Search

From a study of the magnetic field dependence of the linear electric field effect (LEFE) in EPR spectroscopy, we demonstrate that iron-sulfur cluster 3 in air-oxidized, beef heart succinate-ubiquinone oxidoreductase (Complex II) is a 3-iron cluster. This suggests that cluster 3 may arise by oxidative degradation from a 4-iron cluster originally present in the enzyme and may be reconverted back into a 4-iron cluster under reducing conditions. Linear electric field effect studies of succinate-reduced Complex II are in accord with the view that cluster 1 is a 2-iron cluster.

The turnover number of succinate dehydrogenase from mammalian heart determined by the spectrophotometric phenazine methosulfate assay, after complete activation, is approximately 21,000 mol of succinate oxidized/min/mol of histidyl flavin at 38 degrees in relatively intact inner membrane preparations and mitochondria. Reconstitutively active soluble preparations, extracted anaerobically in the presence of succinate from inner membrane preparations show turnover numbers of 11,500 to 14,500 and a significantly lower apparent Km for phenazine methosulfate than the parent particles. The decline of both the turnover number and of the Km occurs during the brief period when the enzyme is detached from the membrane. The observed values represent the activities in the soluble extract of both the reconstitutively active and reconstitutively inactive enzyme. The latter may be from 10 to 40% even in the most carefully prepared enzyme; it has a lower turnover number in the phenazine methosulfate assay than the average for the solution and is devoid of catalytic activity in the "low Km" ferricyanide assay (Vinogradov, A. D., Ackrell, B.A.C., and Singer, T.P. (1975) Biochem. Biophys. Res. Commun. 67, 803-809). The reconstitutively active form of the soluble enzyme has a turnover number of at least 15,000 and an equal activity in the low Km ferricyamide assay. When recombined with the membrane the total activity of the enzyme is increased by over 60% and it regains the original turnover number, Km for phenazine methosulfate, and sensitivity of the phenazine methosulfate reductase activity to thenoyltrifluoroacetone, carboxamides, and cyanide. It appears, therefore, that the membrane environment or some component of it exerts a positive modulating influence on the enzyme even in the fully activated state. In certain particulate sources (Keilin-Hartree preparations, Complex II) the enzyme shows lower turnover numbers (11,000 to 12,500) than in more intact inner membranes. This seems to be due to inactivation in the course of preparation and, in the case of Complex II, in part also to loss of the normal membrane environment or of a membrane component, possibly Q-10, during isolation.

The fumarate reductase complex and soluble enzyme from Escherichia coli have been investigated by low temperature magnetic circular dichroism and electron paramagnetic resonance spectroscopies. The results confirm the presence of one [2Fe-2S] cluster and show that the high potential iron-sulfur center is a 3Fe cluster of the type found in bacterial ferredoxins. Since the 3Fe cluster is present in catalytically competent enzyme and does not appear to be involved in any type of cluster conversion under reducing conditions, we conclude that it is an intrinsic component of the functional enzyme. The significance of the results is discussed in relation to the published amino acid sequence and the iron-sulfur cluster composition of bacterial fumarate reductases.

The reoxidation phase of the catalytic cycle of succinate dehydrogenase was studied in Complex II preparations' by the rapid freeze-electron paramagnetic resonance (epr) technique. With the synthetic water-soluble Q1 analog, 2,3-dimethoxy-5-methyl-6-pentyl-1, 4-benzoquinone (DPB), as the oxidant, the observed reoxidation of the epr-detectable components, previously reduced with dithionite or succinate, came to completion within a few milliseconds, well within the turnover time of the enzyme. Only ~80% of Fe-S center 1 and the HiPIP (the high-potential cluster) Fe-S center reacted rapidly with DPB, however; similarly incomplete reactions were observed previously in our studies of the reduction of the enzyme by succinate. The subsequent addition of ferricyanide, which appears to act as a chemical oxidant in these experiments, caused immediate reoxidation of the Fe-S centers and of the free radical. Ferricyanide and phenazine methosulfate (PMS) reoxidized all epr-detectable components in Complex II as well as in reconstitutively active, soluble preparations in'

Succinate dehydrogenase in the deactivated form becomes activated on incubation at mildly acid pH to an extent dependent on the pH, type of anion present, and the concentration of the anion. The equilibrium level established under one set of conditions can be perturbed by changing either the pH or the anion concentration and the resulting level of activation reflects the new set of conditions. The rate of activation is influenced by the temperature, the anion concentration, and the pH when simple anions (e.g. Br-) are used, but the energy of activation is not affected by either pH or anion concentration. The value found for the activation energy (Ea) is 25 Cal per mole. In contrast, when activation of the enzyme is mediated by succinate, malonate, or reduced coenzyme Q10, the Ea value is 31 to 33 Cal per mole. Although this difference in energies of activation suggests that activation by mild acid and anions involves a different mechanism, no difference in the catalytic activity of preparations activated by these different methods is detected.

The effects of iron deficiency on the NADH- and succinate-oxidizing complexes of rat skeletal muscle mitochondria have been investigated. Both systems were similarly affected: activities were about 30% of normal in dehydrogenase, ubiquinone reductase, and oxidase assays, and similar reductions in the concentration of their respective flavin prosthetic groups were also evident in the iron-deficient membranes. Thus, the turnover numbers of the two enzymes were unchanged in iron deficiency. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed similarly reduced levels of those peptide components of Complexes I and II that could be unequivocally distinguished. Soluble beef heart succinate dehydrogenase added to alkaline-treated rat skeletal muscle mitochondrial membranes attached to binding sites exposed by the treatment, forming a hybrid complex indistinguishable from the original skeletal muscle complex, with restoration of succinoxidase and succinate-ubiquinone reductase activities to the levels observed in the original rat membranes. Iron-deficient particles behaved like the normal in these tests. No unfilled binding sites for the enzyme could be detected prior to alkaline treatment. The data are interpreted as indicating that the lower activities of these two respiratory complexes in iron deficiency are due to lower content of the enzymes rather than to the presence of impaired enzymes in the membrane, that only fully competent complexes are present in these membranes, and that iron-deficient complexes are either not assembled or are lost after assembly.

When succinate dehydrogenase contains oxalacetate in firmly bound form, activity cannof the enzyme results in dissociation of oxalacetate and activation of the enzyme. The course of reductive titrations appears the same whether or not the enzyme contains oxalacetate, and complete reduction as monitored by bleaching of chromophoric groups requires the incorporation of 6 to 7 reducing equivalents in either case. The stoichiometry is that expected from the non-heme iron and flavin content of the enzyme. Activation of the enzyme during reductive titrations occurs predominantly with the incorporation of the second pair of electrons, while determination of activation levels at various poised potentials shows that the group involved is reduced with the uptake of 2 H+ and 2 e-. These characteristics are consistent with titration of the flavin moiety rather than non-heme iron groups. Thus it appears that activation is concurrent with the reduction of flavin to the hydroquinone form. From the measured half-reduction potential for activation, that of the flavin in an oxalacetate-free enzyme has been estimated at -90 to -60 mv at pH 7.

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.

In most procedures for the isolation of membrane-bound or soluble succinate dehydrogenase, the enzyme is obtained largely in the deactivated form. As judged by the quantity of oxalacetate enzymatically determined in perchlorate extracts, the deactivated form contains tightly bound oxalacetate in 1:1 proportion to the enzyme (equated with histidyl flavin content) in particles. A somewhat lower ratio has been obtained for soluble preparations of the enzyme. The oxalacetate is bound very tightly: it is not displaced by succinate or malonate in the cold, nor released by precipitation or gel exclusion of the enzyme, and it does not react with malate dehydrogenase. It is released, however, on denaturation of the enzyme with perchloric acid or under conditions which favor conversion of the enzyme to the activated form. If the enzyme is freed from oxalacetate by activation with bromide and gel exclusion, treatment of the oxalacetate-free fully active enzyme with very low concentrations of l- or d-malate or oxalacetate leads to deactivation and concomitant formation of tightly bound oxalacetate in the same molar ratio found in the enzyme as isolated from heart mitochondria. Dissociation of the complex formed in this manner requires the same experimental conditions as those needed for dissociation of the complex as found in the isolated enzyme. The activation energies for activation and for release of oxalacetate are the same but vary with the type of activator used. The first order rate constant for activation by a variety of agents is about twice that measured for the dissociation of tightly bound oxalacetate. Moreover, with some activators oxalacetate release shows a considerable lag behind recovery of catalytic activity. It is suggested that the presence of oxalacetate in the deactivated enzyme is not the cause of its lack of catalytic activity but a consequence of the deactivated conformation. Further observations are described which suggest that a deactivation-activation cycle may also occur in the absence of oxalacetate.

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.

1. 1. The spontaneous, reversible inactivation of the l -amino acid oxidase of snake venoms has been compared with reversible processes which regulate the activity of other enzymes. 2. 2. Since no change in the size, shape, electrophoretic mobility or solubility of the oxidase has been detected upon extensive inactivation of the enzyme, the process has been interpreted as involving only a limited alteration of the configuration of the native enzyme rather than an extensive unfolding. 3. 3. The mechanism of this reversible process has been interpreted as follows. The active enzyme is in reversible equilibrium with an inactive form, which differs from the active form by the presence of a new acidic group not present in the native enzyme, or an acidic group whose dissociation constant changes by about two pH units as a result of structural alterations in the protein during the inactivation step. The probable nature of this ionizing group has been discussed and its possible identity with an imidazole nitrogen, located at the active center of the enzyme, has been pointed out. 4. 4. On the basis of the above hypothesis, a kinetic development has been presented to account for the fact that the rate of reactivation of the enzyme is a function of pH while the rate of inactivation is not. 5. 5. It has been suggested that the inactivation process may involve the reversible breakage of one or two strong covalent linkages or of several weaker hydrogen bonds and that at least one of these may be joined to an imidazole ring at the active center of the enzyme. 6. 6. The possible nature of the ionizing groups at the active center of the enzyme is discussed from considerations of the influence of pH on the activity and stability of the enzyme.

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 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.

1. 1. The reversible inactivation of l-amino acid oxidase, previously described for the enzyme of moccasin venom, occurs in all species of Crotalidae venoms and in many Viperidae venoms, while Elapidae do not at all undergo this type of inactivation. This indicates a basic difference in the protein component of the enzyme from Crotalidae and from Elapidae. 2. 2. The l-amino acid oxidase activity of 23 species of venoms has been determined under conditions where the enzymes are stable. 3. 3. The following univalent anions protect the enzyme from inactivation to about the same extent: Cl−, Br−, F−, NO3+, CNS−, N3+, and ClO4+; I−, which is somewhat inhibitory to the enzyme, protects less effectively. The singly charged phosphate, metaphosphate, and arsenate anions do not prevent the inactivation. None of the doubly or triply charged anions or undissociated acids tested were protective. The cationic constituents of buffers are without effect on the enzyme at the concentrations tested. 4. 4. In dilute solutions of phosphate and related anions the inactivation and its reversal proceed in the same manner as in distilled water. Phosphate enhances the extent of inactivation only at high concentrations. 5. 5. The effect of the protective univalent anions is on the stability of the enzyme; they are not required for activity. The differences in the behavior of various anions are ascribed to selective binding of certain anions by the protein at those sites where bonds are ruptured during the reversible inactivation process.

1. 1. L-Cysteinesulfinic dehydrogenase, the enzyme which catalyzes the dehydrogenation of L-cysteinesulfinic acid, requires a coenzyme for activity. The coenzyme requirement is not fulfilled by known vitamins, coenzymes, or related substances, but boiled yeast extract is a good source of the cofactor. 2. 2. The new coenzyme, provisionally named coenzyme III, has been extensively purified from baker's yeast. When the dehydrogenase oxidizes cysteinesulfinic acid in the presence of the purified coenzyme, the latter is reduced to a substance with an absorption spectrum characteristic of dihydropyridine nucleotides. Upon addition of L-cysteic acid, the reduced spectrum disappears, indicating reversal of the dehydrogenation. 3. 3. The purified coenzyme is rapidly destroyed by 0.1 N acids at 100° and by nucleotide pyrophosphatase but is not acted upon by 5-nucleotidase or phosphodiesterase. The heavy metal salts of coenzyme III are water soluble. In charcoal chromatography it behaves very much like nicotinamide mononucleotide. 4. 4. On the basis of its behaviour to chemical reagents and to hydrolytic enzymes, the structure of coenzyme III is tentatively suggested to be nicotinamide-ribose-(5)-pyrophosphate. 5. 5. Some of the salient properties of L-cysteinesulfinic dehydrogenase have been investigated. 6. 6. On the basis of a detailed study of the electron transport from coenzyme III to O2, it is concluded that a diaphorase (or cytochrome reductase), concerned with the reoxidation of coenzyme III, is present in the extracts in the apoenzyme form. This protein requires a second new coenzyme, which is not identical with FMN or FAD, but is likely to be a flavin. The latter cofactor is present in yeast extract and is readily separated from coenzyme III. 7. 7. It is pointed out that coenzyme III also functions as the prosthetic group of dehydrogenases of animal origin and that it appears to be of wide distribution.

The disappearance of succinate dehydrogenase activity and of protein-bound histidyl flavin were studied in aerobic yeast cells incubated with high glucose concentrations. The decay of succinate dehydrogenase activity, covalently bound flavin, and of respiration is prevented by cycloheximide but not by chloramphenicol. During this decay there is a large increase in mitochondrial phospholipase D activity; the appearance of this enzyme is also prevented by cycloheximide. It seems possible, therefore, that the formation of phospholipase D may be important in triggering the disappearance of covalently bound flavin, succinate dehydrogenase, and of other mitochondrial enzymes during glucose repression of aerobic yeast cells.

Existing procedures for the isolation of mammalian succinate dehydrogenase yield preparations of high purity or retain reconstitution activity, but not both. A new procedure is described for the isolation in good yield of virtually homogeneous preparations with full reconstitution activity, and retaining iron-sulfur center 3 and the "low Km" reaction site of ferricyanide. On reincorporation of the soluble enzyme into alkali-treated membranes, the same high turnover number (approximately 21,000/min at 38 degrees) is obtained in catalytic assays as with intact inner membrane preparations.

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

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.

Succinate-ubiquinone oxidoreductase (Complex II) is a membrane-bound enzyme that couples the oxidation of succinate to fumarate to the reduction of ubiquinone in the mitochondrial respiratory chain. It is composed of four subunits (1); two hydrophilic subunits, a flavoprotein containing covalently bound FAD (Mr = 70,000) and a smaller iron-sulfur protein (Mr. = 27,000), that together constitute the enzyme succinate dehydrogenase (2), and two small hydrophobic subunits which are associated with a b-type cytochrome (3). The two small peptides are required for anchoring the enzyme to the membrane and for the reduction of ubiquinone, but are not required for the catalytic oxidation of succinate in the presence of artificial electron acceptors (4,5).

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.

INVESTIGATIONS in this laboratory have shown that the various DPNH-cytochrome reductase preparations from heart and liver mitochondria described in the literature are fragments of the respiratory chain-linked DPNH dehydrogenase produced by the extraction method (acid-ethanol-heat or thermal degradation) used in the isolation of cytochrome reductases1–5. By the action of these agents as well as various proteolytic enzymes, urea and under a number of other experimental conditions the purified dehydrogenase is also degraded to lower molecular weight cytochrome-reducing fragments. This conversion involves major changes in many of the molecular and catalytic properties of the enzyme of which the emergence of cytochrome (and DCIP) reductase activity is but one feature. The observations on thermal and proteolytic degradation have been confirmed and extended by Kaniuga7,8, who has independently discovered the urea-catalysed transformation of the enzyme. Acidethanol degraded DPNH dehydrogenase is not distinguishable from the preparations of Mahler et al.9, de Bernard10, and Mackler11, while the thermally degraded enzyme is in every respect identical with the preparation isolated by King and Howard12 of which the major component is the Mahler enzyme9. Although these investigations have directed attention to the fact that DPNH dehydrogenase is unstable under a variety of conditions13 and readily breaks down to fragments with very different properties, very recently yet another DPNH oxidizing preparation derived from heart mitochondria has been described14 which appears to be a breakdown product of the respiratory chain-linked DPNH dehydrogenase. It is the purpose of this communication to show that, in terms of available evidence, the “DPNH dehydrogenase” of Chapman and Jagannathan14 is not a naturally occurring enzyme but a fragment produced by the action of thiourea on DPNH dehydrogenase.

1. 1. Solutions of the l -amino acid oxidase of moccasin venom, in the crude or purified state, undergo an unusual type of spontaneous inactivation. 2. 2. The process is entirely reversible, and the position of the apparent equilibrium between the active and inactive forms of the enzyme is determined by the pH. In the range of about pH 5.5–7.5, the higher the pH, the greater the extent of inactivation at equilibrium. 3. 3. Temperature influences primarily the rate of the process. Below 10 ° no inactivation or reactivation is observed. Between 25 and 45 ° the rate of inactivation increases in a regular fashion, with an energy of activation of 42,500 cal./mole. 4. 4. Protein concentration does not seem to influence the inactivation. 5. 5. The inactivation process follows the kinetics of a first-order reversible reaction. 6. 6. The inactivation is completely prevented by 10 −2 M univalent anions and by low concentrations of riboflavin and those of its analogs which have a high affinity for the oxidase. Substrates and competitive inhibitors of the enzyme are also effective protective agents. At concentrations of 10 −4 to 10 −3 M , Cl − and similar anions effect partial prevention of the inactivation. 7. 7. Concentrations of univalent anions and of flavins which prevent the inactivation effectively, reverse it also. Reactivation, like inactivation, proceeds at a measurable rate only at elevated temperatures. In general, the higher the temperature, the more rapid is the return of activity. Reactivation is also a function of the concentration of univalent anions and of pH. Significant reversal is accomplished without added anions by merely lowering the pH. At 55 ° the return of activity is very rapid under these conditions.

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.

1. 1. Homogenates and soluble extracts of rat liver mitochondrial acetone powder catalyze the rapid, coupled oxidation of cysteinesulfinate (CSA) and fumarate (or malate) to yield pyruvate, aspartate, and inorganic sulfate, as well as an analogous reaction between CSA and α-ketoglutarate to yield pyruvate, glutamate, and sulfate. In preparations from heart mitochondria the same reactions occur, except that the sulfur moiety of CSA accumulates as sulfite. 2. 2. The reactions above have been shown to involve rapid transaminations of CSA with α-ketoglutarate or oxalacetate. β-Sulfinylpyruvate, the product of these transaminations does not accumulate in the preparations but is cleaved to pyruvate and sulfite. The latter is oxidized to sulfate by sulfite oxidase in liver preparations but not by heart mitochondria. 3. 3. The very slow formation of oxalacetate from fumarate or malate by malic dehydrogenase in the mitochondrial system is greatly enhanced by the presence of CSA, by virtue of the rapid and complete removal of oxalacetate in the transamination reaction. 4. 4. Highly purified glutamic-oxalacetic transaminase catalyzes the reaction of CSA and α-ketoglutarate more rapidly than the transamination between aspartate and α-ketoglutarate. It is suggested that the observed transamination of CSA with oxalacetate and α-ketoglutarate may be the result of the action of glutamic-oxalacetic transaminase. 5. 5. Another, apparently non-transaminative, oxidation of CSA to pyruvate, sulfate, and ammonia has been observed in fresh rat liver mitochondrial acetone powders. The reaction is DPN-dependent. It is suggested that the initial steps may be the anaerobic dehydrogenation of CSA by a DPN-linked enzyme to β-sulfinylpyruvate and ammonia and that the further metabolism of the keto acid is identical with that occurring in the coupled reactions described. 6. 6. It is pointed out that these results and other data in the literature confirm the view that the mineralization of the sulfur moiety of cysteine in liver occurs at the oxidation level of CSA and entails cleavage of β-sulfinylpyruvate to sulfite, followed by the action of sulfite oxidase. 7. 7. The central position of transaminations involving CSA in the interrelations of carbohydrate and sulfur metabolism is discussed.

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.

1. 1. The succinic dehydrogenase of baker's yeast has been solubilized and isolated from the particulate fraction of the cell wherein the enzyme is localized. The best preparations obtained were 65 % pure by ultracentrifugal analysis. 2. 2. The enzyme is an amber-brown protein, containing 4 atoms of iron and 1 mole of flavine per mole of protein. No method has been found to remove either the iron or the flavine without denaturation of the protein. The iron was readily liberated in the inorganic form by acid or thermal denaturation, but separation of the flavine from the protein required extensive proteolytic digestion of the latter. The flavine, set free in this manner, was not free FAD but, like that from the beef heart enzyme, appeared to be in bound form, possibly as peptide derivatives. 3. 3. The action of the enzyme is readily reversible. Inactivation on aging usually entailed a much greater loss of succinic dehydrogenase than of fumaric reductase activity. 4. 4. The following aspects of the action of the enzyme have been studied: effects of pH and temperature; specificity for electron carriers; inhibition by sulfhydryl reagents and iron complexors; kinetic constants for succinate, malonate, and fumarate; and turnover number. 5. 5. In most respects the yeast enzyme resembles closely its counterpart in beef heart mitochondria.

IT was observed by Massey and Swoboda1 that on storage at 4° of Keilin–Hartree preparations isolated by centrifugation at pH 5.4 a gradual breakdown of FAD to FMN occurs. Immediately after isolation such preparations contained up to 95 per cent of the acid-extractable flavins in the form of FAD, but this quantity decreased to about 70 per cent over a period of several days. These authors concluded that in strictly fresh Keilin–Hartree preparations and in mitochondria, within experimental error, all the acid-extractable flavin may be in the form of FAD, and suggested, therefore, that FMN enzymes isolated from these sources might be artefacts.