Identification of ubiquinol binding motifs at the Qo-site of the cytochrome bc1 complex (original) (raw)
Related papers
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.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2004
The rate-limiting reaction of the bc 1 complex from Rhodobacter sphaeroides is transfer of the first electron from ubihydroquinone (quinol, QH 2 ) to the [2Fe -2S] cluster of the Rieske iron -sulfur protein (ISP) at the Q o -site. Formation of the ES-complex requires participation of two substrates (S), QH 2 and ISP ox . From the variation of rate with [S], the binding constants for both substrates involved in formation of the complex can be estimated. The configuration of the ES-complex likely involves the dissociated form of the oxidized ISP (ISP ox ) docked at the b-interface on cyt b, in a complex in which N q of His-161 (bovine sequence) forms a H-bond with the quinol UOH. A coupled proton and electron transfer occurs along this H-bond. This brief review discusses the information available on the nature of this reaction from kinetic, structural and mutagenesis studies. The rate is much slower than expected from the distance involved, likely because it is controlled by the low probability of finding the proton in the configuration required for electron transfer. A simplified treatment of the activation barrier is developed in terms of a probability function determined by the Brønsted relationship, and a Marcus treatment of the electron transfer step. Incorporation of this relationship into a computer model allows exploration of the energy landscape. A set of parameters including reasonable values for activation energy, reorganization energy, distances between reactants, and driving forces, all consistent with experimental data, explains why the rate is slow, and accounts for the altered kinetics in mutant strains in which the driving force and energy profile are modified by changes in E m and/or pK of ISP or heme b L .
Observations concerning the quinol oxidation site of the cytochrome bc1 complex
FEBS Letters, 2003
A direct hydrogen bond between ubiquinone/quinol bound at the Q O site and a cluster-ligand histidine of the iron^sulfur protein (ISP) is described as a major determining factor explaining much experimental data on position of the ISP ectodomain, electron paramagnetic resonance (EPR) lineshape and midpoint potential of the iron^sulfur cluster, and the mechanism of the bifurcated electron transfer from ubiquinol to the high and low potential chains of the bc 1 complex.
Journal of Biological Chemistry, 2006
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.
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.
Atomistic determinants of co-enzyme Q reduction at the Qi-site of the cytochrome bc1 complex
Scientific Reports, 2016
The cytochrome (cyt) bc 1 complex is an integral component of the respiratory electron transfer chain sustaining the energy needs of organisms ranging from humans to bacteria. Due to its ubiquitous role in the energy metabolism, both the oxidation and reduction of the enzyme's substrate co-enzyme Q has been studied vigorously. Here, this vast amount of data is reassessed after probing the substrate reduction steps at the Q i-site of the cyt bc 1 complex of Rhodobacter capsulatus using atomistic molecular dynamics simulations. The simulations suggest that the Lys251 side chain could rotate into the Q i-site to facilitate binding of half-protonated semiquinone-a reaction intermediate that is potentially formed during substrate reduction. At this bent pose, the Lys251 forms a salt bridge with the Asp252, thus making direct proton transfer possible. In the neutral state, the lysine side chain stays close to the conserved binding location of cardiolipin (CL). This back-and-forth motion between the CL and Asp252 indicates that Lys251 functions as a proton shuttle controlled by pH-dependent negative feedback. The CL/K/D switching, which represents a refinement to the previously described CL/K pathway, fine-tunes the proton transfer process. Lastly, the simulation data was used to formulate a mechanism for reducing the substrate at the Q i-site.
Pathways for proton release during ubihydroquinone oxidation by the bc1 complex
Proceedings of the National Academy of Sciences, 1999
Quinol oxidation by the bc 1 complex of Rhodobacter sphaeroides occurs from an enzyme-substrate complex formed between quinol bound at the Q o site and the iron-sulfur protein (ISP) docked at an interface on cytochrome b. From the structure of the stigmatellin-containing mitochondrial complex, we suggest that hydrogen bonds to the two quinol hydroxyl groups, from Glu-272 of cytochrome b and His-161 of the ISP, help to stabilize the enzyme-substrate complex and aid proton release. Reduction of the oxidized ISP involves H transfer from quinol. Release of the proton occurs when the acceptor chain reoxidizes the reduced ISP, after domain movement to an interface on cytochrome c 1 . Effects of mutations to the ISP that change the redox potential and͞or the pK on the oxidized form support this mechanism. Structures for the complex in the presence of inhibitors show two different orientations of Glu-272. In stigmatellin-containing crystals, the side chain points into the site, to hydrogen bond with a ring hydroxyl, while His-161 hydrogen bonds to the carbonyl group. In the native structure, or crystals containing myxothiazol or -methoxyacrylate-type inhibitors, the Glu-272 side chain is rotated to point out of the site, to the surface of an external aqueous channel. Effects of mutation at this residue suggest that this group is involved in ligation of stigmatellin and quinol, but not quinone, and that the carboxylate function is essential for rapid turnover. H ؉ transfer from semiquinone to the carboxylate side chain and rotation to the position found in the myxothiazol structure provide a pathway for release of the second proton.
Journal of Biological Chemistry, 2006
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.
Q-cycle bypass reactions at the Qo site of the cytochrome bc1 (and related) complexes
Methods in enzymology, 2004
The Q-Cycle and Its Bypass Reactions The cytochrome (cyt) bc 1 complex plays a central role in chemiosmotic energy conversion in mitochondria and many bacteria, oxidizing quinol (QH 2)-ubihydroquinone (UQH 2) in the case of mitochondria-and reducing a soluble electron carrier (cyt c in mitochondria), while acting as a proton ''shuttle'' or translocator to store energy in an electrochemical proton gradient, or proton motive force (pmf). 1,2 The pmf in turn drives the synthesis of ATP at the F 0-F 1-ATP synthase. The structurally analogous cyt b 6 f complex of chloroplasts plays a similar role-but oxidizes plastohydroquinone (PQH 2)-during oxygenic photosynthesis. The cyt bc 1 /b 6 f enzymes are dimeric integral membrane complexes composed of as few as three subunits in some prokaryotes and up to eleven subunits in mitochondria. The proteins house four essential redox-active, metal centers in two distinct chains. The ''low potential chain'' consists of two b-type hemes, cyt b H and cyt b L with relatively higher and lower redox potentials, housed in a single cyt b protein. The ''high potential chain'' consists of a ''Rieske'' iron-sulfur complex (2Fe2S) in the Rieske iron-sulfur protein (ISP) and another relatively high potential carrier, cyt c 1 in the cyt bc 1 complex, cyt f in the cyt b 6 f complex, and an assortment of other carriers in complexes from distantly related bacteria. 3,4 The cyt bc 1 and b 6 f complexes also possess two quinone/quinol binding sites. One of these, the Q o site is located on the p-side of the membrane (the intermembrane space in mitochondria), at the interface between cyt b L and the ISP and acts during normal turnover to oxidize QH 2 to quinone (Q). The other site, termed the Q i site, is located close to cyt b H , towards the n-side of the membrane, and acts to reduce Q to QH 2. Biochemical studies and X-ray crystal structures of mitochondrial cyt bc 1 complexes show that the Q o site possesses proximal and distal Q o site