cytochromes-c1 and ubiquinol

The electron transfer reactions within wild-type Rhodobacter sphaeroides cytochrome bc

Topics: Cytochromes b; Cytochromes c; Cytochromes c1; Electrons; Iron-Sulfur Proteins; Oxidation-Reduction; Rotation

The respiratory cytochrome bc

Topics: Cytochromes b; Cytochromes c1; Electron Transport; Hydroquinones; Kinetics; Models, Molecular; Mutation; Oxidation-Reduction; Oxygen; Proton-Motive Force; Protons; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; 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

The generation of oxygen radicals in biological systems and their sites of intracellular release have been subject of numerous studies in the last decades. Based on these studies mitochondria are considered to be the major source of intracellular oxygen radicals. Although this finding is more or less accepted, the mechanism of univalent oxygen reduction in mitochondria is still obscure. One of the most critical electron transfer steps in the respiratory chain is the electron bifurcation at the cytochrome bc1 complex. Recent studies with genetically mutated mitochondria have made it clear that electron bifurcation from ubiquinol to the cytochrome bc1 complex requires the free mobility of the head domain of the Rieske iron-sulfur protein. On the other hand, it has been long known that inhibition of electron bifurcation by antimycin A causes leakage of single electrons to dioxygen, which results in the release of superoxide radicals. These findings lead us to study whether hindrance of the interaction of ubiquinol with the cytochrome bc1 complex is the regulator of single electron diversion to oxygen. Hindrance of electron bifurcation was observed following alterations of the physical state of membrane phospholipids in which the cytochrome bc1 complex is inserted. Irrespective of whether the fluidity of the membrane lipids was elevated or decreased, electron flow rates to the Rieske iron-sulfur protein were drastically reduced. Concomitantly superoxide radicals were released from these mitochondria, strongly suggesting an effect on the mobility of the head domain of the Rieske iron-sulfur protein. This revealed the involvement of the ubiquinol cytochrome bc1 redox couple in mitochondrial superoxide formation. The regulator, which controls leakage of electrons to oxygen, appears to be the electron-branching activity of the cytochrome bc1 complex.

Topics: Animals; Antimycin A; Cattle; Cholesterol; Cytochrome b Group; Cytochromes c1; Electron Spin Resonance Spectroscopy; Electron Transport; Electron Transport Complex III; Electrons; Erucic Acids; Hydrogen Peroxide; Iron-Sulfur Proteins; Kinetics; Male; Mitochondria, Heart; NADH Dehydrogenase; Oxidation-Reduction; Oxygen; Rats; Rats, Sprague-Dawley; Submitochondrial Particles; Superoxides; Ubiquinone

Crystallographic structures for the mitochondrial ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) from different sources, and with different inhibitors in cocrystals, have revealed that the extrinsic domain of the iron sulfur subunit is not fixed [Zhang, Z., Huang, L., Shulmeister, V. M., Chi, Y.-I., Kim, K. K., Hung, L.-W., Crofts, A. R., Berry, E. A., and Kim, S.-H. (1998) Nature (London), 392, 677-684], but moves between reaction domains on cytochrome c(1) and cytochrome b subunits. We have suggested that the movement is necessary for quinol oxidation at the Q(o) site of the complex. In this paper, we show that the electron-transfer reactions of the high-potential chain of the complex, including oxidation of the iron sulfur protein by cytochrome c(1) and the reactions by which oxidizing equivalents become available at the Q(o) site, are rapid compared to the rate-determining step. Activation energies of partial reactions that contribute to movement of the iron sulfur protein have been measured and shown to be lower than the high activation barrier associated with quinol oxidation. We conclude that the movement is not the source of the activation barrier. We estimate the occupancies of different positions for the iron sulfur protein from the crystallographic electron densities and discuss the parameters determining the binding of the iron sulfur protein in different configurations. The low activation barrier is consistent with a movement between these locations through a constrained diffusion. Apart from ligation in enzyme-substrate or inhibitor complexes, the binding forces in the native structure are likely to be < = RT, suggesting that the mobile head can explore the reaction interfaces through stochastic processes within the time scale indicated by kinetic measurements.

Topics: Animals; Binding Sites; Crystallography; Cytochrome b Group; Cytochromes c1; Electron Transport Complex III; Iron-Sulfur Proteins; Kinetics; Oxidation-Reduction; Protein Conformation; Temperature; Thermodynamics; Thiazoles; Ubiquinone

Topics: Animals; Binding Sites; Cattle; Crystallization; Crystallography, X-Ray; Cytochromes c1; Diffusion; Dimerization; Electron Transport; Electron Transport Complex III; Hydrogen Bonding; Iron-Sulfur Proteins; Mitochondria, Heart; Oxidation-Reduction; Protein Conformation; Protein Structure, Secondary; Protons; Ubiquinone

Deletion of QCR9, the nuclear gene encoding subunit 9 of the mitochondrial cytochrome bc1 complex in Saccharomyces cerevisiae, results in inactivation of the bc1 complex and inability of the yeast to grow on non-fermentable carbon sources. The loss of bc1 complex activity is due to loss of electron transfer activity at the ubiquinol oxidase site (center P) in the complex. Electron transfer at the ubiquinone reductase site (center N), is unaffected by the loss of subunit 9, but the extent of cytochrome b reduction is diminished. This is the first instance in which a supernumerary polypeptide, lacking a redox prosthetic group, has been shown to be required for an electron transfer reaction within the cytochrome bc1 complex.

Topics: Cytochrome b Group; Cytochromes c1; Electron Transport; Electron Transport Complex III; Intracellular Membranes; Macromolecular Substances; Mitochondria; Oxidation-Reduction; Saccharomyces cerevisiae; Structure-Activity Relationship; Ubiquinone

Myxothiazol, an antibiotic from Myxococcus fulvus, which inhibits mitochondrial respiration in the bc1 complex of the respiratory chain, has effects on the redox components of isolated succinate-cytochrome c reductase complex which suggest that it interacts with both cytochrome b and the iron-sulfur protein of the bc1 complex. The inhibitor appears to increase the midpoint potentials of cytochromes b-562 and b-566, as indicated by an increase in their reducibility by the succinate/fumarate couple. It also causes a red shift in the optical spectrum of ferrocytochrome b-566, as reported previously (Becker, W. F., Von Jagow , G., Anke , T., Steglisch , W. (1981) FEBS Lett. 132, 329-333). This red shift is enhanced by Triton X-100, and there is no shift in the spectrum of b-562. These results are consistent with evidence that mutations conferring myxothiazol resistance in yeast map to the mitochondrial gene for cytochrome b ( Thierbach , G., and Michaelis, G. (1982) Mol. Gen. Genet. 186, 501-506). In addition, myxothiazol has effects on reduction of the cytochromes b and c1 by succinate or ubiquinol which are identical to those caused by removal of the iron-sulfur protein from the bc1 complex. It blocks reduction of cytochrome c1 during single and multiple turnovers of the bc1 complex, but does not block reduction of the b cytochromes. In the presence of antimycin, it blocks reduction of both cytochromes b and c1. In contrast to antimycin, myxothiazol inhibits oxidant-induced reduction of both b cytochromes and does not inhibit their oxidation by fumarate. Myxothiazol also inhibits reduction of the iron-sulfur protein by ubiquinol and shifts the gx resonance in the EPR spectrum of the iron-sulfur protein from g = 1.79 to 1.76. It does not affect the midpoint potential of the iron-sulfur protein, but does eliminate the increase in midpoint potential which is caused by inhibitory hydroxyquinones which bind to the iron-sulfur protein. The effects of myxothiazol are consistent with a protonmotive Q cycle pathway of electron transfer in which myxothiazol binds to cytochrome b and displaces quinone from the iron-sulfur protein of the bc1 complex. These results suggest either that a myxothiazol-induced conformational change in cytochrome b is transmitted to a quinone binding site on the iron-sulfur protein, or that there is a quinone binding site which consists of peptide domains from both cytochrome b and iron-sulfur protein.

Topics: Animals; Antifungal Agents; Cattle; Cytochrome b Group; Cytochromes c1; Electron Transport Complex III; Iron-Sulfur Proteins; Kinetics; Metalloproteins; Methacrylates; Mitochondria, Heart; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Oxygen Consumption; Quinone Reductases; Quinones; Succinates; Succinic Acid; Thiazoles; Ubiquinone