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
It was concluded that the loss of cytochrome f prevents the normal flow of electrons from Photosystem II to NADP and also to a variety of other Hill reaction oxidants. Furthermore, cytochrome f is not required for the reduction of NADP with electron donor systems other than water nor is it an essential component of the mechanism of cyclic photophosphorylation with phenazine methosulphate as cofactor. 相似文献
Similar measurements made with azurin instead of plastocyanin gave k = 6 · 106 and approx. 2 · 107 M−1 · s−1 for reaction of reduced azurin with cytochrome ƒ and algal cytochrome respectively.
Rate constants of 115 and 80 M−1 · s−1 were found for reduction of plastocyanin by ascorbate and hydroquinone at 298 °K and pH 7.0. The rate constants for the oxidation of plastocyanin, cytochrome ƒ, Pseudomonas cytochrome c-551 and red algal cytochrome c-553 by ferricyanide were found to be between 3 · 104 and 8 · 104 M−1 · s−1.
The results are discussed in relation to photosynthetic electron transport. 相似文献
The first type of liposome was reconstituted from a solution of purified cytochrome oxidase, mitochondrial phospholipids and cytochrome c, the latter being enclosed inside liposomes. Cytochrome c bound to the outer surface of the liposome membrane was removed by washing with NaCl. Such liposomes catalyzed oxidation of ascorbate by oxygen in the presence of phenazine methosulfate or N,N,N′,N′-tetramethyl-p-phenylenediamine. The oxidation was found to support the PCB− uptake by liposomes. The PCB− response was prevented and reversed by cyanide, protonophorous uncouplers and external cytochrome c.
Liposomes of the second type were prepared from a solution of mitochondrial phospholipids, coupling factors F1and Fc, and the hydrophobic proteins of the oligomycin-sensitive ATPase. These liposomes catalyzed ATP hydrolysis coupled with the PCB− uptake. The latter effect was prevented and reversed by oligomycin and uncouplers.
The conclusion is made that membrane potential can be independently formed by enzymic reactions of two different kinds: (1) redox (e.g. cytochrome c oxidase) and (2) hydrolytic (ATPase). 相似文献
Light-minus-dark difference spectrum indicated that, in the presence of 10 mM ferrocyanide and 50 μM ferricyanide, only cytochrome c-555 was photooxidized. In the presence of 30 mM ascorbate or at Eh values lower than about 0 mV, both cytochrome c-555 and cytochrome c-552 were photooxidized. The quantum yield of cytochrome c-555 photooxidation was calculated to be about 0.4.
The results obtained in the present study are compared with other investigators' and the possibility of the presence of two types of associations between the cytochromes and reaction-center bacteriochlorophyll is discussed. 相似文献
The kinetic data have been analyzed in terms of the thermodynamics of the interactions. This analysis indicates that the copper protein has a ΔE of about 0.038 V more positive than the heme c component, a value that compares favorably with that of 0.040 V obtained by equilibrium methods. The value of ΔE obtained by the kinetic method for the internal reaction is less precise but is reasonably close to that of 0.070 V determined by an equilibrium technique. 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 superoxide anion radical (O2−) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O2− and ferrocytochrome c.
2. 2. At 20 °C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4 · 106 M−1 · s−1 and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O2− and the form of cytochrome c which exists above pH ≈ 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O2− reacts with the form present below pH 7.45 with k = 1.4 · 106 M−1 · s−1, the form above pH 7.45 with k = 3.0 · 105 M−1 · s−1, and the form present above pH 9.2 with k = 0.
3. 3. The reaction has an activation energy of 20 kJ mol−1 and an enthalpy of activation at 25 °C of 18 kJ mol−1 both above and below pH 7.45. It is suggested that O2− may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex.
4. 4. No reduction of ferricytochrome c by HO2 radicals could be demonstrated at pH 1.2–6.2 but at pH 5.3, HO2 radicals oxidize ferrocytochrome c with a rate constant of about 5 · 105–5 · 106 M−1 · s−1
. 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
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
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.
2. These results demonstrate that upon photodissociation, CO is replaced by azide wheras upon incubation in the dark CO expels azide from its binding site in cytochrome c oxidase.
3. Concomitantly with the binding of CO and dissociation of the azide molecule, and vice versa, electron redistributions occur as inferred from the changes in the intensity of the copper signal at g = 2.
4. The results are explained in a model of cytochrome c oxidase with either a common binding site (cytochrome a3)* for CO and azide or in a model with anti-cooperative interaction between two different sites of binding.
5. Similar types of experiments with cyanide instead of azide show that cyanide is more firmly bound to partially reduced cytochrome c oxidase than CO and azide. The affinity of ligands for partially reduced enzyme decreases in the sequence: cyanide, CO (dark), azide and CO (illuminated). 相似文献
EPR spectra of the aerobic submitochondrial particles in the absence of substrate show the presence of low spin ferric hemes with g values at 3.4 and 3.0, a high spin ferric heme with g = 6, and a g = 2.0 signal characteristic of oxidized copper. In the reduced submitochondrial particles signals of various iron-sulfur centers are observed.
Cytochrome c553 is lost from mitochondria during preparation of the submitochondrial particles. The partially purified cytochrome c553 is a negatively charged protein at neutral pH with an Em7.2 of 0.25 V which binds to the cytochrome c-depleted Tetrahymena mitochondria in the amount of 0.5 nmol/mg protein with a KD of 0.8 · 10−6 M. Reduced cytochrome c553 serves as an efficient substrate in the reaction with its own oxidase. The EPR spectrum of the partially purified cytochrome c553 shows the presence of a low spin ferric heme with the dominant resonance signal at g = 3.28.
A pigment with an absorption maximum at 560 nm can be solubilized from the Tetrahymena cells with butanol. This pigments has a molecular weight of approx. 18 000, and Em7.2 of −0.17 V and exhibits a high spin ferric heme signal at g = 6. 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