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1.
A kinetic study on ubiquinol-cytochrome f reductase (EC 1.10.2.2) has been undertaken either in situ in KCN-inhibited mitochondria and submitochondrial particles, or in the isolated cytochrome b-c1 complex using ubiquinol-1 and exogenous cytochrome c as substrates. The steady-state two-substrate kinetics of the reductase appears to follow a general sequential mechanism, allowing calculation of a Km for ubiquinol-1 of 13.4 μM in mitochondria and of 24.6 μM in the isolated cytochrome b-c1 complex. At low concentrations of cytochrome c, however, the titrations as a function of quinol concentration appear biphasic both in mitochondria and in submitochondrial particles containing trapped cytochrome c inside the vesicle space, fitting two apparent Km values for ubiquinol-1. Relatively high antimycin-sensitive rates of ubiquinol-1-cytochrome c reductase have been found in submitochondrial particles: both the Vmax and the Km for ubiquinol-1 are, however, affected by the overall orientation of the particle preparation, i.e., by the reactivity of cytochrome c with its proper site. The turnover numbers corrected for particle orientation with respect to cytochrome c interaction are at least 2-fold higher in submitochondrial particles than in mitochondria. This is particularly evident using inside-out particles containing trapped cytochrome c in the vesicle space (and therefore reacting with its physiological site). A diffusion step for the quinol substrate appears to be rate limiting in mitochondria and can be removed by addition of deoxycholate, suggesting that the oxidation site of ubiquinol may be more exposed to the matrix side of the inner mitochondrial membrane. 相似文献
2.
An improved method was developed to sequentially fractionate succinate-cytochrome c reductase into three reconstitutive active enzyme systems with good yield: pure succinate dehydrogenase, ubiquinone-binding protein fraction and a highly purified ubiquinol-cytochrome c reductase (cytochrome b-c1 III complex).An extensively dialyzed succinate-cytochrome c reductase was first separated into a succinate dehydrogenase fraction and the cytochrome b-c1 complex by alkali treatment. The resulting succinate dehydrogenase fraction was further purified to homogeneity by the treatment of butanol, calcium phosphate gel adsorption and ammonium sulfate fractionation under anaerobic condition in the presence of succinate and dithiothreitol. The cytochrome b-c1 complex was separated into cytochrome b-c1 III complex and ubiquinone-binding protein fractions by careful ammonium acetate fractionation in the presence of deoxycholate.The purified succinate dehydrogenase contained only two polypeptides with molecular weights of 70 000 and 27 000 as revealed by the sodium dodecyl sulfate polyacrylamide gel electrophoretic pattern. The enzyme has the reconstitutive activity and a low Km ferricyanide reductase activity of 85 μmol succinate oxidized per min per mg protein at 38°C.Chemical composition analysis of cytochrome b-c1 III complex showed that the preparation was completely free of contamination of succinate dehydrogenase and ubiquinone-binding protein and was 30% more pure than the available preparation.When these three components were mixed in a proper ratio, a thenoyl-trifluoroacetone- and antimycin A-sensitive succinate-cytochrome c reductase was reconstituted. 相似文献
3.
Diana S. Beattie Liviu Clejan Yu-Shiaw Chen Ching-I P. Lin Anita Sidhu 《Journal of bioenergetics and biomembranes》1981,13(5-6):357-373
Mitochondria (or mitoplasts) and submitochondrial particles from yeast were treated with [125I] diazobenzenesulfonate to label selectively proteins exposed on the outer or inner surface of the inner mitochondrial membrane. Polyacrylamide gel analysis of the immunoprecipitates formed with antibodies against Complex III or cytochromeb revealed that the two core proteins and cytochromeb were labeled in both mitochondria and submitochondrial particles, suggesting that these proteins span the membrane. Cytochromec
1 and the iron sulfur protein were labeled in mitochondria but not in submitochondrial particles, suggesting that these proteins are exposed on the cytosolic side of the inner membrane. The steady-state reduction of cytochromesb andc
1 was determined with succinate and the decyl analogue of coenzyme Q as substrates. Addition of the coenzyme Q analogue to mitochondria caused reduction of 15–30% of the total dithionite-reducibleb and 100% of the cytochromec
1: Addition of the coenzyme Q analogue to submitochondrial particles led to the reduction of 70% of the total dithionite-reducible cytochromeb but insignificant amounts of cytochromec
1. A model to explain the topography of Complex III in the inner membrane is proposed based on these results.Abbreviations used: DABS, diazobenzene sulfonate; DBH2, reduced form of decyl analogue of coenzyme Q (2,3-dimethoxy-5-methyl-6-n-decyl-1,4-benzoquinone); PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate. 相似文献
4.
The mitochondrial small heat-shock protein protects NADH:ubiquinone oxidoreductase of the electron transport chain during heat stress in plants 总被引:1,自引:0,他引:1
Functional inactivation of the mitochondrial small heat-shock protein (lmw Hsp) in submitochondrial vesicles using protein-specific antibodies indicated that this protein protects NADH:ubiquinone oxidoreductase (complex I), and consequently electron transport from complex I to cytochrome c:O2 oxidoreductase (complex IV). Lmw Hsp function completely accounted for heat acclimation of complex I electron transport in pre-heat-stressed plants. Addition of purified lmw Hsp to submitochondrial vesicles lacking this Hsp increased complex I electron transport rates 100% in submitochondrial vesicles assayed at high temperatures. These results indicate that production of the mitochondrial lmw Hsp is an important adaptation to heat stress in plants. 相似文献
5.
William S. Thayer Emanuel Rubin 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,721(4):328-335
Previous studies have established that hepatic mitochondria and submitochondrial particles from rats, fed ethanol chronically, display diminished respiratory activities and alterations in the contents of specific electron transfer chain components. The latter include a decrease of about 50% in cytochrome b content. Titrations of respiratory activity in submitochondrial particles with antimycin, a stoichiometric inhibitor of electron flow through the cytochrome b-c1 region of the respiratory chain, indicated a comparable decrease (35%) in the amount of antimycin required to elicit maximal inhibition (‘titer’) after chronic ethanol treatment. Measurements of antimycin binding to submitochondrial particles by fluorescence quenching demonstrated a similar diminution in the number of tight binding sites per mg protein. By contrast, hepatocytes isolated from control and ethanol-fed rats exhibited nearly identical rates of oxygen utilization under a variety of conditions. However, antimycin titrations of respiratory activity in isolated hepatocytes revealed a 60% decrease in the antimycin titer, but no change in the maximal extent of inhibition after chronic ethanol treatment. Direct measurements of cytochrome b which could be reduced in the presence of antimycin in hepatocytes confirmed a comparable decrease (42%) after chronic ethanol treatment. The results demonstrate that molecular alterations in the cytochrome b region of the respiratory chain caused by ethanol feeding are present in intact liver cells, but suggest that substrate accessibility, rather than the respiratory chain, limits the rate of oxygen utilization in isolated hepatocytes. The data also suggest that mitochondria account for at least 80% of total oxygen utilization by liver cells from both control and ethanol-fed rats. 相似文献
6.
Efraim Racker 《The Journal of general physiology》1969,54(1):38-49
The problem of the resolution and reconstitution of the inner mitochondrial membrane has been approached at three levels. (1) Starting with phosphorylating submitochondrial particles, a "resolution from without" can be achieved by stripping of surface components. The most extensive resolution was recently obtained with the aid of silicotungstate. Such particles require for oxidative phosphorylation the addition of several coupling factors as well as succinate dehydrogenase. (2) Starting with submitochondrial particles that have been degraded by trypsin and urea a resolution of the inner membrane proper containing an ATPase has been achieved. These experiments show that at least five components are required for the reconstitution of an oligomycin-sensitive ATPase: a particulate component, F
1, Mg++, phospholipids, and Fc. Morphologically, the reconstituted ATPase preparations resemble submitochondrial particles. (3) Starting with intact mitochondria individual components of the oxidation chain have been separated from each other. The following components were required for the reconstitution of succinoxidase: succinate dehydrogenase, cytochrome b\, cytochrome c
1, cytochrome c, cytochrome oxidase, phospholipids and Q
10. The reconstituted complex had properties similar to those of phosphorylating submitochondrial particles; i.e., the oxidation of succinate by molecular oxygen was highly sensitive to antimycin. 相似文献
7.
Peter Nicholls 《BBA》1976,430(1):30-45
1. Beef heart mitochondria have a cytochrome c1 : c : aa3 ratio of 0.65 : 1.0 : 1.0 as isolated; Keilin-Hartree submitochondrial particles have a ratio of 0.65 : 0.4 : 1.0. More than 50% of the submitochondrial particle membrane is in the ‘inverted’ configuration, shielding the catalytically active cytochrome c. The ‘endogenous’ cytochrome c of particles turns over at a maximal rate between 450 and 550 s?1 during the oxidation of succinate or ascorbate plus TMPD; the maximal turnover rate for cytochrome c in mitochondria is 300–400 s?1, at 28° – 30°C, pH 7.4.2. Ascorbate plus N,N,N′,N′-tetramethyl-p-phenylene diamine added to antimycin-treated particles induces anomalous absorption increases between 555 and 565 nm during the aerobic steady state, which disappear upon anaerobiosis; succinate addition abolishes this cycle and permits the partial resolution of cytochrome c1 and cytochrome c steady states at 552.5–547 nm and 550–556.5 nm, respectively.3. Cytochrome c1 is rather more reduced than cytochrome c during the oxidation of succinate and of ascorbate+N,N,N′,N′-tetramethyl-p-phenylene diamine in both mitochondria and submitochondrial particles; a near equilibrium condition exists between cytochromes c1 and c in the aerobic steady state, with a rate constant for the c1 → c reduction step greater than 103 s?1.4. The greater apparent response of the electron transfer step to salts, the hyperbolic inhibition of succinate oxidation by azide and cyanide, and the kinetic behaviour of the succinate-cytochrome c reductase system, are all explicable in terms of a near-equilibrium condition prevailing at the step. Endogenous cytochrome c of mitochondria and submitochondrial particles is apparently largely bound to cytochrome aa3 units in situ. Cytochrome c1 can either reduce the cytochrome c-cytochrome aa3 complex directly, or requires only a small extra amount of cytochrome c to carry the full electron transfer flux. 相似文献
8.
Isabelle Valnot Johanna Kassis Dominique Chretien Pascale de Lonlay Béatrice Parfait Arnold Munnich Jean Kachaner Pierre Rustin Agnès Rötig 《Human genetics》1999,104(6):460-466
Ubiquinol cytochrome c reductase (complex III) deficiency represents a clinically heterogeneous group of mitochondrial respiratory chain disorders
that can theoretically be subject to either a nuclear or a mitochondrial mode of inheritance. In an attempt to elucidate the
molecular bases of the disease, we first determined the nucleotide sequence of three unknown subunits (9.5 kDa, 7.2 kDa, 6.4
kDa) by cyberscreening of human expressed sequence tag data bases and sequenced the 11 cDNA subunits encoding complex III
in five patients with isolated complex III deficiency. No mutation in the nuclearly encoded complex III subunits was observed,
but a mutation in the cd2 helix of the mitochondrial (mt) cytochrome b gene was found to alter the conformation of the bc
1
complex in one patient with severe hypertrophic cardiomyopathy. The present study is highly relevant to genetic counseling
as the absence of mtDNA mutations in all but one patient in our series strongly supports autosomal rather than maternal inheritance
in the majority of patients with complex III deficiency.
Received: 15 January 1999 / Accepted: 31 March 1999 相似文献
9.
A highly purified reduced ubiquinone-cytochrome c reductase preparation (the b-c1III complex) has been made. The b-c1III complex is not reconstitutively active with succinate dehydrogenase. When the complex at about 10 mg/ml is reduced by succinate in the presence of catalytic (nanomolar) amounts of SDH and a ubiquinone protein (required in the succinate dehydrogenase region i.e, OP-S), a ubisemiquinone radical(s) has been detected using EPR measurements. The formation of the radical(s) is concurrent with the reduction of cytochrome b after the complete reduction of cytochrome c1. All these rates are dependent on the amounts of succinate dehydrogenase and QP-S used. The maximal concentration of the radical formed is independent of the amounts of succinate dehydrogenase and QP-S added but dependent on the amount of succinate present. The formation of the radical and the reduction of b and c1 by succinate requires the presence of phospholipids. Addition of thenoyltrifluoroacetone not only prevents the formation of the ubisemiquinone but also abolishes the prior formed radical and causes the reoxidation of b. Antimycin A also diminishes the radical intensity but causes only slight reoxidation of prior reduced cytochrome b. Treatment of the b-c1III complex with α-chymotrypsin results in the diminishing of the radical formation. Consideration of all these results presented collectively indicates the existence of a ubiquinone binding protein in the b-c1III complex preparation. 相似文献
10.
A cytochrome b-c1 complex was isolated from pigeon breast muscle mitochondria and purified to a content of 3 nmol of cytochrome c1 per milligram of protein. Anaerobic suspensions of the preparation were titrated with reducing equivalents (NADH) and oxidizing equivalents (ferricyanide). The oxidation-reduction components of the complex were measured by the number of reducing equivalents accepted or donated per cytochrome c1 and compared with the stoichiometries of the known redox components as measured by independent methods. The preparation accepts or donates 5.2 ± 0.3 equivalents per cytochrome c1, while the measured content of cytochrome c1, cytochrome b561, cytochrome b565, Rieske iron-sulfur protein, ubiquinone, and succinate dehydrogenase accounts for 5.0 ± 0.2 equivalents per cytochrome c1. It is concluded that there are no unknown redox components in the cytochrome b-c1 complex. The cytochrome b-c1 complex (energy transduction site 2) appears to be a structural unit containing equal amounts of cytochrome c1, cytochrome b561, cytochrome b566, and the Rieske iron-sulfur protein. 相似文献
11.
Nocardia asteroides is a pathogenic bacterium that causes severe pulmonary infections and plays a vital role in HIV development. Its electron
transport chain containing cytochromes as electron carriers is still undiscovered. Information regarding cytochromes is important
during drug synthesis based on cytochrome inhibitions. In this study we explored the electron transport of N. asteroides. Spectroscopic analysis of cytoplasm and membranes isolated from N. asteroides indicates the presence of soluble cytochrome-c, complex-II and the modified a
1
c
1
complex as the terminal oxidase. The molecular weight of the respiratory complex-II isolated and purified from the given
bacterium was 103 kDa and was composed of three subunits, of 14, 26 and 63 kDa. Complex-II showed symmetrical α-absorption
peaks at 561 nm in the reduced state. Spectral analysis revealed the presence of only one heme b molecule (14-kDa subunit) in complex-II, which was confirmed by heme staining. Heme b content was found to be 9.5 nmol/mg in complex-II. The electron transport chain of N. asteroides showed the presence of soluble cytochrome-c, cytochrome-a
1
c
1
and cytochrome-b. 相似文献
12.
13.
Kyoko Shinzawa-Itoh Harunobu Shimomura Sachiko Yanagisawa Satoru Shimada Ryoko Takahashi Marika Oosaki Takashi Ogura Tomitake Tsukihara 《The Journal of biological chemistry》2016,291(8):4178-4184
To understand the roles of mitochondrial respiratory chain supercomplexes, methods for consistently separating and preparing supercomplexes must be established. To this end, we solubilized supercomplexes from bovine heart mitochondria with digitonin and then replaced digitonin with amphipol (A8–35), an amphiphilic polymer. Afterward, supercomplexes were separated from other complexes by sucrose density gradient centrifugation. Twenty-six grams of bovine myocardium yielded 3.2 mg of amphipol-stabilized supercomplex. The purified supercomplexes were analyzed based on their absorption spectra as well as Q10 (ubiquinone with ten isoprene units) and lipid assays. The supercomplex sample did not contain cytochrome c but did contain complexes I, III, and IV at a ratio of 1:2:1, 6 molecules of Q10, and 623 atoms of phosphorus. When cytochrome c was added, the supercomplex exhibited KCN-sensitive NADH oxidation; thus, the purified supercomplex was active. Reduced complex IV absorbs at 444 nm, so we measured the resonance Raman spectrum of the reduced amphipol-solubilized supercomplex and the mixture of amphipol-solubilized complexes I1, III2, and IV1 using an excitation wavelength of 441.6 nm, allowing measurement precision comparable with that obtained for complex IV alone. Use of the purified active sample provides insights into the effects of supercomplex formation. 相似文献
14.
Cytochromes b, c, d, and o were identified by spectroscopic analysis of respiratory membrane fragments from Vitreoscilla sp., strain C1. Carbon monoxide difference spectra of the reduced membranes had absorption maxima at 416, 534, and 571 nm (ascribed to cytochrome o) and 632 nm (cytochrome d). Derivative spectra of the pyridine hemochromogen spectra of the membranes identified the presence of b- and c-type cytochromes in Vitreoscilla. The cyanide binding curve of the membranes was biphasic with dissociation constants of 2.14 mM and 10.7 mM which were assigned to cytochrome o and cytochrome d, respectively. Membranes bound carbon monoxide with dissociation constant 3.9 M, which was assigned to cytochrome o. Cytochrome c
556 and a NADH-p-iodonitrotetrazolium violet reductase component were partially purified from Vitreoscilla membranes.Abbreviations INT
p-iodonitrotetrazolium violet
- RMF
respiratory membrane fragments
-
K
d
dissociation constant
- CHAPS
3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate
- DOC
sodium deoxycholate
- PAGE
polyacrylamide gel electrophoresis
- SDS
sodium dodecyl sulfate 相似文献
15.
Cytochrome bd is a terminal quinol oxidase in Escherichia coli. Mitochondrial respiration is inhibited at cytochrome bc1 (complex III) by myxothiazol. Mixing purified cytochrome bd oxidase with myxothiazol-inhibited bovine heart submitochondrial particles (SMP) restores up to 50% of the original rotenone-sensitive NADH oxidase and succinate oxidase activities in the absence of exogenous ubiquinone analogs. Complex III bypassed respiration and is saturated at amounts of added cytochrome bd similar to that of other natural respiratory components in SMP. The cytochrome bd tightly binds to the mitochondrial membrane and operates as an intrinsic component of the chimeric respiratory chain. 相似文献
16.
A membrane-bound cytochrome of the b-type (cytochrome b-560) was success-fully purified from chromatophores of the photosynthetic purple sulfur bacterium Chromatium vinosum by treatment with sodium cholate, sodium deoxycholate, sodium thiocyanate, and bacterial alkaline protease (EC 3·4·21·14) followed by gel filtration.The purified cytochrome b-560 showed the absorption maxima at 279, 412.5 and 533 nm in the oxidized form, and 427, 530 and 560 nm in the reduced form. Reduced-minus-oxidized difference millimolar absorption coefficient was 14.0 for a wavelength pair, 560 minus 540 nm.Isolated cytochrome b-560 was electrophoretically homogeneous, and its minimal molecular weight was estimated to the 13,000 by SDS polyacrylamide gel electrophoresis.The midpoint potential at pH 8.0 was –110mV, and was not dependent on the ambient pH in the pH range of 6.8 to 8.8. 相似文献
17.
Core proteins I (Mr 50 000) and II (Mr 47 000) were isolated from beef heart ubiquinol-cytochrome c reductase, and radioimmunoassays were developed for both. Immunoreplica experiments show that antisera against each protein react with a single peptide in both isolated Complex III and in mitochondria. Thus, core proteins are not aggregated forms of smaller peptides as suggested for the yeast protein (Jeffrey, A., Power, S. and Palmer, G., Biochem. Biophys. Res. Commun. (1979) 86, 271–277). Core proteins were quantitated in Complex III and in mitochondria using radioimmunoassay. Approx. 2 mol core protein II per mol core protein I were found. A molar ratio of 1 : 2 : 2 : 1 is suggested for core protein I : core protein II : cytochrome b : cytochrome c1. Radioimmunoassay shows that the antibodies react as extensively with Complex III-bound core protein as with the isolated core proteins. In spite of this, the antibodies do not inhibit electron transport in submitochondrial particles or isolated Complex III, and they have no oligomycin- or uncoupler-like effects on submitochondrial particles oxidizing NADH. The combined results from radioimmunoassay and immunoreplica experiments strongly suggest, however, that core proteins are specifically associated with Complex III in the mitochondria, implying a specific role there. 相似文献
18.
J. F. Stolz Theresa Gugliuzza Jodi Switzer Blum Ronald Oremland Francisco Martínez Murillo 《Archives of microbiology》1997,167(1):1-5
The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated.
Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE
showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate
contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate.
No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however,
revealed the presence of a cytochrome b
554 (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b
556 (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity
in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for
the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity
with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the
terminal electron acceptor used for growth.
Received: 21 August 1996 / Accepted: 24 October 1996 相似文献
19.
The respiratory chain in the inner mitochondrial membrane contains three large multi‐enzyme complexes that together establish the proton gradient for ATP synthesis, and assemble into a supercomplex. A 19‐Å 3D map of the 1.7‐MDa amphipol‐solubilized supercomplex I1III2IV1 from bovine heart obtained by single‐particle electron cryo‐microscopy reveals an amphipol belt replacing the membrane lipid bilayer. A precise fit of the X‐ray structures of complex I, the complex III dimer, and monomeric complex IV indicates distances of 13 nm between the ubiquinol‐binding sites of complexes I and III, and of 10–11 nm between the cytochrome c binding sites of complexes III and IV. The arrangement of respiratory chain complexes suggests two possible pathways for efficient electron transfer through the supercomplex, of which the shorter branch through the complex III monomer proximal to complex I may be preferred. 相似文献
20.
The binding of cytochrome c to the cytochrome bc1 complex of bovine heart mitochondria was studied. Cytochrome c derivatives, arylazido-labeled at lysine 13 or lysine 22, were prepared and their properties as electron acceptors from the bc1 complex were measured. Mixtures of bc1 complex with cytochrome c derivatives were illuminated with ultraviolet light and afterwards subjected to polyacrylamide gel electrophoresis. The gels were analysed using dualwavelength scanning at 280 minus 300 and 400 minus 430 nm. It was found that illumination with ultraviolet light in the presence of the lysine 13 derivative produced a diminution of the polypeptide of the bc1 complex having molecular weight 30 000 (band IV) and formation of a new polypeptide composed of band IV and cytochrome c. Band IV was identified as cytochrome c1, and it was concluded that this hemoprotein interacts with cytochrome c and contains its binding site in complex III of the mitochondrial respiratory chain. Illumination of the bc1 complex in presence of the lysine 22 derivative did not produce changes of the polypeptide pattern. 相似文献