2. The kinetics of the disappearance of the 648-nm band of cytochrome d with excess cyanide deviates from first-order kinetics at lower temperatures (22 °C) indicating that at least two conformations of the enzyme are involved. At higher temperatures (32 °C) the observed kinetics of the cyanide reaction are first order with a kon = 0.7 M−1·s−1 and with an estimated koff of approximately 5·10−5 s−1.
3. The value of the koff (7·10−4−14·10−4 s−1 at 32 °C) determined from the rate of reduction of cyanocytochrome d by Na2S2O4 or NADH is one order of magnitude larger than the koff value found when the enzyme is in its oxidized state.
4. No effect of cyanide is found on the spectrum of cytochrome a1. 相似文献
2. The transfer of reducing equivalents into isolated mitochondria is stimulated by ATP and by electron transport. The effect of ATP is inhibited by oligomycin. The effect of electron transport is inhibited by uncouplers.
3. Uncoupling of the mitochondria is required for rapid transfer of reducing equivalents out of the mitochondria.
4. A glutamate-stimulated entry of aspartate into energized mitochondria suggests that the malate-aspartate shuttle is to some extent reversible even in a high energy state of the mitochondria.
5. It is concluded that the malate-aspartate shuttle contributes to the formation of the skewed redox situation across the inner mitochondrial membrane, which has a more reduced inside. 相似文献
2. It also inhibits the efflux of intramitochondrial adenine nucleotides induced by this treatment.
3. It is concluded that the inhibitory action of bongkrekic acid on the adenine nucleotide translocator is favoured by the presence of endogenous adenine nucleotides. 相似文献
2. In the presence of Mg2+ the nitreno-ATP is bound to both the and β subunits, mainly (63%) to the subunits.
3. After successive photolabelling of F1 with 8-azido-ATP (no Mg2+) and 8-azido-ADP (with Mg2+) 4 mol label is bound to F1, 2 mol to the and 2 mol to the β subunits.
4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F1 previously labelled with 8-nitreno-ADP. It is concluded that binding to the -subunits hinders binding to the β subunits.
5. F1 that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F1.
6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F1. 相似文献
2. Adenine nucleotide translocation in C. utilis mitochondria is an exchange-diffusion process. The whole pool of internal adenine nucleotides is exchangeable, ADP being the most readily exchangeable nucleotide. The rate of mitochondrial ADP exchange, but not the Km value, depends on growth conditions. At 0 °C, the rate is about 3 to 4 nmoles ADP/min per mg protein for mitochondria obtained from yeast grown in the presence of 1.5% glucose; it rises to 11.5 nmoles when glucose is replaced by 3% ethanol in the growth medium. The Km value for ADP is 2 μM. The Q10 is about 2 between 0 and 20 °C. Among other exchangeable adenine nucleotides are ATP, dADP and the methylene and the hypophosphate analogues of ADP. Unlike mammalian mitochondria, C. utilis mitochondria are able to transport external UDP by a carboxyatractyloside-sensitive process.
3. Under conditions of oxidative phosphorylation (phosphate and substrate present in an aerated medium), added ADP is exchanged with internal ATP. A higher ATP/ADP ratio was found in the extramitochondrial space than in the intramito-chondrial space. The difference between the calculated phosphate potentials in the two spaces was 0.9–1.7 kcal/mole.
4. Atractyloside, carboxyatractyloside, bongkrekic acid and palmityl-CoA inhibit mitochondrial adenine nucleotide translocation in C. utilis as they do in mammalian mitochondria, but 2 to 4 times less efficiently. The inhibition due to atractyloside or palmityl-CoA is competitive with respect to ADP whereas that due to bongkrekic acid and carboxyatractyloside is non-competitive. Carboxyatractyloside and atractyloside inhibitions are additive. The apparent Kd for the binding of [35S]-carboxyatractyloside and [14C]bongkrekic acid is 10–15 nM and the concentration of sites 0.4–0.6 nmole/mg protein in both cases. [35S]Carboxyatractyloside binding is competitively displaced by atractyloside and vice versa.
5. Binding of [14C]ADP has been carried out with mitochondria depleted of their endogenous adenine nucleotides by incubation with phosphate and Mg2+ at 20 °C. The amount of bound [14C]ADP which is atractyloside removable is 0.08–0.16 nmole/mg protein.
6. The rate of ADP transport is quite different in mitochondria isolated from C. utilis, according to whether it is grown on glucose, or on ethanol or in the presence of chloramphenicol; for instance, it decreases by 10 times when 3% ethanol in the growth medium is replaced by 10% glucose and by 5 times when chloramphenicol is added to the medium. These variations are accompanied by parallel variations in cytochrome aa3. The number of atractyloside-sensitive ADP binding sites is not modified by the above conditions of culture, nor the number of [35S]carboxyatractyloside binding sites. The affinity for ADP is apparently not significantly modified, nor the size of the endogenous adenine nucleotide pool. In contrast to glucose repression or chloramphenicol inhibition, semi-anaerobiosis in C. utilis lowers significantly the mitochondrial binding capacity for carboxyatractyloside. Strict anaerobiosis in S. cerevisiae results in a practical loss of the cytochrome oxidase activity, and also of the carboxyatractyloside and ADP binding capacity. Transition from anaerobiosis to aerobiosis restores the cytochrome oxidase activity and the ADP and carboxyatractyloside binding capacities. 相似文献
2. Hydrated electrons do not readily reduce the heme of cytochrome c oxidase. This observation supports our previous conclusion that heme a is not directly exposed to the solvent.
3. In a mixture of cytochrome c and cytochrome c oxidase, cytochrome c is first reduced by hydrated electrons (k = 4 · 1010 M−1 · s−1 at 22 °C and pH 7.2) after which it transfers electrons to cytochrome c oxidase with a rate constant of 6 · 107 M−1 · s−1 at 22 °C and pH 7.2.
4. It was found that two equivalents of cytochrome c are oxidized initially per equivalent of heme a reduced, showing that one electron is accepted by a second electron acceptor, probably one of the copper atoms of cytochrome c oxidase.
5. After the initial reduction, redistribution of electrons takes place until an equilibrium is reached similar to that found in redox experiments of Tiesjema, R. H., Muijsers, A. O. and Van Gelder, B. F. (1973) Biochim. Biophys. Acta 305, 19–28. 相似文献
2. Upon subsequent incubation at 38° in oxygenated medium, these changes were partially reversed. In the hepatoma, the reaccumulation of K+ was equally efficient in phosphate or bicarbonate medium, and in the presence and absence of glucose. Ca2+ was extruded in bicarbonate, but not in phosphate medium, and its extrusion was reduced in the presence of glucose.
3. When respiration was inhibited in the presence of glucose, K+ transport by the hepatoma continued to an extent which varied with the glycolytic activity of the slices, suggesting that the rate of ATP synthesis was a limiting factor under these conditions.
4. In the absence of glucose, the transport of Na+ and K+ was completely stopped by respiratory inhibition. However, more than 50% of the O2 uptake had to be inhibited before any effect on transport was observed, suggesting that the rate of synthesis of ATP from endogenous respiration is in excess of that required to maintain transport.
5. Inhibition of transport by ouabain was accompanied by a 30% fall in the rate of endogenous respiration, and by a fall of 33% in the rate of glycolysis in the presence of cyanide plus glucose.
6. Comparison of the minimum rates of respiration and of glycolysis (in the presence of glucose plus cyanide) required to maintain the maximal extent of K+ transport in the hepatoma slices, suggests that ATP derived from oxidative phosphorylation or from anaerobic glycolysis is equally efficient as a source of energy for ion transport. 相似文献
2. A similar signal found in a number of iron—sulfur containing proteins was found by quantitative EPR estimations to exist in a variable but substantial concentration when compared to the intensity of the reduced g = 1.9 type EPR resonance.
3. Reaction of the phosphorylating particles with excess potassium ferricyanide resulted in an alteration of the initial g = 2.011 iron signal resulting in the detection by microwave power studies of at least two different iron species which exhibited major g-values at 1.992 and 2.027. 相似文献
2. Exogenous NADH is a very good substrate for yeast mitochondrial respiration and apparently has a very low Km. However, one-third of the added NADH is not available for oxidation probably due to some form of compartmentation. Studies of both oxygen uptake and the redox changes of cytochrome b show complete oxidation of two-third of the added NADH.
3. Difference spectra of yeast mitochondria at liquid-nitrogen temperatures show all the characteristic peaks of cytochromes a (600 nm), b (558, 525 and 428 nm), c1 (552 nm) and c (545 and 516 nm).
4. The reduction of cytochrome b by dicumarol in antimycin A inhibited mitochondria provides evidence for an energy conservation site on the substrate side of cytochrome b.
5. In the absence of added ADP, the oxidation of malate and pyruvate occurs in the yeast mitochondria in a new respiratory state (State X) where the oxygen uptake occurs at State 4 rate but the redox level of the flavins, cytochrome b and c are similar to State 3. State X respiration is believed to be due to depletion of the high energy intermediate C I caused by the substrate anions accumulation.
6. The responses of yeast mitochondria to Ca2+ are qualitatively similar to those in rat liver mitochondria, particularly with respect to respiratory stimulation, membrane alkalinization and its accumulation in the mitochondria with succinate as the substrate in the presence and absence of acetate. 相似文献
2. Yeast mitochondria treated with filipin complex, or purified Filipin II, exhibit “uncoupled” succinate oxidation and inhibited -ketoglutarate oxidation. Maximum filipin effect is observed at a concentration of 4 mM Filipin II. Rat-liver mitochondria are more sensitive to filipin than yeast mitochondria, and respiratory inhibition is observed regardless of substrate.
3. In liver mitochondria filipin-inhibited respiration is not relieved by Mg2+, K+, Ca2+ or 2,4-dinitrophenol, but is reversed by cytochrome c.
4. It is proposed that filipin treatment leads to altered membrane permeability and that respiratory inhibition is due to a loss of endogenous respiratory cofactors or an inactivation of primary dehydrogenases. The filipin-uncoupled yeast respiration may likewise be attributed to an altered phosphate permeability of the yeast mitochondrial membranes. 相似文献
2. The rate of the peroxidation reaction is proportional to the concentration of H2O2 and ferricytochrome c but is independent of the concentration of ferrocytochrome c in the concentration ranges studied.
3. Integration of the rate equation, d[c3+]/dt = k[c3+][H2O2], gives a theoretical expression which fits the experimental time courses for the ferrocytochrome c peroxidation reaction.
4. No direct spectral evidence was found for the formation of a catalytically active ferricytochrome c-H2O2 derivative. Kinetic evidence is presented, however, which indicates the existence of such an intermediate.
5. Ferricytochrome c was more susceptible than ferrocytochrome c to an apparent degradation reaction caused by excess H2O2, thus supporting the idea that the cytochrome c heme iron is more accessible in the oxidized form. 相似文献
2. The purified enzyme preparation was free from hydrogenase activity. Either NADH or NADPH served as an electron donor for the reduction of benzyl viologen. This reaction is reversible.
3. The essential thiol groups of the enzyme are protected since they do not react with N-ethylmaleimide and p-chloromercuribenzoate inhibits only after it has been preincubated with the enzyme. 相似文献
2. The change in dye absorbance was linear with the membrane potential change induced either by light excitation or by application of diffusion potential by adding valinomycin in the presence of K+ concentration gradient.
3. It was estimated that chromatophore membrane became 40–60 mV and 110–170 mV inside positive upon single and multiple excitations with single-turnover flashes, respectively, from the responses of the dye and the carotenoid.
4. Electron transfers between cytochrome c-555 or c-552 and reaction center bacteriochlorophyll dimer (BChl2) and between BChl2 and the primary electron acceptor were concluded to be electrogenic from the redox titration of the dye response.
5. No dye response which corresponded to the change of redox level of cytochrome b was observed in the titration curve. Addition of antimycin A slightly decreased the dye response.
6. The dye response was decreased under phosphorylating conditions.
7. From the results obtained localization of the electron transfer components in chromatophore membrane is discussed. 相似文献
2. Preparations of cytochrome b active in reconstitution contained 5–28% native cytochrome b, as adjudged by reducibility with succinate in the reconstituted preparation and by lack of reaction with CO. Preparations of cytochrome b containing no native cytochrome b according to this criterion were inactive in reconstitution.
3. With a fixed amount of cytochrome b, the activity of the reconstituted preparation increased with increasing amounts of cytochrome c1 until a ratio of about 2b (total): 1c1 (allowing for the cytochrome c1 present in the cytochrome b preparation) was reached.
4. The amount of antimycin necessary for maximal inhibition of the reconstituted enzyme is a function of the amount of the cytochrome b and is independent of the amount of cytochrome c1. It is equal to about one half the amount of native cytochrome b.
5. Preparations of intact or reconstituted succinate-cytochrome c reductase or of cytochrome b completely quench the fluorescence of added antimycin, until an amount of antimycin equal to onehalf the amount of native cytochrome b present was added. Antimycin added in excess of this amount fluoresces with normal intensity. The quenching is only partial in the presence of Na2S2O4. Denatured cytochrome b does not quench the fluorescence.
6. Since preparations of cytochrome b active in reconstitution contained cytochrome c1 in an amount exceeding one half the amount of native cytochrome b present in the preparation, there is no evidence that native cytochrome b has been resolved from cytochrome c1. The stimulatory action of cytochrome c1 may be due to the restoration of a damaged membrane conformation.
7. Based on the assumption that the bc1 segment of the respiratory chain contains 2b:1c1:1 antimycin-binding sites, the specific quenching of antimycin fluorescence by binding to cytochrome b enables an accurate determination of the absorbance coefficients of cytochromes b and c1. These are 25.6 and 20.1 mM−1×cm−1 for the wavelength pairs 563–577 nm and 553–539 nm, respectively, in the difference spectrum reduced minus oxidized. 相似文献
2. The oxygen concentration required to provide half-maximal reduction of cytochrome c (p50c) ranges from 0.27 to 0.03 μM (0.2-0.02 Torr) depending upon the metabolic activity. There is a linear increase of the p50c value with increasing respiratory rate.
3. The fraction of the normoxic respiration that is observed at p50c is 70–90% under State 4 conditions, but is 30% under State 3 conditions.
4. The oxygen requirement for half-maximal reduction of pyridine nucleotide (p50PN) varies less than p50c, being 0.08 μM in State 3 and 0.06 μM in the uncoupled state.
5. The ability of the mitochondria to exhibit an energy-linked reduction of pyridine nucleotide by succinate disappears at an oxygen concentration of 0.09 μM (0.06 Torr). Below this oxygen concentration, endogenous Ca2+ begins to be released from the mitochondria. Thus, the critical oxygen concentration for bioenergetic function of mitochondria corresponds approximately to 50% reduction of pyridine nucleotide (p50PN). 相似文献