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Bernard L. Trumpower - One of the best experts on this subject based on the ideXlab platform.
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the rate limiting step in the cytochrome bc1 complex ubiquinol cytochrome c oxidoreductase is not changed by inhibition of cytochrome b dependent deprotonation implications for the mechanism of ubiquinol oxidation at center p of the bc1 complex
Journal of Biological Chemistry, 2009Co-Authors: Raul Covian, Bernard L. TrumpowerAbstract:Abstract Quinol oxidation at center P of the cytochrome bc1 complex involves bifurcated electron transfer to the Rieske iron-sulfur Protein and cytochrome b. It is unknown whether both electrons are transferred from the same domain close to the Rieske Protein, or if an unstable semiquinone anion intermediate diffuses rapidly to the vicinity of the bL heme. We have determined the pre-steady state rate and activation energy (Ea) for quinol oxidation in purified yeast bc1 complexes harboring either a Y185F mutation in the Rieske Protein, which decreases the redox potential of the FeS cluster, or a E272Q cytochrome b mutation, which eliminates the proton acceptor in cytochrome b. The rate of the bifurcated reaction in the E272Q mutant (<10% of the wild type) was even lower than that of the Y185F enzyme (∼20% of the wild type). However, the E272Q enzyme showed the same Ea (61 kJ mol-1) with respect to the wild type (62 kJ mol-1), in contrast with the Y185F mutation, which increased Ea to 73 kJ mol-1. The rate and Ea of the slow reaction of quinol with oxygen that are observed after cytochrome b is reduced were unaffected by the E272Q substitution, whereas the Y185F mutation modified only its rate. The Y185F/E272Q double mutation resulted in a synergistic decrease in the rate of quinol oxidation (0.7% of the wild type). These results are inconsistent with a sequential “movable semiquinone” mechanism but are consistent with a model in which both electrons are transferred simultaneously from the same domain in center P.
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regulatory interactions between ubiquinol oxidation and ubiquinone reduction sites in the dimeric cytochrome bc1 complex
Journal of Biological Chemistry, 2006Co-Authors: Raul Covian, Bernard L. TrumpowerAbstract:We have obtained evidence for conformational communication between ubiquinol oxidation (center P) and ubiquinone reduction (center N) sites of the yeast bc1 complex dimer by analyzing antimycin binding and heme bH reduction at center N in the presence of different center P inhibitors. When stigmatellin was occupying center P, concentration-dependent binding of antimycin occurred only to half of the center N sites. The remaining half of the bc1 complex bound antimycin with a slower rate that was independent of inhibitor concentration, indicating that a slow conformational change needed to occur before half of the enzyme could bind antimycin. In contrast, under conditions where the Rieske Protein was not fixed proximal to heme bL at center P, all center N sites bound antimycin with fast and concentration-dependent kinetics. Additionally, the extent of fast cytochrome b reduction by menaquinol through center N in the presence of stigmatellin was approximately half of that observed when myxothiazol was bound at center P. The reduction kinetics of the bH heme by decylubiquinol in the presence of stigmatellin or myxothiazol were also consistent with a model in which fixation of the Rieske Protein close to heme bL in both monomers allows rapid binding of ligands only to one center N. Decylubiquinol at high concentrations was able to abolish the biphasic binding of antimycin in the presence of stigmatellin but did not slow down antimycin binding rates. These results are discussed in terms of half-of-the-sites activity of the dimeric bc1 complex.
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Elimination of the disulfide bridge in the Rieske iron-sulfur Protein allows assembly of the [2Fe-2S] cluster into the Rieske Protein but damages the ubiquinol oxidation site in the cytochrome bc1 complex.
Biochemistry, 2003Co-Authors: Torsten Merbitz-zahradnik, Klaus Zwicker, Jurgen H Nett, Thomas A. Link, Bernard L. TrumpowerAbstract:The (2Fe-2S) cluster of the Rieske iron-sulfur Protein is held between two loops of the Protein that are connected by a disulfide bridge. We have replaced the two cysteines that form the disulfide bridge in the Rieske Protein of Saccharomyces cereVisiae with tyrosine and leucine, and tyrosine and valine, to evaluate the effects of the disulfide bridge on assembly, stability, and thermodynamic properties of the Rieske iron-sulfur cluster. EPR spectra of the Rieske Proteins lacking the disulfide bridge indicate the iron-sulfur cluster is assembled in the absence of the disulfide bridge, but there are significant shifts in all g values, indicating a change in the electronic structure of the (2Fe-2S) iron -sulfur center. In addition, the midpoint potential of the iron-sulfur cluster is lowered from 265 mV in the Rieske Protein from wild-type yeast to 150 mV in the Protein from the C164Y/C180L mutant and to 160 mV in the Protein from the C164Y/C180V mutant. Ubiquinol-cytochrome c reductase activities of the bc1 complexes with Rieske Proteins lacking the disulfide bridge are less than 1% of the activity of the bc1 complex from wild-type yeast, even though normal amounts of the iron-sulfur Protein are present as judged by Western blot analysis. These activities are lower than the 105 -115 mV decrease in the midpoint potential of the Rieske iron-sulfur cluster can account for. Pre-steady-state reduction of the bc1 complexes with menadiol indicates that quinol is not oxidized through center P but is oxidized through center N. In addition, the levels of stigmatellin and UHDBT binding are markedly diminished, while antimycin binding is unaffected, in the bc1 complexes with Rieske Proteins lacking the disulfide bridge. Taken together, these results indicate that the ubiquinol oxidation site at center P is damaged in the bc1 complexes with Rieske Proteins lacking the disulfide bridge even though the iron-sulfur cluster is assembled into the Rieske Protein.
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structure of the yeast cytochrome bc1 complex with a hydroxyquinone anion qo site inhibitor bound
Journal of Biological Chemistry, 2003Co-Authors: Hildur Palsdottir, Bernard L. Trumpower, Carlos Gomez Lojero, Carola HunteAbstract:Abstract Bifurcated electron transfer during ubiquinol oxidation is the key reaction of cytochrome bc1 complex catalysis. Binding of the competitive inhibitor 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole to the Qo site of the cytochrome bc1 complex from Saccharomyces cerevisiae was analyzed by x-ray crystallography. This alkylhydroxydioxobenzothiazole is bound in its ionized form as evident from the crystal structure and confirmed by spectroscopic analysis, consistent with a measured pKa = 6.1 of the hydroxy group in detergent micelles. Stabilizing forces for the hydroxyquinone anion inhibitor include a polarized hydrogen bond to the iron-sulfur cluster ligand His181 and on-edge interactions via weak hydrogen bonds with cytochrome b residue Tyr279. The hydroxy group of the latter contributes to stabilization of the Rieske Protein in the b-position by donating a hydrogen bond. The reported pH dependence of inhibition with lower efficacy at alkaline pH is attributed to the protonation state of His181 with a pKa of 7.5. Glu272, a proposed primary ligand and proton acceptor of ubiquinol, is not bound to the carbonyl group of the hydroxydioxobenzothiazole ring but is rotated out of the binding pocket toward the heme bL propionate A, to which it is hydrogen-bonded via a single water molecule. The observed hydrogen bonding pattern provides experimental evidence for the previously proposed proton exit pathway involving the heme propionate and a chain of water molecules. Binding of the alkyl-6-hydroxy-4,7-dioxobenzothiazole is discussed as resembling an intermediate step of ubiquinol oxidation, supporting a single occupancy model at the Qo site.
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failure to insert the iron sulfur cluster into the Rieske iron sulfur Protein impairs both center n and center p of the cytochrome bc 1 complex
Journal of Biological Chemistry, 2002Co-Authors: Emma Berta Gutierrezcirlos, Torsten Merbitzzahradnik, Bernard L. TrumpowerAbstract:Abstract Mutation of a serine that forms a hydrogen bond to the iron-sulfur cluster of the Rieske iron-sulfur Protein to a cysteine results in a respiratory-deficient yeast strain due to formation of iron-sulfur Protein lacking the iron-sulfur cluster. The Rieske apoProtein lacking the iron-sulfur cluster is inserted into both monomers of the dimeric cytochrome bc 1 complex and processed to mature size, but the Protein lacking iron-sulfur cluster is more susceptible to proteolysis. In addition, the Protein environment of center P in one half of the dimer is affected by failure to insert the iron-sulfur cluster as indicated by the fact that only one molecule of myxothiazol can be bound to the cytochromebc 1 dimer. Although thebc 1 complex lacking the Rieske iron-sulfur cluster cannot oxidize ubiquinol through center P, rates of reduction of cytochrome b by menaquinol through center N are normal. However, less cytochrome b is reduced through center N, and only one molecule of antimycin can be bound at center N in the bc 1 dimer lacking iron-sulfur cluster. These results indicate that failure to insert the [2Fe-2S] cluster impairs assembly of the Rieske Protein into the bc 1 complex and that this interferes with proper assembly of both center P and center N in one half of the dimeric enzyme.
Matthias Rögner - One of the best experts on this subject based on the ideXlab platform.
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Structural and functional characterisation of the cyanobacterial PetC3 Rieske Protein family
Biochimica biophysica acta (BBA) - Bioenergetics, 2016Co-Authors: Sebastian Veit, Kazuki Takeda, Yuichi Tsunoyama, Matthias Rögner, Kunio Miki, Reinat Nevo, Ziv Reich, Sascha RexrothAbstract:The cyanobacterium Synechocystis PCC 6803 possesses three Rieske isoforms: PetC1, PetC2 and PetC3. While PetC1 and PetC2 have been identified as alternative subunits of the cytochrome b6f complex (b6f), PetC3 was localized exclusively within the plasma membrane. The spatial separation of PetC3 from the photosynthetic and respiratory Protein complexes raises doubt in its involvement in bioenergetic electron transfer. Here we report a detailed structural and functional characterization of the cyanobacterial PetC3 Protein family indicating that PetC3 is not a component of the b6f and the photosynthetic electron transport as implied by gene annotation. Instead PetC3 has a distinct function in cell envelope homeostasis. Especially proteomic analysis shows that deletion of petC3 in Synechocystis PCC 6803 primarily affects cell envelope Proteins including many nutrient transport systems. Therefore, the observed downregulation in the photosynthetic electron transport - mainly caused by photosystem 2 inactivation - might constitute a stress adaptation. Comprehensive in silico sequence analyses revealed that PetC3 Proteins are periplasmic lipoProteins tethered to the plasma membrane with a subclass consisting of soluble periplasmic Proteins, i.e. their N-terminal domain is inconsistent with their integration into the b6f. For the first time, the structure of PetC3 was determined by X-ray crystallography at an atomic resolution revealing significant high similarities to non-b6f Rieske subunits in contrast to PetC1. These results suggest that PetC3 affects processes in the periplasmic compartment that only indirectly influence photosynthetic electron transport. For this reason, we suggest to rename "Photosynthetic electron transport Chain 3" (PetC3) Proteins as "periplasmic Rieske Proteins" (Prp).
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Structure of a thermophilic cyanobacterial b6f-type Rieske Protein.
Acta crystallographica. Section D Biological crystallography, 2012Co-Authors: Sebastian Veit, Kazuki Takeda, Yuichi Tsunoyama, Dorothea Rexroth, Matthias Rögner, Kunio MikiAbstract:The `Rieske Protein' PetC is one of the key subunits of the cytochrome b(6)f complex. Its Rieske-type [2Fe-2S] cluster participates in the photosynthetic electron-transport chain. Overexpression and careful structure analysis at 2.0 Å resolution of the extrinsic soluble domain of PetC from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 enabled in-depth spectroscopic and structural characterization and suggested novel structural features. In particular, both the Protein structure and the positions of the internal water molecules unexpectedly showed a higher similarity to eukaryotic PetCs than to other prokaryotic PetCs. The structure also revealed a deep pocket on the PetC surface which is oriented towards the membrane surface in the whole complex. Its surface properties suggest a binding site for a hydrophobic compound and the complete conservation of the pocket-forming residues in all known PetC sequences indicates the functional importance of this pocket in the cytochrome b(6)f complex.
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dynamics of the cyanobacterial photosynthetic network communication and modification of membrane Protein complexes
European Journal of Cell Biology, 2010Co-Authors: Marc M Nowaczyk, Dorothea Rexroth, Julia Sander, Nicole Grasse, Kai U Cormann, Gabor Bernat, Matthias RögnerAbstract:Cyanobacterial photosystem 2 and cytochrome b(6)f complexes have been structurally resolved up to the molecular level while the adjustment of their function in response to environmental and intracellular parameters is based on various modifications of these complexes which have not yet been resolved in detail. This minireview summarizes recent results on two central modifications for each complex: (a) for the cytochrome b(6)f complex the implication of PetP, a new subunit, and of three copies of PetC, the Rieske Protein, for the fine-tuning of the photosynthetic electron transport is evaluated; (b) for photosystem 2, the heterogeneity of the D1 subunit and the role of subunit Psb27 is discussed in relation to stress response and the biogenesis/repair cycle. The presented "dynamic" models for both complexes should illustrate the need to complement structural by more extensive functional models which consider the flexibility of individual complexes in the physiological context - beyond structure.
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multiple Rieske Proteins enable short and long term light adaptation of synechocystis sp pcc 6803
Journal of Biological Chemistry, 2009Co-Authors: Yuichi Tsunoyama, Gabor Bernat, Nina Gwendolyn Dyczmons, Dirk Schneider, Matthias RögnerAbstract:In contrast to eukaryotes, most cyanobacteria contain several isoforms of the Rieske iron-sulfur Protein, PetC, resulting in heterogeneity in the composition of the cytochrome b6f complexes. Of three isoforms in the mesophilic cyanobacterium Synechocystis PCC 6803, PetC1 is the major Rieske Protein in the cytochrome b6f complex, whereas the physiological function of PetC2 and PetC3 is still uncertain. Comparison of wild type and various petC-deficient strains under selected light conditions revealed distinct functional differences: high-light exposure of wild type cells resulted in a significantly enhanced petC2 transcript level, whereas a ΔpetC1 mutant showed a low cytochrome b6f content, low electron flux, and a considerably increased accumulation of cytochrome-bd oxidase. In contrast to wild type and ΔpetC1, ΔpetC2 and ΔpetC3 strains still grew fast under high-light conditions although all three Rieske Proteins are required for maximal electron transport rates. Although the presence of PetC3 appears to be required for activation of the cyclic electron transport, state transitions were more effective in the absence of PetC2 and/or PetC3. In summary, our data suggest defined roles of the various PetC Proteins in short- and long-term light adaptation.
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PetC1 is the major Rieske iron-sulfur Protein in the cytochrome b6f complex of Synechocystis sp. PCC 6803
The Journal of biological chemistry, 2004Co-Authors: Dirk Schneider, Andreas Seidler, Stephan Berry, Thomas Volkmer, Matthias RögnerAbstract:Many of the completely sequenced cyanobacterial genomes contain a gene family that encodes for putative Rieske iron-sulfur Proteins. The Rieske Protein is one of the large subunits of the cytochrome bc-type complexes involved in respiratory and photosynthetic electron transfer. In contrast to all other subunits of this complex that are encoded by single genes, the genome of the cyanobacterium Synechocystis PCC 6803 contains three petC genes, all encoding potential Rieske subunits. Most interestingly, any of the petC genes can be deleted individually without altering the Synechocystis phenotype dramatically. In contrast, double deletion experiments revealed that petC1 and petC2 cannot be deleted in combination, whereas petC3 can be deleted together with any of the other two petC genes. Further results suggest a different physiological function for each of the Rieske Proteins. Whereas PetC2 can partly replace the dominating Rieske isoform PetC1, PetC3 is unable to functionally replace either PetC1 or PetC2 and may have a special function involving a special donor with a lower redox potential than plastoquinone. A predominant role of PetC1, which is (partly) different from PetC2, is suggested by the mutational analysis and a detailed characterization of the electron transfer reactions in the mutant strains.
Francis-andré Wollman - One of the best experts on this subject based on the ideXlab platform.
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The Chloroplast Rieske Iron-Sulfur Protein AT THE CROSSROAD OF ELECTRON TRANSPORT AND SIGNAL TRANSDUCTION
The Journal of biological chemistry, 2004Co-Authors: Catherine De Vitry, Giovanni Finazzi, Yexin Ouyang, Francis-andré Wollman, Toivo KallasAbstract:We have addressed the functional and structural roles of three domains of the chloroplast Rieske iron-sulfur Protein; that is, the flexible hinge that connects the transmembrane helix to the soluble cluster-bearing domain, the N-terminal stromal protruding domain, and the transmembrane helix. To this aim mutants were generated in the green alga Chlamydomonas reinhardtii. Their capacities to assemble the cytochrome b6f complex, perform plastoquinol oxidation, and signal redox-induced activation of the light-harvesting complex II kinase during state transition were tested in vivo. Deletion of one residue and extensions of up to five residues in the flexible hinge had no significant effect on complex accumulation or electron transfer efficiency. Deletion of three residues (Delta3G) dramatically decreased reaction rates by a factor of approximately 10. These data indicate that the chloroplast iron-sulfur Protein-linking domain is much more flexible than that of its counterpart in mitochondria. Despite greatly slowed catalysis in the Delta3G mutant, there was no apparent delay in light-harvesting complex II kinase activation or state transitions. This indicates that conformational changes occurring in the Rieske Protein did not represent a limiting step for kinase activation within the time scale tested. No phenotype could be associated with mutations in the N-terminal stromal-exposed domain. In contrast, the N17V mutation in the Rieske Protein transmembrane helix resulted in a large decrease in the cytochrome f synthesis rate. This reveals that the Rieske Protein transmembrane helix plays an active role in assembly-mediated control of cytochrome f synthesis. We propose a structural model to interpret this phenomenon based on the C. reinhardtii cytochrome b6f structure.
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contrasted effects of inhibitors of cytochromeb 6 f complex on state transitions inchlamydomonas reinhardtii the role of qo site occupancy in lhcii kinase activation
Journal of Biological Chemistry, 2001Co-Authors: Giovanni Finazzi, Francesca Zito, Romina Paola Barbagallo, Francis-andré WollmanAbstract:Abstract We have investigated the relationship between the occupancy of the Qo site in the cytochromeb 6 f complex and the activation of the LHCII Protein kinase that controls state transitions. To this aim, fluorescence emission and LHCII phosphorylation patterns were studied in whole cells of Chlamydomonas reinhardtii treated with different plastoquinone analogues. The analysis of fluorescence induction at room temperature indicates that stigmatellin consistently prevented transition to State 2, whereas 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone behaved as an inhibitor of state transitions only after the cells were preilluminated. The same effects were observed on the phosphorylation patterns of the LHCII Proteins, while subunit V of the cytochromeb 6 f complex showed a different behavior. These findings are discussed on the basis of a dynamic structural model of cytochrome b 6 fthat relates the activation of the LHCII kinase to the occupancy of the Qo site and the movement of the Rieske Protein.
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Contrasted effects of inhibitors of cytochrome b6f complex on state transitions in Chlamydomonas reinhardtii: the role of Qo site occupancy in LHCII kinase activation.
The Journal of biological chemistry, 2000Co-Authors: Giovanni Finazzi, Francesca Zito, Romina Paola Barbagallo, Francis-andré WollmanAbstract:We have investigated the relationship between the occupancy of the Q(o) site in the cytochrome b(6)f complex and the activation of the LHCII Protein kinase that controls state transitions. To this aim, fluorescence emission and LHCII phosphorylation patterns were studied in whole cells of Chlamydomonas reinhardtii treated with different plastoquinone analogues. The analysis of fluorescence induction at room temperature indicates that stigmatellin consistently prevented transition to State 2, whereas 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone behaved as an inhibitor of state transitions only after the cells were preilluminated. The same effects were observed on the phosphorylation patterns of the LHCII Proteins, while subunit V of the cytochrome b(6)f complex showed a different behavior. These findings are discussed on the basis of a dynamic structural model of cytochrome b(6)f that relates the activation of the LHCII kinase to the occupancy of the Q(o) site and the movement of the Rieske Protein.
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evidence for a role of clpp in the degradation of the chloroplast cytochrome b 6 f complex
The Plant Cell, 2000Co-Authors: Wojciech Majeran, Francis-andré Wollman, Olivier VallonAbstract:In the green alga Chlamydomonas reinhardtii, the ClpP protease is encoded by an essential chloroplast gene. Mutating its AUG translation initiation codon to AUU reduced ClpP accumulation to 25 to 45% of that of the wild type. Both the mature Protein and the putative precursor containing its insertion sequence were present in reduced amounts. Attenuation of ClpP did not affect growth rates under normal conditions but restricted the ability of the cells to adapt to elevated CO2 levels. It also affected the rate of degradation of the cytochrome b6f complex of the thylakoid membrane in two experimental situations: (1) during nitrogen starvation, and (2) in mutants deficient in the Rieske iron–sulfur Protein. The ClpP level also controls the steady state accumulation of a mutated version of the Rieske Protein. In contrast, attenuation of ClpP did not rescue the fully unassembled subunits in other cytochrome b6f mutants. We conclude that proteolytic disposal of fully or partially assembled cytochrome b6f is controlled by the Clp protease.
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evidence for a role of clpp in the degradation of the chloroplast cytochrome b6f complex
The Plant Cell, 2000Co-Authors: Wojciech Majeran, Francis-andré Wollman, Olivier VallonAbstract:In the green alga Chlamydomonas reinhardtii, the ClpP protease is encoded by an essential chloroplast gene. Mutating its AUG translation initiation codon to AUU reduced ClpP accumulation to 25 to 45% of that of the wild type. Both the mature Protein and the putative precursor containing its insertion sequence were present in reduced amounts. Attenuation of ClpP did not affect growth rates under normal conditions but restricted the ability of the cells to adapt to elevated CO2 levels. It also affected the rate of degradation of the cytochrome b6f complex of the thylakoid membrane in two experimental situations: (1) during nitrogen starvation, and (2) in mutants deficient in the Rieske iron–sulfur Protein. The ClpP level also controls the steady state accumulation of a mutated version of the Rieske Protein. In contrast, attenuation of ClpP did not rescue the fully unassembled subunits in other cytochrome b6f mutants. We conclude that proteolytic disposal of fully or partially assembled cytochrome b6f is controlled by the Clp protease.
Giovanni Finazzi - One of the best experts on this subject based on the ideXlab platform.
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The Chloroplast Rieske Iron-Sulfur Protein AT THE CROSSROAD OF ELECTRON TRANSPORT AND SIGNAL TRANSDUCTION
The Journal of biological chemistry, 2004Co-Authors: Catherine De Vitry, Giovanni Finazzi, Yexin Ouyang, Francis-andré Wollman, Toivo KallasAbstract:We have addressed the functional and structural roles of three domains of the chloroplast Rieske iron-sulfur Protein; that is, the flexible hinge that connects the transmembrane helix to the soluble cluster-bearing domain, the N-terminal stromal protruding domain, and the transmembrane helix. To this aim mutants were generated in the green alga Chlamydomonas reinhardtii. Their capacities to assemble the cytochrome b6f complex, perform plastoquinol oxidation, and signal redox-induced activation of the light-harvesting complex II kinase during state transition were tested in vivo. Deletion of one residue and extensions of up to five residues in the flexible hinge had no significant effect on complex accumulation or electron transfer efficiency. Deletion of three residues (Delta3G) dramatically decreased reaction rates by a factor of approximately 10. These data indicate that the chloroplast iron-sulfur Protein-linking domain is much more flexible than that of its counterpart in mitochondria. Despite greatly slowed catalysis in the Delta3G mutant, there was no apparent delay in light-harvesting complex II kinase activation or state transitions. This indicates that conformational changes occurring in the Rieske Protein did not represent a limiting step for kinase activation within the time scale tested. No phenotype could be associated with mutations in the N-terminal stromal-exposed domain. In contrast, the N17V mutation in the Rieske Protein transmembrane helix resulted in a large decrease in the cytochrome f synthesis rate. This reveals that the Rieske Protein transmembrane helix plays an active role in assembly-mediated control of cytochrome f synthesis. We propose a structural model to interpret this phenomenon based on the C. reinhardtii cytochrome b6f structure.
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contrasted effects of inhibitors of cytochromeb 6 f complex on state transitions inchlamydomonas reinhardtii the role of qo site occupancy in lhcii kinase activation
Journal of Biological Chemistry, 2001Co-Authors: Giovanni Finazzi, Francesca Zito, Romina Paola Barbagallo, Francis-andré WollmanAbstract:Abstract We have investigated the relationship between the occupancy of the Qo site in the cytochromeb 6 f complex and the activation of the LHCII Protein kinase that controls state transitions. To this aim, fluorescence emission and LHCII phosphorylation patterns were studied in whole cells of Chlamydomonas reinhardtii treated with different plastoquinone analogues. The analysis of fluorescence induction at room temperature indicates that stigmatellin consistently prevented transition to State 2, whereas 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone behaved as an inhibitor of state transitions only after the cells were preilluminated. The same effects were observed on the phosphorylation patterns of the LHCII Proteins, while subunit V of the cytochromeb 6 f complex showed a different behavior. These findings are discussed on the basis of a dynamic structural model of cytochrome b 6 fthat relates the activation of the LHCII kinase to the occupancy of the Qo site and the movement of the Rieske Protein.
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Contrasted effects of inhibitors of cytochrome b6f complex on state transitions in Chlamydomonas reinhardtii: the role of Qo site occupancy in LHCII kinase activation.
The Journal of biological chemistry, 2000Co-Authors: Giovanni Finazzi, Francesca Zito, Romina Paola Barbagallo, Francis-andré WollmanAbstract:We have investigated the relationship between the occupancy of the Q(o) site in the cytochrome b(6)f complex and the activation of the LHCII Protein kinase that controls state transitions. To this aim, fluorescence emission and LHCII phosphorylation patterns were studied in whole cells of Chlamydomonas reinhardtii treated with different plastoquinone analogues. The analysis of fluorescence induction at room temperature indicates that stigmatellin consistently prevented transition to State 2, whereas 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone behaved as an inhibitor of state transitions only after the cells were preilluminated. The same effects were observed on the phosphorylation patterns of the LHCII Proteins, while subunit V of the cytochrome b(6)f complex showed a different behavior. These findings are discussed on the basis of a dynamic structural model of cytochrome b(6)f that relates the activation of the LHCII kinase to the occupancy of the Q(o) site and the movement of the Rieske Protein.
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Analysis of the Nucleus-Encoded and Chloroplast-Targeted Rieske Protein by Classic and Site-Directed Mutagenesis of Chlamydomonas
The Plant Cell, 1999Co-Authors: Catherine De Vitry, Giovanni Finazzi, Toivo KallasAbstract:Three mutants of the alga Chlamydomonas reinhardtii affected in the nuclear PETC gene encoding the Rieske iron-sulfur Protein 2Fe-2S subunit of the chloroplast cytochrome b(6)f complex have been characterized. One has a stable deletion that eliminates the Protein; two others carry substitutions Y87D and W163R that result in low accumulation of the Protein. Attenuated expression of the stromal protease ClpP increases accumulation and assembly into b(6)f complexes of the Y87D and W163R mutant Rieske Proteins in quantities sufficient for analysis. Electron-transfer kinetics of these complexes were 10- to 20-fold slower than those for the wild type. The deletion mutant was used as a recipient for site-directed mutant petC alleles. Six glycine residues were replaced by alanine residues (6G6A) in the flexible hinge that is critical for domain movement; substitutions were created near the 2Fe-2S cluster (S128 and W163); and seven C-terminal residues were deleted (G171och). Although the 6G6A and G171och mutations affect highly conserved segments in the chloroplast Rieske Protein, photosynthesis in the mutants was similar to that of the wild type. These results establish the basis for mutational analysis of the nuclear-encoded and chloroplast-targeted Rieske Protein of photosynthesis
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function directed mutagenesis of the cytochrome b6f complex in chlamydomonas reinhardtii involvement of the cd loop of cytochrome b6 in quinol binding to the q o site
Biochemistry, 1997Co-Authors: Giovanni Finazzi, C De Vitry, Pierre Joliot, Sylvie Buschlen, Fabrice Rappaport, F A WollmanAbstract:The FUD2 mutant from the green alga Chlamydomonas reinhardtii expresses a cytochrome b6 variant of higher apparent molecular mass [Lemaire et al. (1986) Biochim. Biophys. Acta 851, 239-248]. Here, we show that the mutation corresponds to a 36 base pair duplication in the chloroplast petB gene, which corresponds to a 12 amino acid duplication in the cd loop of cytochrome b6. The resulting Protein still binds its heme cofactors and assembles into cytochrome b6f complexes, which accumulate in wild type amounts in exponentially growing cells of FUD2. However, these cytochrome b6f complexes show loosened binding of the Rieske Protein and are more prone to degradation in aging cells. Electron transfer through the cytochrome b6f complexes is about 8 times slower in FUD2 than in wild type cells. This is due to a slower oxidation of plastoquinol at the Q(o) site, the folding of which is most likely altered by the duplication. By varying the redox state of the plastoquinone pool in vivo, we show that there is a dramatic decrease in the affinity of the Q(o) site for plastoquinols, which is about 100 times lower in FUD2 than in wild type cells. Our results show that the value of the binding constant of plastoquinol to the Q(o) site (2 x 10(4) M(-1)) derived in [Kramer et al. (1994) Biochim. Biophys. Acta 1184, 251-262] may be extrapolated to in vivo conditions.
Toivo Kallas - One of the best experts on this subject based on the ideXlab platform.
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The Chloroplast Rieske Iron-Sulfur Protein AT THE CROSSROAD OF ELECTRON TRANSPORT AND SIGNAL TRANSDUCTION
The Journal of biological chemistry, 2004Co-Authors: Catherine De Vitry, Giovanni Finazzi, Yexin Ouyang, Francis-andré Wollman, Toivo KallasAbstract:We have addressed the functional and structural roles of three domains of the chloroplast Rieske iron-sulfur Protein; that is, the flexible hinge that connects the transmembrane helix to the soluble cluster-bearing domain, the N-terminal stromal protruding domain, and the transmembrane helix. To this aim mutants were generated in the green alga Chlamydomonas reinhardtii. Their capacities to assemble the cytochrome b6f complex, perform plastoquinol oxidation, and signal redox-induced activation of the light-harvesting complex II kinase during state transition were tested in vivo. Deletion of one residue and extensions of up to five residues in the flexible hinge had no significant effect on complex accumulation or electron transfer efficiency. Deletion of three residues (Delta3G) dramatically decreased reaction rates by a factor of approximately 10. These data indicate that the chloroplast iron-sulfur Protein-linking domain is much more flexible than that of its counterpart in mitochondria. Despite greatly slowed catalysis in the Delta3G mutant, there was no apparent delay in light-harvesting complex II kinase activation or state transitions. This indicates that conformational changes occurring in the Rieske Protein did not represent a limiting step for kinase activation within the time scale tested. No phenotype could be associated with mutations in the N-terminal stromal-exposed domain. In contrast, the N17V mutation in the Rieske Protein transmembrane helix resulted in a large decrease in the cytochrome f synthesis rate. This reveals that the Rieske Protein transmembrane helix plays an active role in assembly-mediated control of cytochrome f synthesis. We propose a structural model to interpret this phenomenon based on the C. reinhardtii cytochrome b6f structure.
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Analysis of the Nucleus-Encoded and Chloroplast-Targeted Rieske Protein by Classic and Site-Directed Mutagenesis of Chlamydomonas
The Plant Cell, 1999Co-Authors: Catherine De Vitry, Giovanni Finazzi, Toivo KallasAbstract:Three mutants of the alga Chlamydomonas reinhardtii affected in the nuclear PETC gene encoding the Rieske iron-sulfur Protein 2Fe-2S subunit of the chloroplast cytochrome b(6)f complex have been characterized. One has a stable deletion that eliminates the Protein; two others carry substitutions Y87D and W163R that result in low accumulation of the Protein. Attenuated expression of the stromal protease ClpP increases accumulation and assembly into b(6)f complexes of the Y87D and W163R mutant Rieske Proteins in quantities sufficient for analysis. Electron-transfer kinetics of these complexes were 10- to 20-fold slower than those for the wild type. The deletion mutant was used as a recipient for site-directed mutant petC alleles. Six glycine residues were replaced by alanine residues (6G6A) in the flexible hinge that is critical for domain movement; substitutions were created near the 2Fe-2S cluster (S128 and W163); and seven C-terminal residues were deleted (G171och). Although the 6G6A and G171och mutations affect highly conserved segments in the chloroplast Rieske Protein, photosynthesis in the mutants was similar to that of the wild type. These results establish the basis for mutational analysis of the nuclear-encoded and chloroplast-targeted Rieske Protein of photosynthesis
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reconstitution of the 2fe 2s center and g 1 89 electron paramagnetic resonance signal into overproduced nostoc sp pcc 7906 Rieske Protein
Biochemistry, 1996Co-Authors: Beatrice Holton, Richard Malkin, David Kramer, A I Tsapin, Toivo KallasAbstract:The Rieske 2Fe-2S Protein is a distinguishing subunit of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plas...
