Abbreviations: HOQNO, 2-heptyl-4-hydroxy quinoline-N-oxide; PMS, phenazine methosulfate 相似文献
1. Circular dichroism spectra of the cytochromes in membrane fragments derived from sonicated beef heart mitochondria have been obtained in the wavelength region 400–480 nm in which the major absorbance maxima of the heme prosthetic groups are found.
2. 2. Cytochrome oxidase in the mitochondrial membrane fragments has a band of positive ellipticity at 426 nm in the oxidized form and a pronounced band of positive ellipticity at 445 nm in the reduced form. The reduced-minus-oxidized difference molar ellipticity at 445 nm, Δ[θ]445 is 3.0·105 degree·cm−2·dmole−1 heme a for membrane-bound oxidase compared to 1.6·105 degree·cm−2·dmole−1 heme a for the purified oxidase. The membrane-bound oxidase in the reduced form also appears to have a band of negative ellipticity at 426 nm not found in the purified oxidase.
3. 3. When reduced with succinate in the presence of cyanide and oxygen, cytochrome oxidase in the membrane fragments has a positive band at 442 nm very similar to that observed with the purified oxidase.
4. 4. Cytochrome c, which has a positive band at 426 nm in the purified form when reduced, appears to have a negative band at this wavelength in the mito-chondrial membrane fragments which contributes to the pronounced negative band at 426 nm observed in the membrane fragments reduced with succinate in anaerobiosis. There is no evidence for a contribution to the CD spectra of the membrane fragments from cytochrome c1 or from cytochrome b561 in either the oxidized or the reduced form.
5. 5. Cytochrome b566 in the mitochondrial membrane fragments has no detectable CD spectrum in the oxidized form, but has a small positive band at 427 nm and a small negative band at 436 nm in the reduced form. The same CD spectrum is observed with cytochrome b566 reduced with succinate in the presence of antimycin A or 2-heptyl-4-hydroxyquinoline-N-oxide. The same increase in positive ellipticity is observed at 427 nm in the mitochondrial membrane fragments, treated with oligomycin to restore energy coupling, when cytochrome b566 is reduced with succinate in the energized membrane, as is observed in the inhibitor-treated membrane fragments. The absence of a pronounced conformational change in cytochrome b566 on energization, as revealed by its CD spectrum, favors the concept that its reduction by succinate in the energized state is due to reversed electron transport rather than an intrinsic shift in the cytochrome's midpoint redox potential.
1. 1. Cycles of oxidation followed by reduction at pH 7.2 have been induced in uncoupled anaerobic mung bean mitochondria treated with succinate and malonate by addition of oxygen-saturated medium. Under the conditions used, cytochromes b557, b553, c549 (corresponding to c1 in mammalian mitochondria) and ubiquinone are completely oxidized in the aerobic state, but become completely reduced in anaerobiosis.
2. 2. The time course of the transition from fully oxidized to fully reduced in anaerobiosis was measured for cytochromes c549, b557, and b553. The intramitochondrial redox potential (IMPh) was calculated as a function of time for each of the three cytochromes from the time course of the oxidized-to-reduced transition and the known midpoint potentials of the cytochromes at pH 7.2. The three curves so obtained are superimposable, showing that the three cytochromes are in redox equilibrium under these conditions during the oxidized-to-reduced transition.
3. 3. This result shows that the slow reduction of cytochrome b557 under these conditions, heretofore considered anomalous, is merely a consequence of its more negative midpoint potential of +42 mV at pH 7.2, compared to +75 mV for cytochrome b553 and +235 mV for cytochrome c549. Cytochrome b557 is placed on the low potential side of coupling site II and transfers electrons to cytochrome c549 via the coupling site.
4. 4. The time course of the transition from fully oxidized to fully reduced was also measured for ubiquinone. Using the change in intramitochondrial potential IMPh with time obtained from the three cytochromes, the change in redox state of ubiquinone with IMPh was calculated. When replotted as IMPh versus the logarithm of the ratio (fraction oxidized)/(fraction reduced), two redox components with n = 2 were found. The major component is ubiquinone with a midpoint potential Em7.2 = + 70 mV. The minor component has a midpoint potential Em7.2 = − 12 mV; its nature is unknown.
Abbreviations: IMPh, intramitochondrial potential, referred to the normal hydrogen electrode; Em7.2, midpoint potential at pH 7.2 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.
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
Properties of cytochrome b6: Cytochrome b6 migrated in undenatured form as a single band in disc electrophoresis (pH 8, 7 or 8.9). None of the limited, accepted properties of the cytochrome in particles was altered by the purification procedure: Reduced b6 has absorption maxima (22 °C) at 434, 536, and 563 nm; at −199 °C the a absorption region shows two peaks of equal intensity at 561 and 557 nm. Cytochrome b6 is reduced by dithionite (not by ascorbate) and is autoxidizable. The prosthetic group of b6 is protohaemin and is fully extractable by acid-acetone. No non-haem iron is present. The millimolar extinction coefficient of reduced b6 (563–600nm) per mole of haem is 21. The protein equivalent weight is 40000 g per mole of haem. Cytochrome b4 is an intrinsically aggregatable molecule. The reduced cytochrome does not react with CO except when Triton X-100 is present. 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. 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
H2O → IIbhv → C550 → cyt. b559 → PC → IIahv → Fd → NADP+
Photoreaction IIb involves an electron transfer from water to C550 that does not require plastocyanin and is the first known System II photoreaction resistant to inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) and o-phenanthroline. Cytochrome b559 is reduced by C550 in a reaction that is readily inhibited by DCMU or o-phenanthroline. Thus, the site of DCMU (and o-phenanthroline) inhibition of System II appears to lie between C550 and cytochrome b559. Photoreaction IIa involves an electron transfer from cytochrome b559 and plastocyanin to ferredoxin-NADP+. 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. 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
At − 196 °C, continuous illumination results in a parallel reduction of C-550 and oxidation of cytochrome b559 high potential. With flash excitation, C-550 is reduced, but only a small fraction of cytochrome b559 is oxidized. The specific effect of flash illumination is suppressed if the chloroplasts are preilluminated by one flash at 0 °C.
At − 50 °C, continuous illumination results in the reduction of C-550 but little oxidation of cytochrome b559. However, complete oxidation is obtained if the chloroplasts have been preilluminated by one flash at 0 °C. The effect of preillumination is not observed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.
A model is discussed for the reaction center, with two electron donors, cytochrome b559 and Z, acting in competition. Their respective efficiency is dependent on temperature and on their states of oxidation. The specific effect of flash excitation is attributed to a two-photon reaction, possibly based on energy-trapping properties of the oxidized trap chlorophyll. 相似文献