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Bernard L. Trumpower - One of the best experts on this subject based on the ideXlab platform.
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anti cooperative oxidation of ubiquinol by the yeast Cytochrome bC1 complex
Journal of Biological Chemistry, 2004Co-Authors: Raul Covian, Emma Berta Gutierrezcirlos, Bernard L. TrumpowerAbstract:Abstract We have investigated the interaction between monomers of the dimeric yeast Cytochrome bC1 complex by analyzing the pre-steady and steady state activities of the isolated enzyme in the presence of antimycin under conditions that allow the first turnover of ubiquinol oxidation to be observable in Cytochrome C1 reduction. At pH 8.8, where the redox potential of the iron-sulfur protein is ∼200 mV and in a bC1 complex with a mutated iron-sulfur protein of equally low redox potential, the amount of Cytochrome C1 reduced by several equivalents of decyl-ubiquinol in the presence of antimycin corresponded to only half of that present in the bC1 complex. Similar experiments in the presence of several equivalents of Cytochrome c also showed only half of the bC1 complex participating in quinol oxidation. The extent of Cytochrome b reduced corresponded to two bH hemes undergoing reduction through one center P per dimer, indicating electron transfer between the two Cytochrome b subunits. Antimycin stimulated the ubiquinol-Cytochrome c reductase activity of the bC1 complex at low inhibitor/enzyme ratios. This stimulation could only be fitted to a model in which half of the bC1 dimer is inactive when both center N sites are free, becoming active upon binding of one center N inhibitor molecule per dimer, and there is electron transfer between the Cytochrome b subunits of the dimer. These results are consistent with an alternating half-of-the-sites mechanism of ubiquinol oxidation in the bC1 complex dimer.
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evidence for a concerted mechanism of ubiquinol oxidation by the Cytochrome bc 1 complex
Journal of Biological Chemistry, 2000Co-Authors: Christopher H Snyder, Emma Berta Gutierrezcirlos, Bernard L. TrumpowerAbstract:To better understand the mechanism of divergent electron transfer from ubiquinol to the iron-sulfur protein and Cytochrome bL within the Cytochrome bC1 complex, we have examined the effects of antimycin on the presteady state reduction kinetics of the bC1 complex in the presence or absence of endogenous ubiquinone. When ubiquinone is present, antimycin slows the rate of Cytochrome C1 reduction by ;10-fold but had no effect upon the rate of Cytochrome C1 reduction in bC1 complex lacking endogenous ubiquinone. In the absence of endogenous ubiquinone Cytochrome C1, reduction was slower than when ubiquinone was present and was similar to that in the presence of ubiquinone plus antimycin. These results indicate that the low potential redox components, Cytochrome bH and bL, exert negative control on the rate of reduction of Cytochrome C1 and the Rieske iron-sulfur protein at center P. If electrons cannot equilibrate from Cytochrome bH and bL to ubiquinone, partial reduction of the low potential components slows reduction of the high potential components. We also examined the effects of decreasing the midpoint potential of the iron-sulfur protein on the rates of Cytochrome b reduction. As the midpoint potential decreased, there was a parallel decrease in the rate of b reduction, demonstrating that the rate of b reduction is dependent upon the rate of ubiquinol oxidation by the iron-sulfur protein. Together these results indicate that ubiquinol oxidation is a concerted reaction in which both the low potential and high potential redox components control ubiquinol oxidation at center P, consistent with the protonmotive Q cycle mechanism.
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aromatic amino acids in the rieske iron sulfur protein do not form an obligatory conduit for electron transfer from the iron sulfur cluster to the heme of Cytochrome C1 in the Cytochrome bC1 complex
Biochimica et Biophysica Acta, 1999Co-Authors: Christopher H Snyder, Elke Denke, Bernard L. TrumpowerAbstract:Abstract We have changed nine conserved aromatic amino acids by site-directed mutagenesis of the cloned iron–sulfur protein gene to determine if any of these residues form an obligatory conduit for electron transfer within the iron–sulfur protein of the yeast Cytochrome bC1 complex. The residues include W111, F117, W152, F173, W176, F177, H184, Y205 and F207. Greater than 70% of the catalytic activity was retained for all of the mutated iron–sulfur proteins, except for those containing a W152L and a W176L–F177L double mutation, for which the activity was ∼45%. The crystal structures of the bC1 complex indicate that F177 and H184 are at the surface of the iron–sulfur protein near the surface of Cytochrome C1, but not directly in a linear pathway between the iron–sulfur cluster and the C1 heme. The pre-steady-state rates of reduction of Cytochromes b and C1 in mutants in which F177 and H184 were changed to non-aromatic residues were approximately 70–85% of the wild-type rates. There was a large decrease in iron–sulfur protein levels in mitochondrial membranes resulting from the W152L mutation and the W176L–F177L double mutation, and a small decrease for the Y205L, W176L and F177L mutations. This indicates that the decreases in activity resulting from these amino acid changes are due to instability of the altered proteins. These results show that these aromatic amino acids are unnecessary for electron transfer, but several are required for structural stability.
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deletion of qcr6 the gene encoding subunit six of the mitochondrial Cytochrome bC1 complex blocks maturation of Cytochrome C1 and causes temperature sensitive petite growth in saccharomyces cerevisiae
Journal of Biological Chemistry, 1994Co-Authors: Meijia Yang, Bernard L. TrumpowerAbstract:It was previously reported that disruption or deletion of QCR6, the nuclear gene encoding subunit 6 of the Cytochrome bC1 complex, does not impair growth of yeast on non-fermentable carbon sources (Schoppink, P. J., Hemrika, W., Reyne, J. M., Grivell, L.A., and Berden, J. A. (1988) Eur. J. Biochem. 113, 115-122; Crivellone, M. D., Wu, M. M., and Tzagoloff, A. (1988) J. Biol. Chem. 262, 14323-14333; Schmitt, M. E., and Trumpower, B. L. (1990) J. Biol. Chem. 265, 17005-17011). We have discovered that deletion of QCR6 results in a temperature-sensitive petite phenotype, manifested at 37 degrees C, and that this phenotype can be masked by spontaneously arising suppressor mutations. Mitochondrial membranes from the deletion strain grown at 37 degrees C lack ubiquinol-Cytochrome c reductase activity, and optical spectra reveal an extensive decrease in Cytochrome b absorption, but little or no decrease in Cytochrome C1 absorption. Immunoblots of mitochondrial membrane proteins from the deletion strain indicate that processing of Cytochrome C1 from intermediate to mature size is blocked coincident with the loss of subunit 6. This is the first example where mutation of a subunit within the bC1 complex blocks maturation of Cytochrome C1.
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acidic regions of Cytochrome C1 are essential for ubiquinol Cytochrome c reductase activity in yeast cells lacking the acidic qcr6 protein
Journal of Biochemistry, 1993Co-Authors: Masato Nakai, Bernard L. Trumpower, Toshiya Endo, Toshiharu Hase, Yoshikazu Tanaka, Haruko Ishiwatari, Akiko Asada, Mayumi Bogaki, Hiroshi MatsubaraAbstract:It has been suggested that the two acidic regions around residue 70 and residue 170 in yeast Cytochrome C1, a subunit of ubiquinol-Cytochrome c reductase (complex III), interact with Cytochrome c in the electron transfer reaction and that the QCR6 protein, the acidic subunit of yeast complex III, enhances this interaction. In order to determine the roles of the acidic regions of Cytochrome C1 more precisely, we introduced several mutations in the two acidic regions and examined their effects on the ability of modified Cytochrome C1 to complement the respiration deficiency of yeast cells lacking only Cytochrome C1 or both Cytochrome C1 and the QCR6 protein. The mutant Cytochrome C1 with the deletion of the first acidic region (delta 68-80) was still functional in the Cytochrome C1-deficient strain. Mutant Cytochrome C1 with the deletion of the second acidic region (delta 168-179) caused a decrease in the complementing ability, but this is probably due to failure in its proteolytic maturation and/or correct assembly into complex III. Mutant Cytochrome C1 with altered charge distribution in the acidic regions (Asp170Asp171-->Asn170Asn171 or Asp170Asp171-->Asn170Lys171) made the Cytochrome C1-deficient cells respiration-competent. On the other hand, mutant Cytochrome C1 with the deletion of the first acidic region (delta 68-80) or altered charge distribution in the second region (Asp170Asp171-->Asn170Lys171) did not restore the respiration deficiency of the cells lacking not only Cytochrome C1 but also the QCR6 protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Antony R Crofts - One of the best experts on this subject based on the ideXlab platform.
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physicochemical aspects of the movement of the rieske iron sulfur protein during quinol oxidation by the bC1 complex from mitochondria and photosynthetic bacteria
Biochemistry, 1999Co-Authors: Antony R Crofts, Zhaolei Zhang, Sangjin Hong, E A BerryAbstract:Crystallographic structures for the mitochondrial ubihydroquinone:Cytochrome c oxidoreductase (bC1 complex) from different sources, and with different inhibitors in cocrystals, have revealed that the extrinsic domain of the iron sulfur subunit is not fixed (Zhang, Z., Huang, L., Shulmeister, V. M., Chi, Y.-I., Kim, K. K., Hung, L.-W., Crofts, A. R., Berry, E. A., and Kim, S.-H. (1998) Nature (London), 392, 677-684), but moves between reaction domains on Cytochrome C1 and Cytochrome b subunits. We have suggested that the movement is necessary for quinol oxidation at the Qo site of the complex. In this paper, we show that the electron-transfer reactions of the high-potential chain of the complex, including oxidation of the iron sulfur protein by Cytochrome C1 and the reactions by which oxidizing equivalents become available at the Qo site, are rapid compared to the rate-determining step. Activation energies of partial reactions that contribute to movement of the iron sulfur protein have been measured and shown to be lower than the high activation barrier associated with quinol oxidation. We conclude that the movement is not the source of the activation barrier. We estimate the occupancies of different positions for the iron sulfur protein from the crystallographic electron densities and discuss the parameters determining the binding of the iron sulfur protein in different configurations. The low activation barrier is consistent with a movement between these locations through a constrained diffusion. Apart from ligation in enzyme- substrate or inhibitor complexes, the binding forces in the native structure are likely to be eRT, suggesting that the mobile head can explore the reaction interfaces through stochastic processes within the time scale indicated by kinetic measurements.
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electron transfer by domain movement in Cytochrome bC1
Nature, 1998Co-Authors: Zhaolei Zhang, Edward A. Berry, Antony R Crofts, Lishar Huang, Vladimir M Shulmeister, Young In Chi, Kyeong Kyu Kim, Liwei Hung, Sunghou KimAbstract:The Cytochrome bC1 is one of the three major respiratory enzyme complexes residing in the inner mitochondrial membrane. Cytochrome bC1 transfers electrons from ubiquinol to Cytochrome c and uses the energy thus released to form an electrochemical gradient across the inner membrane. Our X-ray crystal structures of the complex from chicken, cow and rabbit in both the presence and absence of inhibitors of quinone oxidation, reveal two different locations for the extrinsic domain of one component of the enzyme, an iron-sulphur protein. One location is close enough to the supposed quinol oxidation site to allow reduction of the Fe-S protein by ubiquinol. The other site is close enough to Cytochrome C1 to allow oxidation of the Fe-S protein by the Cytochrome. As neither location will allow both reactions to proceed at a suitable rate, the reaction mechanism must involve movement of the extrinsic domain of the Fe-S component in order to shuttle electrons from ubiquinol to Cytochrome C1. Such a mechanism has not previously been observed in redox protein complexes.
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preparation and characterization of the water soluble heme binding domain of Cytochrome C1 from the rhodobacter sphaeroides bC1 complex
Journal of Biological Chemistry, 1991Co-Authors: K Konishi, S R Van Doren, David Kramer, Antony R Crofts, Robert B GennisAbstract:The ubiquinol:Cytochrome c2 oxidoreductase (bC1 complex) of Rhodobacter sphaeroides consists of four subunits. One of these subunits, Cytochrome C1, is the site of interaction with Cytochrome c2, a periplasmic protein. In addition, the sequences of the fbcC gene and of the Cytochrome C1 subunit that it encodes suggest that the protein should be located on the periplasmic side of the cytoplasmic membrane and that it is anchored to the membrane by a single membrane-spanning alpha-helix located at the carboxyl-terminal end of the polypeptide. Site-directed mutagenesis of the fbcC gene was used to alter the codon for Gln228 to a stop codon. This results in the production of a truncated version of the Cytochrome C1 subunit that lacks the membrane anchor at the carboxyl terminus. The bC1 complex fails to assemble properly as a result of this mutation, but the Rb. sphaeroides cells expressing the altered gene contain a water-soluble form of Cytochrome C1 in the periplasm. The water-soluble Cytochrome C1 was purified and characterized. The amino-terminal sequence is identical with that of the membrane-bound subunit, indicating the signal sequence is properly processed. High pressure liquid chromatography gel filtration chromatography indicates it is monomeric (28 kDa). The heme content and electrochemical properties are similar to those of the intact subunit within the complex. Flash-induced electron transfer kinetics measured using whole cells demonstrated that the water-soluble Cytochrome C1 is competent as a reductant for Cytochrome c2 within the periplasmic space. These data show that the isolated water-soluble Cytochrome C1 retains many of the properties of the membrane-bound subunit of the bC1 complex and, therefore, will be useful for further structural and functional characterization.
Artur Osyczka - One of the best experts on this subject based on the ideXlab platform.
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functional flexibility of electron flow between quinol oxidation qo site of Cytochrome bC1 and Cytochrome c revealed by combinatory effects of mutations in Cytochrome b iron sulfur protein and Cytochrome C1
Biochimica et Biophysica Acta, 2018Co-Authors: Arkadiusz Borek, Robert Ekiert, Artur OsyczkaAbstract:Transfer of electron from quinol to Cytochrome c is an integral part of catalytic cycle of Cytochrome bC1. It is a multi-step reaction involving: i) electron transfer from quinol bound at the catalytic Qo site to the Rieske iron-sulfur ([2Fe-2S]) cluster, ii) large-scale movement of a domain containing [2Fe-2S] cluster (ISP-HD) towards Cytochrome C1, iii) reduction of Cytochrome C1 by reduced [2Fe-2S] cluster, iv) reduction of Cytochrome c by Cytochrome C1. In this work, to examine this multi-step reaction we introduced various types of barriers for electron transfer within the chain of [2Fe-2S] cluster, Cytochrome C1 and Cytochrome c. The barriers included: impediment in the motion of ISP-HD, uphill electron transfer from [2Fe-2S] cluster to heme C1 of Cytochrome C1, and impediment in the catalytic quinol oxidation. The barriers were introduced separately or in various combinations and their effects on enzymatic activity of Cytochrome bC1 were compared. This analysis revealed significant degree of functional flexibility allowing the cofactor chains to accommodate certain structural and/or redox potential changes without losing overall electron and proton transfers capabilities. In some cases inhibitory effects compensated one another to improve/restore the function. The results support an equilibrium model in which a random oscillation of ISP-HD between the Qo site and Cytochrome C1 helps maintaining redox equilibrium between all cofactors of the chain. We propose a new concept in which independence of the dynamics of the Qo site substrate and the motion of ISP-HD is one of the elements supporting this equilibrium and also is a potential factor limiting the overall catalytic rate.
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visualizing changes in electron distribution in coupled chains of Cytochrome bC1 by modifying barrier for electron transfer between the fes cluster and heme C1
Biochimica et Biophysica Acta, 2010Co-Authors: Ewelina Cieluch, Krzysztof Pietryga, Marcin Sarewicz, Artur OsyczkaAbstract:Cytochrome C1 of Rhodobacter (Rba.) species provides a series of mutants which change barriers for electron transfer through the cofactor chains of Cytochrome bC1 by modifying heme C1 redox midpoint potential. Analysis of post-flash electron distribution in such systems can provide useful information about the contribution of individual reactions to the overall electron flow. In Rba. capsulatus, the non-functional low-potential forms of Cytochrome C1 which are devoid of the disulfide bond naturally present in this protein revert spontaneously by introducing a second-site suppression (mutation A181T) that brings the potential of heme C1 back to the functionally high levels, yet maintains it some 100 mV lower from the native value. Here we report that the disulfide and the mutation A181T can coexist in one protein but the mutation exerts a dominant effect on the redox properties of heme C1 and the potential remains at the same lower value as in the disulfide-free form. This establishes effective means to modify a barrier for electron transfer between the FeS cluster and heme C1 without breaking disulfide. A comparison of the flash-induced electron transfers in native and mutated Cytochrome bC1 revealed significant differences in the post-flash equilibrium distribution of electrons only when the connection of the chains with the quinone pool was interrupted at the level of either of the catalytic sites by the use of specific inhibitors, antimycin or myxothiazol. In the non-inhibited system no such differences were observed. We explain the results using a kinetic model in which a shift in the equilibrium of one reaction influences the equilibrium of all remaining reactions in the cofactor chains. It follows a rather simple description in which the direction of electron flow through the coupled chains of Cytochrome bC1 exclusively depends on the rates of all reversible partial reactions, including the Q/QH2 exchange rate to/from the catalytic sites.
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atr ftir spectroscopy studies of iron sulfur protein and Cytochrome C1 in the rhodobacter capsulatus Cytochrome bC1 complex
Biochemistry, 2004Co-Authors: Masayo Iwaki, Artur Osyczka, Christopher C Moser, Leslie P Dutton, Peter R RichAbstract:Redox transitions in the Rhodobacter capsulatus Cytochrome bC1 complex were investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy combined with synchronous visible spectroscopy, in both the wild type and a Cytochrome C1 point mutant, M183K, in which the midpoint potential of heme was lowered from the wild-type value of 320 mV to 60 mV. Overall redox difference spectra of the wild type and M183K mutant were essentially identical, indicating that the mutation did not cause any major structural perturbation. Spectra were compared with data on the bovine bC1 complex, and tentative assignments of several bands could be made by comparison with available data on model compounds and crystallographic structures. The bacterial spectra showed contributions from ubiquinone that were much larger than in the bovine enzyme, arising from additional bound and adventitious ubiquinone. The M183K mutant enabled selective reduction of the iron−sulfur protein which in ...
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novel cyanide inhibition at Cytochrome C1 of rhodobacter capsulatus Cytochrome bC1
Biochimica et Biophysica Acta, 2004Co-Authors: Artur Osyczka, Christopher C Moser, Leslie P DuttonAbstract:Abstract Oxidized Cytochrome c 1 in photosynthetic bacterium Rhodobacter capsulatus Cytochrome bc 1 reversibly binds cyanide with surprisingly high, micromolar affinity. The binding dramatically lowers the redox midpoint potential of heme c 1 and inhibits steady-state turnover activity of the enzyme. As Cytochrome c 1 , an auxiliary redox center of the high-potential chain of Cytochrome bc 1 , does not interact directly with the catalytic quinone/quinol binding sites Q o and Q i , cyanide introduces a novel, Q-site independent locus of inhibition. This is the first report of a reversible inhibitor that manipulates the energetics and electron transfers of the high-potential redox chain of Cytochrome bc 1 , while maintaining quinone substrate catalytic sites in an intact form.
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spectroscopic and oxidation reduction properties of rhodobacter capsulatus Cytochrome C1 and its m183k and m183h variants
Biochimica et Biophysica Acta, 2002Co-Authors: Elisabeth Darrouzet, Artur Osyczka, Fevzi Daldal, Ish K Dhawan, Michael K Johnson, David B KnaffAbstract:Abstract Two variants of the Cytochrome c 1 component of the Rhodobacter capsulatus Cytochrome bc 1 complex, in which Met 183 (an axial heme ligand) was replaced by lysine (M183K) or histidine (M183H), have been analyzed. Electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectra of the intact complex indicate that the histidine/methionine heme ligation of the wild-type Cytochrome is replaced by histidine/lysine ligation in M183K and histidine/histidine ligation in M183H. Variable amounts of histidine/histidine axial heme ligation were also detected in purified wild-type Cytochrome c 1 and its M183K variant, suggesting that a histidine outside the CSACH heme-binding domain can be recruited as an alternative ligand. Oxidation–reduction titrations of the heme in purified Cytochrome c 1 revealed multiple redox forms. Titrations of the purified Cytochrome carried out in the oxidative or reductive direction differ. In contrast, titrations of Cytochrome c 1 in the intact bc 1 complex and in a subcomplex missing the Rieske iron–sulfur protein were fully reversible. An E m7 value of −330 mV was measured for the single disulfide bond in Cytochrome c 1 . The origins of heme redox heterogeneity, and of the differences between reductive and oxidative heme titrations, are discussed in terms of conformational changes and the role of the disulfide in maintaining the native structure of Cytochrome c 1 .
Robert B Gennis - One of the best experts on this subject based on the ideXlab platform.
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preparation and characterization of the water soluble heme binding domain of Cytochrome C1 from the rhodobacter sphaeroides bC1 complex
Journal of Biological Chemistry, 1991Co-Authors: K Konishi, S R Van Doren, David Kramer, Antony R Crofts, Robert B GennisAbstract:The ubiquinol:Cytochrome c2 oxidoreductase (bC1 complex) of Rhodobacter sphaeroides consists of four subunits. One of these subunits, Cytochrome C1, is the site of interaction with Cytochrome c2, a periplasmic protein. In addition, the sequences of the fbcC gene and of the Cytochrome C1 subunit that it encodes suggest that the protein should be located on the periplasmic side of the cytoplasmic membrane and that it is anchored to the membrane by a single membrane-spanning alpha-helix located at the carboxyl-terminal end of the polypeptide. Site-directed mutagenesis of the fbcC gene was used to alter the codon for Gln228 to a stop codon. This results in the production of a truncated version of the Cytochrome C1 subunit that lacks the membrane anchor at the carboxyl terminus. The bC1 complex fails to assemble properly as a result of this mutation, but the Rb. sphaeroides cells expressing the altered gene contain a water-soluble form of Cytochrome C1 in the periplasm. The water-soluble Cytochrome C1 was purified and characterized. The amino-terminal sequence is identical with that of the membrane-bound subunit, indicating the signal sequence is properly processed. High pressure liquid chromatography gel filtration chromatography indicates it is monomeric (28 kDa). The heme content and electrochemical properties are similar to those of the intact subunit within the complex. Flash-induced electron transfer kinetics measured using whole cells demonstrated that the water-soluble Cytochrome C1 is competent as a reductant for Cytochrome c2 within the periplasmic space. These data show that the isolated water-soluble Cytochrome C1 retains many of the properties of the membrane-bound subunit of the bC1 complex and, therefore, will be useful for further structural and functional characterization.
Christopher H Snyder - One of the best experts on this subject based on the ideXlab platform.
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evidence for a concerted mechanism of ubiquinol oxidation by the Cytochrome bc 1 complex
Journal of Biological Chemistry, 2000Co-Authors: Christopher H Snyder, Emma Berta Gutierrezcirlos, Bernard L. TrumpowerAbstract:To better understand the mechanism of divergent electron transfer from ubiquinol to the iron-sulfur protein and Cytochrome bL within the Cytochrome bC1 complex, we have examined the effects of antimycin on the presteady state reduction kinetics of the bC1 complex in the presence or absence of endogenous ubiquinone. When ubiquinone is present, antimycin slows the rate of Cytochrome C1 reduction by ;10-fold but had no effect upon the rate of Cytochrome C1 reduction in bC1 complex lacking endogenous ubiquinone. In the absence of endogenous ubiquinone Cytochrome C1, reduction was slower than when ubiquinone was present and was similar to that in the presence of ubiquinone plus antimycin. These results indicate that the low potential redox components, Cytochrome bH and bL, exert negative control on the rate of reduction of Cytochrome C1 and the Rieske iron-sulfur protein at center P. If electrons cannot equilibrate from Cytochrome bH and bL to ubiquinone, partial reduction of the low potential components slows reduction of the high potential components. We also examined the effects of decreasing the midpoint potential of the iron-sulfur protein on the rates of Cytochrome b reduction. As the midpoint potential decreased, there was a parallel decrease in the rate of b reduction, demonstrating that the rate of b reduction is dependent upon the rate of ubiquinol oxidation by the iron-sulfur protein. Together these results indicate that ubiquinol oxidation is a concerted reaction in which both the low potential and high potential redox components control ubiquinol oxidation at center P, consistent with the protonmotive Q cycle mechanism.
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aromatic amino acids in the rieske iron sulfur protein do not form an obligatory conduit for electron transfer from the iron sulfur cluster to the heme of Cytochrome C1 in the Cytochrome bC1 complex
Biochimica et Biophysica Acta, 1999Co-Authors: Christopher H Snyder, Elke Denke, Bernard L. TrumpowerAbstract:Abstract We have changed nine conserved aromatic amino acids by site-directed mutagenesis of the cloned iron–sulfur protein gene to determine if any of these residues form an obligatory conduit for electron transfer within the iron–sulfur protein of the yeast Cytochrome bC1 complex. The residues include W111, F117, W152, F173, W176, F177, H184, Y205 and F207. Greater than 70% of the catalytic activity was retained for all of the mutated iron–sulfur proteins, except for those containing a W152L and a W176L–F177L double mutation, for which the activity was ∼45%. The crystal structures of the bC1 complex indicate that F177 and H184 are at the surface of the iron–sulfur protein near the surface of Cytochrome C1, but not directly in a linear pathway between the iron–sulfur cluster and the C1 heme. The pre-steady-state rates of reduction of Cytochromes b and C1 in mutants in which F177 and H184 were changed to non-aromatic residues were approximately 70–85% of the wild-type rates. There was a large decrease in iron–sulfur protein levels in mitochondrial membranes resulting from the W152L mutation and the W176L–F177L double mutation, and a small decrease for the Y205L, W176L and F177L mutations. This indicates that the decreases in activity resulting from these amino acid changes are due to instability of the altered proteins. These results show that these aromatic amino acids are unnecessary for electron transfer, but several are required for structural stability.
