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. The near-ultraviolet difference spectrum of whole cells reveals an absorption peak at 315 mμ with a shoulder around 350 mμ.
3. Both the endogenous respiration and motility of spermatozoa are completely blocked by 0.2 mM CN− and by 0.2 μM antimycin A. 2,4-Dinitrophenol and pentachlorophenol completely inhibit motility at the maximal stimulation of respiration. Rotenone strongly inhibits NADH oxidase of spermatozoa, although it has no effect on the respiration of whole cells.
4. It is concluded that the motility of mussel spermatozoa is tightly coupled to respiration, and the respiratory chain phosphorylating process is the only energy-supplying system for motility. 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
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
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
1. It is shown that removal of oxygen at the end of the exponential phase of growth (after completion of mitochondria formation) causes a decrease in activity of the respiratory enzymes. The activity of the complete respiratory system decreases much more rapidly than the activities of its fragments (NADH: ferricyanide reductase, succinate:ferricyanide reductase, NADH:cytochrome c reductase, succinate:cytochrome c reductase and cytochrome oxidase). The activities are restored to their initial level upon aeration of the cell suspension. The addition of Tween-80 and ergosterol to the medium prior to deaeration does not prevent inactivation of the respiratory system.
All the changes in mitochondria described occurred under conditions where cell division was insignificant.
2. Deaeration of the medium decreases the content of cytochromes b and aa3 in the mitochondrial fraction, cytochrome aa3 “disappearing” more quickly. The concentration of cytochromes in this fraction increases upon subsequent aeration of the cells. The total cytochromal content of the cells remains practically unchanged under the same conditions.
3. According to electron microscopic data, anaerobiosis causes a certain disorganization of mitochondrial cristal membranes. The mitochondrial structures are recovered upon aeration of the yeast cell suspension. It may be reasoned that inactivation and reactivation of the respiratory system are associated with reversible changes in mitochondrial membrane structure.
4. The effect of protein synthesis inhibitors on the restoration of mitochondria was investigated. It is shown that chloramphenicol does not suppress this process. In the presence of cycloheximide, oxygen induces reactivation of the respiratory system and simultaneously the appearance of particles resembling mitochondria. However, these particles gradually undergo morphological changes and the respiratory activity of the mitochondrial fraction decreases. Cycloheximide added to yeast cells that had not been deaerated, did not affect their mitochondria.
5. The results described suggest that the functions of oxygen in the formation of mitochondria are not restricted to the induction of mitochondrial protein synthesis and to the participation in the synthesis of certain non protein membrane components. Evidently, oxygen has a direct effect on the assembly of the respiratory system and mitochondrial membranes as a whole. 相似文献
These membrane preparations contain very small quantities of cytochromes c, b and cytochrome oxidase. The cytochrome c is not extracted by any method attempted. The cytochrome b is reducible only by dithionite and is believed not to be involved in the direct transfer of electrons during the oxidation of NADH by these preparations. The NADH oxidase activity of the membrane is inhibited by high concentrations of cyanide and also by 2-(n-heptyl)-4-hydroxyquinoline-N-oxide (HQNO). The cytochrome oxidase of the membrane contains both cytochromes a and a3 and is present in low concentrations relative to cytochrome c. The cytochrome a3 component was identified by characteristic complexes with both CO and cyanide and shows a γ-band absorption maximum at a slightly lower wavelength than the cytochrome oxidase of mammalian mitochondria (442 nm vs. 445 nm). The functional activity of the cytochrome oxidase is indicated by the inhibition of reoxidation of reduced cytochromes c and a in the presence of cyanide. 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
1. 1. Cyanide inhibits the catalytic activity of cytochrome aa3 in both polarographic and spectrophotometric assay systems with an apparent velocity constant of 4·103 M−1·s−1 and a Ki that varies from 0.1 to 1.0 μM at 22 °C, pH 7·3.
2. 2. When cyanide is added to the ascorbate-cytochrome c-cytochromeaa3−O2 system a biphasic reduction of cytochrome c occurs corresponding to an initial Ki of 0.8 μM and a final Ki of about 0.1 μM for the cytochrome aa3−cyanide reaction.
3. 3. The inhibited species (a2+a33+HCN) is formed when a2+a33+ reacts with HCN, when a2+a32+HCN reacts with oxygen, or when a3+a33+HCN (cyano-cytochrome aa3) is reduced. Cyanide dissociates from a2+a33+HCN at a rate of 2·10−3 s−1 at 22 °C, pH 7.3.
4. 4. The results are interpreted in terms of a scheme in which one mole of cyanide binds more tightly and more rapidly to a2+a33+ than to a3+a33+.
Abbreviations: TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine 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