity. The present study demonstrates activation of the enzyme by succinyl CoA, which occurs at about the rate and to the extent observed with the same concentration of succinate and represents an example of feed-forward regulation. Further, in membranal and purified, soluble preparations lowering the pH in the range of 7.5 to 5.5 results in “spontaneous” activation: the lower the pH, the more extensive activation is observed. On raising the pH of SD thus activated, deactivation occurs to the level characteristic of the new pH. A number of anions (Cl−, Br−, SO=4, formate, CIO4 increase the extent of activation observed on lowering the pH and appear to act by stabilizing the active conformation of SD.