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...

Enzymes in the mitochondrial respiratory chain are involved in various physiological events in addition to their essential role in the production of ATP by oxidative phosphorylation. The use of specific and potent inhibitors of complex I (NADH-ubiquinone reductase) and complex III (ubiquinol-cytochrome c reductase), such as rotenone and antimycin, respectively, has allowed determination of the role of these enzymes in physiological processes. However, unlike complexes I, III, and IV (cytochrome c oxidase), there are few potent and specific inhibitors of complex II (succinate-ubiquinone reductase) that have been described. In this article, we report that atpenins potently and specifically inhibit the succinate-ubiquinone reductase activity of mitochondrial complex II. Therefore, atpenins may be useful tools for clarifying the biochemical and structural properties of complex II, as well as for determining its physiological roles in mammalian tissues. T he use of specific and potent inhibitors of respiration has enabled the investigation of how the respiratory enzymes function in physiological processes. However, unlike other enzyme complexes in the respiratory chain, there has been a lack of potent and specific inhibitors of complex II [succinateubiquinone reductase (SQR)]. Although carboxin (5,6-dihydro-2-methyl-N-phenyl-1,4-oxathiin-3-carboxamide), TTFA [4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione], and HQNO (2-heptyl-4-hydroxyquinoline N-oxide) have long been known as complex II inhibitors and have been used extensively to elucidate the structure-function relationships of complex II, rather higher concentration is required for the inhibition (1). This result has hampered the study of the structure-function relationship of the complex II enzyme, as well as its roles in physiological processes.Complex II catalyzes the oxidation of succinate in the inner membrane of mitochondria and in the cytoplasmic membrane of bacteria (1-3). In addition to its function as a dehydrogenase in the respiratory system, complex II plays an important role in the tricarboxylic acid cycle. Mitochondrial complex II is an integral membrane protein consisting of four subunits (Fig. 1). The largest subunit is the 70-kDa, FAD-containing flavoprotein subunit (Fp). The dehydrogenase catalytic portion of complex II is formed by Fp and an Ϸ30-kDa iron-sulfur protein subunit (Ip) containing three different types of iron-sulfur clusters. The small hydrophobic subunits, SDHC or CybL (Ϸ15 kDa) and SDHD or CybS (Ϸ13 kDa), anchor the catalytic portion to the membrane and are also required for electron transfer to quinones. In contrast to mitochondrial complex IIs, some bacterial complex IIs contain only one larger hydrophobic polypeptide as a membrane anchor (see ref. 4 for reviews).In addition to its essential role in energy production, various recent findings suggest that mutant variants of complex II are involved in causing diverse physiological disorders. For instance, a mutation in the CybL subunit in Caenorhabditis elegans (mev-1 mutant) resu...

Sea anemones are a rich source of two classes of peptide toxins, sodium channel toxins and potassium channel toxins, which have been or will be useful tools for studying the structure and function of specific ion channels. Most of the known sodium channel toxins delay channel inactivation by binding to the receptor site 3 and most of the known potassium channel toxins selectively inhibit Kv1 channels. The following peptide toxins are functionally unique among the known sodium or potassium channel toxins: APETx2, which inhibits acid-sensing ion channels in sensory neurons; BDS-I and II, which show selectivity for Kv3.4 channels and APETx1, which inhibits human ether-a-go-go-related gene potassium channels. In addition, structurally novel peptide toxins, such as an epidermal growth factor (EGF)-like toxin (gigantoxin I), have also been isolated from some sea anemones although their functions remain to be clarified.

Acetylcholinesterase is a critical enzyme that regulates neurotransmission by degrading the neurotransmitter acetylcholine in synapses of the nervous system. It is an important target for both therapeutic drugs that treat Alzheimer's disease and chemical warfare agents that cripple the nervous system and cause death through paralysis. The enzyme has both catalytic and peripheral sites to which inhibitors may bind. Structures of recombinant human acetylcholinesterase in complex with the natural product inhibitors dihydrotanshinone I and territrem B reveal dihydrotanshinone I binding that is specific to only the peripheral site and territrem B binding that spans both sites and distorts the protein backbone in the peripheral site. These inhibitors may function as important molecular templates for therapeutics used for treatment of disease and protection against nerve agents.