Edna B. Kearney

[47] Mammalian succinate dehydrogenase

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.

Studies on the respiratory chain-linked reduced nicotinamide adenine dinucleotide dehydrogenase: XVI. Characteristics of the membrane-bound dehydrogenase in Candida utilis and Saccharomyces cerevisiae☆

Abstract The respiratory chain-linked reduced nicotinamide adenine dinucleotide dehydrogenase of S. cerevisiae and C. utilis in mitochondrial and in membrane preparations has been investigated. Among methods tested, the highest activities for the dehydrogenase are obtained in the NADH-coenzyme Q1 reaction in S. cerevisiae, while the NADH-ferricyanide reaction provides the best activity determination for the enzyme from C. utilis. In several respects, including specificity for electron acceptor and substrate and inhibition by amytal, rotenone, and piericidin A, the dehydrogenase from C. utilis resembles its counterpart in mammalian mitochondria much more closely than does the enzyme from S. cerevisiae. The latter enzyme appears to be extensively solubilized on digestion of membrane preparations with N. naja venom phospholipase A, as is the case with the mammalian enzyme, whereas the dehydrogenase from C. utilis is not extracted by this treatment. The failure of phospholipase A to extract the C. utilis enzyme is not due to unusual phosphatide composition as shown by phosphatide analyses and by the fact that lecithin isolated from C. utilis mitochondria is readily hydrolyzed by phospholipase A, but may be due to the inaccessibility of the phospholipase to mitochondrial lipids in situ. NADH dehydrogenase from both types of yeasts is extensively inactivated by the heat-acid-ethanol method previously used by other workers for the isolation of “NADH dehydrogenase” from S. cerevisiae. During the O2-induced mitochondrial biogenesis in S. cerevisiae the respiratory chain-linked enzyme is formed considerably more rapidly than the overall NADH oxidase system; thus, the assembly of the components may be the rate-limiting step. On exposure of aerobic cells of S. cerevisiae to high glucose concentration, NADH dehydrogenase appears to undergo active destruction.

Structure of the covalently bound flavin of monoamine oxidase

Abstract 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.

Interrelations of reconstitution activity, reactions with electron acceptors, and iron-sulfur centers in succinate dehydrogenase

Abstract Reconstitutively active, soluble preparations of succinate dehydrogenase have been examined for the stoichiometric relationship of the covalently bound flavin to the electron paramagnetic resonance (epr) signals of the iron-sulfur centers known to be present in membrane-bound forms of the enzyme. The Fe-S center ( g = 2.01), which is paramagnetic in the oxidized state (the high-potential Fe-S cluster of the enzyme, HiPIP) and which was identified in earlier studies as a component of succinate-ubiquinone reductase (Beinert et al. , 1975, Eur. J. Biochem. 54 , 185), can be demonstrated in reconstitutively active, soluble preparations (0.20 to 0.35 equiv/mol of enzyme) at a significantly higher level than in enzyme preparations extracted without succinate. Studies of the kinetics of the reduction of the epr-detectable Fe-S centers revealed no difference between reconstitutively active and inactive preparations in the behavior of centers 1 and 2 and, of the oxidized Fe-S center detected in reconstitutively active preparations, only a part or none at all was reduced by succinate at catalytically significant rates. The failure to demonstrate the HiPIP center in stoichiometric amounts even when the enzyme is extracted and purified under anaerobic conditions in the presence of succinate may be due to the experimental conditions needed to convert this center to the paramagnetic state for observation, i.e., the obligatory use of artificial electron acceptors, such as ferricyanide. A procedure devised to circumvent this problem by incorporating the soluble enzyme into alkali-treated membranes resulted in a reconstituted preparation which was more stable to oxidizing agents. After incorporation into the membrane, between 50 and 100% of the HiPIP center was then detectable, suggesting a relationship between this Fe-S component and reconstitution activity. It has also been noted that, under aerobic conditions, the rates of decay of this epr signal, the reconstitution activity, and the catalytic activity of soluble preparations with low concentrations of ferricyanide as electron acceptor go parallel and may, indeed, be functions of the integrity of the same property of the protein. During this time, about half of the phenazine methosulfate reductase activity also declines, and the process is accompanied by a major increase in K m for phenazine methosulfate. The content of the two ferredoxin-type Fe-S cehters, one of which is reduced by the substrate (center 1) and the other by dithionite but not by succinate (center 2), in reconstitutively active preparations did not differ significantly from that of reconstitutively inactive, soluble preparations. On the basis of these data, the notion that the ferredoxin-type Fe-S centers or signals of reconstitutively active enzyme have properties very different from those of other preparations is considered lacking in experimental support.

Enzymic transformation of L-cysteinesulfinic acid☆

1. 1. A soluble enzyme preparation, obtained from Proteus vulgaris by ultrasonic disintegration, in the presence of suitable cofactors, catalyzes the rapid oxidation of L-cysteinesulfinic acid. The metabolism of the latter compound is shown to proceed by two simultaneous and competing pathways. In pathway A it is oxidized to cysteic acid and the latter is transaminated with α-ketoglutarate. Pathway B is initiated by transamination of cysteinesulfinate with α-ketoglutarate (or oxaloacetate). The resulting β-sulfinylpyruvic acid is desulfinated to pyruvate and SO3= and the latter is oxidized to SO4=, while α-ketoglutarate is regenerated under the influenceof a TPN-specific glutamic dehydrogenase. 2. 2. Three new enzymes are described: L-cysteinesulfinic dehydrogenase (the enzyme responsible for the formation of cysteic acid), oxaloacetic-cysteinesulfinic and α-ketoglutaric-cysteinesulfinic transaminases. The properties of the latter are discussed in some detail. 3. 3. β-Sulfinylpyruvic acid, an analogue of oxaloacetic acid, which has not hitherto been implicated in intermediary metabolism, has been accumulated by transaminase action. This compound is desulfinated to SO3= and pyruvate, under the influence of Mn++, analogously to the β-decarboxylation of oxaloacetic acid by Mn++. The desulfination is a rapid, non-enzymic reaction, as is the oxidation of SO3= to SO4= by Mn++. 4. 4. The possibility is discussed that enzymes may exist for the desulfination reaction and that the reversal of their action (SO2 fixation into pyruvate) may be a mechanism for the incorporation of inorganic sulfur into organic linkages. 5. 5. L-Cysteinesulfinic dehydrogenase requires a previously unrecognized, pyridine nucleotide coenzyme. 6. 6. The applicability of the metabolic scheme to intact bacterial cells and to animal tissues is discussed.

Tightly bound oxalacetate and the activation of succinate dehydrogenase.

Abstract 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.

Epr studies on the mechanism of action of succinate dehydrogenase in activated preparations

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.

Sequence and structure of a cysteinyl flavin peptide from monoamine oxidase

Abstract 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:

Multiple control mechanisms for succinate dehydrogenase in mitochondria

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.

On the need for regulation of succinate dehydrogenase

It has been known since 1955 that substrates and competitive inhibitors of succinate dehydrogenase activate the enzyme by a process which is believed to involve a conformation change in the enzyme [ 1, 21. The activation is completely reversible, since on removal of the substrate the activated enzyme rapidly reverts to the unactivated (inactive) form [3]. This type of activation has been observed in soluble and particulate preparations, in inner mem- brane preparations and in mitochondria [2-41. More recently a second type of reversible activation of the enzyme was discovered [5, 61. It was noted that in inner membrane preparations (ETP or ETPB) NADH also activates the enzyme reversibly. Studies with suitable inhibitors indicated that NADH itself is not the direct activator, but merely serves to reduce endo- genous ubiquinone, so that the reduced quinone ap- pears to be the immediate activating agent. This con- clusion was verified by two experimental approaches: (1) reduced ubiquinone (CoQ,,H,) is at least as good an activator as NADH in membrane preparations and (2) in pentane-extracted preparations, which are de- void of CoQie, NADH no longer activates, but succi- nate or malonate still do; on reincorporation of the CoQie activation by NADH is restored [S, 61, Acti- vation via CoQ,,H2 is characterized by the same ki- netic and thermodynamic parameters as by substrates, and thus might involve the same type of conformation- al alteration. Since the reduced/oxidized CoQic ratio undergoes