Kinetic studies of succinate dehydrogenase by electron paramagnetic resonance spectroscopy (original) (raw)
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Mechanism of the reductive activation of succinate dehydrogenase
Journal of Biological Chemistry, 1975
When succinate dehydrogenase contains oxalacetate in firmly bound form, activity cannot be expressed without special pretreatment ("activation") of the enzyme. Reduction of 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.
On the need for regulation of succinate dehydrogenase
FEBS Letters, 1971
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 . This type of activation has been observed in soluble and particulate preparations, in inner membrane 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 endogenous ubiquinone, so that the reduced quinone appears to be the immediate activating agent. This conclusion was verified by two experimental approaches:
Analytical Biochemistry, 2006
The Fe(II)/2-oxoglutarate-dependent dioxygenases are a catalytically diverse family of nonheme iron enzymes that oxidize their primary substrates while decomposing the 2-oxoglutarate cosubstrate to form succinate and CO 2 . We report a generic assay for these enzymes that uses succinyl-coenzyme A synthetase, pyruvate kinase, and lactate dehydrogenase to couple the formation of the product succinate to the conversion of reduced nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide. We demonstrate the utility of this new method by measuring the kinetic parameters of two bacterial Fe(II)/2-oxoglutarate-dependent dioxygenases. SigniWcantly, this method can be used to investigate both the productive turnover reactions and the nonproductive "uncoupled" decarboxylation reactions of this enzyme family, as demonstrated by using wild-type and variant forms of 2-oxoglutarate-dependent taurine dioxygenase. This assay is amenable to miniaturization and easily adapted to a format suitable for high-throughput screening; thus, it will be a valuable tool to study Fe(II)/2-oxoglutarate-dependent dioxygenases.
Journal of Magnetic Resonance, Series B, 1996
Burkholderia cepacia AC1100 (formerly classified as a sulfur clusters, providing direct data about their type, oxidation state, and nearest surroundings (8, 9). Among the dis-Pseudomonas) degrades 2,4,5-trichlorophenoxyacetate (2,4,5-T), an herbicide used in the Vietnam War for defolia-tinguishing characteristics of [2Fe-2S] clusters in the reduced state are the principal values of the g tensor. In all tion. A multiple component oxygenase is responsible for the conversion of 2,4,5-T to 2,4,5-trichlorophenol (2,4,5-TCP) reported cases (10-14), the reduced Rieske clusters exhibit a characteristic g tensor (g 1 á 2.01-2.02, g 2 á 1.90-1.92, (1, 2). The genes encoding the oxygenase component have been cloned and sequenced (1). The oxygenase component g 3 á 1.76-1.8) with larger anisotropy and considerably lower g av á 1.91 than those of the plant-ferredoxin-type has a native molecular weight of 140,000, and it is composed of two 49 kDa a subunits and two 24 kDa b subunits. This cluster (g 1 á 2.04-2.05, g 2 á 1.96, g 3 á 1.87-1.88) or adrenal ferredoxin (g Å 2.025, g ⊥ Å 1.932) both with g av component is red and its spectrum in the visible region has maxima at 430 and 560 nm (shoulder), whereas upon reducá 1.96 (15, 16). Conclusive determination of the ligands in the Rieske-tion it has maxima at 420 (shoulder) and 530 nm. Each ab heterodimer contains three irons and two labile sulfides. type cluster was obtained by application of the solid-state, high-resolution EPR techniques of electron-nuclear double-These properties suggest the presence of a [2Fe-2S] cluster. At present two types of [2Fe-2S] clusters have been resonance (ENDOR) and electron-spin-echo envelope-modulation (ESEEM) spectroscopies. X-and Q-band ENDOR characterized. One is the widely distributed plant-ferredoxintype cluster where each iron atom is coordinated by the studies of the reduced Rieske-type cluster in phthalate dioxygenase from B. cepacia (17) and in ubiquinol-cytochrome sulfur atoms of two cysteine residues as shown in the available X-ray structures (3, 4). The other type of [2Fe-2S] c 2 oxidoreductase from Rhodobactor capsulatus (18) established that two histidine ligands are coordinated to one iron cluster with considerably higher redox potential was first described in the so-called Rieske protein isolated from the in place of two cysteines as in the plant ferredoxins and adrenodoxin. The ESEEM experiments performed on the ubiquinol-cytochrome c oxidoreductase of bovine heart mitochondria (5, 6). Similar proteins were found in other types reduced Rieske clusters in the cytochrome b 6 f complex of spinach chloroplast; in cytochrome bc 1 complexes of Rho-of membrane electron-transfer chains and aromatic dioxygenases (7). dospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria (19); and in Complex III of bovine Analysis of the deduced amino acid sequences of the a and b subunits in 2,4,5-T monooxygenase shows homology heart mitochondrial membranes (20) all show coordination with two histidine ligands. The reported values of hyperfine to the a and b subunits of other multicomponent aromatic dioxygenases. The a subunit has two conserved cysteine-constants of the two coordinated 14 N histidine nitrogens vary between 3.6 and 4.5 MHz and 4.6 and 5.5 MHz. In contrast, histidine pairs at the identical positions (Cys-X-His-17 amino acids-Cys-X-X-His) as in the other a subunits. This ENDOR and ESEEM investigations of plant-ferredoxin-type clusters show only interaction with nitrogens of the peptide motif is also repeated in Rieske iron-sulfur proteins. Electron paramagnetic resonance spectroscopy plays a backbone chain, with maximum hyperfine coupling Ç1 MHz (21-26). particularly important role in the characterization of iron-289