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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.

Riboflavin-requiring mutants of Saccharomyces cerevisiae are able to transport 14C-labeled riboflavin into the cell, although no significant transport is seen in commercial yeast or in the parent strain from which the mutants were derived. Transport activity is greatest in the early to mid-log phase of anaerobic growth and declines sharply in the late log phase. In aerobically grown cells activity is substantially lower at all stages of growth. In the assay devised for its measurement, transport activity shows a sharp pH optimum at pH 7.5, a strong temperature dependence (EA = 23,100 cal/mol), and saturation kinetics with respect to riboflavin (Km = 15 muM), characteristics consistent with a carrier-mediated mechanism. Monovalent inorganic cations, particularly K+ and Rb+, stimulate riboflavin uptake, while certain organic cations are inhibitory. Besides riboflavin only 7-methylriboflavin, 8-methylriboflavin, and 5-deazaflavin have been found to serve as substrates, while lumiflavin, tetraacetylriboflavin, and N10-[4'-carboxybutyl]-7,8-dimethylisoalloxazine do not, although a number of flavin analogs in which the ribityl side chain is modified are good competitive inhibitors of riboflavin uptake. Compounds resembling the ribityl side chain, such as sugars and sugar alcohols, do not inhibit. An apparent inhibition of uptake by D-glucose, D-mannose, and D-fructose, which develops in the course of assay, proved to result from stimulation of an opposing process, the release of riboflavin from the cells.

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.

Electron paramagnetic resonance studies of Complex II from the mitochondrial respiratory chain and soluble preparations of succinate dehydrogenase have, for the first time, identified a signal arising from a [4Fe-4S]1+ cluster, S2, in dithionite-reduced samples. Redox titrations, monitored by electron paramagnetic resonance spectroscopy demonstrate that this signal appears at the same midpoint potential as the enhancement of the spin relaxation properties of the [2Fe-2S]1+ center, S1, in both Complex II and reconstitutively active soluble enzyme. The results complement recent magnetic circular dichroism studies of succinate dehydrogenase (Johnson, M. K., Morningstar, J. E., Bennett, D. E., Ackrell, B. A. C., and Kearney, E. B. (1985) J. Biol. Chem. 260, 7368-7378) which assigned cluster S2 as a [4Fe-4S]2+,1+ center and provide evidence for spin interaction between the paramagnetic reduced forms of centers S1 and S2.

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.

Reconstitutively active and inactive succinate dehydrogenase have been investigated by low temperature magnetic circular dichroism (MCD) and EPR spectroscopy and room temperature CD and absorption spectroscopy. Reconstitutively active succinate dehydrogenase is found to contain three spectroscopically distinct Fe-S clusters: S1, S2, and S3. In agreement with previous studies, MCD and CD spectroscopy confirm that center S1 is a succinate-reducible [2Fe-2S]2+,1+ center. The MCD characteristics of center S2 identify it as a dithionite-reducible [4Fe-4S]2+,1+ similar to those in bacterial ferredoxins. EPR power saturation studies and the weakness of the EPR signal from reduced S2 indicate that there is a weak magnetic interaction between centers S1 and S2 in their paramagnetic, S = 1/2, reduced states. Center S3 is identified both by the form of the MCD spectrum and the characteristic magnetization behavior as a reduced [3Fe-xS] center in both succinate- and dithionite-reduced reconstitutively active succinate dehydrogenase. Arguments are presented in favor of centers S2 and S3 being separate centers rather than interconversion products of the same cluster. Reconstitutively inactive succinate dehydrogenase is found to be deficient in center S3. These results resolve many of the controversies concerning the Fe-S cluster content of succinate dehydrogenase and reconcile published EPR data with analytical and core extrusion studies. Moreover, they indicate that center S3 is a necessary requirement for reconstitutive activity and suggest that it is able to sustain ubiquinone reductase activity as a [3Fe-xS] center.

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)

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.

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

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

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.

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.

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.