The cDNA sequence of beef heart CII-3, a membrane-intrinsic subunit of succinate-ubiquinone oxidoreductase.

We provide the first full-length cDNA and amino acid sequences for beef heart CII-3, one of two hydrophobic subunits that bind succinate dehydrogenase to the mitochondrial inner membrane to form succinate-ubiquinone oxidoreductase (EC 1.3.99.1). Other low molecular weight proteins present in preparations of the isolated complex, including three possible forms of the second anchor polypeptide CII-4, have been identified by amino terminal sequencing.

Deficiencies of NADH and succinate dehydrogenases in degenerative diseases and myopathies.

This paper examines the experimental foundations of reports in the literature on mitochondrial diseases involving Complexes I and II of the respiratory chain. Many of the reports may be questioned on the basis of the assay conditions used which disregard established knowledge of the precautions required for valid activity measurements. In addition, some findings are open to question because of the experimental material chosen for the study, such as the measurement of NADH oxidase activity in platelets in Parkinson's disease, which affects selectively the dopamine neurons, or the use of autopsy material stored for prolonged periods during which post-mortem changes may have occurred. Deficiencies claimed to involve several components of the respiratory chain may reflect indirect effects, such as defects in the synthesis of iron-sulfur clusters or in the availability of iron, rather than mutations in the genes coding for the deficient enzymes. Nevertheless, there are a few instances reported of Complex II deficiency free from such criticisms. As to Complex I, idiopathic Parkinsonism appears to involve a documentable decline in the activity of this enzyme. Using the model system provided by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces biochemical, pharmacological, and clinical syndromes closely resembling Parkinsonism, the etiology of the disease is examined.

The cDNA sequence of the flavoprotein subunit of human heart succinate dehydrogenase.

We report the full-length cDNA sequence for the flavoprotein subunit of human heart succinate dehydrogenase (succinate: (acceptor) oxidoreductase EC 1.3.99.1). Identical sequence was obtained for part of the cDNA of the human placental flavoprotein, in contrast to a previously published sequence. The human sequence, like the bovine one, contains a cysteine triplet and at the active site there is an additional cysteine when compared with yeast or prokaryotes.

Voltammetric studies of bidirectional catalytic electron transport in Escherichia coli succinate dehydrogenase: comparison with the enzyme from beef heart mitochondria.

The succinate dehydrogenases (SDH: soluble, membrane-extrinsic subunits of succinate:quinone oxidoreductases) from Escherichia coli and beef heart mitochondria each adsorb at a pyrolytic graphite 'edge' electrode and catalyse the interconversion of succinate and fumarate according to the electrochemical potential that is applied. E. coli and beef heart mitochondrial SDH share only ca. 50% homology, yet the steady-state catalytic activities, when measured over a continuous potential range, display very similar catalytic operating potentials and energetic biases (the relative ability to catalyse succinate oxidation vs. fumarate reduction). Importantly, E. coli SDH also exhibits the interesting 'tunnel-diode' behaviour previously reported for the mitochondrial enzyme. Thus as the potential is lowered below ca. -60 mV (pH 7, 38 degrees C) the rate of catalytic fumarate reduction decreases abruptly despite an increase in driving force. Since the homology relates primarily to residues associated with active site regions, the marked similarity in the voltammetry reaffirms our previous conclusions that the tunnel-diode behaviour is a characteristic property of the enzyme active site. Thus, succinate dehydrogenase is an excellent fumarate reductase, but its activity in this direction is limited to a very specific range of potential.

The 64-kilodalton eye muscle protein is the flavoprotein subunit of mitochondrial succinate dehydrogenase: the corresponding serum antibodies are good markers of an immune-mediated damage to the eye muscle in patients with Graves' hyperthyroidism.

Thyroid-associated ophthalmopathy (TAO) is a progressive eye disorder associated with thyroid autoimmunity, particularly Graves' hyperthyroidism, which is generally considered to have an autoimmune etiology. Eye muscle membrane proteins reportedly of 55 and 64 kDa are the best markers of the ophthalmopathy. The main focus of our recent studies has been to purify the pertinent proteins from porcine eye muscle membranes and characterize them. The 64-kDa protein is now shown from a partial sequence and by Western blotting using specific antibody probes to be the flavoprotein (Fp) subunit of succinate dehydrogenase and to have a correct molecular mass of 67 kDa. The protein was purified and cleaved with cyanogen bromide, and the N-terminal region of an immunoreactive partial peptide was determined. The 20-amino acid porcine sequence so obtained matched one within the Fp subunits of human and bovine succinate dehydrogenases in 20 and 18 of these positions, respectively. Succinate dehydrogenase is both a citric acid cycle enzyme and a component (complex II) of the mitochondrial respiratory chain. It is thus essential for aerobic energy production and is highly conserved. The mature human and bovine Fp subunits are 92% homologous and have a molecular mass of approximately 67 kDa, the same as our redetermined value for the 64-kDa marker protein. Sera from patients with TAO and from those with Graves' hyperthyroidism without evident ophthalmopathy highlighted the 64-kDa marker protein in crude porcine eye muscle membranes and the Fp subunit of highly purified bovine succinate dehydrogenase at the identical position on Western blots. Anti-beef Fp antibodies were detected in sera from 67% of patients with active TAO of more than 1-yr duration, in 30% with stable TAO of more than 3-yr duration, and in 30% of patients with Graves' hyperthyroidism without ophthalmopathy, but in only 7% of age- and sex-matched normal subjects. As succinate dehydrogenase is bound to the matrix (inside) surface of the mitochondrial inner membrane, it is unlikely to be accessible to circulating autoantibodies. We would postulate that eye muscle damage in ophthalmopathy is probably caused by cytotoxic antibodies or CD+ T lymphocytes targeting a cell membrane antigen, such as the thyroid and eye muscle shared protein G2s, and that presentation of succinate dehydrogenase is secondary. On the other hand, an autoantibody response to succinate dehydrogenase may be a good marker of immune-mediated damage to the eye muscle fiber and may support the idea that the extraocular muscles are targets of the autoimmune reactions of TAO.

Serum antibodies against the flavoprotein subunit of succinate dehydrogenase are sensitive markers of eye muscle autoimmunity in patients with Graves' hyperthyroidism.

Thyroid-associated ophthalmopathy is an autoimmune disorder of the extraocular muscles and orbital connective tissue, which is usually associated with Graves' hyperthyroidism. Well-studied markers of ophthalmopathy are eye muscle membrane antigens, reportedly of approximately 64-kDa molecular mass. One, originally identified only as the 64-kDa protein, has recently been shown to be the flavoprotein (Fp) subunit of mitochondrial succinate dehydrogenase, which has a correct molecular mass of 67 kDa. We have used purified beef heart Fp as antigen in an enzyme-linked immunosorbent assay for cross-reactive human autoantibodies. Sera have been screened from patients with thyroid-associated ophthalmopathy classified according to activity and presence or not of eye muscle disease, and from those with Graves' hyperthyroidism without eye involvement. Also examined were serum samples taken periodically from 20 patients with Graves' hyperthyroidism during 24 months of treatment of their hyperthyroidism with antithyroid drugs. Four of these patients had ophthalmopathy at the onset, 12 developed ophthalmopathy, and 4 did not develop any eye signs during treatment. Anti-Fp subunit antibodies were detected in 73% of patients with active ophthalmopathy and evidence of eye muscle involvement but only in 25% if there was only congestive ophthalmopathy. These values were 0% and 11% for patients with chronic ophthalmopathy, with or without eye muscle dysfunction, respectively. The antibodies were also detected in 14% of patients with Graves' hyperthyroidism without evident ophthalmopathy, 11% of patients with nonimmunologic thyroid disorders, 12% of type I diabetics, and 12% of age- and sex-matched normal subjects. Significantly, appearance of anti-Fp antibodies predicted the development of ophthalmopathy in 5 of the 6 patients with Graves' hyperthyroidism, who developed eye muscle dysfunction after treatment of the hyperthyroidism, and coincided with the onset of eye muscle signs in the other patient. Antibodies were not detected in any of 6 patients who developed congestive ophthalmopathy without evidence of eye muscle damage or in 4 patients who did not develop any eye signs. In conclusion, we have shown a close relationship between eye muscle disease and serum antibodies against the Fp subunit of succinate dehydrogenase in patients with Graves' hyperthyroidism.

Progress in understanding structure-function relationships in respiratory chain complex II.

Complex II (succinate:quinone oxidoreductase) of aerobic respiratory chains oxidizes succinate to fumarate and passes the electrons directly into the quinone pool. It serves as the only direct link between activity in the citric acid cycle and electron transport in the membrane. Finer details of these reactions and interactions are but poorly understood. However, complex II has extremely similar structural and catalytic properties to quinol:fumarate oxidoreductases of anaerobic organisms, for which X-ray structures have recently become available. These offer new insights into structure-function relationships of this class of flavoenzymes, including evidence favoring protein movement during catalysis.

Classification of fumarate reductases and succinate dehydrogenases based upon their contrasting behaviour in the reduced benzylviologen/fumarate assay.

Reduction of fumarate by soluble beef heart succinate dehydrogenase has been shown previously by voltammetry to become increasingly retarded as the potential is lowered below a threshold potential of -80 mV at pH 7.5. The behaviour resembles that of a tunnel diode, an electronic device exhibiting the property of negative resistance. The enzyme thus acts to oppose fumarate reduction under conditions of high thermodynamic driving force. We now provide independent evidence for this phenomenon from spectrophotometric kinetic assays. With reduced benzylviologen as electron donor, we have studied the reduction of fumarate catalysed by various enzymes classified either as succinate dehydrogenases or fumarate reductases. For succinate dehydrogenases, the rate increases as the concentration of reduced dye (driving force) decreases during the reaction. In contrast, authentic fumarate reductases of anaerobic cells (and 'succinate dehydrogenase' from Bacillus subtilis) neither exhibit the electrochemical effect nor deviate from simple kinetic behaviour in the cuvette assay. The 'tunnel-diode' effect may thus represent an evolutionary adaptation to aerobic metabolism.

Thyroid-associated ophthalmopathy in black South African patients with Graves' disease: relationship to antiflavoprotein antibodies.

Thyroid-associated ophthalmopathy (TAO) is a progressive eye disorder associated with Graves' hyperthyroidism, which is generally considered to have an autoimmune etiology. Eye muscle membrane proteins of 64 kd are good markers of ophthalmopathy in patients with thyroid autoimmunity. The 64-kd protein is now shown from a partial sequence to be the flavoprotein subunit (Fp) of mitochondrial succinate dehydrogenase. Hyperthyroidism due to Graves' disease is increasing in incidence among urban black female Africans, possibly because of exposure to environmental risk factors such as increased dietary iodine ingestion and stress. Ophthalmopathy is frequently observed in this clinical context, but its association with serum autoantibodies reactive with Fp has not been examined. We studied 19 black South African patients with Graves' disease during the course of prolonged antithyroid drug administration, of whom 10 had congestive ophthalmopathy, but no clinical evidence for eye muscle damage at the onset. Anti-Fp antibodies were detected in 2 of these patients, as well as in 2 of the 9 patients who did not have overt eye disease. Additionally, the antibodies became positive in 3 patients with ophthalmopathy in whom tests were negative initially, remained positive in 1 patient throughout the study period and became negative in 1 patient with positive tests initially. Ophthalmopathy did not develop in any of the 9 patients who lacked this complication on presentation. The reasons why we failed to demonstrate a close relationship between anti-Fp antibodies and the eye muscle component of ophthalmopathy are unclear although one possibility is that ocular myopathy is an uncommon manifestation in African thyrotoxic patients compared with those of Caucasian origin. The relationship between anti-Fp antibodies and eye muscle inflammation in patients with thyroid autoimmunity of different ethnic origins and environmental settings, needs to be addressed in a large prospective study.

Interaction of ubiquinone and vitamin K3 with mitochondrial succinate-ubiquinone oxidoreductase.

Two new methods have been devised for measuring fumarate reduction by beef heart succinate-ubiquinone oxidoreductase with quinols as electron donors. In one assay the quinone is maintained in the reduced state by coupling fumarate reduction with the DT-diaphorase reaction, in the other assay by the presence of excess dithionite. The advantages of these methods are discussed, along with preliminary characterization of the quinol-protein interaction.

Interactions of oxaloacetate with Escherichia coli fumarate reductase.

Fumarate reductase of Escherichia coli is converted to a deactivated state when tightly bound by oxaloacetate (OAA). Incubation of the inhibited enzyme with anions or reduction of the enzyme by substrate restores both the activity of the enzyme and its sensitivity to thiol reagents. In these respects the enzyme behaves like cardiac succinate dehydrogenase. Close to an order of magnitude difference was found to exist between the affinities of OAA for the oxidized (KD approximately 0.12 microM) and reduced (KD approximately 0.9 microM) forms of fumarate reductase. Redox titrations of deactivated fumarate reductase preparations have confirmed that reductive activation, as in cardiac succinate dehydrogenase (B. A. C. Ackrell, E. B. Kearney, and D. Edmondson (1975) J. Biol. Chem. 250, 7114-7119), is the result of reduction of the covalently bound FAD moiety and not the non-heme iron clusters of the enzyme. However, the processes differed for the two enzymes; activation of fumarate reductase involved 2e- and 1H+, consistent with reduction of the flavin to the anionic hydroquinone form, whereas the process requires 2e- and 2H+ in cardiac succinate dehydrogenase. The reason for the difference is not known. The redox potential of the FAD/FADH2 couple in FRD (Em approximately -55 mV) was also slightly more positive than that in cardiac succinate dehydrogenase (-90 mV).

Carboxin resistance in Paracoccus denitrificans conferred by a mutation in the membrane-anchor domain of succinate:quinone reductase.

Succinate:quinone reductase is a membrane-bound enzyme of the citric acid cycle and the respiratory chain. Carboxin is a potent inhibitor of the enzyme of certain organisms. The bacterium Paracoccus denitrificans was found to be sensitive to carboxin in vivo, and mutants that grow in the presence of 3'-methyl carboxin were isolated. Membranes of the mutants showed resistant succinate:quinone reductase activity. The mutation conferring carboxin resistance was identified in four mutants. They contained the same missense mutation in the sdhD gene, which encodes one of two membrane-intrinsic polypeptides of the succinate:quinone reductase complex. The mutation causes an Asp to Gly replacement at position 89 in the SdhD polypeptide. P. denitrificans strains that overproduced wild-type or mutant enzymes were constructed. Enzymic properties of the purified enzymes were analyzed. The apparent Km for quinone (DPB) and the sensitivity to thenoyltrifluoroacetone was normal for the carboxin-resistant enzyme, but the succinate:quinone reductase activity was lower than for the wild-type enzyme. Mutations conferring carboxin resistance indicate the region on the enzyme where the inhibitor binds. A previously reported His to Leu replacement close to the [3Fe-4S] cluster in the iron-sulfur protein of Ustilago maydis succinate:quinone reductase confers resistance to carboxin and thenoyltrifluoroacetone. The Asp to Gly replacement in the P. denitrificans SdhD polypeptide, identified in this study to confer resistance to carboxin but not to thenoyltrifluoroacetone, is in a predicted cytoplasmic loop connecting two transmembrane segments. It is likely that this loop is located in the neighborhood of the [3Fe-4S] cluster.

Multiple forms of bacterial hydrogenases.

Ackrell, B. A. C. (University of Hawaii, Honolulu), R. N. Asato, and H. F. Mower. Multiple forms of bacterial hydrogenases. J. Bacteriol. 92:828-838. 1966.-Extracts of certain bacterial species have been shown by disc electrophoresis on polyacrylamide gel to contain multiple hydrogenase systems. The hydrogenase enzymes comprising these systems have different electrophoretic mobilities and produce a band pattern that is unique for each bacterial species. Of 20 bacterial species known to possess hydrogenase activity and which were examined by this technique, only the activities of Clostridium tetanomorphum and C. thermosaccharolyticum could be attributed, at pH 8.3, to a single hydrogenase enzyme. This multiplicity of hydrogenase forms was found both in bacteria which contain mostly soluble hydrogenases and in those where the hydrogenase is predominantly associated with particulate material. When solubilization of this particulate material could be effected, at least two solubilized hydrogenases were released, and, of these, one would have the same electrophoretic properties (i.e., R(F)) as one of the soluble hydrogenases already present in small amounts within the cell. Different growth conditions for various types of bacteria, such as the nitrogen source, the degree of aeration, and photosynthetic versus aerobic growth in the dark, as well as the conditions under which the cells were stored, markedly affected the hydrogenase activity of the cells, but not their hydrogenase band pattern. The disc electrophoresis technique proved to be 10 times more sensitive than the manometric technique in detecting hydrogenase activity.

Effect of membrane environment on succinate dehydrogenase activity.

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.