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 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.
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. Sorghum mesophyll chloroplasts have a chlorophyll (chl) and carotenoid composition similar to that of spinach chloroplasts. In contrast, the sorghum bundle sheath chloroplasts have a higher chl a/chl b ratio; they are enriched in β-carotene and contain relatively less xanthophylls as compared to sorghum mesophyll or spinach chloroplasts.
3. Sorghum mesophyll chloroplasts with 1 cytochrome f, 2 cytochrome b6 and 2 cytochrome b-559 per 430 chlorophylls have a cytochrome composition similar to spinach chloroplasts. Sorghum bundle sheath chloroplasts contain cytochrome f and cytochrome b6 in the same molar ratios as for the mesophyll chloroplasts, but cytochrome b-559 is barely detectable.
4. The chl/P700 ratios of mesophyll chloroplasts of S. bicolor and mesophyll and bundle sheath chloroplasts of Atriplex spongiosa are similar to that of spinach chloroplasts suggesting that these chloroplasts possess an identical photosynthetic unit size to that of spinach. The agranal bundle sheath chloroplasts of S. bicolor possess a photosynthetic unit which contains only about half as many chlorophyll molecules per P700 as found in the grana-containing chloroplasts.
5. The similarity of the composition of the bundle sheath chloroplasts of S. bicolor with that of the Photosystem I subchloroplast fragments, together with their smaller photosynthetic unit and low Photosystem II activities suggests that these chloroplasts are highly deficient in the pigment assemblies of Photosystem II. 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
1. 1. The kinetics of light-induced absorbance changes due to oxidation and reduction of cytochromes were measured in a suspension of intact cells of the unicellular red alga Porphyridium aerugineum. Absorbance changes in the region 540–570 nm upon alternating far-red light and darkness indicated the oxidation of cytochrome ƒ and reduction of cytochrome b563 upon illumination. The relative efficiencies of far-red and orange light indicated that both reactions were driven by Photosystem I.
2. 2. Experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), with anaerobic cells and in alternating far-red and orange light indicated that cytochrome b563 reacts in a cyclic chain around Photosystem I, and that the reduced cytochrome does not react with oxygen or with another oxidized product of Photosystem II. The quantum requirement for the photoreduction was about 6 quanta/equiv at 700 nm. A low concentration of N-methylphenazonium methosulphate (PMS) enhanced the rate of reoxidation of cytochrome b563 in the dark. In the presence of higher concentrations of PMS a photooxidation, driven by Photosystem I, instead of reduction was observed. These observations suggest that PMS enhances the rate of reactions between reduced cytochrome b563 and oxidized products of Photosystem I.
3. 3. In the presence of carbonylcyanide m-chlorophenylhydrazone (CCCP) a light-induced decrease of absorption at 560 nm occurred. Spectral evidence suggested the photooxidation of cytochrome b559 under these conditions. Inhibition by DCMU and a relatively efficient action of orange light suggested that this photooxidation is driven by Photosystem II.
Abbreviations: DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; CCCP, carbonylcyanide m-chlorophenylhydrazone; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; P700, chlorophyllous pigment absorbing at 700 nm, primary electron donor of Photosystem I; PMS, N-methylphenazonium methosulphate 相似文献
Incubation of chloroplasts with KCN or HgCl2 decreased the amplitude of the 20 μs phase. This provides evidence for a function of plastocyanin as the immediate electron donor of P-700.
At low concentrations of salt and sugar the fast phases of P-700+ reduction were largely inhibited. Increasing concentrations of MgCl2, KCl and sorbitol (up to 5, 150 and 200 mM, respectively) were found to increase the relative amplitudes of the fast phases to about one-third of the total P-700 signal. Addition of both 3 mM MgCl2 and 200 mM sorbitol increased the relative amplitude of the 20 μs phase to 70%. The interaction between P-700 and plastocyanin is concluded to be favoured by a low internal volume of the thylakoids and compensation of surface charges of the membrane.
The half-time of 20 μs was not changed when the amplitude of this phase was altered either by salt and sorbitol, or by inhibition of plastocyanin. This is evidence for the existence of a complex between plastocyanin and P-700 with a lifetime long compared to the measuring time. The 200 μs phase exhibited changes in its half-time that indicated the participation of a more mobile pool of plastocyanin.
Cytochrome f was oxidized with a biphasic time course with half-times of 70–130 μs and 440–860 μs at different salt and sorbitol concentrations. The half-time of the faster phase and a short lag of 30–50 μs in the beginning of the kinetics indicate an oxidation of cytochrome f via the 20 μs electron transfer to P-700. An inhibition of this oxidation by MgCl2 suggests that the electron transfer from cytochrome f to complexed plastocyanin is not controlled by negative charges in contrast to that from plastocyanin to P-700. 相似文献
1. 1. From a correlation of these kinetics it can be concluded that at least 85% of the electrons from plastohydroquinone are transferred to chlorophyll aI.
2. 2. After one flash 93% of the oxidized chlorophyll aI is reduced. This suggests a high equilibrium constant between chlorophyll aI and its donor as well as an equilibration between different chlorophyll aI molecules.
3. 3. Cytochrome f is also reduced by plastohydroquinone. A ratio of active cytochrome f to chlorophyll aI of 0.4:1 is observed. The half-life time of the reduction of cytochrome f is 17 ms. The time course indicates that in the dark cytochrome f does not transfer electrons to chlorophyll aI and that no more than 15% of the electron transport passes cytochrome f. Therefore cytochrome f should be situated in a side path of the linear electron transport.
4. 4. The electrons which are released from plastohydroquinone and are not accepted by oxidized cytochrome f and chlorophyll aI have been calculated. From this difference properties of an electron carrier, as yet not identified, between plastoquinone and chlorophyll aI are predicted.
Abbreviations: Tricine; N-tris(hydroxymethyl)methylglycine 相似文献
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