flavin-adenine-dinucleotide and fumaric-acid

A membrane protein complex, succinate dehydrogenase (SQR) from Escherichia coli has been purified and crystallised. This enzyme is composed of four subunits containing FAD, three iron-sulphur clusters and one haem b as prosthetic groups. The obtained crystals belong to the hexagonal space group P6(3) with the unit-cell dimensions of a=b=123.8 A and c=214.6 A. An asymmetric unit of the crystals contains one SQR monomer (M(r) 120 kDa). A data set is now available at 4.0 A resolution with 88.1% completeness and 0.106 R(merge). We have obtained a molecular replacement solution that shows sensible molecular packing, using the soluble domain of E. coli QFR (fumarate reductase) as a search model. The packing suggests that E. coli SQR is a crystallographic trimer rather than a dimer as observed for the E. coli QFR.

Topics: Crystallography; Electron Transport Complex II; Escherichia coli; Flavin-Adenine Dinucleotide; Fumarates; Heme; Intracellular Membranes; Iron-Sulfur Proteins; Membrane Proteins; Models, Molecular; Multienzyme Complexes; Oxidoreductases; Quinone Reductases; Succinate Dehydrogenase; Succinic Acid

The Saccharomyces cerevisiae succinate-ubiquinone reductase or succinate dehydrogenase (SDH) is a tetramer of non-equivalent subunits encoded by the SDH1, SDH2, SDH3, and SDH4 genes. In most organisms, SDH contains one or two endogenous b-type hemes. However, it is widely believed that the yeast SDH does not contain heme. In this report, we demonstrate the presence of a stoichiometric amount of cytochrome b562 in the yeast SDH. The cytochrome is detected as a peak present in fumarate-oxidized, dithionite-reduced mitochondria. The peak is centered at 562 nm and is present at a heme:covalent FAD molar ratio of 0.92+/-0.11. The cytochrome is not detectable in mitochondria isolated from SDH3 and SDH4 deletion strains. These observations strongly support our conclusion that cytochrome b562 is a component of the yeast SDH.

Topics: Animals; Cytochrome b Group; Dithionite; Electron Transport Complex II; Escherichia coli Proteins; Flavin-Adenine Dinucleotide; Fumarates; Fungal Proteins; Gene Deletion; Intracellular Membranes; Lactic Acid; Malonates; Mice; Mitochondria; Multienzyme Complexes; Oxidation-Reduction; Oxidoreductases; Saccharomyces cerevisiae; Spectrum Analysis; Succinate Dehydrogenase

The integral membrane protein fumarate reductase catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The homologous enzyme succinate dehydrogenase also plays a prominent role in cellular energetics as a member of the Krebs cycle and as complex II of the aerobic respiratory chain. Fumarate reductase consists of four subunits that contain a covalently linked flavin adenine dinucleotide, three different iron-sulfur clusters, and at least two quinones. The crystal structure of intact fumarate reductase has been solved at 3.3 angstrom resolution and demonstrates that the cofactors are arranged in a nearly linear manner from the membrane-bound quinone to the active site flavin. Although fumarate reductase is not associated with any proton-pumping function, the two quinones are positioned on opposite sides of the membrane in an arrangement similar to that of the Q-cycle organization observed for cytochrome bc1.

Topics: Aerobiosis; Anaerobiosis; Binding Sites; Cell Membrane; Crystallization; Crystallography, X-Ray; Electron Transport; Energy Metabolism; Escherichia coli; Flavin-Adenine Dinucleotide; Fumarates; Iron-Sulfur Proteins; Models, Molecular; Oxidation-Reduction; Oxygen Consumption; Protein Conformation; Protein Folding; Quinones; Succinate Dehydrogenase

Fumarate respiration is one of the most widespread types of anaerobic respiration. The soluble fumarate reductase of Shewanella putrefaciens MR-1 is a periplasmic tetraheme flavocytochrome c. The crystal structures of the enzyme were solved to 2.9 A for the uncomplexed form and to 2.8 A and 2.5 A for the fumarate and the succinate-bound protein, respectively. The structures reveal a flexible capping domain linked to the FAD-binding domain. A catalytic mechanism for fumarate reduction based on the structure of the complexed protein is proposed. The mechanism for the reverse reaction is a model for the homologous succinate dehydrogenase (complex II) of the respiratory chain. In flavocytochrome c fumarate reductase, all redox centers are in van der Waals contact with one another, thus providing an efficient conduit of electrons from the hemes via the FAD to fumarate.

Topics: Amino Acid Oxidoreductases; Amino Acid Sequence; Binding Sites; Catalytic Domain; Crystallization; Crystallography, X-Ray; Cytochrome c Group; Electrons; Escherichia coli; Escherichia coli Proteins; Flavin-Adenine Dinucleotide; Fumarates; Heme; Models, Molecular; Molecular Sequence Data; Oxidation-Reduction; Oxidoreductases; Protein Folding; Protein Structure, Secondary; Shewanella putrefaciens; Succinate Dehydrogenase; Succinic Acid

Succinate dehydrogenase of the bacterial or inner mitochondrial membrane catalyses the oxidation of succinate to fumarate and directs reducing equivalents into the electron-transport chain. The enzyme is also able to catalyse the reverse reaction, the reduction of fumarate to succinate. The enzyme is composed of four subunits. These subunits include a catalytic dimer composed of a flavoprotein subunit with a covalently bound FAD, and an iron-sulfur protein subunit with three different iron-sulfur centres, which is anchored to the membrane by two smaller integral membrane proteins. The FAD moiety is attached to the flavoprotein subunit by an 8 alpha-[N(3)-histidyl]FAD linkage at a conserved histidine residue, His90 of the Saccharomyces cerevisiae succinate dehydrogenase. By mutating His90 to a serine residue, we have constructed a flavoprotein subunit that is unable to covalently bind FAD. The mutant flavoprotein is targeted to mitochondria, translocated across the mitochondrial membranes, and is assembled with the other subunits where it binds FAD non-covalently. The resulting holoenzyme has no succinate-dehydrogenase activity but retains fumarate reductase activity. The covalent attachment of FAD is therefore necessary for succinate oxidation but is dispensable for both fumarate reduction and for the import and assembly of the flavoprotein subunit.

Topics: Binding Sites; Electron Spin Resonance Spectroscopy; Electron Transport; Electrophoresis, Polyacrylamide Gel; Flavin-Adenine Dinucleotide; Flavoproteins; Fumarates; Iron-Sulfur Proteins; Mitochondria; Mutagenesis, Site-Directed; Oxidation-Reduction; Saccharomyces cerevisiae; Succinate Dehydrogenase; Succinates; Succinic Acid

Fumarate reductase (Escherichia coli) can be immobilized in an extremely electroactive state at an electrode, with retention of native catalytic properties. The membrane-extrinsic FrdAB component adsorbs to monolayer coverage at edge-oriented pyrolytic graphite and catalyzes reduction of fumarate or oxidation of succinate, depending upon the electrode potential. In the absence of substrates, reversible redox transformations of centers in the enzyme are observed by cyclic voltammetry. The major component of the voltammograms is a pair of narrow reduction and oxidation signals corresponding to a pH-sensitive couple with formal reduction potential E degree' = -48 mV vs SHE at pH 7.0 (25 degrees C). This is assigned to two-electron reduction and oxidation of the active-site FAD. A redox couple with E degree' = -311 mV at pH 7 is assigned to center 2 ([4Fe-4S]2+/1+). Voltammograms for fumarate reduction at 25 degrees C, measured with a rotating-disk electrode, show high catalytic activity without the low-potential switch-off that is observed for the related enzyme succinate dehydrogenase. The catalytic electrochemistry is interpreted in terms of a basic model incorporating mass transport of substrate, interfacial electron transfer, and intrinsic kinetic properties of the enzyme, each of these becoming a rate-determining factor under certain conditions. Electrochemical reversibility is approached under conditions of low turnover rate, for example, as the supply of substrate to the active site is limited. In this situation, electrocatalytic half-wave potentials, E1/2, are similar for oxidation of bulk succinate and reduction of bulk fumarate and coincide closely with the E degree' value assigned to the FAD. At 25 degrees C and pH 7, the apparent KM for fumarate reduction is 0.16 mM, and kcat is 840 s-1. Accordingly the second-order rate constant, kcat/KM, is 5.3 x 10(6) M-1 s-1. Under the same conditions, oxidation of succinate is much slower. As the supply of fumarate to the enzyme is raised to increase turnover, the electrochemical reaction eventually becomes limited by the rate of electron transfer from the electrode. Under these conditions a second catalytic wave becomes evident, the E1/2 value of which corresponds to the reduction potential of the redox couple suggested to be center 2. This small boost to the catalytic current indicates that the low-potential [4Fe-4S] cluster can function as a second center for relaying electrons to the FAD.

Topics: Adsorption; Catalysis; Electrochemistry; Electrodes; Electron Transport; Enzymes, Immobilized; Flavin-Adenine Dinucleotide; Fumarates; Hydrogen-Ion Concentration; Kinetics; Oxidation-Reduction; Succinate Dehydrogenase; Succinates; Succinic Acid

Modification by covalent FAD attachment to a histidine residue via an 8 alpha-(N3-histidyl)-riboflavin linkage occurs in several flavoenzymes. Among them is 6-hydroxy-D-nicotine oxidase (6-HDNO) of Arthrobacter oxidans and the flavoprotein subunits of the fumarate reductase and succinate dehydrogenase complex of Escherichia coli and other bacterial and eukaryotic cells. We found that 6-HDNO holoenzyme formation from apo-6-HDNO, monitored by [14C]FAD incorporation and increase in enzyme activity, can be mediated not only by phosphoenolpyruvate [Nagursky, H., Bichler, V. and Brandsch, R. (1988) Eur. J. Biochem. 177, 319-325], but also by one of the glycolytic intermediates glyceraldehyde-3-P, glycerate-3-P, or the intermediate in glycerol utilization by bacteria, glycerol-3-P. Apoflavoprotein of fumarate reductase and succinate dehydrogenase was obtained in an E. coli riboflavin-requiring strain (E. coli RR28rf) overexpressing the frdABCD or the sdhCDAB operon from the recombinant plasmids pGS39 and pGS141, respectively. In extracts obtained from these cells, flavoprotein flavinylation, analyzed as covalent [14C]FAD incorporation into the apoflavoprotein polypeptide by polyacrylamide gel electrophoresis and fluorography, was stimulated severalfold by the citric acid cycle intermediates citrate, isocitrate, succinate and fumarate. Our results suggest that covalent modification and thus activation of these enzymes is dependent on specific metabolic intermediates which may act as allosteric effectors in the reaction.

Topics: Citrates; Citric Acid Cycle; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Escherichia coli; Flavin-Adenine Dinucleotide; Flavoproteins; Fumarates; Isocitrates; Plasmids; Pyruvates; Spectrometry, Fluorescence; Succinate Dehydrogenase; Succinates