ubiquinone and stigmatellin

The primary energy conversion (Qo) site of the cytochrome bc1 complex is flanked by both high- and low-potential redox cofactors, the [2Fe-2S] cluster and cytochrome bL, respectively. From the sensitivity of the reduced [2Fe-2S] cluster electron paramagnetic resonance (EPR) spectral g(x)-band and line shape to the degree and type of Qo site occupants, we have proposed a double-occupancy model for the Qo site by ubiquinone in Rhodobacter capsulatus membrane vesicles containing the cytochrome bc1 complex. Biophysical and biochemical experiments have confirmed the double occupancy model and from a combination of these results and the available cytochrome bc1 crystal structures we suggest that the two ubiquinone molecules in the Qo site serve distinct catalytic roles. We propose that the strongly bound ubiquinone, termed Qos, is close to the [2Fe-2S] cluster, where it remains tightly associated with the Qo site during turnover, serving as a catalytic cofactor; and the weaker bound ubiquinone, Qow, is distal to the [2Fe-2S] cluster and can exchange with the membrane Qpool on a time scale much faster than the turnover, acting as the substrate. The crystallographic data demonstrates that the FeS subunit can adopt different positions. Our own observations show that the equilibrium position of the reduced FeS subunit is proximal to the Qo site. On the basis of this, we also report preliminary results modeling the electron transfer reactions that can occur in the cytochrome bc1 complex and show that because of the strong distance dependence of electron transfer, significant movement of the FeS subunit must occur in order for the complex to be able to turn over at the experimental observed rates.

Topics: Animals; Bacterial Proteins; Binding Sites; Catalysis; Catalytic Domain; Crystallography, X-Ray; Diphenylamine; Electron Spin Resonance Spectroscopy; Electron Transport; Electron Transport Complex III; Mitochondria; Models, Chemical; Mutagenesis, Site-Directed; Oxidation-Reduction; Polyenes; Protein Structure, Tertiary; Rhodobacter capsulatus; Stilbenes; Ubiquinone

The ubiquinol: cytochrome c oxidoreductase, or the bc1 complex, is a key component of both respiratory and photosynthetic electron transfer and contributes to the formation of an electrochemical gradient necessary for ATP synthesis. Numerous bacteria harbor a bc1 complex comprised of three redox-active subunits, which bear two b-type hemes, one c-type heme, and one [2Fe-2S] cluster as prosthetic groups. Photosynthetic bacteria like Rhodobacter species provide powerful models for studying the function and structure of this enzyme and are being widely used. In recent years, extensive use of spontaneous and site-directed mutants and their revertants, new inhibitors, discovery of natural variants of this enzyme in various species, and engineering of novel bc1 complexes in species amenable to genetic manipulations have provided us with a wealth of information on the mechanism of function, nature of subunit interactions, and assembly of this important enzyme. The recent resolution of the structure of various mitochondrial bc1 complexes in different crystallographic forms has consolidated previous findings, added atomic-scale precision to our knowledge, and raised new issues, such as the possible movement of the Rieske Fe-S protein subunit during Qo site catalysis. Here, studies performed during the last few years using bacterial bc1 complexes are reviewed briefly and ongoing investigations and future challenges of this exciting field are mentioned.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Bacterial Proteins; Binding Sites; Catalysis; Electron Transport; Electron Transport Complex III; Heme; Iron-Sulfur Proteins; Ligands; Models, Molecular; Molecular Sequence Data; Oxidation-Reduction; Polyenes; Protein Conformation; Protein Engineering; Protein Folding; Protein Structure, Tertiary; Rhodobacter capsulatus; Structure-Activity Relationship; Ubiquinone

Yeast Ndi1 is a monotopic alternative NADH dehydrogenase. Its crystal structure in complex with the electron acceptor, ubiquinone, has been determined. However, there has been controversy regarding the ubiquinone binding site. To address these points, we identified the first competitive inhibitor of Ndi1, stigmatellin, along with new mixed-type inhibitors, AC0-12 and myxothiazol, and thereby determined the crystal structures of Ndi1 in complexes with the inhibitors. Two separate binding sites of stigmatellin, STG-1 and STG-2, were observed. The electron density at STG-1, located at the vicinity of the FAD cofactor, further demonstrated two binding modes: STG-1a and STG-1b. AC0-12 and myxothiazol are also located at the vicinity of FAD. The comparison of the binding modes among stigmatellin at STG-1, AC0-12, and myxothiazol revealed a unique position for the aliphatic tail of stigmatellin at STG-1a. Mutations of amino acid residues that interact with this aliphatic tail at STG-1a reduced the affinity of Ndi1 for ubiquinone. In conclusion, the position of the aliphatic tail of stigmatellin at STG-1a provides a structural basis for its competitive inhibition of Ndi1. The inherent binding site of ubiquinone is suggested to overlap with STG-1a that is distinct from the binding site for NADH.

Topics: Amino Acid Sequence; Binding Sites; Binding, Competitive; Coenzymes; Crystallography, X-Ray; Electron Transport Complex I; Flavin-Adenine Dinucleotide; Gene Expression; Kinetics; Methacrylates; Models, Molecular; Mutation; Polyenes; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Recombinant Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Thermodynamics; Thiazoles; Ubiquinone

The modified Q cycle mechanism accounts for the proton and charge translocation stoichiometry of the bc(1) complex, and is now widely accepted. However the mechanism by which the requisite bifurcation of electron flow at the Q(o) site reaction is enforced is not clear. One of several proposals involves conformational gating of the docking of the Rieske ISP at the Q(o) site, controlled by the stage of the reaction cycle. Effects of different Q(o)-site inhibitors on the position of the ISP seen in crystals may reflect the same conformational mechanism, in which case understanding how different inhibitors control the position of the ISP may be a key to understanding the enforcement of bifurcation at the Q(o) site (Table 1). Here we examine the available structures of cytochrome bc(1) with different Q(o)-site inhibitors and different ISP positions to look for clues to this mechanism. The effect of ISP removal on binding affinity of the inhibitors stigmatellin and famoxadone suggest a "mutual stabilization" of inhibitor binding and ISP docking, however this thermodynamic observation sheds little light on the mechanism. The cd(1) helix of cytochrome b moves in such a way as to accommodate docking when inhibitors favoring docking are bound, but it is impossible with the current structures to say whether this movement of α-cd(1) is a cause or result of ISP docking. One component of the movement of the linker between E and F helices also correlates with the type of inhibitor and ISP position, and seems to be related to the H-bonding pattern of Y279 of cytochrome b. An H-bond from Y279 to the ISP, and its possible modulation by movement of F275 in the presence of famoxadone and related inhibitors, or its competition with an alternate H-bond to I269 of cytochrome b that may be destabilized by bound famoxadone, suggest other possible mechanisms. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.

Topics: Binding Sites; Catalytic Domain; Crystallography, X-Ray; Cytochromes; Cytochromes c; Databases, Protein; Electron Spin Resonance Spectroscopy; Electron Transport Complex III; Enzyme Inhibitors; Hydrogen Bonding; Methacrylates; Models, Molecular; Molecular Structure; Mutation; Oxazoles; Polyenes; Principal Component Analysis; Protein Binding; Protein Conformation; Protein Structure, Secondary; Strobilurins; Tyrosine; Ubiquinone

Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia (Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc(1) complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Q(i) site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Q(o) site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A(3) in a fashion similar to the Q(o) site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Q(i) and the Q(o) sites of the fungal cytochrome bc(1) complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron-sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc(1) complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Q(o) and Q(i) sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc(1) complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Q(i) and Q(o) sites. Moreover, crystal structure of chicken cytochrome bc(1) complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Q(i) and Q(o) sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc(1) inhibitor that acts at both of the active sites of this enzyme.

Topics: Alkenes; Animals; Anti-Bacterial Agents; Antimycin A; Catalytic Domain; Cattle; Chickens; Crystallography, X-Ray; Cytochromes b; Cytochromes c; Electron Spin Resonance Spectroscopy; Electron Transport; Electron Transport Complex III; Male; Mitochondria, Liver; Mitochondrial Proteins; Oxidoreductases; Phenols; Pichia; Plant Proteins; Polyenes; Rats; Rats, Wistar; Respiration; Rhodobacter capsulatus; Superoxides; Ubiquinone

The protonation state of residues around the Q(o) binding site of the cytochrome bc(1) complex from Paracoccus denitrificans and their interaction with bound quinone(s) was studied by a combined electrochemical and FTIR difference spectroscopic approach. Site-directed mutations of two groups of conserved residues were investigated: (a) acidic side chains located close to the surface and thought to participate in a water chain leading up to the heme b(L) edge, and (b) residues located in the vicinity of this site. Interestingly, most of the mutants retain a high degree of catalytic activity. E295Q, E81Q and Y297F showed reduced stigmatellin affinity. On the basis of electrochemically induced FTIR difference spectra, we suggest that E295 and D278 are protonated in the oxidized form or that their mutation perturbs protonated residues. Mutations Y302, Y297, E81 and E295, directly perturb signals from the oxidized quinone and of the protein backbone. By monitoring the interaction with the inhibitor stigmatellin for the wild-type enzyme at various redox states, interactions of the bound stigmatellin with amino acid side chains such as protonated acidic residues and the backbone were observed, as well as difference signals arising from the redox active inhibitor itself and the replaced quinone. The infrared difference spectra of the above Q(o) site mutations in the presence of stigmatellin confirm the previously established role of E295 as a direct interaction partner in the enzyme from P.denitrificans as well. The protonated residue E295 is proposed to change the hydrogen-bonding environment upon stigmatellin binding in the oxidized form, and is deprotonated in the reduced form. Of the residues located close to the surface, D278 remains protonated and unperturbed in the oxidized form but its frequency shifts in the reduced form. The mechanistic implications of our observations are discussed, together with previous inhibitor binding data, and referred to the published X-ray structures.

Topics: Binding Sites; Electron Transport Complex III; Mutagenesis, Site-Directed; Oxidation-Reduction; Paracoccus denitrificans; Polyenes; Spectroscopy, Fourier Transform Infrared; Tyrosine; Ubiquinone

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc1 complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c1 position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c1 [S141C(ISP)/G180C(cyt c1)]. The formation of a disulfide bond between ISP and cyt c1 in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with beta-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc1 complex. Formation of the disulfide bond between ISP and cyt c1 shortens the distance between the [2Fe-2S] cluster and heme c1, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.

Topics: Anti-Bacterial Agents; Binding Sites; Cysteine; Cytochromes c1; Disulfides; Electron Spin Resonance Spectroscopy; Electron Transport; Electron Transport Complex III; Hydrogen-Ion Concentration; Iron-Sulfur Proteins; Mercaptoethanol; Models, Molecular; Mutation; Oxidation-Reduction; Photosynthesis; Polyenes; Protein Binding; Protein Conformation; Protein Engineering; Protein Subunits; Rhodobacter sphaeroides; Sulfhydryl Reagents; Ubiquinone

We describe in detail the conformations of the inhibitor stigmatellin in its free form and bound to the ubiquinone-reducing (Q(B)) site of the reaction center and to the ubiquinol-oxidizing (Q(o)) site of the cytochrome bc(1) complex. We present here the first structures of a stereochemically correct stigmatellin in complexes with a bacterial reaction center and the yeast cytochrome bc1 complex. The conformations of the inhibitor bound to the two enzymes are not the same. We focus on the orientations of the stigmatellin side-chain relative to the chromone head group, and on the interaction of the stigmatellin side-chain with these membrane protein complexes. The different conformations of stigmatellin found illustrate the structural variability of the Q sites, which are affected by the same inhibitor. The free rotation about the chi1 dihedral angle is an essential factor for allowing stigmatellin to bind in both the reaction center and the cytochrome bc1 pocket.

Topics: Electron Transport Complex III; Models, Biological; Models, Molecular; Photosynthetic Reaction Center Complex Proteins; Polyenes; Protein Binding; Protein Conformation; Rhodopseudomonas; Saccharomyces cerevisiae; Ubiquinone

In Rhodobacter sphaeroides reaction centers containing the mutation Ala M260 to Trp (AM260W), transmembrane electron transfer along the A-branch of cofactors is prevented by the loss of the QA ubiquinone. Reaction centers that contain this AM260W mutation are proposed to photoaccumulate the P(+)QB- radical pair following transmembrane electron transfer along the B-branch of cofactors (Wakeham, M. C., Goodwin, M. G., McKibbin, C., and Jones, M. R. (2003) Photoaccumulation of the P(+)QB- radical pair state in purple bacterial reaction centers that lack the QA ubiquinone. FEBS Lett. 540, 234-240). The yield of the P(+)QB- state appears to depend upon which additional mutations are present. In the present paper, Fourier transform infrared (FTIR) difference spectroscopy was used to demonstrate that photooxidation of the reaction center's primary donor in QA-deficient reaction centers results in formation of a semiquinone at the QB site by B-branch electron transfer. Reduction of QB by the B-branch pathway still occurs at 100 K, with a yield of approximately 10% relative to that at room temperature, in contrast to the QA- to QB reaction in the wild-type reaction center, which is not active at cryogenic temperatures. These FTIR results suggest that the conformational changes that "gate" the QA- to QB reaction do not necessarily have the same influence on QB reduction when the electron donor is the HB anion, at least in a minority of reaction centers.

Topics: Alanine; Anti-Bacterial Agents; Benzoquinones; Binding Sites; Electron Transport; Freezing; Light; Mutagenesis, Site-Directed; Oxidation-Reduction; Photosynthetic Reaction Center Complex Proteins; Polyenes; Rhodobacter sphaeroides; Spectroscopy, Fourier Transform Infrared; Tryptophan; Ubiquinone

The mitochondrial permeability transition (MPT) pore is a calcium-sensitive channel in the mitochondrial inner membrane that plays a crucial role in cell death. Here we show that cytochrome bc(1) regulates the MPT in isolated rat liver mitochondria and in CEM and HL60 cells by two independent pathways. Glutathione depletion activated the MPT via increased production of reactive oxygen species (ROS) generated by cytochrome bc(1). The ROS producing mechanism in cytochrome bc(1) involves movement of the "Rieske" iron-sulfur protein subunit of the enzyme complex, because inhibition of cytochrome bc(1) by pharmacologically blocking iron-sulfur protein movement completely abolished ROS production, MPT activation, and cell death. The classical inhibitor of the MPT, cyclosporine A, had no protective effect against MPT activation. In contrast, the calcium-activated, cyclosporine A-regulated MPT in rat liver mitochondria was also blocked with inhibitors of cytochrome bc(1). These results indicate that electron flux through cytochrome bc(1) regulates two distinct pathways to the MPT, one unregulated and involving mitochondrial ROS and the other regulated and activated by calcium.

Topics: Animals; Antimycin A; Calcium; Catalytic Domain; Cell Death; Electron Transport Complex III; Flow Cytometry; Glutathione; Humans; Ion Channels; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Polyenes; Rats; Reactive Oxygen Species; Ubiquinone

Complex III in the mitochondrial electron transport chain is a proposed site for the enhanced production of reactive oxygen species that contribute to aging in the heart. We describe a defect in the ubiquinol binding site (Q(O)) within cytochrome b in complex III only in the interfibrillar population of cardiac mitochondria during aging. The defect is manifested as a leak of electrons through myxothiazol blockade to reduce cytochrome b and is observed whether cytochrome b in complex III is reduced from the forward or the reverse direction. The aging defect increases the production of reactive oxygen species from the Q(O) site of complex III in interfibrillar mitochondria. A greater leak of electrons from complex III during the oxidation of ubiquinol is a likely mechanism for the enhanced oxidant production from mitochondria that contributes to aging in the rat heart.

Topics: Aging; Animals; Antimycin A; Binding Sites; Cytochrome b Group; Electron Transport; Electron Transport Complex III; Enzyme Activation; Hydroquinones; In Vitro Techniques; Male; Methacrylates; Mitochondria, Heart; Mitochondrial Diseases; Myofibrils; Oxidation-Reduction; Polyenes; Rats; Reactive Oxygen Species; Thiazoles; Ubiquinone

Atovaquone is a substituted 2-hydroxynaphthoquinone that is used therapeutically to treat Plasmodium falciparum malaria, Pneumocystis carinii pneumonia, and Toxoplasma gondii toxoplasmosis. It is thought to act on these organisms by inhibiting the cytochrome bc1 complex. We have examined the interaction of atovaquone with the bc1 complex isolated from Saccharomyces cerevisiae, a surrogate, nonpathogenic fungus. Atovaquone inhibits the bc1 complex competitively with apparent Ki = 9 nm, raises the midpoint potential of the Rieske iron-sulfur protein from 285 to 385 mV, and shifts the g values in the EPR spectrum of the Rieske center. These results indicate that atovaquone binds to the ubiquinol oxidation pocket of the bc1 complex, where it interacts with the Rieske iron-sulfur protein. A computed energy-minimized structure for atovaquone liganded to the yeast bc1 complex suggests that a phenylalanine at position 275 of cytochrome b in the bovine bc1 complex, as opposed to leucine at the equivalent position in the yeast enzyme, is responsible for the decreased sensitivity of the bovine bc1 complex (Ki = 80 nm) to atovaquone. When a L275F mutation was introduced into the yeast cytochrome b, the sensitivity of the yeast enzyme to atovaquone decreased (Ki = 100 nm) with no loss in activity, confirming that the L275F exchange contributes to the differential sensitivity of these two species to atovaquone. These results provide the first molecular description of how atovaquone binds to the bc1 complex and explain the differential inhibition of the fungal versus mammalian enzymes.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Atovaquone; Binding Sites; Binding, Competitive; Electron Transport Complex III; Molecular Sequence Data; Naphthoquinones; Oxidation-Reduction; Polyenes; Protein Structure, Secondary; Protein Structure, Tertiary; Saccharomyces cerevisiae; Ubiquinone

The mitochondrial permeability transition (MPT) is a key event in apoptotic and necrotic cell death and is controlled by the cellular redox state. To further investigate the mechanism(s) involved in regulation of the MPT, we used diethylmaleate to deplete GSH in HL60 cells and increase mitochondrial reactive oxygen species (ROS) production. The site of mitochondrial ROS production was determined to be mitochondrial respiratory complex III (cytochrome bc1), because 1). stigmatellin, a Qo site inhibitor, blocked ROS production and prevented the MPT and cell death and 2). cytochrome bc1 activity was abolished in cells protected from the redox-dependent MPT by stigmatellin. We next investigated the effect of pretreating cells with coenzyme Q10 analogs decylubiquinone (dUb) and ubiquinone 0 (Ub0) on the redox-dependent MPT. Pretreatment of HL60 cells with dUb blocked ROS production induced by GSH depletion and prevented activation of the MPT and cell death, whereas Ub0 did not. Since we also found that dUb did not inhibit cytochrome bc1 activity, the mechanism of protection against redox-dependent MPT by dUb may depend on its ability to scavenge ROS generated by cytochrome bc1. These results indicate that dUb, like the clinically used ubiquinone analog idebenone, may serve as a candidate antioxidant compound for the development of pharmacological agents to treat diseases where there is an oxidative stress component.

Topics: Electron Transport Complex III; Enzyme Inhibitors; Glutathione; HL-60 Cells; Humans; Oxidation-Reduction; Polyenes; Reactive Oxygen Species; Ubiquinone

The rotenone-insensitive NADH dehydrogenase isolated from mitochondria of the procyclic form of Trypanosoma brucei has the ability to produce superoxide anions (Biochemistry 41 (2002) 3065). Superoxide production by the purified enzyme was 60% inhibited by diphenyl iodonium (DPI), stimulated significantly by ubiquinone analogues, and unaffected by metal ions. Production of reactive oxygen species (ROS) in intact cells was not affected by addition of rotenone with proline and malate as substrates; however, addition of rotenone inhibited 41% ROS production with succinate as substrate. These results suggest that complex I is not involved in production of ROS and that succinate-linked reversed electron transport occurs in trypanosome mitochondria. Superoxide formation in mitochondria with NADH as substrate was stimulated by antimycin A but was unaffected by myxothiazol plus stigmatellin, indicating that bc(1) complex is not a source of superoxide. DPI and fumarate inhibited by 68 and 36%, respectively, the rate of superoxide production with NADH as substrate. Addition of both fumarate and DPI blocked 70% superoxide production in mitochondria, a total inhibition similar to that observed with DPI addition alone. These results suggest that the rotenone-insensitive NADH dehydrogenase in addition to NADH fumarate reductase is a potential source of superoxide production in procyclic trypanosome mitochondria.

Topics: Animals; Anti-Bacterial Agents; Antimycin A; Biphenyl Compounds; Fumarates; Malates; Methacrylates; Mitochondria; NAD; NADH Dehydrogenase; Onium Compounds; Polyenes; Proline; Rotenone; Substrate Specificity; Succinic Acid; Superoxides; Thiazoles; Trypanosoma brucei brucei; Ubiquinone; Uncoupling Agents

The cytochrome bc(1) complex is a dimeric enzyme that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is re-reduced at a second center, referred to as center N. To understand better the mechanism of ubiquinol oxidation, we have examined the interaction of several inhibitory analogs of ubiquinol with the yeast cytochrome bc(1) complex. Stigmatellin and methoxyacrylate stilbene, two inhibitors that block ubiquinol oxidation at center P, inhibit the yeast enzyme with a stoichiometry of 0.5 per bc(1) complex, indicating that one molecule of inhibitor is sufficient to fully inhibit the dimeric enzyme. This stoichiometry was obtained when the inhibitors were titrated in cytochrome c reductase assays and in reactions of quinol with enzyme in which the inhibitors block pre-steady state reduction of cytochrome b. As an independent measure of inhibitor binding, we titrated the red shift in the optical spectrum of ferrocytochrome b with methoxyacrylate stilbene and thus confirmed the results of the inhibition of activity titrations. The titration curves also indicate that the binding is anti-cooperative, in that a second molecule of inhibitor binds with much lower affinity to a dimer in which an inhibitor molecule is already bound. Because these inhibitors bind to the ubiquinol oxidation site in the bc(1) complex, we propose that the yeast cytochrome bc(1) complex oxidizes ubiquinol by an alternating, half-of-the-sites mechanism.

Topics: Anti-Bacterial Agents; Antimycin A; Electron Transport Complex III; Fungal Proteins; Oxidation-Reduction; Polyenes; Saccharomyces cerevisiae; Stilbenes; Ubiquinone

NADH:ubiquinone oxidoreductase (complex I) is the first, largest and most complicated enzyme of the mitochondrial electron transport chain. Photoaffinity labeling with the highly potent and specific inhibitor trifluoromethyldiazirinyl-[(3)H]pyridaben ([(3)H]TDP) labels only the PSST and ND1 subunits of complex I in electron transport particles. PSST is labeled at a high-affinity site responsible for inhibition of enzymatic activity while ND1 is labeled at a low-affinity site not related to enzyme inhibition. In this study we found, as expected, that 13 complex I inhibitors decreased labeling at the PSST site without effect on ND1 labeling. However, there were striking exceptions where an apparent interaction was found between the PSST and ND1 subunits: preincubation with NADH increases PSST labeling and decreases ND1 labeling; the very weak complex I inhibitor 1-methyl-4-phenylpyridinium ion (MPP(+)) and the semiquinone analogue stigmatellin show the opposite effect with increased labeling at ND1 coupled to decreased labeling at PSST in a concentration- and time-dependent manner. MPP(+), stigmatellin and ubisemiquinone have similarly positioned centers of highly negative and positive electrostatic potential surfaces. Perhaps the common action of MPP(+) and stigmatellin on the functional coupling of the PSST and ND1 subunits is initiated by binding at a semiquinone binding site in complex I.

Topics: 1-Methyl-4-phenylpyridinium; Binding Sites; Electron Transport Complex I; Enzyme Inhibitors; Enzyme Stability; Hot Temperature; Molecular Structure; Multienzyme Complexes; NAD; NADH, NADPH Oxidoreductases; Photoaffinity Labels; Polyenes; Pyridazines; Rotenone; Structure-Activity Relationship; Tritium; Ubiquinone

Topics: Animals; Anti-Bacterial Agents; Antifungal Agents; Atovaquone; Female; Naphthoquinones; Oxidoreductases; Parabens; Pneumocystis; Polyenes; Rats; Ubiquinone

Crystal structures of the cytochrome bc1 complex indicate that the catalytic domain of the Rieske iron-sulfur protein, which carries the [2Fe-2S] cluster, is connected to a transmembrane anchor by a flexible linker region. This flexible linker allows the catalytic domain to move between two positions, proximal to cytochrome b and cytochrome c1. Addition of an alanine residue to the flexible linker region of the Rieske protein lowers the ubiquinol-cytochrome c reductase activity of the mitochondrial membranes by one half and causes the apparent Km for ubiquinol to decrease from 9.3 to 2.6 microM. Addition of two alanine residues lowers the activity by 90% and the apparent Km decreases to 1.9 microM. Deletion of an alanine residue lowers the activity by approximately 40% and the apparent Km decreases to 5.0 microM. Addition or deletion of an alanine residue also causes a pronounced decrease in efficacy of inhibition of ubiquinol-cytochrome c reductase activity by stigmatellin, which binds analogous to reaction intermediates of ubiquinol oxidation. These results indicate that the length of the flexible linker region is critical for interaction of ubiquinol with the bc1 complex, consistent with electron transfer mechanisms in which ubiquinol must simultaneously interact with the iron-sulfur protein and cytochrome b.

Topics: Alanine; Amino Acid Sequence; Antimycin A; Aspartic Acid; Blotting, Western; Catalysis; Crystallography, X-Ray; Electron Transport Complex III; Electrons; Intracellular Membranes; Iron-Sulfur Proteins; Kinetics; Mitochondria; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; NADH Dehydrogenase; Polyenes; Protein Structure, Tertiary; Saccharomyces cerevisiae; Sequence Homology, Amino Acid; Ubiquinone

Native structures of ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) from different sources, and structures with inhibitors in place, show a 16-22 A 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.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Binding Sites; Chickens; Computer Simulation; Crystallography; Cytochrome b Group; Electron Transport Complex III; Enzyme Inhibitors; Iron-Sulfur Proteins; Ligands; Mitochondria, Heart; Molecular Sequence Data; Mutation; Oxidation-Reduction; Polyenes; Protein Engineering; Protein Structure, Secondary; Sequence Alignment; Stilbenes; Thiazoles; Ubiquinone

Structures of mitochondrial ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) from several animal sources have provided a basis for understanding the functional mechanism at the molecular level. Using structures of the chicken complex with and without inhibitors, we analyze the effects of mutation on quinol oxidation at the Q(o) site of the complex. We suggest a mechanism for the reaction that incorporates two features revealed by the structures, a movement of the iron sulfur protein between two separate reaction domains on cytochrome c(1) and cytochrome b and a bifurcated volume for the Q(o) site. The volume identified by inhibitor binding as the Q(o) site has two domains in which inhibitors of different classes bind differentially; a domain proximal to heme b(L), where myxothiazole and beta-methoxyacrylate- (MOA-) type inhibitors bind (class II), and a distal domain close to the iron sulfur protein docking interface, where stigmatellin and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiaole (UHDBT) bind (class I). Displacement of one class of inhibitor by another is accounted for by the overlap of their volumes, since the exit tunnel to the lipid phase forces the hydrophobic "tails" to occupy common space. We conclude that the site can contain only one "tailed" occupant, either an inhibitor or a quinol or one of their reaction products. The differential sensitivity of strains with mutations in the different domains is explained by the proximity of the affected residues to the binding domains of the inhibitors. New insights into mechanism are provided by analysis of mutations that affect changes in the electron paramagnetic resonance (EPR) spectrum of the iron sulfur protein, associated with its interactions with the Q(o)-site occupant. The structures show that all interactions with the iron sulfur protein must occur at the distal position. These include interactions between quinone, or class I inhibitors, and the reduced iron sulfur protein and formation of a reaction complex between quinol and oxidized iron sulfur protein. The step with high activation energy is after formation of the reaction complex, likely in formation of the semiquinone and subsequent dissociation of the complex into products. We suggest that further progress of the reaction requires a movement of semiquinone to the proximal position, thus mapping the bifurcated reaction to the bifurcated volume. We suggest that such a movement, together with a change in conformation of the site,

Topics: Animals; Binding Sites; Chickens; Electron Transport Complex III; Mitochondria, Heart; Oxidation-Reduction; Polyenes; Thiazoles; Ubiquinone

In a reaction of central importance to the energetics of photosynthetic bacteria, light-induced electron transfer in the reaction centre (RC) is coupled to the uptake of protons from the cytoplasm at the binding site of the secondary quinone (QB). In the original structure of the RC from Rhodopseudomonas viridis (PDB entry code 1PRC), the QB site was poorly defined because in the standard RC crystals it was only approximately 30% occupied with ubiquinone-9 (UQ9). We report here the structural characterization of the QB site by crystallographic refinement of UQ9-depleted RCs and of complexes of the RC either with ubiquinone-2 (UQ2) or the electron-transfer inhibitor stigmatellin in the QB site.. The structure of the RC complex with UQ2, refined at 2.45 A resolution, constitutes the first crystallographically reliably defined binding site for quinones from the bioenergetically important quinone pool of biological, energy-transducing membranes. In the UQ9-depleted QB site of the RC structure, refined at 2.4 A resolution, apparently five (and possibly six) water molecules are bound instead of the ubiquinone head group, and a detergent molecule binds in the region of the isoprenoid tail. All of the protein-cofactor interactions implicated in the binding of the ubiquinone head group are also implicated in the binding of the stigmatellin head group. In the structure of the stigmatellin-RC complex, refined at 2.4 A resolution, additional hydrogen bonds stabilize the binding of stigmatellin over that of ubiquinone. The tentative position of UQ9 in the QB site in the original data set (1PRC) was re-examined using the structure of the UQ9-depleted RC as a reference. A modified QB site model, which exhibits greater similarity to the distal ubiquinone-10 (UQ10) positioning in the structure of the RC from Rhodobacter sphaeroides (PDB entry code 1PCR), is suggested as the dominant binding site for native UQ9.. The structures reported here can provide models of quinone reduction cycle intermediates. The binding pattern observed for the stigmatellin complex, where the ligand donates a hydrogen bond to Ser L223 (where 'L' represents the L subunit of the RC), can be viewed as a model for the stabilization of a monoprotonated reduced intermediate (QBH or QBH-). The presence of Ser L223 in the QB site indicates that the QB site is not optimized for QB binding, but for QB reduction to the quinol.

Topics: Bacteriochlorophylls; Binding Sites; Crystallography, X-Ray; Electron Transport; Energy Metabolism; Hydrogen Bonding; Light; Light-Harvesting Protein Complexes; Membrane Proteins; Models, Molecular; Molecular Structure; Photosynthetic Reaction Center Complex Proteins; Polyenes; Protein Binding; Protons; Rhodopseudomonas; Ubiquinone; Water

The binding of specific inhibitors to the ubiquinol oxidation pocket ("QP center") of cytochrome c reductase was analyzed before and after removal of bound phospholipid and the "Rieske" iron-sulfur protein using optical spectroscopy and fluorescence quench binding assays. The enzyme lacking iron-sulfur protein showed almost unchanged, tight binding of the E-beta-methoxyacrylate inhibitors oudemansin A and MOA-stilbene, whereas binding of the chromone inhibitor stigmatellin was almost completely abolished. The affinity of the weak inhibitor 3-undecyl-2-hydroxy-naphthoquinone was decreased. Oudemansin A binding to the defective pocket of the iron-sulfur protein-depleted enzyme was lowered by added phospholipid. It was deduced from these results that the QP center is a spacious pocket formed by domains of cytochrome b, bearing the E-beta-methoxcyacrylate binding site, and the iron-sulfur protein, bearing the stigmatellin binding site. Moreover, removal of the iron-sulfur protein leaves this pocket defective but essentially unchanged in its remaining binding capability. The affinity of three preparations of cytochrome c reductase, the complete, the delipidated, and the iron-sulfur depleted enzyme for E-beta-methoxyacrylate-stilbene, was analyzed for different redox states of the catalytic centers of cytochrome c reductase. The apparent Kd values for the different redox states were interpreted in terms of two conformational states. It is suggested that these changes reflect the two states of the "catalytic switch" proposed recently for the QP pocket of cytochrome c reductase (Brandt, U., and von Jagow, G. (1991) Eur. J. Biochem. 195, 163-170). According to the refined model presented in this work, changeover to the "b" state is triggered by reduction of the iron-sulfur cluster, and changeover back to the "FeS" state is triggered by electron transfer from the low potential onto the high potential heme b center. Our interpretation implies that the stability of the two states is affected by the redox states of the enzyme, but that additionally changing the redox states of the two centers is required for "switching" on a catalytic time scale.

Topics: Acrylates; Animals; Binding Sites; Catalysis; Cattle; Electron Transport Complex III; Iron-Sulfur Proteins; Mitochondria, Heart; Oxidation-Reduction; Polyenes; Spectrometry, Fluorescence; Stilbenes; Ubiquinone

Seven single-site mutants in six residues of the cyt b polypeptide of Rhodobacter capsulatus selected for resistance to the Qo site inhibitors stigmatellin, myxothiazol, or mucidin [Daldal, F., Tokito, M.K., Davidson, E., & Faham, M. (1989) EMBO J. 8, 3951-3961] have been characterized by using optical and EPR spectroscopy and single-turnover kinetic analysis. The strains were compared with wild-type strain MT1131 and with the Ps- strain R126 (G158D), which is dysfunctional in its Qo site [Robertson, D.E., Davidson, E., Prince, R.C., van den Berg, W.H., Marrs, B.L., & Dutton, P.L. (1986) J. Biol. Chem. 261, 584-591]. Mutants selected for stigmatellin resistance induced a weakening in the binding of the inhibitor without discernible loss of ubiquinone(Q)/ubiquinol(QH2) binding affinity to the Qo site or kinetic impairment to catalysis. Mutants selected for myxothiazol or mucidin resistance, inducing weakening of inhibitor binding, all displayed impaired rates of Qo site catalysis: The most severe cases (F144L, F144S) displayed loss of affinity for Q, and evidence suggests that parallel loss of affinity for the substrate QH2 was incurred in these strains. The results provide a view of the nature of the interaction of Q and QH2 of the Qpool with the Qo site. Consideration of the mutational substitutions and their structural positions along with comparisons with the QA and QB sites of the photosynthetic reaction center suggests a model for the structure of the Qo site.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Cytochrome b Group; Electron Spin Resonance Spectroscopy; Electron Transport; Electron Transport Complex III; Fatty Acids, Unsaturated; Kinetics; Methacrylates; Models, Structural; Molecular Sequence Data; Mutation; Oxidation-Reduction; Polyenes; Protein Conformation; Rhodobacter capsulatus; Strobilurins; Thiazoles; Ubiquinone

Stigmatellin, a chromone inhibitor acting at the Q0 center of the bc1 complex, binds to the heme b-566 domain of cytochrome b as well as to the Fe2S2 protein. Its binding induces a shift of the alpha-band of heme b-566 to 568 nm. It does not influence the ligand field of the heme b-562 center. Concomitant with the red shift, stigmatellin gives rise to an alteration of the EPR line shape of the Fe2S2 cluster, namely linewidth narrowing and g value shifts at all 3 principal values. The midpoint redox potential of the Fe2S2 protein is shifted from 290 to 540 mV.

Topics: Animals; Binding Sites; Binding, Competitive; Cattle; Electron Spin Resonance Spectroscopy; Electron Transport Complex III; Intracellular Membranes; Iron-Sulfur Proteins; Methacrylates; Mitochondria; Multienzyme Complexes; Oxidation-Reduction; Polyenes; Protein Binding; Quinone Reductases; Thiazoles; Ubiquinone