ubiquinone and 3-methoxy-2-(2-styrylphenyl)propenic-acid-methyl-ester

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

A key issue concerning the primary conversion (Q(O)) site function in the cytochrome bc(1) complex is the stoichiometry of ubiquinone/ubihydroquinone occupancy. Previous evidence suggests that the Q(O) site is able to accommodate two ubiquinone molecules, the double occupancy model [Ding, H., Robertson, D. E., Daldal, F., and Dutton, P. L. (1992) Biochemistry 31, 3144-3158]. In the recently reported crystal structures of the cytochrome bc(1) complex, no electron density was identified in the Q(O) site that could be ascribed to ubiquinone. To provide further insight into this issue, we have manipulated the cytochrome bc(1) complex Q(O) site occupancy in photosynthetic membranes from Rhodobacter capsulatus by using inhibitor titrations and ubiquinone extraction to modulate the amount of ubiquinone bound in the site. The nature of the Q(O) site occupants was probed via the sensitivity of the reduced [2Fe-2S] cluster electron paramagnetic resonance (EPR) spectra to modulation of Q(O) site occupancy. Diphenylamine (DPA) and methoxyacrylate (MOA)-stilbene are known Q(O) site inhibitors of the cytochrome bc(1) complex. Addition of stoichiometric concentrations of MOA-stilbene or excess DPA to cytochrome bc(1) complexes with natural levels of ubiquinone elicits the same change in the [2Fe-2S] cluster EPR spectra; the g(x)() resonance broadens and shifts from 1. 800 to 1.783. This is exactly the same signal as that obtained when there is only one ubiquinone present in the Q(O) site. Furthermore, addition of MOA-stilbene or DPA to the cytochrome bc(1) complex depleted of ubiquinone does not alter the [2Fe-2S] cluster EPR spectral line shapes, which remain indicative of one ubiquinone or zero ubiquinones in the Q(O) site, with broad g(x)() resonances at 1. 783 or 1.765, respectively. The results are quite consistent with the Q(O) site double occupancy model, in which MOA-stilbene and DPA inhibit by displacing one, but not both, of the Q(O) site ubiquinones.

Topics: Diphenylamine; Electron Spin Resonance Spectroscopy; Electron Transport Complex III; Enzyme Inhibitors; Models, Chemical; Oxidation-Reduction; Rhodobacter capsulatus; Stilbenes; Structure-Activity Relationship; Ubiquinone

The unique bifurcated oxidation of ubiquinol at center P (Qo) of the cytochrome bc1 complex is the reaction within the Q-cycle reaction scheme that is most critical for the link between electron transfer and vectorial proton translocation. While there is a general consensus about the overall reaction at center P, the nature of the intermediates and the way the reaction is controlled to ensure obligatory bifurcation is still controversial. By reducing the reaction to its essential steps, a kinetic net rate model is developed in which the activation barrier is associated with the deprotonation of ubiquinol, but the steady state rate is kinetically controlled by the occupancy of the ubiquinol anion and the semiquinone state. This concept is used to interpret experimental data and is discussed in terms of various mechanistic models that are under discussion. It is outlined how other aspects of the center P mechanism like the proposed "prosthetic" ubiquinone and the moving domain of the "Rieske" protein could be incorporated in the kinetic framework.

Topics: Animals; Electron Transport; Electron Transport Complex III; Enzyme Inhibitors; Heme; Iron-Sulfur Proteins; Mitochondria; Models, Chemical; Models, Molecular; Motion; Protein Conformation; Protein Structure, Tertiary; Protons; Stilbenes; Structure-Activity Relationship; Thermodynamics; 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

We determined the sites of artificial electron transfer onto 2-nitrosofluorene (NOF), a metabolite of carcinogenic 2- acetylaminofluorene in mitochondria and isolated cytochrome bc1 complex. NOF-induced O2 consumption in mitochondria was sensitive to antimycin A, but insensitive to myxothiazol. In the isolated cytochrome bc1 complex, NOF induced rapid MOA-stilbene-insensitive reoxidation of cytochrome b, whereas in the presence of antimycin A, reoxidation was very slow. The corresponding hydroxylamine, N-hydroxy-2-aminofluorene (N-OH-AF), reduced cytochrome b specifically through center N of the cytochrome bc1 complex. We conclude that NOF and N-OH-AF bind to center N of the cytochrome bc1 complex and act as electron acceptor and donor, respectively. The N-OH-AF/NOF interconversion is considered to be involved in the cytotoxicity of 2-acetylaminofluorene in vivo.

Topics: Animals; Antimycin A; Cytochrome b Group; Dithionite; Electron Transport Complex III; Fluorenes; Male; Methacrylates; Mitochondria, Liver; Nitroso Compounds; Oxidation-Reduction; Oxygen Consumption; Potassium Cyanide; Rats; Rats, Wistar; Stilbenes; Tetramethylphenylenediamine; Thiazoles; Ubiquinone; Uncoupling Agents