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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.

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.

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.

The reduced nicotinamide dinucleotide dehydrogenase of the respiratory chain has been further purified by gradient centrifugations at pH 10. The best preparations obtained are approximately 70% pure by sedimentation analysis and show a single flavoprotein component. The molecular weight is estimated to be of the order of 550,000, and the turnover number 800,000 per minute at 30 degrees. Provisional data are given for the NADH dehydrogenase content of heart mitochondria, Keilin-Hartree preparations, and electron transport particles. It is shown that NADH dehydrogenase contributes to the acid-extractable flavin of these preparations only to a very minor extent. (Author)

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.

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.

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 fact that the d -α-hydroxy acid dehydrogenase is formed best under anaerobic conditions of growth and disappears on environmental adaptation of the cells to O2, while l (+) lactic dehydrogenase is absent in anaerobic cells and is synthesized during O2 adaptation, has repeatedly led to suggestions in the literature that d -α-hydroxy acid dehydrogenase is a precursor of l (+) lactic dehydrogenase. In the present study it is shown that the disappearance of d -α-hydroxy acid dehydrogenase during O2 adaptation is the consequence of the fact that the enzyme is rapidly destroyed in yeast cells once active growth ceases and that the destruction is as rapid on anaerobic incubation of the cells as aerobically. Thus the destruction of d -α-hydroxy acid dehydrogenase can proceed without any synthesis of l (+) lactic dehydrogenase. Further, under suitable conditions, extensive synthesis of l (+) lactic dehydrogenase occurs without the disappearance of d -α-hydroxy acid dehydrogenase . These facts and the extensive differences in constitution and catalytic properties between d -α-hydroxy acid dehydrogenase and l (+) lactic dehydrogenase suggest that a precursor-product relationship between the two enzymes is highly unlikely.

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.

Resolution of Some Problems Concerning the Reduced Diphosphopyridine Nucleotide Dehydrogenase of the Respiratory Chain

Extensively or completely activated preparations of beef heart succinate dehydrogenase have been investigated by electron paramagnetic resonance (EPR) techniques at 6 to 97 K. Reductive titrations with dithionite and rapid kinetic studies were performed with various types of soluble and membrane-bound preparations of the enzyme. The following components were detected and their behavior analyzed: a free radical, presumably arising from the covalently bound flavin on reduction, two iron-sulfur centers of the ferredoxin type, the signals of which appear on reduction, and a high-potential iron-sulfur component, detectable in the oxidized state. The high-potential component was only detected in complex II and inner-membrane preparations. This component and one of the ferredoxin-type centers were present in amounts close to stoichiometric with the flavin and were reduced by substrate. The other ferredoxin-type center was present in amounts between 0.1 and 0.5 times that of the flavin and was reduced only by dithionite. Of the components reduced by succinate. however, only a fraction (up to 50% of the high-potential iron-sulfur center and 40–60% of the ferredoxin-type iron-sulfur center) was reduced within the turnover time of the enzymes. In complex II not more than about 10% of the flavin appeared in the semiquinone form at any time. Soluble, purified preparations behaved similarly except that the high-potential component was nearly or completely absent and extensive accumulation of the free radical occurred (up to 70 to 80% of the flavin) in titration and kinetic experiments. No significant difference was observed between the rates of semiquinone formation and the reduction of the ferredoxin-type or high-potential centers by the substrate. Also no qualitative differences in the properties studied in this work became apparent between preparations containing 4 or 8 iron atoms, respectively.

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.

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.

The cytoplasmic membrane of the H 37 Ra strain of Mycobacterium tuberculosis has been isolated free of cell wall. These membrane preparations contain very small quantities of cytochromes c , b and cytochrome oxidase. The cytochrome c is not extracted by any method attempted. The cytochrome b is reducible only by dithionite and is believed not to be involved in the direct transfer of electrons during the oxidation of NADH by these preparations. The NADH oxidase activity of the membrane is inhibited by high concentrations of cyanide and also by 2-( n -heptyl)-4-hydroxyquinoline- N -oxide (HQNO). The cytochrome oxidase of the membrane contains both cytochromes a and a 3 and is present in low concentrations relative to cytochrome c . The cytochrome a 3 component was identified by characteristic complexes with both CO and cyanide and shows a γ-band absorption maximum at a slightly lower wavelength than the cytochrome oxidase of mammalian mitochondria (442 nm vs. 445 nm). The functional activity of the cytochrome oxidase is indicated by the inhibition of reoxidation of reduced cytochromes c and a in the presence of cyanide.

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.