BBA - Bioenergetics (v.1797, #12)

Quinone binding and catalysis by Fraser MacMillan; Carola Hunte (1841).

The two spatially distant quinone-binding sites of the ubihydroquinone: cytochrome c oxidoreductase (cyt bc 1) complex have been shown to influence one another in some fashion. This transmembrane communication alters cofactor and redox partner binding interactions and could potentially influence the timing or ‘concerted’ steps involved in the steady-state turnover of the homodimeric enzymes. Yet, despite several lines of evidence corroborating the coupling of the quinone binding active sites to one another, little to no testable hypothesis has been offered to explain how such a “signal” might be transmitted across the presumably rigid hydrophobic domain of the enzyme. Recently, it has been shown that this interquinone binding sites communication influences the steady-state position of the mobile [2Fe–2S] cluster containing iron sulfur protein (Sarewicz M., Dutka M., Froncisz W., Osyczka A. (2009) Biochemistry 48, 5708–5720) as mediated by at least one transmembrane helix of the b-type cyt containing subunit (Cooley, J. W., Lee, D. W., and Daldal, F. (2009) Biochemistry 48, 1988–1999). Here we provide an overview of the evidence supporting the structural coupling of these sites and provide a theoretical framework for how the redox state of a quinone at one cofactor binding site might influence the cofactor–, inhibitor–, and/or protein–protein interactions at the structurally distant opposing Q binding site.
Keywords: Antimycin A; Cytochrome bc 1; Complex III; Rhodobacter capsulatus; Photosynthesis and respiration; Energy transduction;

How does antimycin inhibit the bc 1 complex? A part-time twin by Stéphane Ransac; Jean-Pierre Mazat (1849-1857).
Using a stochastic simulation without any other hypotheses, we recently demonstrated the natural emergence of the modified Mitchell Q-cycle in the functioning of the bc 1 complex, with few short-circuits and a very low residence time of the reactive semiquinone species in the Qo site. However, this simple model fails to explain both the inhibition by antimycin of the bc 1 complex and the accompanying increase in ROS production. To obtain inhibition, we show that it is necessary to block the return of the electron from the reduced haem bL to Qo. With this added hypothesis we obtain a sigmoid inhibition curve due to the fact that when only one antimycin is bound per bc 1 dimer, the electron of the inhibited monomer systematically crosses the dimer interface from bL to bL to reduce a quinone or a semiquinone species in the other (free) Qi site. Because this step is not limiting, the activity is unchanged (compared to the activity of the free dimer). Interestingly, this bL–bL pathway is almost exclusively taken in this half-bound antimycin dimer. In the free dimer, the natural faster pathway is bL–bH on the same monomer. The addition of the assumption of half-of-the-sites reactivity to the previous hypothesis leads to a transient activation in the antimycin titration curve preceding a quasi-complete inhibition at antimycin saturation.
Keywords: Gillespie algorithm; bc 1 complex; Antimycin A; Stochastic modelling;

Cytochrome bc 1-complexes of animals and bacteria (hereafter bc 1), as well as related cytochrome b 6 f complexes of plants and cyanobacteria (hereafter bf) are dimeric quinol:cytochrome c/plastocyanin oxidoreductases capable of translocating protons across energy-converting membranes. The commonly accepted Q-cycle mechanism suggests that these enzymes oxidize two quinol molecules in their catalytic centers P to yield one quinol molecule in another catalytic center N. Earlier, based upon data on flash-induced redox changes of cytochromes b and c 1, voltage generation, and proton transfer in membrane vesicles of Rhodobacter capsulatus, we have put forward a scheme of an “activated Q-cycle” for the bc 1. The scheme suggests that the bc 1 dimers, being “activated” by injection of electrons from the membrane ubiquinol pool via centers N, steadily contain two electrons in their cytochrome b moieties under physiological conditions, most likely, as a bound semiquinone in center N of one monomer and a reduced high-potential heme b in the other monomer. Then the oxidation of each ubiquinol molecule in centers P of an activated bc 1 should result in a complete catalytic cycle leading to the formation of a ubiquinole molecule in the one of enzyme's centers N and to voltage generation. Here it is argued that a similar pre-loading by two electrons can explain the available data on flash-induced reactions in cytochrome b 6 f-complexes of green plants and cyanobacteria.
Keywords: Photosynthesis; Electron transfer; Proton transfer; Ubiquinol:cytochrome c oxidoreductase; Ubiquinone; Plastoquinone; Cytochrome b; Rhodobacter capsulatus; Rhodobacter sphaeroides;

The alternative complex III: A different architecture using known building modules by Patrícia N. Refojo; Filipa L. Sousa; Miguel Teixeira; Manuela M. Pereira (1869-1876).
Until recently cytochrome bc 1 complexes were the only enzymes known to be able to transfer electrons from reduced quinones to cytochrome c. However, a complex with the same activity and with a unique subunit composition was purified from the membranes of Rhodothermus marinus. This complex, named alternative complex III (ACIII) was then biochemical, spectroscopic and genetically characterized. Later it was observed that the presence of ACIII was not exclusive of R. marinus being the genes coding for ACIII widespread, at least in the Bacteria domain. In this work, a comprehensive description of the current knowledge on ACIII is presented. The relation of ACIII with members of the complex iron-sulfur molybdoenzyme family is investigated by analyzing all the available completely sequenced genomes. It is concluded that ACIII is a new complex composed by a novel combination of modules already identified in other respiratory complexes.
Keywords: Alternative complex III; bc 1complex; Molybdoenzymes; Complex I; Quinone;

The quinone-binding and catalytic site of complex II by Elena Maklashina; Gary Cecchini (1877-1882).
The complex II family of proteins includes succinate:quinone oxidoreductase (SQR) and quinol:fumarate oxidoreductase (QFR). In the facultative bacterium Escherichia coli both are expressed as part of the aerobic (SQR) and anaerobic (QFR) respiratory chains. SQR from E. coli is homologous to mitochondrial complex II and has proven to be an excellent model system for structure/function studies of the enzyme. Both SQR and QFR from E. coli are tetrameric membrane-bound enzymes that couple succinate/fumarate interconversion with quinone/quinol reduction/oxidation. Both enzymes are capable of binding either ubiquinone or menaquinone, however, they have adopted different quinone binding sites where catalytic reactions with quinones occur. A comparison of the structures of the quinone binding sites in SQR and QFR reveals how the enzymes have adapted in order to accommodate both benzo- and napthoquinones. A combination of structural, computational, and kinetic studies of members of the complex II family of enzymes has revealed that the catalytic quinone adopts different positions in the quinone-binding pocket. These data suggest that movement of the quinone within the quinone-binding pocket is essential for catalysis.
Keywords: Complex II; Quinone-binding; Succinate dehydrogenase; Fumarate reductase; Succinate-quinone oxidoreductase; Menaquinol-fumarate oxidoreductase;

Quinone binding and reduction by respiratory complex I by Maja A. Tocilescu; Volker Zickermann; Klaus Zwicker; Ulrich Brandt (1883-1890).
Complex I (NADH:ubiquinone oxidoreductase) has a central function in oxidative phosphorylation and hence for efficient ATP production in most prokaryotic and eukaryotic cells. This huge membrane protein complex transfers electrons from NADH to ubiquinone and couples this exergonic redox reaction to endergonic proton pumping across bioenergetic membranes. Although quinone reduction seems to be critical for energy conversion, this part of the reaction is least understood. Here we summarize and discuss experimental evidence indicating that complex I contains an extended ubiquinone binding pocket at the interface of the 49-kDa and PSST subunits. Close to iron–sulfur cluster N2, the proposed immediate electron donor for ubiquinone, a highly conserved tyrosine constitutes a critical element of the quinone reduction site. A possible quinone exchange path leads from cluster N2 to the N-terminal β-sheet of the 49-kDa subunit. We discuss the possible functions of a highly conserved HRGXE motif and a redox–Bohr group associated with cluster N2. Resistance patterns observed with a large number of point mutations suggest that all types of hydrophobic complex I inhibitors also act at the interface of the 49-kDa and the PSST subunit. Finally, current controversies regarding the number of ubiquinone binding sites and the position of the site of ubiquinone reduction are discussed.
Keywords: Mitochondria; Complex I; Ubiquinone; Inhibitor resistance; Mutagenesis; Yarrowia lipolytica;

In many energy transducing systems which couple electron and proton transport, for example, bacterial photosynthetic reaction center, cytochrome bc 1-complex (complex III) and E. coli quinol oxidase (cytochrome bo 3 complex), two protein-associated quinone molecules are known to work together. T. Ohnishi and her collaborators reported that two distinct semiquinone species also play important roles in NADH-ubiquinone oxidoreductase (complex I). They were called SQNf (fast relaxing semiquinone) and SQNs (slow relaxing semiquinone). It was proposed that QNf serves as a “direct” proton carrier in the semiquinone-gated proton pump (Ohnishi and Salerno, FEBS Letters 579 (2005) 4555), while QNs works as a converter between one-electron and two-electron transport processes. This communication presents a revised hypothesis in which QNf plays a role in a “direct” redox-driven proton pump, while QNs triggers an “indirect” conformation-driven proton pump. QNf and QNs together serve as (1e/2e) converter, for the transfer of reducing equivalent to the Q-pool.
Keywords: Quinone-induced conformation-driven proton pump; Quinone-gated proton pump; Direct proton pump; Indirect proton pump;

Spin labeling of the Escherichia coli NADH ubiquinone oxidoreductase (complex I) by Thomas Pohl; Thomas Spatzal; Müge Aksoyoglu; Erik Schleicher; Arpad Mihai Rostas; Helga Lay; Udo Glessner; Corinne Boudon; Petra Hellwig; Stefan Weber; Thorsten Friedrich (1894-1900).
The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy revealed the two-part structure of the complex with a peripheral arm involved in electron transfer and a membrane arm most likely involved in proton translocation. It was proposed that the quinone binding site is located at the joint of the two arms. Most likely, proton translocation in the membrane arm is enabled by the energy of the electron transfer reaction in the peripheral arm transmitted by conformational changes. For the detection of the conformational changes and the localization of the quinone binding site, we set up a combination of site-directed spin labeling and EPR spectroscopy. Cysteine residues were introduced to the surface of the Escherichia coli complex I. The spin label (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate (MTSL) was exclusively bound to the engineered positions. Neither the mutation nor the labeling had an effect on the NADH:decyl-ubiquinone oxidoreductase activity. The characteristic signals of the spin label were detected by EPR spectroscopy, which did not change by reducing the preparation with NADH. A decyl-ubiquinone derivative with the spin label covalently attached to the alkyl chain was synthesized in order to localize the quinone binding site. The distance between a MTSL labeled complex I variant and the bound quinone was determined by continuous-wave (cw) EPR allowing an inference on the location of the quinone binding site. The distances between the labeled quinone and other complex I variants will be determined in future experiments to receive further geometry information by triangulation.
Keywords: Complex I; NADH:ubiquinone oxidoreductase; Quinone binding site; Spin labeling; EPR spectroscopy; Escherichia coli;

Considerable disagreement still exists concerning the superoxide generation sites in the purified bovine heart NADH-ubiquinone oxidoreductase (complex I). Majority of investigators agree that superoxide is generated at the flavin site. Here we present a new hypothesis that the generation of superoxide reflects a dynamic balance between the flavosemiquinone (semiflavin or SF) and the semiquinone (SQ), like a “tug-of-war” through electrons. All preparations of bovine heart complex I, which have been isolated at Yoshikawa's laboratory, have one protein-bound endogenous ubiquinone per complex I (Shinzawa-Itoh et al., Biochemistry, 49 (2010) 487–492). Using these preparations, we measured (i) EPR signals of the SF, the SQ and iron–sulfur cluster N2 simultaneously with cryogenic EPR and (ii) superoxide production with both the room temperature spin-trapping technique and the partially acetylated cytochrome c method. Our experimental evidence was (1) without added decylubiquinone (DBQ), no catalytic oxidation of NADH occurs. The NADH addition produced mostly SF and it generated superoxide as reported by Kussmaul and Hirst (PNAS, 103 (2006) 7607–7612). (2) During catalytic electron transfer from NADH to DBQ, the superoxide generation site was mostly shifted to the SQ. (3) A quinone-pocket binding inhibitor (rotenone or piericidin A) inhibits the catalytic formation of the SQ, and it enhances the formation of SF and increases the overall superoxide generation. This suggests that if electron transfer was inhibited under pathological conditions, superoxide generation from the SF would be increased.
Keywords: NADH-ubiquinone oxidoreductase, complex I; Electron transport; Superoxide generation; Semiflavin radical; Iron–sulfur cluster N2; Semiquinone radical; Quinone-pocket inhibitors;

The electron transfer flavoprotein: Ubiquinone oxidoreductases by Nicholas J. Watmough; Frank E. Frerman (1910-1916).
Electron transfer flavoprotein:ubiqionone oxidoreductase (ETF-QO) is a component of the mitochondrial respiratory chain that together with electron transfer flavoprotein (ETF) forms a short pathway that transfers electrons from 11 different mitochondrial flavoprotein dehydrogenases to the ubiquinone pool. The X-ray structure of the pig liver enzyme has been solved in the presence and absence of a bound ubiquinone. This structure reveals ETF-QO to be a monotopic membrane protein with the cofactors, FAD and a [4Fe–4S]+ 1+2 cluster, organised to suggests that it is the flavin that serves as the immediate reductant of ubiquinone. ETF-QO is very highly conserved in evolution and the recombinant enzyme from the bacterium Rhodobacter sphaeroides has allowed the mutational analysis of a number of residues that the structure suggested are involved in modulating the reduction potential of the cofactors. These experiments, together with the spectroscopic measurement of the distances between the cofactors in solution have confirmed the intramolecular pathway of electron transfer from ETF to ubiquinone. This approach can be extended as the R. sphaeroides ETF-QO provides a template for investigating the mechanistic consequences of single amino acid substitutions of conserved residues that are associated with a mild and late onset variant of the metabolic disease multiple acyl-CoA dehydrogenase deficiency (MADD).►ETF:QO is an iron-sulfur flavoprotein linking central metabolism with the respiratory chain. ►Structural and mutagenesis studies suggest that FAD is the electron donor to ubiquinone (UQ). ►Mutations in the UQ binding domain can cause a mild lipid storage myopathy that is riboflavin responsive.
Keywords: Fatty acid oxidation; Flavin; Iron–sulfur cluster; Ubiquinone; Electron-transfer; Superoxide;

A study of cytochrome bo 3 in a tethered bilayer lipid membrane by Sophie A. Weiss; Richard J. Bushby; Stephen D. Evans; Lars J.C. Jeuken (1917-1923).
An assay has been developed in which the activity of an ubiquinol oxidase from Escherichia coli, cytochrome bo 3 (cbo 3), is determined as a function of the hydrophobic substrate ubiquinol-10 (UQ-10) in tethered bilayer lipid membranes (tBLMs). UQ-10 was added in situ, while the enzyme activity and the UQ-10 concentration in the membrane have been determined by cyclic voltammetry. Cbo 3 is inhibited by UQ-10 at concentrations above 5–10 pmol/cm2, while product inhibition is absent. Cyclic voltammetry has also been used to characterise the effects of three inhibitors; cyanide, inhibiting oxygen reduction; 2-n-Heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibiting the quinone oxidation and Zn(II), thought to block the proton channels required for oxygen reduction and proton pumping activity. The electrochemical behaviour of cbo 3 inhibited with HQNO and Zn(II) is almost identical, suggesting that Zn(II) ions inhibit the enzyme reduction by quinol, rather than oxygen reduction. This suggests that at Zn(II) concentration below 50 µM the proton release of cbo 3 is inhibited, but not the proton uptake required to reduce oxygen to water.
Keywords: Ubiquinol oxidase; Cytochrome c oxidase; Electrochemistry; Tethered bilayer lipid membrane; Enzyme mechanism;

The quinone-binding sites of the cytochrome bo 3 ubiquinol oxidase from Escherichia coli by Lai Lai Yap; Myat T. Lin; Hanlin Ouyang; Rimma I. Samoilova; Sergei A. Dikanov; Robert B. Gennis (1924-1932).
Cytochrome bo3 is the major respiratory oxidase located in the cytoplasmic membrane of Escherichia coli when grown under high oxygen tension. The enzyme catalyzes the 2-electron oxidation of ubiquinol-8 and the 4-electron reduction of dioxygen to water. When solubilized and isolated using dodecylmaltoside, the enzyme contains one equivalent of ubiquinone-8, bound at a high affinity site (QH). The quinone bound at the QH site can form a stable semiquinone, and the amino acid residues which hydrogen bond to the semiquinone have been identified. In the current work, it is shown that the tightly bound ubiquinone-8 at the QH site is not displaced by ubiquinol-1 even during enzyme turnover. Furthermore, the presence of high affinity inhibitors, HQNO and aurachin C1–10, does not displace ubiquinone-8 from the QH site. The data clearly support the existence of a second binding site for ubiquinone, the QL site, which can rapidly exchange with the substrate pool. HQNO is shown to bind to a single site on the enzyme and to prevent formation of the stable ubisemiquinone, though without displacing the bound quinone. Inhibition of the steady state kinetics of the enzyme indicates that aurachin C1–10 may compete for binding with quinol at the QL site while, at the same time, preventing formation of the ubisemiquinone at the QH site. It is suggested that the two quinone binding sites may be adjacent to each other or partially overlap.
Keywords: Ubiquinone; Oxidase; E. coli; Respiration; EPR;

The alternative oxidase (AOX) is a non-protonmotive ubiquinol oxidase that is found in all plants, some fungi, green algae, bacteria and pathogenic protozoa. The lack of AOX in the mammalian host renders this protein an important potential therapeutic target in the treatment of pathogenic protozoan infections. Bioinformatic searches revealed that, within a putative ubiquinol-binding crevice in AOX, Gln242, Asn247, Tyr253, Ser256, His261 and Arg262 were highly conserved. To confirm that these amino-acid residues are important for ubiquinol-binding and hence activity substitution mutations were generated and characterised. Assessment of AOX activity in isolated Schizosaccharomyces pombe mitochondria revealed that mutation of either Gln242, Ser256, His261 and Arg262 resulted in >90% inhibition of antimycin A-insensitive respiration suggesting that hydroxyl, guanidino, imidazole groups, polar and charged residues in addition to the size of the amino-acid chain are important for ubiquinone-binding. Substitution of Asn247 with glutamine or Tyr253 with phenylalanine had little effect upon the respiratory rate indicating that these residues are not critical for AOX activity. However replacement of Tyr253 by alanine resulted in a 72% loss of activity suggesting that the benzoquinone group and not hydroxyl group is important for quinol binding. These results provide important new insights into the ubiquinol-binding site of the alternative oxidase, the identity of which maybe important for future rational drug design.
Keywords: Alternative oxidase; Ubiquinone-binding; Structure–function relations; Site-directed mutagenesis; Schizosaccharomyces pombe mitochondria;