Inhibitory Analogs of Ubiquinol Act Anti-cooperatively on the Yeast Cytochrome bc1 Complex. EVIDENCE FOR AN ALTERNATING, HALF-OF-THE-SITES MECHANISM OF UBIQUINOL OXIDATION (original) (raw)
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Anti-cooperative Oxidation of Ubiquinol by the Yeast Cytochrome bc1 Complex
Journal of Biological Chemistry, 2004
We have investigated the interaction between monomers of the dimeric yeast cytochrome bc 1 complex by analyzing the pre-steady and steady state activities of the isolated enzyme in the presence of antimycin under conditions that allow the first turnover of ubiquinol oxidation to be observable in cytochrome c 1 reduction. At pH 8.8, where the redox potential of the iron-sulfur protein is ϳ200 mV and in a bc 1 complex with a mutated iron-sulfur protein of equally low redox potential, the amount of cytochrome c 1 reduced by several equivalents of decyl-ubiquinol in the presence of antimycin corresponded to only half of that present in the bc 1 complex. Similar experiments in the presence of several equivalents of cytochrome c also showed only half of the bc 1 complex participating in quinol oxidation. The extent of cytochrome b reduced corresponded to two b H hemes undergoing reduction through one center P per dimer, indicating electron transfer between the two cytochrome b subunits. Antimycin stimulated the ubiquinolcytochrome c reductase activity of the bc 1 complex at low inhibitor/enzyme ratios. This stimulation could only be fitted to a model in which half of the bc 1 dimer is inactive when both center N sites are free, becoming active upon binding of one center N inhibitor molecule per dimer, and there is electron transfer between the cytochrome b subunits of the dimer. These results are consistent with an alternating half-of-the-sites mechanism of ubiquinol oxidation in the bc 1 complex dimer.
Biochemistry, 1999
Native structures of ubihydroquinone:cytochrome c oxidoreductase (bc 1 complex) from different sources, and structures with inhibitors in place, show a 16-22 Å displacement of the [2Fe-2S] cluster and the position of the C-terminal extrinsic domain of the iron sulfur protein. None of the structures shows a static configuration that would allow catalysis of all partial reactions of quinol oxidation. We have suggested that the different conformations reflect a movement of the subunit necessary for catalysis. The displacement from an interface with cytochrome c 1 in native crystals to an interface with cytochrome b is induced by stigmatellin or 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and involves ligand formation between His-161 of the [2Fe-2S] binding cluster and the inhibitor. The movement is a rotational displacement, so that the same conserved docking surface on the iron sulfur protein interacts with cytochrome c 1 and with cytochrome b. The mobile extrinsic domain retains essentially the same tertiary structure, and the anchoring N-terminal tail remains in the same position. The movement occurs through an extension of a helical segment in the short linking span. We report details of the protein structure for the two main configurations in the chicken heart mitochondrial complex and discuss insights into mechanism provided by the structures and by mutant strains in which the docking at the cytochrome b interface is impaired. The movement of the iron sulfur protein represents a novel mechanism of electron transfer, in which a tethered mobile head allows electron transfer through a distance without the entropic loss from free diffusion. † We acknowledge with gratitude the support for this research provided by NIH Grants GM 35438 (to A.R.C.) and DK 44842 (to E.paramagnetic resonance; [2Fe-2S], iron sulfur cluster of the Rieske-type iron sulfur protein; ISP, Rieske-type iron sulfur protein; ISP ox and ISP red , oxidized and reduced states of the iron sulfur protein; ISPB or ISPC, iron sulfur protein with the mobile extrinsic domain docked at cytochrome b or cytochrome c1 interface, respectively; LH1, light-harvesting complex 1 of bacterial photosynthesis; MOA-, -methoxyacrylate, or similar group, acting as the pharmacophore of a class of inhibitors acting at the Qo site; QH2, ubihydroquinone or quinol; Q, ubiquinone or quinone; Qi site, quinone reducing site; Qo site, quinol oxidizing site; Qos and Qow, postulated strongly and weakly binding quinone molecules bound to the quinol oxidizing site; -PEWY-, highly conserved span with this sequence in single-letter amino acid code; UHDBT, 5-n-undecyl-6-hydroxy-4,7dioxobenzothiazole.
Structure of the yeast cytochrome bc1 complex with a hydroxyquinone anion Qo site inhibitor bound
Journal of Biological …, 2003
Bifurcated electron transfer during ubiquinol oxidation is the key reaction of cytochrome bc 1 complex catalysis. Binding of the competitive inhibitor 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole to the Q o site of the cytochrome bc 1 complex from Saccharomyces cerevisiae was analyzed by x-ray crystallography. This alkylhydroxydioxobenzothiazole is bound in its ionized form as evident from the crystal structure and confirmed by spectroscopic analysis, consistent with a measured pK a ؍ 6.1 of the hydroxy group in detergent micelles. Stabilizing forces for the hydroxyquinone anion inhibitor include a polarized hydrogen bond to the iron-sulfur cluster ligand His 181 and on-edge interactions via weak hydrogen bonds with cytochrome b residue Tyr 279. The hydroxy group of the latter contributes to stabilization of the Rieske protein in the b-position by donating a hydrogen bond. The reported pH dependence of inhibition with lower efficacy at alkaline pH is attributed to the protonation state of His 181 with a pK a of 7.5. Glu 272 , a proposed primary ligand and proton acceptor of ubiquinol, is not bound to the carbonyl group of the hydroxydioxobenzothiazole ring but is rotated out of the binding pocket toward the heme b L propionate A, to which it is hydrogen-bonded via a single water molecule. The observed hydrogen bonding pattern provides experimental evidence for the previously proposed proton exit pathway involving the heme propionate and a chain of water molecules. Binding of the alkyl-6-hydroxy-4,7-dioxobenzothiazole is discussed as resembling an intermediate step of ubiquinol oxidation, supporting a single occupancy model at the Q o site.
Three Molecules of Ubiquinone Bind Specifically to Mitochondrial Cytochrome bc1 Complex
Journal of Biological Chemistry, 2001
Bifurcated electron flow to high potential "Rieske" iron-sulfur cluster and low potential heme b L is crucial for respiratory energy conservation by the cytochrome bc 1 complex. The chemistry of ubiquinol oxidation has to ensure the thermodynamically unfavorable electron transfer to heme b L. To resolve a central controversy about the number of ubiquinol molecules involved in this reaction, we used high resolution magic-angle-spinning nuclear magnetic resonance experiments to show that two out of three n-decyl-ubiquinones bind at the ubiquinol oxidation center of the complex. This substantiates a proposed mechanism in which a charge transfer between a ubiquinol/ubiquinone pair explains the bifurcation of electron flow.
Probing the Ubiquinol-Binding Site in Cytochrome bd by Site-Directed Mutagenesis †
Biochemistry, 2006
To probe the structure of the quinol oxidation site in loop VI/VII of the Escherichia coli cytochrome bd, we substituted three conserved residues (Gln249, Lys252, and Glu257) in the N-terminal region and three glutamates (Glu278, Glu279, and Glu280) in the first internal repeat. We found that substitutions of Glu257 by Ala or Gln, and Glu279 and Glu280 by Gln, severely reduced the oxidase activity and the expression level of cytochrome bd. In contrast, Lys252 mutations reduced only the oxidase activity. Blue shifts in the 440 and 630 nm peaks of the reduced Lys252 mutants and in the 561 nm peak of the reduced Glu257 mutants indicate the proximity of Lys252 to the heme b 595 -d binuclear center and Glu257 to heme b 558 , respectively. Perturbations of reduced heme b 558 upon binding of aurachin D support structural changes in the quinol-binding site of the mutants. Substitutions of Lys252 and Glu257 caused large changes in kinetic parameters for the ubiquinol-1 oxidation. These results indicate that Lys252 and Glu257 in the N-terminal region of the Q-loop are involved in the quinol oxidation by bd-type terminal oxidase.
Identification of ubiquinol binding motifs at the Qo-site of the cytochrome bc1 complex
The journal of physical chemistry. B, 2015
Enzymes of the bc1 complex family power the biosphere through their central role in respiration and photosynthesis. These enzymes couple the oxidation of quinol molecules by cytochrome c to the transfer of protons across the membrane, to generate a proton-motive force that drives ATP synthesis. Key for the function of the bc1 complex is the initial redox process that involves a bifurcated electron transfer in which the two electrons from a quinol substrate are passed to different electron acceptors in the bc1 complex. The electron transfer is coupled to proton transfer. The overall mechanism of quinol oxidation by the bc1 complex is well enough characterized to allow exploration at the atomistic level, but details are still highly controversial. The controversy stems from the uncertain binding motifs of quinol at the so-called Qo active site of the bc1 complex. Here we employ a combination of classical all atom molecular dynamics and quantum chemical calculations to reveal the bindi...
A New Ubiquinone Metabolite and Its Activity at the Mitochondrial bc 1 Complex
Chemical Research in Toxicology, 2007
Ubichromanol, a reductive cyclization product of ubiquinone, acts as radical scavenging antioxidant and is similarly effective as R-tocopherol. However, nothing is known so far on the two-electron oxidation product of this antioxidant and its bioactivity. This study demonstrates that ubichromanol yields a ubiquinone-like compound with a hydroxyl-substituted side chain (UQOH) on oxidation. HPLC/MS and HPLC/ECD measurements revealed its natural presence in bovine liver mitochondria. The bioactivity of this formerly unknown compound as substrate for mitochondrial complex III was tested by measurements of the quinol:cytochrome c oxidoreductase activity in bovine submitochondrial particles and isolated mitochondrial bc 1 complex. Consistently in both model systems, reduced UQOH exhibited substrate efficiencies below that of native ubiquinone but a significantly higher efficiency than R-tocopheryl quinone. Model calculations revealed that on binding of reduced UQOH to the bc 1 complex the polar hydroxyl group was located close to hydrophobic amino acid residues. This fact could in part explain the lower efficiency of reduced UQOH in comparison to ubiquinone as a substrate for the mitochondrial bc 1 complex. Therefore, the hydroxylation of the aliphatic or isoprenoid side chains of bioquinones, which is typical for quinoid oxidation products of chromanols, such as R-tocopherol and ubichromanol, disturbs substrate binding at the mitochondrial electron-transfer complexes, which usually interact with ubiquinone.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1986
An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-l,4-benzoquinone, was used to study the ubiquinone-protein interaction and to identify the ubiquinone-binding proteins in yeast mitochondrial ubiquinone-cytochrome c reductase. The phospholipids and Q6 in purified reductase were removed by repeated ammonium sulfate precipitation in the presence of 0.5% sodium cholate. The resulting phospholipid-and ubiquinone-depleted reductase shows no enzymatic activity; activity can be completely restored by the addition of phospholipids and Q6 or Q2. The ubiquinone-and phospholipid-replenisbed ubiquinonol-cytochrome c reductase is also fully active upon reconstituting with bovine succinate-ubiquinone reductase to form succinate-cytochrome c reductase. When an azido-ubiquinone derivative was added to the ubiquinone and phospbolipid-depleted reductase in the dark, followed by the addition of phospholipids, partial reconstitutive activity was restored, while full ubiquinol-cytochrome C reductase activity was observed when Q2H2 was used as substrate in the assay mixture. Apparently, the large amount of Q2H2 present in the assay mixture displaces the azido-ubiquinone in the system. Photolysis of the azido-Q-treated reductase with long-wavelength ultraviolet light abolishes about 70% of both the restored reconstitutive activity and Q2H2-cytochrome c reductase activity. The activity loss is directly proportional to the covalent binding of [3Hlazido-ubiquinone to the reductase protein. When the photolyzed, [3H]azido-ubiquinone-treated sample was subjected to SDS-polyacrylamide gel electrophoresis followed by analysis of the distribution of radioactivity among the subunits, the cytochrome b protein and a protein with an apparent molecular weight of 14 000 were heavily labeled. The amount of radioactive labeling in both these proteins was affected by the presence of pbospholipids.
Archives of Biochemistry and Biophysics, 2005
Famoxadone (FAM) is a newly commercialized antibiotic for use against plant pathogenic fungi. It inhibits mitochondria ubiquinol:cytochrome c oxidoreductase (EC 1.10.2.2, bc 1 complex) function by binding to the proximal niche of the quinol oxidation site on the enzyme. FAM has effects on the enzyme characteristic of both type Ia (E-b-methoxyacrylates) and type Ic (stigmatellin) inhibitors. Steady-state and tight-binding inhibition kinetics; as well as direct binding measurements with famoxadone (FAM) and methoxyacrylate stilbene (MOAS), indicated that FAM is a non-competitive inhibitor of the enzyme while methoxyacrylate stilbene (MOAS) is better described as a mixed-competitive inhibitor with respect to substrate. Mixed-competitive and non-competitive inhibition kinetics predicts a ternary enzyme-substrate-inhibitor (ESI) intermediate in the reaction sequence. Current views of the Qo domain architecture propose substrate binding niches in both distal and proximal regions of the domain. Since both inhibitors bind within the proximal niche, the formation of an ESI complex implicates substrate binding within the distal niche near the iron-sulfur protein (ISP) and cytochrome c 1 (C1). In the presence of saturating FAM, addition of substrate led to a slow, nearly stoichiometric reduction of C1 that was enzyme dependent, and independent of O À 2 production. Similar experiments with saturating MOAS led to a slow, sub-stoichiometric reduction of C1 by substrate. A comparison of the stoichiometries of reduction, and the apparent second order rate constants (K cat /K m) indicated that saturating MOAS elicits two distinct enzyme-inhibitor (EI) intermediates. One form does not bind substrate, but the other does. In contrast, saturating FAM leads to a predominant EI form capable of binding substrate. We suggest that these differences can be correlated to the respective effects of each inhibitor on the position of the ISP, and the integrity of a distal substrate binding site. The results also indicate that binding of these inhibitory substrate analogues to the proximal niche of the Qo domain significantly increases the DG à for reduction of C1.