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1.
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 dual-wavelength scanning at 280 minus 300 and 400 minus 430 nm. It was found that illumination with ultraviolet light in the presence of the lysine 12 derivative produced a diminution of the polypeptide of the bc1 coplex 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   

2.
Specific protein-lipid interactions have been identified in X-ray structures of membrane proteins. The role of specifically bound lipid molecules in protein function remains elusive. In the current study, we investigated how phospholipids influence catalytic, spectral and electrochemical properties of the yeast respiratory cytochrome bc1 complex and how disruption of a specific cardiolipin binding site in cytochrome c1 alters respiratory supercomplex formation in mitochondrial membranes. Purified yeast cytochrome bc1 complex was treated with phospholipase A2. The lipid-depleted enzyme was stable but nearly catalytically inactive. The absorption maxima of the reduced b-hemes were blue-shifted. The midpoint potentials of the b-hemes of the delipidated complex were shifted from − 52 to − 82 mV (heme bL) and from + 113 to − 2 mV (heme bH). These alterations could be reversed by reconstitution of the delipidated enzyme with a mixture of asolectin and cardiolipin, whereas addition of the single components could not reverse the alterations. We further analyzed the role of a specific cardiolipin binding site (CLi) in supercomplex formation by site-directed mutagenesis and BN-PAGE. The results suggested that cardiolipin stabilizes respiratory supercomplex formation by neutralizing the charges of lysine residues in the vicinity of the presumed interaction domain between cytochrome bc1 complex and cytochrome c oxidase. Overall, the study supports the idea, that enzyme-bound phospholipids can play an important role in the regulation of protein function and protein-protein interaction.  相似文献   

3.
Several components of the respiratory chain of the eubacterium Thermus thermophilus have previously been characterized to various extent, while no conclusive evidence for a cytochrome bc1 complex has been obtained. Here, we show that four consecutive genes encoding cytochrome bc1 subunits are organized in an operon-like structure termed fbcCXFB. The four gene products are identified as genuine subunits of a cytochrome bc1 complex isolated from membranes of T. thermophilus. While both the cytochrome b and the FeS subunit show typical features of canonical subunits of this respiratory complex, a further membrane-integral component (FbcX) of so far unknown function copurifies as a subunit of this complex. The cytochrome c1 carries an extensive N-terminal hydrophilic domain, followed by a hydrophobic, presumably membrane-embedded helical region and a typical heme c binding domain. This latter sequence has been expressed in Escherichia coli, and in vitro shown to be a kinetically competent electron donor to cytochrome c552, mediating electron transfer to the ba3 oxidase. Identification of this cytochrome bc1 complex bridges the gap between the previously reported NADH oxidation activities and terminal oxidases, thus, defining all components of a minimal, mitochondrial-type electron transfer chain in this evolutionary ancient thermophile.  相似文献   

4.
Ting Su  Lothar Esser  Di Xia  Chang-An Yu  Linda Yu 《BBA》2012,1817(2):298-305
Cytochrome bc1 complex catalyzes the reaction of electron transfer from ubiquinol to cytochrome c (or cytochrome c2) and couples this reaction to proton translocation across the membrane. Crystallization of the Rhodobacter sphaeroides bc1 complex resulted in crystals containing only three core subunits. To mitigate the problem of subunit IV being dissociated from the three-subunit core complex during crystallization, we recently engineered an R. sphaeroides mutant in which the N-terminus of subunit IV was fused to the C-terminus of cytochrome c1 with a 14-glycine linker between the two fusing subunits, and a 6-histidine tag at the C-terminus of subunit IV (c1-14Gly-IV-6His). The purified fusion mutant complex shows higher electron transfer activity, more structural stability, and less superoxide generation as compared to the wild-type enzyme. Preliminary crystallization attempts with this mutant complex yielded crystals containing four subunits and diffracting X-rays to 5.5 Å resolution.  相似文献   

5.
The effect of Zn2+ on the rates of electron transfer and of voltage generation in the cytochrome bc1 complex (bc1) was investigated under excitation of Rhodobacter capsulatus chromatophores with flashing light. When added, Zn2+ retarded the oxidation of cytochrome b and allowed to monitor (at 561-570 nm) the reduction of its high potential heme bh (in the absence of Zn2+ this reaction was masked by the fast re-oxidation of the heme). The effect was accompanied by the deceleration of both the cytochrome c1 reduction (as monitored at 552-570 nm) and the generation of transmembrane voltage (monitored by electrochromism at 522 nm). At Zn2+ <100 μM the reduction of heme bh remained 10 times faster than other reactions. The kinetic discrepancy was observed even after an attenuated flash, when bc1 turned over only once. These observations (1) raise doubt on the notion that the transmembrane electron transfer towards heme bh is the main electrogenic reaction in the cytochrome bc1 complex, (2) imply an allosteric link between the site of heme bh oxidation and the site of cytochrome c1 reduction at the opposite side of the membrane, and (3) indicate that the internal redistribution of protons might account for the voltage generation by the cytochrome bc1 complex.  相似文献   

6.
Cytochrome c1, the electron donor for cytochrome c, is a subunit of the mitochondrial cytochrome bc1 complex (complex III, cytochrome c reductase). To test if cytochrome c1 is the cytochrome c-binding subunit of the bc1 complex, binding of cytochrome c to the complex and to isolated cytochrome c1 was compared by a gel-filtration method under non-equilibrium conditions (a bc1 complex lacking the Rieske ironsulfur protein was used; von Jagow et al. (1977) Biochim. Biophys. Acta 462, 549–558). The approximate stoichiometries and binding affinities were found to be very similar. Binding of cytochrome c to isolated cytochrome b which is another subunit of the reductase was not detectable by the gel-filtration method. Further, the same lysine residues of cytochrome c were shielded towards chemical acetylation in the complexes c:c1 and c:bc1. From this we conclude that the same surface area of cytochrome c is in direct contact with cytochrome bc1 and with cytochrome c1 in the respective complexes and that therefore cytochrome c is most probably the structural ligand for cytochrome c in mitochondrial cytochrome c reductase.  相似文献   

7.
In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c1, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c1 and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a “floating boat bridge” of cytochrome c molecules (between complexes III and IV) in plant respirasome.  相似文献   

8.
Cytochrome c derivatives labeled with a 3-nitrophenylazido group at lysine 13, at lysine 22, or at both residues have been prepared. The interaction of the cytochrome c derivatives with beef heart cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) in the presence of ultrviolet light results in formation of a covalent complex between cytochrome c and the oxidase. Using the lysine 22 derivative, the polypeptide composition of the oxidase is not modified, nor is its catalytic activity, whereas with the lysine 13 derivative, the gel electrophoretic pattern is altered and the catalytic activity of the complex diminished. The data are consisten with a specfic covalent interaction of the lysine 13 derivative of cytochrome c with the polypeptide of molecular weight 23,700 (Subunit II) of cytochrome c oxidase.  相似文献   

9.
Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The cytochrome bc1 complex (cytbc1) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc1 complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc1 complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc1 to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc1 complexes retain their native dimeric structure and co-purify with 56 ± 6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the cytochrome b6f complex (cytb6f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc1/cytb6f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q0 and Qi sites.  相似文献   

10.
Extinction coefficients for cytochrome b and c1 in the isolated cytochrome bc1 complex from Rhodopseudomonas sphaeroides GA have been determined. They are 25 mM?1.cm?1 at 561 nm for cytochrome b and 17.4 mM?1.cm?1 at 553 nM for cytochrome c1 for the difference between the reduced and the oxidized state. Cytochrome b is present in two forms in the complex. One form has an Em7 of 50 mV, an α-peak of 557 nm at liquid N2 temperature and of 561 nm at RT, which is red-shifted by antimycin A. The other form has an Em7 of ?90 mV, a double α-peak of 555 and 561 nm at liquid N2 temperature corresponding to 559 and 566 nm at RT. The absorption at 566 nm is red-shifted by myxothiazol. The two shifts are independent of each other. Both midpoint potentials of cytochromes b are pH-dependent. The redox center compositions of the cytochrome bc1 complexes from Rhodopseudomonas sphaeroides and from mitochondria are identical.  相似文献   

11.
Ametoctradin is an agricultural fungicide that selectively inhibits the cytochrome bc1 complex of oomycetes. Previous spectrophotometric studies using the purified cytochrome bc1 complex from Pythium sp. showed that Ametoctradin binds to the Qo-site of the enzyme. However, as modeling studies suggested a binding mode like that of the substrate ubiquinol, the possibility for a dual Qo- and Qi-site binding mode was left open.In this work, binding studies and enzyme assays with mitochondrial membrane preparations from Pythium sp. and an S. cerevisiae strain with a modified Qi-site were used to investigate further the binding mode of Ametoctradin. The results obtained argue that the compound could bind to both the Qo- and Qi-sites of the cytochrome bc1 complex and that its position or binding pose in the Qi-site differs from that of Cyazofamid and Amisulbrom, the two Qi-site-targeting, anti-oomycetes compounds. Furthermore, the data support the argument that Ametoctradin prefers binding to the reduced cytochrome bc1 complex. Thus, Ametoctradin has an unusual binding mode and further studies with this compound may offer the opportunity to better understand the catalytic cycle of the cytochrome bc1 complex.  相似文献   

12.
The preparation, purification, and characterization of four new derivatives of cytochrome c trifluoroacetylated at lysines 72, 79, 87, and 88 are reported. The redox reaction rates of these derivatives with cytochrome b5, cytochrome c1 and cytochrome oxidase indicated that the interaction domain on cytochrome c for all three proteins involves the lysines immediately surrounding the heme crevice. Modification of lysines 72, 79, and 87 had a large effect on the rate of all three reactions, while modification of lysine 88 had a very small effect. Even though lysines 87 and 88 are adjacent to one another, lysine 87 is at the top left of the heme crevice oriented towards the front of cytochrome c, while lysine 88 is oriented more towards the back. Since the interaction sites for cytochrome c1 and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.  相似文献   

13.
Hydroxy-naphthoquinones are competitive inhibitors of the cytochrome bc1 complex that bind to the ubiquinol oxidation site between cytochrome b and the iron-sulfur protein and presumably mimic a transition state in the ubiquinol oxidation reaction catalyzed by the enzyme. The parameters that affect efficacy of binding of these inhibitors to the bc1 complex are not well understood. Atovaquone®, a hydroxy-naphthoquinone, has been used therapeutically to treat Pneumocystis carinii and Plasmodium infections. As the pathogens have developed resistance to this drug, it is important to understand the molecular basis of the drug resistance and to develop new drugs that can circumvent the drug resistance. We previously developed the yeast and bovine bc1 complexes as surrogates to model the interaction of atovaquone with the bc1 complexes of the target pathogens and human host. As a first step to identify new cytochrome bc1 complex inhibitors with therapeutic potential and to better understand the determinants of inhibitor binding, we have screened a library of 2-hydroxy-naphthoquinones with aromatic, cyclic, and non-cyclic alkyl side-chain substitutions at carbon-3 on the hydroxy-quinone ring. We found a group of compounds with alkyl side-chains that effectively inhibit the yeast bc1 complex. Molecular modeling of these into the crystal structure of the yeast cytochrome bc1 complex provides structural and quantitative explanations for their binding efficacy to the target enzyme. In addition we also identified a 2-hydroxy-naphthoquinone with a branched side-chain that has potential for development as an anti-fungal and anti-parasitic therapeutic.  相似文献   

14.
Edward A. Berry  Dong-Woo Lee  Kazuo Nagai 《BBA》2010,1797(3):360-7281
Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia (Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc1 complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Qi site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Qo site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A3 in a fashion similar to the Qo site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Qi and the Qo sites of the fungal cytochrome bc1 complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron-sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc1 complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Qo and Qi sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc1 complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Qi and Qo sites. Moreover, crystal structure of chicken cytochrome bc1 complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Qi and Qo sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc1 inhibitor that acts at both of the active sites of this enzyme.  相似文献   

15.
S.P.J. Brooks  P. Nicholls 《BBA》1982,680(1):33-43
Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrateferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the Km (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the ‘association’ rate but also by increasing the ‘dissociation’ rate for bound cytochrome c converting the ‘primary’ (T) site at high salt concentrations into a site similar kinetically to the ‘secondary’ (L) site at low ionic strength. A finite Km of 170 μM at very high ionic strength indicates a ratio of KMK0M of about 5000. It is proposed that anions either modify the E10 of cytochrome c bound at the primary (T) site or that they perturb an equilibrium between two forms of bound c in favour of a less active form.  相似文献   

16.
A water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, has been used to cross-link horse heart cytochrome c to spinach chloroplast plastocyanin. The complex was formed in yields up to 90%, and was found to have a stoichiometry of 1 mol plastocyanin per mol cytochrome c. The cytochrome c in the complex was fully reducible by ascorbate and potassium ferrocyanide, and had a redox potential only 25 mV less than that of native cytochrome c. The complex was nearly completely inactive towards succinate-cytochrome c reductase and cytochrome c oxidase, suggesting that the heme crevice region of cytochrome c was blocked. We propose that the carbodiimide promoted the formation of amide cross-links between lysine amino groups surrounding the heme crevice of cytochrome c and complementary carboxyl groups on plastocyanin. It is of interest that the high-affinity site for cytochrome c binding on bovine heart cytochrome c oxidase has recently been found to involve a sequence of subunit II with some homology to the copper-binding sequence of plastocyanin.  相似文献   

17.
J. Wilms  J. Lub  R. Wever 《BBA》1980,589(2):324-335
1. The steady-state oxidation of ferrocytochrome c by dioxygen catalyzed by cytochrome c oxidase, is inhibited non-competitively towards cytochrome c by methanethiol, ethanethiol, 1-propanethiol and 1-butanethiol with Ki values of 4.5, 91, 200 and 330 μM, respectively.2. The inhibition constant Ki of ethanethiol is found to be constant between pH 5 and 8, which suggests that only the neutral form of the thiol inhibits the enzyme.3. The absorption spectrum of oxidized cytochrome c oxidase in the Soret region shows rapid absorbance changes upon addition of ethanethiol to the enzyme. This process is followed by a very slow reduction of the enzyme. The fast reaction, which represents a binding reaction of ethanethiol to cytochrome c oxidase, has a k1 of 33 M?1 · s?1 and dissociation constant Kd of 3.9 mM.4. Ethanethiol induces fast spectral changes in the absorption spectrum of cytochrome c, which are followed by a very slow reduction of the heme. The rate constant for the fast ethanethiol reaction representing a bimolecular binding step is 50 M?1 · s?1 and the dissociation constant is about 2 mM. Addition of up to 25 mM ethanethiol to ferrocytochrome c does not cause spectral changes.5. EPR (electron paramagnetic resonance) spectra of cytochrome c oxidase, incubated with methanethiol or ethanethiol in the presence of cytochrome c and ascorbate, show the formation of low-spin cytochrome a3-mercaptide compounds with g values of 2.39, 2.23, 1.93 and of 2.43, 2.24, 1.91, respectively.  相似文献   

18.
A.L. Ducluzeau 《BBA》2008,1777(9):1140-1146
Heliobacteria have a Rieske/cytochrome b complex composed of a Rieske protein, a cytochrome b6, a subunit IV and a di-heme cytochrome c. The overall structure of the complex seems close to the b6f complex from cyanobacteria and chloroplasts to the exception of the di-heme cytochrome. We show here by biochemical and biophysical studies that a heme ci is covalently attached to the Rieske/cytochrome b complex from Heliobacteria. We studied the EPR signature of this heme in two different species, Heliobacterium modesticaldum and Heliobacillus mobilis. In contrast to the case of b6f complex, a strong axial ligand to the heme is present, most probably a protonatable amino acid residue.  相似文献   

19.
Astrid R. Klingen  Carola Hunte 《BBA》2007,1767(3):204-221
Cytochrome bc1 is a major component of biological energy conversion that exploits an energetically favourable redox reaction to generate a transmembrane proton gradient. Since the mechanistic details of the coupling of redox and protonation reactions in the active sites are largely unresolved, we have identified residues that undergo redox-linked protonation state changes. Structure-based Poisson-Boltzmann/Monte Carlo titration calculations have been performed for completely reduced and completely oxidised cytochrome bc1. Different crystallographically observed conformations of Glu272 and surrounding residues of the cytochrome b subunit in cytochrome bc1 from Saccharomyces cerevisiae have been considered in the calculations. Coenzyme Q (CoQ) has been modelled into the CoQ oxidation site (Qo-site). Our results indicate that both conformational and protonation state changes of Glu272 of cytochrome b may contribute to the postulated gating of CoQ oxidation. The Rieske iron-sulphur cluster could be shown to undergo redox-linked protonation state changes of its histidine ligands in the structural context of the CoQ-bound Qo-site. The proton acceptor role of the CoQ ligands in the CoQ reduction site (Qi-site) is supported by our results. A modified path for proton uptake towards the Qi-site features a cluster of conserved lysine residues in the cytochrome b (Lys228) and cytochrome c1 subunits (Lys288, Lys289, Lys296). The cardiolipin molecule bound close to the Qi-site stabilises protons in this cluster of lysine residues.  相似文献   

20.
We have measured the rates of superoxide anion generation by cytochrome bc1 complexes isolated from bovine heart and yeast mitochondria and by cytochrome bc1 complexes from yeast mutants in which the midpoint potentials of the cytochrome b hemes and the Rieske iron-sulfur cluster were altered by mutations in those proteins. With all of the bc1 complexes the rate of superoxide anion production was greatest in the absence of bc1 inhibitor and ranged from 3% to 5% of the rate of cytochrome c reduction. Stigmatellin, an inhibitor that binds to the ubiquinol oxidation site in the bc1 complex, eliminated superoxide anion formation, while myxothiazol, another inhibitor of ubiquinol oxidation, allowed superoxide anion formation at a low rate. Antimycin, an inhibitor that binds to the ubiquinone reduction site in the bc1 complex, also allowed superoxide anion formation and at a slightly greater rate than myxothiazol. Changes in the midpoint potentials of the cytochrome b hemes had no significant effect on the rate of cytochrome c reduction and only a small effect on the rate of superoxide anion formation. A mutation in the Rieske iron-sulfur protein that lowers its midpoint potential from +285 to +220 mV caused the rate of superoxide anion to decline in parallel with a decline in cytochrome c reductase activity. These results indicate that superoxide anion is formed by similar mechanisms in mammalian and yeast bc1 complexes. The results also show that changes in the midpoint potentials of the redox components that accept electrons during ubiquinol oxidation have only small effects on the formation of superoxide anion, except to the extent that they affect the activity of the enzyme.  相似文献   

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