Abbreviations: ETP, non-phosphorylating electron transport particle preparation; ETPH, phosphorylating electron transport particle preparation; TMPD, tetramethylphenylenediamine; Complexes I, preparations of NADH-ubiquinone reductase; Complexes II, succinate-ubiquinone reductase; Complexes III, reduced ubiquinone-cytochrome c reductase; Complexes I-III, NADH-cytochrome c reductase; Complexes II-III, succinate-cytochrome c reductase 相似文献
2. In contrast to the wild type, inhibition of electron transport in the mutant has a much higher antimycin titre than effects on cytochromes b (viz., aerobic steadystate reduction; reduction in the presence of substrate, cyanide and oxygen; the ‘red shift’ and lowering of E′0 of cytochrome b-562). Moreover, the titration curve of electron transport is hyperbolic whereas the curves for the reduction are sigmoidal. The conclusion is, that in both mutant and wild type, the actions of antimycin on electron transport and cytochromes b are separable.
3. The red shift in the mutant is more extensive than in the wild type.
4. Cytochrome b-558 and cytochrome b-566 (that absorbs in mutant and wild type at 564.5 nm) do not respond simultaneously to addition of antimycin, indicating that they are two separate cytochromes.
5. The difference between the effect of antimycin on electron transport and cytochromes b reduction is also found in intact cells of the mutant.
6. A model is suggested for the wild-type respiratory chain in which (i) the cytochromes b lie, in an uncoupled system, out of the main electron-transfer chain, (ii) antimycin induces a conformation change in QH2-cytochrome c reductase resulting in effects on cytochrome b and inhibition of electron transport, (iii) a second antimycinbinding site with low affinity to the antibiotic is present, capable of inhibiting electron transport. 相似文献
The b-type cytochrome in Chromatium has an -band maximum at 560 nm and a midpoint oxidation-reduction potential of −5 mV at pH 8.0. The b-type cytochrome in Chlorobium has an -band maximum at 564 nm and an apparent midpoint oxidation-reduction potential near −90 mV.
Chromatophores isolated from both Chromatium and Chlorobium cells catalyze a photoreduction of cytochrome b that is enhanced in the presence of antimycin A. Antimycin A and 2-n-heptyl-4-hydroxyquinoline-N-oxide inhibit endogenous (but not phenazine methosulfate-mediated) cyclic photophosphorylation in Chromatium chromatophores and non-cyclic electron flow from Na2S to NADP in Chlorobium chromatophores. These observations suggest that b-type cytochromes may function in electron transport reactions in photosynthetic sulfur bacteria. 相似文献
2. In the presence of a Co/N2 mixture, the apparent E′0 of cytochrome b270 shifts markedly towards higher potentials (+355 mV); a similar but less pronounced shift is apparent also for cytochrome b150. The effect of CO on the midpoint potential of cytochrome b270 is absent in the respiration deficient mutant M6 which possesses a specific lesion in the CO-sensitive segment of the branched respiratory chain present in the wild type strain.
3. Preparations of spheroplasts with lysozyme digestion lead to the release of a large amount of cytochrome c2 and of virtually all cytochrome cc′. These preparations show a respiratory chain impaired in the electron pathway sensitive to low KCN concentration, in agreement with the proposed role of cytochrome c2 in this branch; on the contrary, the activity of the CO-sensitive branch remains unaffected, indicating that neither cytochrome c2 nor the CO-binding cytochrome cc′ are involved in this pathway.
4. Membranes prepared from spheroplasts still possess a CO-binding pigment characterized by maxima at 420.5, 543 and 574 nm and minima at 431, 560 nm in CO-difference spectra and with an band at 562.5 nm in reduced minus oxidized difference spectra. This membrane-bound cytochrome, which is coincident with cytochrome b270, can be classified as a typical cytochrome “o” and considered the alternative CO-sensitive oxidase. 相似文献
1. 1. Three b-type cytochromes (b557.5, b560, and b562.5), plus a chromophore with an absorption peak at 558 nm at 77 °K, have been found to be associated with the electron transport system of bovine heart mitochondria. The reduced minus oxidized spectra of these components at 77 °K, as well as that of cytochrome c1, have been recorded with a wavelength accuracy of ± 0.1 nm and presented to the nearest 0.5 nm. All the major and β absorption peaks of cytochromes b557.5, b560, b562.5, c1 and c have been shown by fourth derivative analysis to be present in the dithionite-reduced minus oxidized spectra of mitochondria and submitochondrial particles.
2. 2. The distribution of the above components has been studied in the four electron transfer complexes of the respiratory chain. Cytochromes b560, b562.5 and c1, as well as chromophore-558, were found to fractionate into Complex III (reduced ubiquinone-cytochrome c reductase), whereas cytochrome b557.5 was found in Complex II (succinate-ubiquinone reductase).
3. 3. Cytochrome b560 was readily reduced by NADH or succinate, but b562.5 was not reduced by substrates unless the preparation was treated with antimycin A. In antimycin-treated preparations pre-reduction of c1 with ascorbate inhibited the subsequent reduction of b562.5 by substrates. These results indicate that b560 and b562.5 correspond, respectively, to bK and bT previously described by Chance et al.14 (1970, Proc. Natl. Acad. Sci. U.S. 66, 1175–1182).
4. 4. Similar to b560, chromophore-558 can be reduced by substrates in the absence or presence of antimycin A. However, in antimycin-treated preparations, pre-reduction of c1 inhibits its subsequent reduction by substrates. This property is similar to that of b562.5.
5. 5. Cytochrome b557.5, which occurs in Complex II, appears to have a low mid-point potential. It can be reduced with dithionite and oxidized by fumarate or ubiquinone. CO treatment of dithionite-reduced b557.5 neither modified the spectrum of this cytochrome nor diminished the extent of b557.5 reoxidation by fumarate.
6. 6. Antimycin A treatment does not appear to alter the spectra of the above cytochromes. However, small amounts (< 4%) of ethanol or methanol, which are usually added to particles as solvent for antimycin A, have a pronounced effect on the peaks of cytochrome c1. The spectrum of cytochrome c1 at 77 °K as modified by 3% (v/v) ethanol is shown.
The cytochrome b maxima observed in the presence of succinate plus antimycin A were shifted from the 431 and 561 nm positions observed at 23 °C to 427 and 557 nm at 77 °K. Multiple b cytochromes were not apparent.
Unlike other soluble c-type cytochromes, the maximum of cytochrome c555 was not shifted at 77 °K although it was split to give a 551 nm shoulder adjacent to the 555 nm maximum. This lack of a low-temperature blue shift was true for partially purified hemoprotein preparations as well as in situ in the mitochondrial membrane.
Using cytochrome c555-depleted mitochondria, a cytochrome c1 pigment was observed with a maximum at 420 nm and multiple maxima at 551, 556, and 560 nm. After extraction of non-covalently bound heme, the pyridine hemochromogen difference spectrum of cytochrome c555-depleted preparations exhibited an maximum at 553 nm at room temperature.
The reduced rate of succinate oxidation by cytochrome c555-depleted mitochondria and the ferricyanide requirement for the reoxidation of cytochrome c1, even in the presence of antimycin, indicated that cytochrome c555-mediated electron transfer between cytochromes c1 and a+a3 in a manner analagous to that of cytochrome c in mammalian mitochondria. 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
1. 1. Difference spectra of whole cells and of a particulate fraction of a streptomycin-bleached strain of Euglena gracilis showed the presence of a b-type cytochrome, cytochrome b (561 Euglena), and an a-type cytochrome, cytochrome a-type (609 Euglena). The cytochromes were characterized by pyridine hemochromogen formation and were found associated with a particulate fraction enriched with mitochondria.
2. 2. Both b-type and a-type cytochromes were reduced by succinate, oxidized by oxygen and reacted with a soluble c-type cytochrome, cytochrome c-type (556 Euglena), in reversible oxidation-reduction reactions. The steady-state level of reduction for each cytochrome was 92, 22 and 5% of the anaerobic level for the b-type, c-type and a-type cytochrome, respectively.
3. 3. Oxidation of c-type and a-type cytochromes was completely inhibited by cyanide, although respiration of a particulate fraction was only 60% inhibited by the same concentration of cyanide. Antimycin A inhibited respiration by up to 70% but completely inhibited reduction of the c-type cytochrome.
4. 4. The data suggest that electron transfer in the respiratory pathway of Euglena involves the b-, c- and a-type cytochrome in a direct sequence. The cyanide and antimycin A-insensitive oxidation pathway is considered to involve a more direct oxidation of the b-type cytochrome.
Abbreviations: STE medium, 250 mM sucrose, 24 mM Tris-HCI buffer (pH 7.6) and 0.1 mM EDTA 相似文献
1. 1. The functional terminal oxidase of the light-anaerobically grown Rhodopseudomonas spheroides cells was found to be the o-type cytochrome, whereas that of the dark-aerobically grown cells was the a-type cytochrome. When the dark-aerobically grown cells were further incubated under a semianaerobic condition in the dark, the content of the o-type cytochrome was increased in these cells, while the synthesis of the a-type cytochrome appeared to be repressed. In Rhodospirillum rubrum cells, grown either aerobically in the dark or anaerobically in the light, cytochrome o was the sole functional terminal oxidase.
2. 2. Reactions with the a-type and o-type cytochromes from Rhodopseudomonas spheroides and also with the o-type cytochrome from Rhodospirillum rubrum were compared using reduced yeast cytochrome c as substrate. The reaction with the a-type cytochrome was far less sensitive to NaN3 and hydroxylamine than those with the o-type cytochromes, whereas all the reactions were inhibited by KCN in apparently the same manner.
Abbreviations: Rps, Rhodopseudomonas; Rsp, Rhodospirillum; DCIP, 2,6-dichlorophenol-indophenol 相似文献
2. The amounts of cytochromes c1 and aa3 are similar in the mutant and wild type. Cytochrome b-566 could not be detected in low-temperature spectra after reduction with various substrates or dithionite. A b-558 is, however, present.
3. The b-cytochromes in the mutant are not reduced by NADH or succinate during the steady state even after addition of ubiquinone-1. QH2-3: cytochrome c reductase activity is very low and succinate oxidation is highly stimulated by phenazine methosulphate.
4. Antimycin does not bind to either oxidized or reduced mitochondrial particles of the mutant.
5. In contrast to the b-cytochromes of the wild type, b-558 in the mutant reacts with CO.
6. Cytochromes aa3, c and c1 are partly reduced in aerated submitochondrial particles isolated from the mutant and the EPR signal of Cu (II), measured at 35°K, is detectable only after the addition of ferricyanide. In the mutant, a signal with a trough at g = 2.01 is found, in addition to the signal at g = 1.98 found in the wild type.
7. The ATPase activity of particles isolated from the mutant is much lower than in the wild type but is still inhibited by oligomycin. 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
In intact cells, cytochrome bT is reduced immediately after anaerobiosis or cyanide treatment, and rapidly oxidized when uncoupler is added. Addition of antimycin A instead of uncoupler to the anaerobic cells causes oxidation of mainly cytochrome bT while addition of antimycin A to the aerobic cells results in a reduction of the cytochrome bT. 相似文献
2. The antimycin-induced extra reduction of cytochrome b is always dependent on the initial presence of an oxidant such as oxygen. After removal of the oxidant this effect remains or is partially (under some conditions even completely) abolished depending on the redox potential of the substrate used and the leak through the antimycin-inhibited site.
3. The increased reduction of cytochrome b induced by oxidant in the presence of antimycin involves all three spectroscopically resolvable b components (b-562, b-566 and b-558.
4. Redox mediators with an actual redox potential of less than 100–170 mV cause the oxidation of cytochrome b reduced under the influence of antimycin and oxidant.
5. Redox titrations of cytochrome b with the succinate/fumarate couple were performed aerobically in the presence of cyanide. In the presence of antimycin two b components are separated potentiometrically, one with an apparent midpoint potential above 80 mV (at pH 7.0), outside the range of the succinate/fumurate couple, and one with an apparent midpoint potential of 40 mV and an n value of 2. In the absence of antimycin cytochrome b titrates essentially as one species with a midpoint potential of 39 mV (at pH 7.0) and n = 1.14.
6. The increased reducibility of cytochrome b induced by antimycin plus oxidant is considered to be the result of two effects: inhibition of oxidation of ferrocytochrome b by ferricytochrome c1 (the effect of antimycin), and oxidation of the semiquinone form of a two-equivalent redox couple such as ubiquinone/ubiquinol by the added oxidant, leading to a decreased redox potential of the QH2/QH• couple and reduction of cytochrome b. 相似文献
2. A shoulder on this spectrum around 662 nm is due to a component different from chlorophyll b. This component may well be identical with the chlorophyll a form, chlorophyll a (665).
3. The 77°K chlorophyll b absorption spectra in the nonfractionated photosyn-thetic pigment apparatus and in fractions mainly representing Photosystems 1 or 2 are not significantly different.
4. The aerobic irreversible photobleaching of chlorophyll b was studied in the intact pigment complex as well as in fractions mainly consisting of Photosystem 1 or 2. A two-step photobleaching was observed in all cases. The time-course of this bleaching was not significantly different for chlorophyll b in both fractions.
5. These results do not indicate that more than a single chlorophyll b complex occurs in vivo. 相似文献
The cytochromes b in lyophilized beef-heart mitochondria are more readily accessible to electrons from NADH, and in the presence of antimycin and NADH a complete and stable reduction is obtained under both aerobic and anaerobic conditions. Gradual addition of rotenone under these conditions causes re-oxidation of cytochromes b in which oxidation of cytochromes b-558 and b-566 precedes that of cytochrome b-562.
It is concluded that (1) the effect of antimycin in the presence of oxygen involves all three cytochromes b, (2) the reducibility of the cytochromes b in the aerobic steady state of antimycin-treated mitochondria is dependent upon the potential of the substrate redox couple registered on the cytochromes, and (3) the midpoint potential of cytochrome b-562 in the presence of antimycin is higher than that of cytochrome b-558 or b-566. 相似文献
Cytochrome oxidase Escherichia coli Cytochrome b-d complex Bacterial electron transport Low-temperature technique 相似文献
2. Through an electrostatic interaction of the bacteriochlorophyll reaction-center complex and mammalian cytochrome c, an intimate contact between the two components resulted, and a collision-independent electron-transfer with a halftime of 25 μsec can be attained by laser-flash excitation. The absorbance changes at 870 and 550 nm indicated a good stoichiometry of the reaction. The oxidation of the c-type cytochrome in cells of Rps. spheroides (R-26 mutant) has a halftime of 12 μsec.
3. The portion of P870 which recovered rapidly was closely related to the mole ratio of cytochrome/P870. Complete recovery with a halftime of 25 μsec occurred when the cytochrome/P870 ratio was above approx. 10. At cytochrome/P870 ratios lower than 10, only the fraction of the reaction-center complex which have cytochromes bound at the active site can recover with the rapid decay time. Ultrafiltration measurements showed that each particle of the reaction-center complex can bind approx. 24 cytochrome molecules.
4. An electro static interaction is expected simply from the large difference between the isoelectric points of cytochrome c ( 10) and that of the reaction-center complex (4.1 measured by electro-focusing). The electro static interaction was further evidenced by the effects of pH, ionic strength, and by polylysine displacement of binding sites on the coupled oxidation of ferrocytochrome c by P870. From the limiting polylysine concentration giving complete blocking of cytochrome coupling, it was calculated that each reaction-center complex with a particle weight of 6.5×105 contained approx. 500 negative charges.
5. Arrhenius plot of the first-order rate constants vs. the reciprocal absolute temperature yielded an activation energy of 12 kcal/mole for the cytochrome/P870 reaction, which is presumably the energy needed for cytochrome to achieve the most favorable orientation for the rapid electron transfer. Below the freezing temperature of the sample, the cytochrome reaction appeared to be uncoupled. The temperature dependence is consistent with the effect of viscosity on the reaction rate.
6. Double flash excitations spaced 200 μsec apart showed that at a cytochrome/P870 ratio of 24, the first flash caused maximum oxidation, indicating that all the reaction-center particles have at least one cytochrome attached to the active site. However, only 60% of the particles have a second cytochrome closely attached and capable of undergoing the rapid electron transport. 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