Thermodynamic properties of the semiquinone and its binding site in the ubiquinol-cytochrome c (c2) oxidoreductase of respiratory and photosynthetic systems (original) (raw)
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The Journal of biological chemistry, 1979
Although the energy conserving membranes of the photosynthetic bacterium Rhodopseudomonas sphaeroides contain a 25 (+/- 3)-fold molar excess of ubiquinone over the photochemical reaction center, the activity of the ubiquinone-cytochrome b-c2 oxidoreductase is unaffected by quinone extraction until only 3, or at most 4, ubiquinones remain; only then does further extraction prevent the function of the oxidoreductase. Since 2 of these last ubiquinones are integral parts of the photochemical reaction center, we conclude that the ubiquinone-cytochrome b-c2 oxidoreductase requires only 1, or at most 2, molecules of ubiquinone-10 for its function. Earlier kinetic data identified a major electron donor to ferricytochrome c2 as a single molecule (known as Z) which requires 2 electrons and 2 protons for its equilibrium reduction. Hence, we identify a single molecule of quinone, probably ubiquinone-10 in a special environment, as a major electron donor to ferricytochrome c2 in the ubiquinone c...
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1986
- Ubiquinone-10 can be extracted from iyophilized chromatophores of Rhodobacter sphaeroides (previously called Rhodopseudomonas sphaeroides) without significant losses in other components of the electron-transfer chain or irreversible damages in the membrane structure. The pool of ubiquinone can be restored with exogenous UQ-10 to sizes larger than the ones in unextracted membranes. (2) The decrease in the pool size has marked effects on the kinetics of reduction of cytochrome b-561 induced by a single flash of light and measured in the presence of antimycin. The initial rate of reduction, which in unextracted preparations increases on reduction of the suspension over the E h range between 170 and 100 mV (pH 7), is also stimulated in partially UQ-depleted membranes, although at more negative Eh's. When the UQ pool is completely extracted the rate of cytochrome (Cyt) b-561 reduction is low and unaffected by the redox potential.
Journal of Biological Chemistry, 1999
Activation energies for partial reactions involved in oxidation of quinol by the bc 1 complex were independent of pH in the range 5.5-8.9. Formation of enzyme-substrate complex required two substrates, ubihydroquinone binding from the lipid phase and the extrinsic domain of the iron-sulfur protein. The activation energy for ubihydroquinone oxidation was independent of the concentration of either substrate, showing that the activated step was in a reaction after formation of the enzyme-substrate complex. At all pH values, the partial reaction with the limiting rate and the highest activation energy was oxidation of bound ubihydroquinone. The pH dependence of the rate of ubihydroquinone oxidation reflected the pK on the oxidized iron-sulfur protein and requirement for the deprotonated form in formation of the enzyme-substrate complex. We discuss different mechanisms to explain the properties of the bifurcated reaction, and we preclude models in which the high activation barrier is in the second electron transfer or is caused by deprotonation of QH 2 . Separation to products after the first electron transfer and movement of semiquinone formed in the Q o site would allow rapid electron transfer to heme b L . This would also insulate the semiquinone from oxidation by the iron-sulfur protein, explaining the efficiency of bifurcation. . The abbreviations used are: bc 1 complex, ubihydroquinone:cytochrome c (or c 2 ) oxidoreductase; b H , higher potential cyt b heme; b L , lower potential cyt b heme; cyt, cytochrome; DAD, 2,3,5,6-tetramethylp-phenylenediamine (E m 245 mV); DQ, duroquinone; ISP, iron sulfur protein; ISP ox , ISP red , oxidized and reduced states of the iron sulfur protein; HEPPS, N-2-hydroxyethylpiperazine-NЈ-3-propane sulfonic acid; HEPES, N-2-hydroxyethylpiperazine-NЈ-2-ethane sulfonic acid; MES, 4-morpholineethanesulfonic acid; MOA-, -methoxyacrylate, or a similar group, the pharmacophore of a class of inhibitors acting at the Q o -site; MOPS, 4-morpholinepropane sulfonic acid; 1,2 NQ, 1,2 naphthoquinone (E m 145 mV); 1,4 NQ, 1,4 naphthoquinone (E m 145 mV); 2-OH-1,4 NQ, 2-hydroxy, 1,4 naphthoquinone (E m 145 mV); p-BQ, p-
PLANT PHYSIOLOGY, 1985
The functional and thermodynamic characteristics of the ubiquinolcytochrome (Cyt) c oxidoreductase in a Cyt b/c,-enriched fraction (defined S-1) isolated from Jerusalem artichoke mitochondria (JAM) (Helianthus tuberosus), have been analyzed. Fraction S-1, obtained through deoxycholate-KCI fractionation procedure, contained one Cyt ofc type (formally cl with Em7.0 of +240 millivolts), two b type Cyt with Em7.. values of +100 and-25 millivolts, ferredoxin-like centers presumably linked to succinic-and NADH-dehydrogenases, and a Rieske-type iron sulfur center (gy = 1.89). The ubiquinol-dependent Cyt c reduction by fraction S-I showed sensitivity to antimycin A, myxothiazol, and n-2-hepthyl-1hydroxyquinoline N-oxide with I50 of 12 nanomolar, 30 nanomolar, and 0.1 micromblar, respectively. Oxidation-induced extra b type reduction, a widespread phenomenon of bacterial and mitochondrial respiratory systems, has also been observed in both intact mitochondria and S-1 fraction. The data seem to blur previous experiments in which both spectral and functional differences between higher plant and mammalian mitochondria have been underlined.
European Journal of Biochemistry, 1994
The dependence of electron flux through quinone-reducing and quinol-oxidizing pathways on the redox state of the ubiquinone (Q) pool was investigated in plant mitochondria isolated from potato (Solanurn tuberosum cv. Bintje, fresh tissue and callus), sweet potato (Ipornoea batatas) and Arum italicurn. We have determined the redox state of the Q pool with two different methods, the Q-electrode and Q-extraction techniques. Although results from the two techniques agree well, in all tissues tested (with the exception of fresh potato) an inactive pool of QH, was detected by the extraction technique that was not observed with the electrode. In potato callus mitochondria, an inactive Q pool was also found. An advantage of the extraction method is that it permits determination of the Q redox state in the presence of substances that interfere with the Q-electrode, such as benzohydroxamate and NADH. We have studied the relation between rate and Q redox state for both quinol-oxidizing and quinone-reducing pathways under a variety of metabolic conditions including state 3, state 4, in the presence of myxothiazol, and benzohydroxamate. Under state 4 conditions or in the presence of myxothiazol, a non-linear dependence of the rate of respiration on the Q-redox state was observed in potato callus mitochondria and in sweet potato mitochondria. The addition of benzohydroxamate, under state 4 conditions, removed this non-linearity confirming that it is due to activity of the cyanide-resistant pathway. The relation between rate and Q redox state for the external NADH dehydrogenase in potato callus mitochondria was found to differ from that of succinate dehydrogenase. It is suggested that the oxidation of cytoplasmic NADH in vivo uses the cyanide-resistant pathway more than the pathway involving the oxidation of succinate. A model is used to predict the kinetic behaviour of the respiratory network. It is shown that titrations with inhibitors of the alternative oxidase cannot be used to demonstrate a pure overflow function of the alternative oxidase.
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.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1977
We have examined the thermodynamic properties of the physiological electron donor to ferricytochrome c2 in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides. This donor (Z), which is capable of reducing the ferricytochrome with a halftime of 1-2 ms under optimal conditions, has an oxidationreduction midpoint potential of close to 150 mV at pH 7.0, and apparently requires two electrons and two protons for its equilibrium reduction. The state of reduction of Z, which may be a quinone • protein complex near the inner (cytochrome c2) side of the membrane, appears to govern the rate at which the cyclic photosynthetic electron transport system can operate. If Z is oxidized prior to the flash-oxidation of cytochrome c2, the re-reduction of the cytochrome takes hundreds of milliseconds and no third phase of the carotenoid bandshift occurs. In contrast if Z is reduced before flash activation, the cytochrome is rereduced within milliseconds and the third phase of the carotenoid bandshift occurs. The prior reduction of Z also has a dramatic effect on the uncoupler sensitivity of the rate of electron flow; if it is oxidized prior to activation, uncoupler can stimulate the cytochrome rereduction after several turnovers by less than tenfold, but if it is reduced prior to activation, the stimulation after several turnovers can be as dramatic as a thousandfold. The results suggest that Z plays a central role in controlling electron and proton movements in the ubiquinone cytochrome b-c2 oxido-reductase.