Identification of quinone-binding and heme-ligating residues of the smallest membrane-anchoring subunit (QPs3) of bovine heart mitochondrial succinate:ubiquinone reductase.
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The smallest membrane-anchoring subunit (QPs3) of bovine heart succinate:ubiquinone reductase was overexpressed in Escherichia coli JM109 as a glutathione S-transferase fusion protein using the expression vector pGEX2T/QPs3. The yield of soluble active recombinant glutathione S-transferase-QPs3 fusion protein was isopropyl-1-thio-beta-D-galactopyranoside concentration-, induction growth time-, temperature-, and medium-dependent. Maximum yield of soluble recombinant fusion protein was obtained from cells harvested 3.5 h post-isopropyl-1-thio-beta-D-galactopyranoside (0.4 mM)-induction growth at 25 degrees C in 2.0% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose (SOC medium) containing 440 mM sorbitol and 2.5 mM betaine. QPs3 was released from the fusion protein by proteolytic cleavage with thrombin. Isolated recombinant QPs3 shows one protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that corresponds to subunit V of mitochondrial succinate:ubiquinone reductase. Although purified recombinant QPs3 is dispersed in 0.01% dodecylmaltoside, it is in a highly aggregated form, with an apparent molecular mass of more than 1 million. The recombinant QPs3 binds ubiquinone, causing a spectral blue shift. Upon titration of the recombinant protein with ubiquinone, a saturation behavior is observed, suggesting that the binding is specific and that recombinant QPs3 may be in the functionally active state. Two amino acid residues, serine 33 and tyrosine 37, in the putative ubiquinone binding domain of QPs3 are involved in ubiquinone binding because the S33A- or Y37A-substituted recombinant QPs3s do not cause the spectral blue shift of ubiquinone. Although recombinant QPs3 contains little cytochrome b560 heme, the spectral characteristics of cytochrome b560 are reconstituted upon addition of hemin chloride. Reconstituted cytochrome b560 in recombinant QPs3 shows a EPR signal at g = 2.92. Histidine residues at positions 46 and 60 are responsible for heme ligation because the H46N- or H60N-substituted QPs3 fail to restore cytochrome b560 upon addition of hemin chloride. (+info)
Stopped-flow studies of the binding of 2-n-heptyl-4-hydroxyquinoline-N-oxide to fumarate reductase of Escherichia coli.
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We have studied the kinetics of binding of the menaquinol analog 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) by fumarate reductase (FrdABCD) using the stopped-flow method. The results show that the fluorescence of HOQNO is quenched when HOQNO binds to FrdABCD. The observed quenching of HOQNO fluorescence has two phases and it can be best fitted to a double exponential equation. A two-step equilibrium model is applied to describe the binding process in which HOQNO associates with FrdABCD by a fast bimolecular step to form a loosely bound complex; this is subsequently converted into a tightly bound complex by a slow unimolecular step. The rates of the forward and the reverse reactions for the first equilibrium (k1 and k2) are determined to be k1 = (1.1 +/- 0.1) x 10(7) M-1.s-1, and k2 = 6.0 +/- 0.6 s-1, respectively. The dissociation constants of the first equilibrium (Kd1 = k2/k1) is calculated to be about 550 nM. The overall dissociation constant for the two-step equilibrium, Kd overall = Kd1/[1+ (1/Kd2)], is estimated to be < or = 7 nM. Comparison of the kinetic parameters of HOQNO binding by FrdABCD and by dimethyl sulfoxide reductase provides important information on menaquinol binding by these two enzymes. (+info)
Characterization of succinic dehydrogenase mutants of Bacillus subtilis by crossed immunoelectrophoresis.
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Eleven succinic dehydrogenase (SDH) mutants in Bacillus subtilis were analyzed by crossed immunoelectrophoresis with antiserum prepared against wild-type B. subtilis cytoplasmic membrane. A precipitate which stained for SDH was found in Triton X-100-solubilized wild-type membranes and in membranes from two of the SDH mutants. The remaining nine mutants did not show an SDH-staining precipitate. The respective mutations in these nine mutants all map in one locus, citF (Ohne et al., J. Bacteriol. 115:738-745, 1973). An SDH-specific antiserum was prepared by immunizing rabbits with the SDH precipitate obtained in crossed immunoelectrophoresis with solubilized wild-type membrane. Using this antiserum, it was shown that all of the nine citF mutants lack an SDH-specific antigen in the membrane but five of the citF mutants have a soluble SDH-specific antigen. No major differences were found in sodium dodecyl sulfatepolyacrylamide gels of membrane proteins from wild-type B. subtilis and from SDH mutants. A model for the organization of SDH in B. subtilis is proposed. (+info)
Enzyme histochemical study of germanium dioxide-induced mitochondrial myopathy in rats.
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The purpose of this study were 1) to determine the earliest pathological changes of germanium dioxide (GeO2)-induced myopathy; 2) to determine the pathomechanism of GeO2-induced myopathy; and 3) to determine the minimal dose of GeO2 to induce myopathy in rats. One hundred and twenty five male and female Sprague-Dawley rats, each weighing about 150 gm, were divided into seven groups according to daily doses of GeO2. Within each group, histopathological studies were done at 4, 8, 16, and 24 weeks of GeO2 administration. Characteristic mitochondrial myopathy was induced in the groups treated daily with 10 mg/kg of GeO2 or more. In conclusion, the results were as follows: 1) The earliest pathological change on electron microscope was the abnormalities of mitochondrial shape, size and increased number of mitochondria; 2) The earliest pathological change on light microscope was the presence of ragged red fibers which showed enhanced subsarcolemmal succinate dehydrogenase and cytochrome c oxidase reactivity; 3) GeO2 seemed to affect the mitochondrial oxidative metabolism of muscle fibers; 4) GeO2 could induce mitochondrial myopathy with 10 mg/kg of GeO2 for 4 weeks or less duration in rats. (+info)
Significance of serum antibodies reactive with flavoprotein subunit of succinate dehydrogenase in thyroid associated orbitopathy.
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AIMS: Thyroid associated orbitopathy (TAO) is an autoimmune disorder of extraocular muscles and orbital connective tissue. Identification of the principal target antigens would help the understanding of the pathogenesis of the disease and possibly lead to the development of specific therapies in the future. The purpose of this study was to measure serum antibodies against the flavoprotein subunit of succinate dehydrogenase in patients with TAO and correlate their presence with factors of TAO. METHODS: Sera of patients with active TAO of 6 months' duration or less were tested for antibodies against the flavoprotein subunit of succinate dehydrogenase. Clinical data were obtained by retrospective review of patients' charts. Enzyme linked immunosorbent assay was used to test sera for serum antibodies against purified succinate dehydrogenase. RESULTS: 38 patients with TAO and 32 healthy age and sex matched controls were included in the study. Anti-flavoprotein antibodies were detected in 24 out of 38 patients with TAO (63.16%) and in five out of 32 healthy controls (15.63%) (p<0.01). Neither age, sex, duration of thyroid disease, thyroid status, treatment of thyroid disease, smoking history, duration of orbitopathy, activity of orbitopathy, nor the presence of lid retraction were significantly associated with the presence of serum anti-flavoprotein antibodies (p>0.05). However, the total number of rectus muscles affected in both eyes of the patients was significantly correlated with the finding of a positive antibody test (p<0.05). CONCLUSIONS: Serum antibodies reactive with the flavoprotein subunit of succinate dehydrogenase are associated with extraocular muscle involvement in active TAO of recent onset. (+info)
The unusual iron sulfur composition of the Acidianus ambivalens succinate dehydrogenase complex.
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The succinate dehydrogenase complex of the thermoacidophilic archaeon Acidianus ambivalens was investigated kinetically and by EPR spectroscopy in its most intact form, i.e., membrane bound. Here it is shown that this respiratory complex has an unusual iron-sulfur cluster composition in respect to that of the canonical succinate dehydrogenases known. The spectroscopic studies show that center S3, the succinate responsive [3Fe-4S]1+/0 cluster of succinate dehydrogenases, is not present in membranes prepared from aerobically grown A. ambivalens, nor in partially purified complex fractions. On the other hand, EPR features associated to the remaining centers, clusters S1 ([2Fe-2S]1+/2+) and S2 ([4Fe-4S]2+/1+), could be observed. Similar findings were made in other archaea, namely Acidianus infernus and Sulfolobus solfataricus. Kinetic investigations showed that the A. ambivalens enzyme is reversible, capable of operating as a fumarate reductase - a required activity if this obligate autotroph performs CO2 fixation via a reductive citric acid cycle. Sequencing of the sdh operon confirmed the spectroscopic data. Center S3 ([3Fe-4S]) is indeed replaced by a second [4Fe-4S] center, by incorporation of an additional cysteine, at the cysteine cluster binding motif (CxxYxxCxxxC-->CxxCxxCxxxC). Genomic analysis shows that genes encoding for succinate dehydrogenases similar to the ones here outlined are also present in bacteria, which may indicate a novel family of succinate/fumarate oxidoreductases, spread among the Archaea and Bacteria domains. (+info)
The Saccharomyces cerevisiae succinate dehydrogenase anchor subunit, Sdh4p: mutations at the C-terminal lys-132 perturb the hydrophobic domain.
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The yeast succinate dehydrogenase (SDH) is a tetramer of non-equivalent subunits, Sdh1p-Sdh4p, that couples the oxidation of succinate to the transfer of electrons to ubiquinone. One of the membrane anchor subunits, Sdh4p, has an unusual 30 amino acid extension at the C-terminus that is not present in SDH anchor subunits of other organisms. We identify Lys-132 in the Sdh4p C-terminal region as necessary for enzyme stability, ubiquinone reduction, and cytochrome b562 assembly in SDH. Five Lys-132 substituted SDH4 genes were constructed by site-directed mutagenesis and introduced into an SDH4 knockout strain. The mutants, K132E, K132G, K132Q, K132R, and K132V were characterized in vivo for respiratory growth and in vitro for ubiquinone reduction, enzyme stability, and cytochrome b562 assembly. Only the K132R substitution, which conserves the positive charge of Lys-132, produces a wild-type enzyme. The remaining four mutants do not affect the ability of SDH to oxidize succinate in the presence of the artificial electron acceptor, phenazine methosulfate, but impair quinone reductase activity, enzyme stability, and heme insertion. Our results suggest that the presence of a positive charge on residue 132 in the C-terminus of Sdh4p is critical for establishing a stable conformation in the SDH hydrophobic domain that is compatible with ubiquinone reduction and cytochrome b562 assembly. In addition, our data suggest that heme does not play an essential role in quinone reduction. (+info)
Cloning and characterization of the gene encoding Pasteurella haemolytica FnrP, a regulator of the Escherichia coli silent hemolysin sheA.
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A Pasteurella haemolytica A1 gene was identified from a recombinant library clone that expressed hemolysis in host Escherichia coli cells. The gene, designated fnrP, had sequence identity to E. coli fnr, a global transcriptional regulator of genes required for conversion to anaerobic growth. FnrP complemented anaerobic deficiencies of a fnr-null mutant strain of E. coli and increased expression of the Fnr-dependent, anaerobic terminal reductase gene, frdA. FnrP was purified, identified by immunoblotting, and shown to be nonhemolytic. When FnrP was expressed in E. coli DeltasheA, a null mutant of the cryptic hemolysin SheA, the transformants were nonhemolytic, indicating that FnrP activates this silent hemolysin. (+info)