The Quaternary Structure of the Saccharomyces cerevisiae Succinate Dehydrogenase

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245

The transfer of electrons and protons between membranebound respiratory complexes is facilitated by lipid-soluble redox-active quinone molecules (Q). This work presents a structural analysis of the quinone-binding site (Q-site) identified in succinate:ubiquinone oxidoreductase (SQR) from Escherichia coli. SQR, often referred to as Complex II or succinate dehydrogenase, is a functional member of the Krebs cycle and the aerobic respiratory chain and couples the oxidation of succinate to fumarate with the reduction of quinone to quinol (QH 2 ). The interaction between ubiquinone and the Q-site of the protein appears to be mediated solely by hydrogen bonding between the O1 carbonyl group of the quinone and the side chain of a conserved tyrosine residue. In this work, SQR was co-crystallized with the ubiquinone binding-site inhibitor Atpenin A5 (AA5) to confirm the binding position of the inhibitor and reveal additional structural details of the Q-site. The electron density for AA5 was located within the same hydrophobic pocket as ubiquinone at, however, a different position within the pocket. AA5 was bound deeper into the site prompting further assessment using proteinligand docking experiments in silico. The initial interpretation of the Q-site was re-evaluated in the light of the new SQR-AA5 structure and protein-ligand docking data. Two binding positions, the Q 1 -site and Q 2 -site, are proposed for the E. coli SQR quinone-binding site to explain these data. At the Q 2 -site, the side chains of a serine and histidine residue are suitably positioned to provide hydrogen bonding partners to the O4 carbonyl and methoxy groups of ubiquinone, respectively. This allows us to propose a mechanism for the reduction of ubiquinone during the catalytic turnover of the enzyme.The Complex II family is comprised of two homologous integral membrane proteins (1-4). Succinate:quinone oxidoreductase (SQR), 4 or succinate dehydrogenase (SDH), is a functional member of the Krebs cycle and the aerobic respiratory chain coupling the oxidation of succinate to fumarate with the reduction of quinone (Q) to quinol (QH 2 ). Quinol:fumarate oxidoreductase (QFR), or fumarate reductase, catalyzes the reverse reaction to SQR during anaerobic respiration. Both enzymes have a similar subunit and cofactor composition (1-4). The hydrophilic subunits, composed of a flavoprotein (SdhA) and an ironsulfur protein (SdhB), have a high degree of sequence homology across species. The SdhAB catalytic subunits are anchored to the membrane by the hydrophobic SdhCD subunits, which in the case of mammalian and Escherichia coli Complex II form a cytochrome b that contains one heme b. The two-electron oxidation of succinate at the substrate binding site in the SdhA subunit is coupled to the two-electron reduction of quinone in the membrane domain of Complex II. Electrons are sequentially transferred to ubiquinone (in the case of mammalian and E. coli Quinones can undergo 2e Ϫ /2H ϩ oxidation-reduction reactions, an essential feature of respiration, allowing the t...

Section: Discussionmentioning

confidence: 99%

Structural and Computational Analysis of the Quinone-binding Site of Complex II (Succinate-Ubiquinone Oxidoreductase)

Horsefield

Yankovskaya

Sexton

et al. 2006

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245

“…The plates were incubated at 30°C and photographed after overnight incubation. (6,46,59). In E. coli, the equivalent histidine residue is believed to play an essential role in coordinating ubiquinone prior to electron transfer and protonation (60).…”

Section: Discussionmentioning

confidence: 99%

“…SDH is an iron-sulfur flavoprotein that resides in the mitochondrial inner membrane and functions to oxidize succinate to fumarate and reduce ubiquinone (Q) to ubiquinol (1)(2)(3). The yeast SDH, like its mammalian counterpart, consists of four nuclear encoded subunits (Sdh1p-Sdh4p in yeast and SDHA-SDHD in mammals) (2), a covalently attached flavin adenine dinucleotide (FAD) (4), three iron-sulfur centers, a b-type heme, and two ubiquinone-binding sites referred to as the proximal and distal sites (Q P and Q D ), respectively (5,6). Sdh1p and Sdh2p form the catalytic domain, the site of succinate oxidation.…”

mentioning

confidence: 99%

Ubiquinone-binding Site Mutations in the Saccharomyces cerevisiae Succinate Dehydrogenase Generate Superoxide and Lead to the Accumulation of Succinate

Szeto¹,

Reinke

Sykes³

et al. 2007

Self Cite

The mitochondrial succinate dehydrogenase (SDH) is an essential component of the electron transport chain and of the tricarboxylic acid cycle. Also known as complex II, this tetrameric enzyme catalyzes the oxidation of succinate to fumarate and reduces ubiquinone. Mutations in the human SDHB, SDHC, and SDHD genes are tumorigenic, leading to the development of several types of tumors, including paraganglioma and pheochromocytoma. The mechanisms linking SDH mutations to oncogenesis are still unclear. In this work, we used the yeast SDH to investigate the molecular and catalytic effects of tumorigenic or related mutations. We mutated Arg 47 of the Sdh3p subunit to Cys, Glu, and Lys and Asp 88 of the Sdh4p subunit to Asn, Glu, and Lys. Both Arg 47 and Asp 88 are conserved residues, and Arg 47 is a known site of cancer causing mutations in humans. All of the mutants examined have reduced ubiquinone reductase activities. The SDH3 R47K, SDH4 D88E, and SDH4 D88N mutants are sensitive to hyperoxia and paraquat and have elevated rates of superoxide production in vitro and in vivo. We also observed the accumulation and secretion of succinate. Succinate can inhibit prolyl hydroxylase enzymes, which initiate a proliferative response through the activation of hypoxia-inducible factor 1␣. We suggest that SDH mutations can promote tumor formation by contributing to both reactive oxygen species production and to a proliferative response normally induced by hypoxia via the accumulation of succinate.The mitochondrial succinate dehydrogenase (SDH, also known as complex II or succinate:ubiquinone oxidoreductase) 4 is a critical enzyme linking the mitochondrial respiratory chain and the tricarboxylic acid cycle. SDH is an iron-sulfur flavoprotein that resides in the mitochondrial inner membrane and functions to oxidize succinate to fumarate and reduce ubiquinone (Q) to ubiquinol (1-3). The yeast SDH, like its mammalian counterpart, consists of four nuclear encoded subunits (Sdh1p-Sdh4p in yeast and SDHA-SDHD in mammals) (2), a covalently attached flavin adenine dinucleotide (FAD) (4), three iron-sulfur centers, a b-type heme, and two ubiquinone-binding sites referred to as the proximal and distal sites (Q P and Q D ), respectively (5, 6). Sdh1p and Sdh2p form the catalytic domain, the site of succinate oxidation. Electrons flow through the catalytic domain to the membrane domain, consisting of Sdh3p and Sdh4p, where quinone reduction occurs.Mutations in the human SDHA gene can result in Leigh syndrome, an infantile-onset progressive neurodegenerative disease (7,8). Mutations in the human SDHB, SDHC, and SDHD genes lead to inheritable forms of cancer such as pheochromocytomas (catechol-secreting tumors commonly occurring in the adrenal medulla), paragangliomas (benign vascularized tumors in the head and neck), and renal cell carcinoma (9 -11). More recently, it was reported that loss of the SDHD gene may contribute to gastric and colon cancers (12). These observations highlight the importance of SDH genes as mitochondrial tumor suppre...

“…Complex II (succinate-ubiquinone oxidoreductase) is the only enzyme that participates in both the citric acid cycle and the electron transport chain (12). Complex II is composed of a succinate dehydrogenase, which is a citric acid cycle enzyme, and a membrane-anchoring protein fraction, which houses ubiquinone.…”

mentioning

confidence: 99%

Cross-talk between Mitochondrial Malate Dehydrogenase and the Cytochrome bc1 Complex

Wang

2010

The interactions between the mitochondrial cytochrome bc 1 complex and matrix-soluble proteins were studied by a precipitation pulldown technique. Purified, detergent-dispersed bc 1 complex was incubated with mitochondrial matrix proteins followed by dialysis in the absence of detergent. The interacting protein(s) was co-precipitated with bc 1 complex upon centrifugation. One of the matrix proteins pulled down by bc 1 complex was identified as mitochondrial malate dehydrogenase (MDH) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and confirmed by Western blotting with anti-MDH antibody. Using a cross-linking technique, subunits I, II (core I and II), and V of the bc 1 complex were identified as the interacting sites for MDH. Incubating purified MDH with the detergent dispersed bc 1 complex results in an increase of the activities of both the bc 1 complex and MDH. The effect of the bc 1 complex on the activities of MDH is unidirectional (oxaloacetate 3 malate). These results suggest that the novel cross-talk between citric acid cycle enzymes and electron transfer chain complexes might play a regulatory role in mitochondrial bioenergetics.The mitochondrial cytochrome bc 1 complex catalyzes electron transfer from ubiquinol to cytochrome c with the concomitant translocation of protons across the membrane to generate a proton gradient and membrane potential for ATP synthesis (1). Three-dimensional structures of mitochondrial bc 1 complexes from various sources have become available in recent years (2-11). All mitochondrial cytochrome bc 1 complexes contain three essential subunits that house four redox prosthetic groups and seven to eight non-redox prosthetic groups containing supernumerary subunits. The three essential subunits are: the cytochrome b subunit, housing two b-type hemes (b H and b L ); the cytochrome c 1 subunit, housing one c-type heme (c 1 ); the Rieske iron-sulfur protein (ISP), 2 housing a high potential [2Fe-2S] cluster.Although the structural and functional studies of the mitochondrial bc 1 complex have been intensive, few studies of the interactions between the bc 1 complex and the soluble matrix enzymes have been available. Complex II (succinate-ubiquinone oxidoreductase) is the only enzyme that participates in both the citric acid cycle and the electron transport chain (12). Complex II is composed of a succinate dehydrogenase, which is a citric acid cycle enzyme, and a membrane-anchoring protein fraction, which houses ubiquinone. Complex II catalyzes the oxidation of succinate to fumarate and the reduction of ubiquinone to ubiquinol. Other enzymes of citric acid cycle are known to couple to the electron transfer chain through their product NADH at complex I (13). Structurally the mitochondrial bc 1 complex can be divided into three regions: matrix, transmembrane, and the intermembrane space regions. The matrix region contains subunits I (core I), II (core II), VI, IX, and the N-terminal part of subunits V (ISP) and VII (2, 4). Although this region contains the m...