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SummaryThe influence of the level of 6 common minerals on the susceptibility of Swiss mice to Theiler's GDVII virus has been investigated in more than 1100 animals.No demonstrable effect on the susceptibility to this infection was found by varying the level of calcium, magnesium or chlorine in the diet, some effect was noted with sodium, while striking results were obtained with potassium and phosphorus.A progressively decreased resistance was observed as the amount of potassium or of phosphorus was increased in the diets up to the optimal levels.

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'

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.

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.

Riboflavin uptake and membrane-associated riboflavin-binding activity have been investigated in Bacillus subtilis. The uptake and binding activity of the vitamin were found to be repressed coordinately by riboflavin present in the growth medium. The uptake or riboflavin has been shown to have properties of a carrier-mediated process, and membrane vesicles have been shown to demonstrate riboflavin counterflow and exchange. The membrane-associated binding activity for riboflavin has been solubilized with detergents, and a procedure for the partial purification of this component is described. The partially purified riboflavin-binding component has properties expected for a carrier involved in riboflavin uptake, as it shows saturation kinetics and is inhibited by riboflavin analogues. Evidence is also presented showing that reduced riboflavin binds to a greater extent than oxidized riboflavin, and the possible role of the reduced riboflavin in riboflavin uptake is discussed.

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.

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.

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.

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.

Electron paramagnetic resonance spectral studies of protein-bound 8α-substituted FMN neutral semiquinones (in a complex with Azotobacter apoflavodoxin) show peak-to-peak linewidths (~ 20–23 G) similar to those of normal flavoenzymes which form neutral semiquinones. The anionic form of the 8α-substituted flavin semiquinone of thiamine dehydrogenase shows a narrower peak-to-peak linewidth (12 G) than normal anionic flavin semiquinones (14–15 G). The electron paramagnetic resonance spectral properties of the 8α-N(3)-histidyl FAD semiquinone of succinate dehydrogenase showed a peak-to-peak linewidth of 12 G through the pH range of 6.1 to 9.1 and remained unchanged whether the buffer medium was H2O or D2O. From these observations, it is concluded that the covalent flavin semiquinone in succinate dehydrogenase exists in the anionic (red) form.

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.

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).

Identification of the rate-limiting step in the catalytic cycle of succinate dehydrogenase and the related problem of defining the factors which determine its turnover number have for a long time occupied the interest of many investigators. During the past two years a collaboration among several laboratories has been initiated in order to use a variety of experimental approaches to these problems. The present report summarizes the progress which has been achieved.

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.