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Rafael Picorel - One of the best experts on this subject based on the ideXlab platform.
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in vivo reconstitution of a homodimeric Cytochrome B559 like structure the role of the n terminus α subunit from synechocystis sp pcc 6803
Journal of Photochemistry and Photobiology B-biology, 2015Co-Authors: Maria Lujan, Mercedes Roncel, José M. Ortega, Jesus I Martinez, Pablo J Alonso, Alejandro Torrado, Javier Sancho, Rafael PicorelAbstract:Abstract The Cytochrome B559 is a heme-bridged heterodimeric protein with two subunits, α and β. Both subunits from Synechocystis sp. PCC 6803 have previously been cloned and overexpressed in Escherichia coli and in vivo reconstitution experiments have been carried out. The formation of homodimers in the bacterial membrane with endogenous heme was only observed in the case of the β-subunit (β/β) but not with the full length α-subunit. In the present work, reconstitution of a homodimer (α/α) Cytochrome B559 like structure was possible using a chimeric N-terminus α-subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic α-helix that lays on the surface of the membrane. Overexpression and in vivo reconstitution in the bacteria was clearly demonstrated by the brownish color of the culture pellet and the use of a commercial monoclonal antibody against the fusion protein carrier, the maltoside binding protein, and polyclonal antibodies against a synthetic peptide of the α-subunit from Thermosynechococcus elongatus. Moreover, a simple partial purification after membrane solubilization with Triton X-100 confirmed that the overexpressed protein complex corresponded with the maltoside binding protein-chimeric α-subunit Cytochrome B559 like structure. The features of the new structure were determined by UV–Vis, electron paramagnetic resonance and redox potentiometric techniques. Ribbon representations of all possible structures are also shown to better understand the mechanism of the Cytochrome B559 maturation in the bacterial cytoplasmic membrane.
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hyscore spectroscopy in the Cytochrome B559 of the photosystem ii reaction center
Journal of the American Chemical Society, 2003Co-Authors: Ines Garciarubio, Inmaculada Yruela, Rafael Picorel, Jesus I Martinez, Pablo J AlonsoAbstract:A HYSCORE investigation of the heme center in the Cytochrome B559 is presented. To assign the observed signals to specific nuclei, bis-imidazol coordinated heme compounds that model the iron environment in Cytochrome B559 are also studied. In the model compounds selective isotopic substitution of nitrogen atoms has been performed. The HYSCORE spectra allow us to obtain the hyperfine and quadrupolar coupling tensors of heme and imidazol bonding nitrogen atoms. The results can be interpreted in terms of the structure and the electronic distribution of the active center. The hyperfine tensors indicate that the unpaired electron is confined in a nonbonding iron orbital with a negligible nitrogen p orbital contribution. Quadrupolar coupling tensors suggest that the orientation of the semioccupied orbital is driven by the orientation of the two parallel imidazol rings of the axial histidine side chains. The results are discussed in terms of the structure−function relationship of Cytochromes.
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Cytochrome B559 content in isolated photosystem ii reaction center preparations
FEBS Journal, 2003Co-Authors: Inmaculada Yruela, Francisca Miota, Elena Torrado, Michael Seibert, Rafael PicorelAbstract:The Cytochrome B559 content was examined in five types of isolated photosystem II D1-D2-Cytochrome B559 reaction center preparations containing either five or six chlorophylls per reaction center. The reaction center complexes were obtained following isolation procedures that differed in chromatographic column material, washing buffer composition and detergent concentration. Two different types of Cytochrome B559 assays were performed. The absolute heme content in each preparation was obtained using the oxidized-minus-reduced difference extinction coefficient of Cytochrome B559 at 559 nm. The relative amount of D1 and Cytochrome B559α-subunit polypeptide was also calculated for each preparation from immunoblots obtained using antibodies raised against the two polypeptides. The results indicate that the Cytochrome B559 heme content in photosystem II reaction center complexes can vary with the isolation procedure, but the variation of the Cytochrome B559α-subunit/D1 polypeptide ratio was even greater. This variation was not found in the PSII-enriched membrane fragments used as the RC-isolation starting material, as different batches of membranes obtained from spinach harvested at different seasons of the year or those from sugar beets grown in a chamber under controlled environmental conditions lack variation in their α-subunit/D1 polypeptide ratio. A precise determination of the ratio using an RC1-control sample calibration curve gave a ratio of 1.25 Cytochrome B559α-subunit per 1.0 D1 polypeptide in photosystem II membranes. We conclude that the variations found in the reaction center preparations were due to the different procedures used to isolate and purify the different reaction center complexes.
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detergent effect on Cytochrome B559 electron paramagnetic resonance signals in the photosystem ii reaction centre
Photochemical and Photobiological Sciences, 2003Co-Authors: Inmaculada Yruela, Mercedes Roncel, José M. Ortega, Ines Garciarubio, Jesus I Martinez, M V Ramiro, Pablo J Alonso, Rafael PicorelAbstract:The detergent effect on Cytochrome B559 from spinach photosystem II was studied by electron paramagnetic resonance (EPR) spectroscopy in D1–D2–Cyt B559 complex preparations. Various n-dodecyl-β-D-maltoside concentrations from 0 to 0.2% (w/v) were used to stabilise the D1–D2–Cyt B559 complexes. Low spin heme EPR spectra were obtained but the gz feature positions changed depending on the detergent conditions. Redox potentiometric titrations showed a unique redox potential Cytochrome B559 form (E′m = +123–150 mV) in all the D1–D2–Cyt B559 complex preparations indicating that detergent does not affect this property of the protein in those conditions. A similar effect on Cytochrome B559 EPR spectrum was observed in more intact photosystem II preparations independently of their aggregation state. This finding indicates that changes due to detergent could be a common phenomenon in photosystem II complexes. Results are discussed in terms of the environment each detergent provides to the protein.
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EFFECT OF THE PH ON THE ABSORPTION SPECTRUM OF THE ISOLATED D1-D2-Cytochrome B559 COMPLEX OF PHOTOSYSTEM II
Journal of Photochemistry and Photobiology B-biology, 1999Co-Authors: Inmaculada Yruela, Raquel Tomás, Miguel Alfonso, Rafael PicorelAbstract:The effect of pH on the Qy absorption band has been studied in the isolated D1-D2-Cytochrome B559 complex. The pH treatments are done on an ion-exchange chromatographic column. The absorption spectra at 77 K of the complex treated with acidic pH show irreversible loss of absorbance at both the blue and the red sides of the Qy absorption band, with minima at 664.5 and 683.5 nm, respectively. These absorption changes are not accompanied by modifications in the Qx absorption region characteristic of pheophytin pigments. Furthermore, the pigment composition of the D1-D2-Cytochrome B559 complex remains unchanged after this treatment. The effects of basic pH are similar to those of acidic pH, but somewhat more pronounced. These results suggest that chlorophyll pigments absorbing at 664.5 and 683.5 nm are located on or close to the surface of the complex. Freezing/thawing cycle treatment first affects the band absorbing at 683.6 nm, indicating that it corresponds to the chlorophyll most exposed to the medium in the D1-D2-Cytochrome B559 complex. At pH
José M. Ortega - One of the best experts on this subject based on the ideXlab platform.
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Site-Directed Mutagenesis of Cytochrome B559 in the Cyanobacterium Thermosynechococcus elongatus
Photosynthesis. Energy from the Sun, 2020Co-Authors: Fernando Guerrero, Diana Kirilovsky, Mercedes Roncel, José M. OrtegaAbstract:Cytochrome B559 is an intrinsic and essential component of the photosystem II reaction centre in all photosynthetic oxygen- evolving organisms, but its function, although widely investigated, still remains unresolved. Most of the functional hypotheses propose that Cytochrome B559 may participate in secondary electron transfer pathways protecting photosystem II against oxidative damage. Mutational studies have not succeeded in demonstrating this redox function because most of the Cytochrome B559 mutants obtained are impaired in the functional assembly of photosystem II holocomplex. We have constructed a series of sitedirected mutants of Cytochrome B559, each carrying a single amino acid substitution, in the thermophilic cyanobacterium Thermosynechococcus elongatus, in order to modify the redox potential of the heme without altering the assembly properties of photosystem II. We have obtained 19 mutant strains of Cytochrome B559. All the mutants grew photoautotrophically at a rate similar to that of the wild-type. Some of these mutants showed relevant differences in redox properties of Cytochrome B559 compared to wild-type. The α-R18S mutant strain showed the most important effects on redox potential and photosystem II activity.
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mutations of Cytochrome B559 and psbj on and near the qc site in photosystem ii influence the regulation of short term light response and photosynthetic growth of the cyanobacterium synechocystis sp pcc 6803
Biochemistry, 2016Co-Authors: Jineyung Huang, José M. Ortega, Yifang Chiu, Hsingting Wang, Tiensheng Tseng, Shyuechu Ke, Mercedes RoncelAbstract:The characteristic features of two types of short-term light adaptations of the photosynthetic apparatus of the cyanobacterium Synechocystis sp. PCC 6803, state transition and blue-green light-induced fluorescence quenching, were compared in wild-type and Cytochrome B559 and PsbJ mutant cells with mutations on and near the QC site in photosystem II (PSII). All mutant cells grew photoautotrophically and assembled stable PSII. Thermoluminescence emission experiments showed a decrease in the stability of the S3QB–/S2QB– charge pairs in the A16FJ, S28Aβ, and V32Fβ mutant cells. When dark-adapted wild-type and mutant cells were illuminated by medium-intensity blue light, the increase in the PSII fluorescence yield (indicating a transition to state 1) was more prominent in mutant than wild-type cells. Strong blue-light conditions induced a quenching of fluorescence corresponding to nonphotochemical fluorescence quenching (NPQ). The extension of NPQ decreased significantly in the mutants, and the kinetics appear...
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in vivo reconstitution of a homodimeric Cytochrome B559 like structure the role of the n terminus α subunit from synechocystis sp pcc 6803
Journal of Photochemistry and Photobiology B-biology, 2015Co-Authors: Maria Lujan, Mercedes Roncel, José M. Ortega, Jesus I Martinez, Pablo J Alonso, Alejandro Torrado, Javier Sancho, Rafael PicorelAbstract:Abstract The Cytochrome B559 is a heme-bridged heterodimeric protein with two subunits, α and β. Both subunits from Synechocystis sp. PCC 6803 have previously been cloned and overexpressed in Escherichia coli and in vivo reconstitution experiments have been carried out. The formation of homodimers in the bacterial membrane with endogenous heme was only observed in the case of the β-subunit (β/β) but not with the full length α-subunit. In the present work, reconstitution of a homodimer (α/α) Cytochrome B559 like structure was possible using a chimeric N-terminus α-subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic α-helix that lays on the surface of the membrane. Overexpression and in vivo reconstitution in the bacteria was clearly demonstrated by the brownish color of the culture pellet and the use of a commercial monoclonal antibody against the fusion protein carrier, the maltoside binding protein, and polyclonal antibodies against a synthetic peptide of the α-subunit from Thermosynechococcus elongatus. Moreover, a simple partial purification after membrane solubilization with Triton X-100 confirmed that the overexpressed protein complex corresponded with the maltoside binding protein-chimeric α-subunit Cytochrome B559 like structure. The features of the new structure were determined by UV–Vis, electron paramagnetic resonance and redox potentiometric techniques. Ribbon representations of all possible structures are also shown to better understand the mechanism of the Cytochrome B559 maturation in the bacterial cytoplasmic membrane.
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the role of the high potential form of the Cytochrome B559 study of thermosynechococcus elongatus mutants
Biochimica et Biophysica Acta, 2014Co-Authors: Fernando Guerrero, Diana Kirilovsky, Mercedes Roncel, Jose Lino Zurita, José M. OrtegaAbstract:Abstract Cytochrome B559 is an essential component of the photosystem II reaction center in photosynthetic oxygen-evolving organisms, but its function still remains unclear. The use of photosystem II preparations from Thermosynechococcus elongatus of high integrity and activity allowed us to measure for the first time the influence of Cytochrome B559 mutations on its midpoint redox potential and on the reduction of the Cytochrome B559 by the plastoquinone pool (or QB). In this work, five mutants having a mutation in the α-subunit (I14A, I14S, R18S, I27A and I27T) and one in the β-subunit (F32Y) of Cytochrome B559 have been investigated. All the mutations led to a destabilization of the high potential form of the Cytochrome B559. The midpoint redox potential of the high potential form was significantly altered in the αR18S and αI27T mutant strains. The αR18S strain also showed a high sensitivity to photoinhibitory illumination and an altered oxidase activity. This was suggested by measurements of light induced oxidation and dark re-reduction of the Cytochrome B559 showing that under conditions of a non-functional water oxidation system, once the Cytochrome is oxidized by P680+, the yield of its reduction by QB or the PQ pool was smaller and the kinetic slower in the αR18S mutant than in the wild-type strain. Thus, the extremely positive redox potential of the high potential form of Cytochrome B559 could be necessary to ensure efficient oxidation of the PQ pool and to function as an electron reservoir replacing the water oxidation system when it is not operating.
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detergent effect on Cytochrome B559 electron paramagnetic resonance signals in the photosystem ii reaction centre
Photochemical and Photobiological Sciences, 2003Co-Authors: Inmaculada Yruela, Mercedes Roncel, José M. Ortega, Ines Garciarubio, Jesus I Martinez, M V Ramiro, Pablo J Alonso, Rafael PicorelAbstract:The detergent effect on Cytochrome B559 from spinach photosystem II was studied by electron paramagnetic resonance (EPR) spectroscopy in D1–D2–Cyt B559 complex preparations. Various n-dodecyl-β-D-maltoside concentrations from 0 to 0.2% (w/v) were used to stabilise the D1–D2–Cyt B559 complexes. Low spin heme EPR spectra were obtained but the gz feature positions changed depending on the detergent conditions. Redox potentiometric titrations showed a unique redox potential Cytochrome B559 form (E′m = +123–150 mV) in all the D1–D2–Cyt B559 complex preparations indicating that detergent does not affect this property of the protein in those conditions. A similar effect on Cytochrome B559 EPR spectrum was observed in more intact photosystem II preparations independently of their aggregation state. This finding indicates that changes due to detergent could be a common phenomenon in photosystem II complexes. Results are discussed in terms of the environment each detergent provides to the protein.
Mercedes Roncel - One of the best experts on this subject based on the ideXlab platform.
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Site-Directed Mutagenesis of Cytochrome B559 in the Cyanobacterium Thermosynechococcus elongatus
Photosynthesis. Energy from the Sun, 2020Co-Authors: Fernando Guerrero, Diana Kirilovsky, Mercedes Roncel, José M. OrtegaAbstract:Cytochrome B559 is an intrinsic and essential component of the photosystem II reaction centre in all photosynthetic oxygen- evolving organisms, but its function, although widely investigated, still remains unresolved. Most of the functional hypotheses propose that Cytochrome B559 may participate in secondary electron transfer pathways protecting photosystem II against oxidative damage. Mutational studies have not succeeded in demonstrating this redox function because most of the Cytochrome B559 mutants obtained are impaired in the functional assembly of photosystem II holocomplex. We have constructed a series of sitedirected mutants of Cytochrome B559, each carrying a single amino acid substitution, in the thermophilic cyanobacterium Thermosynechococcus elongatus, in order to modify the redox potential of the heme without altering the assembly properties of photosystem II. We have obtained 19 mutant strains of Cytochrome B559. All the mutants grew photoautotrophically at a rate similar to that of the wild-type. Some of these mutants showed relevant differences in redox properties of Cytochrome B559 compared to wild-type. The α-R18S mutant strain showed the most important effects on redox potential and photosystem II activity.
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mutations of Cytochrome B559 and psbj on and near the qc site in photosystem ii influence the regulation of short term light response and photosynthetic growth of the cyanobacterium synechocystis sp pcc 6803
Biochemistry, 2016Co-Authors: Jineyung Huang, José M. Ortega, Yifang Chiu, Hsingting Wang, Tiensheng Tseng, Shyuechu Ke, Mercedes RoncelAbstract:The characteristic features of two types of short-term light adaptations of the photosynthetic apparatus of the cyanobacterium Synechocystis sp. PCC 6803, state transition and blue-green light-induced fluorescence quenching, were compared in wild-type and Cytochrome B559 and PsbJ mutant cells with mutations on and near the QC site in photosystem II (PSII). All mutant cells grew photoautotrophically and assembled stable PSII. Thermoluminescence emission experiments showed a decrease in the stability of the S3QB–/S2QB– charge pairs in the A16FJ, S28Aβ, and V32Fβ mutant cells. When dark-adapted wild-type and mutant cells were illuminated by medium-intensity blue light, the increase in the PSII fluorescence yield (indicating a transition to state 1) was more prominent in mutant than wild-type cells. Strong blue-light conditions induced a quenching of fluorescence corresponding to nonphotochemical fluorescence quenching (NPQ). The extension of NPQ decreased significantly in the mutants, and the kinetics appear...
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in vivo reconstitution of a homodimeric Cytochrome B559 like structure the role of the n terminus α subunit from synechocystis sp pcc 6803
Journal of Photochemistry and Photobiology B-biology, 2015Co-Authors: Maria Lujan, Mercedes Roncel, José M. Ortega, Jesus I Martinez, Pablo J Alonso, Alejandro Torrado, Javier Sancho, Rafael PicorelAbstract:Abstract The Cytochrome B559 is a heme-bridged heterodimeric protein with two subunits, α and β. Both subunits from Synechocystis sp. PCC 6803 have previously been cloned and overexpressed in Escherichia coli and in vivo reconstitution experiments have been carried out. The formation of homodimers in the bacterial membrane with endogenous heme was only observed in the case of the β-subunit (β/β) but not with the full length α-subunit. In the present work, reconstitution of a homodimer (α/α) Cytochrome B559 like structure was possible using a chimeric N-terminus α-subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic α-helix that lays on the surface of the membrane. Overexpression and in vivo reconstitution in the bacteria was clearly demonstrated by the brownish color of the culture pellet and the use of a commercial monoclonal antibody against the fusion protein carrier, the maltoside binding protein, and polyclonal antibodies against a synthetic peptide of the α-subunit from Thermosynechococcus elongatus. Moreover, a simple partial purification after membrane solubilization with Triton X-100 confirmed that the overexpressed protein complex corresponded with the maltoside binding protein-chimeric α-subunit Cytochrome B559 like structure. The features of the new structure were determined by UV–Vis, electron paramagnetic resonance and redox potentiometric techniques. Ribbon representations of all possible structures are also shown to better understand the mechanism of the Cytochrome B559 maturation in the bacterial cytoplasmic membrane.
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the role of the high potential form of the Cytochrome B559 study of thermosynechococcus elongatus mutants
Biochimica et Biophysica Acta, 2014Co-Authors: Fernando Guerrero, Diana Kirilovsky, Mercedes Roncel, Jose Lino Zurita, José M. OrtegaAbstract:Abstract Cytochrome B559 is an essential component of the photosystem II reaction center in photosynthetic oxygen-evolving organisms, but its function still remains unclear. The use of photosystem II preparations from Thermosynechococcus elongatus of high integrity and activity allowed us to measure for the first time the influence of Cytochrome B559 mutations on its midpoint redox potential and on the reduction of the Cytochrome B559 by the plastoquinone pool (or QB). In this work, five mutants having a mutation in the α-subunit (I14A, I14S, R18S, I27A and I27T) and one in the β-subunit (F32Y) of Cytochrome B559 have been investigated. All the mutations led to a destabilization of the high potential form of the Cytochrome B559. The midpoint redox potential of the high potential form was significantly altered in the αR18S and αI27T mutant strains. The αR18S strain also showed a high sensitivity to photoinhibitory illumination and an altered oxidase activity. This was suggested by measurements of light induced oxidation and dark re-reduction of the Cytochrome B559 showing that under conditions of a non-functional water oxidation system, once the Cytochrome is oxidized by P680+, the yield of its reduction by QB or the PQ pool was smaller and the kinetic slower in the αR18S mutant than in the wild-type strain. Thus, the extremely positive redox potential of the high potential form of Cytochrome B559 could be necessary to ensure efficient oxidation of the PQ pool and to function as an electron reservoir replacing the water oxidation system when it is not operating.
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detergent effect on Cytochrome B559 electron paramagnetic resonance signals in the photosystem ii reaction centre
Photochemical and Photobiological Sciences, 2003Co-Authors: Inmaculada Yruela, Mercedes Roncel, José M. Ortega, Ines Garciarubio, Jesus I Martinez, M V Ramiro, Pablo J Alonso, Rafael PicorelAbstract:The detergent effect on Cytochrome B559 from spinach photosystem II was studied by electron paramagnetic resonance (EPR) spectroscopy in D1–D2–Cyt B559 complex preparations. Various n-dodecyl-β-D-maltoside concentrations from 0 to 0.2% (w/v) were used to stabilise the D1–D2–Cyt B559 complexes. Low spin heme EPR spectra were obtained but the gz feature positions changed depending on the detergent conditions. Redox potentiometric titrations showed a unique redox potential Cytochrome B559 form (E′m = +123–150 mV) in all the D1–D2–Cyt B559 complex preparations indicating that detergent does not affect this property of the protein in those conditions. A similar effect on Cytochrome B559 EPR spectrum was observed in more intact photosystem II preparations independently of their aggregation state. This finding indicates that changes due to detergent could be a common phenomenon in photosystem II complexes. Results are discussed in terms of the environment each detergent provides to the protein.
Stenbjorn Styring - One of the best experts on this subject based on the ideXlab platform.
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Spin conversion of Cytochrome B559 in photosystem II induced by exogenous high potential quinone
Chemical Physics, 2003Co-Authors: T. N. Kropacheva, Fikret Mamedov, Stenbjorn Styring, W. Onno Feikema, Yashar Feyziyev, Arnold J. HoffAbstract:The spin-state of Cytochrome B559 (Cyt B559) was studied in photosystem II (PSII) membrane fragments by low-temperature EPR spectroscopy. Treatment of the membranes with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) converts the native low-spin (LS) form of Cyt B559 to the high-spin (HS) form characterized with the g= 6.19 and g= 5.95 split signal. The HS Cyt B559 was pH dependent with the amplitude increasing toward more acidic pH values (pH 5.5-8.5). The HS state was not photochemically active upon 77 and 200 K continuous illumination under our conditions and was characterized by a low reduction potential (=
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spin conversion of Cytochrome B559 in photosystem ii induced by exogenous high potential quinone
Chemical Physics, 2003Co-Authors: T. N. Kropacheva, Fikret Mamedov, Stenbjorn Styring, Yashar Feyziyev, Onno W Feikema, Arnold J. HoffAbstract:The spin-state of Cytochrome B559 (Cyt B559) was studied in photosystem II (PSII) membrane fragments by low-temperature EPR spectroscopy. Treatment of the membranes with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) converts the native low-spin (LS) form of Cyt B559 to the high-spin (HS) form characterized with the g= 6.19 and g= 5.95 split signal. The HS Cyt B559 was pH dependent with the amplitude increasing toward more acidic pH values (pH 5.5-8.5). The HS state was not photochemically active upon 77 and 200 K continuous illumination under our conditions and was characterized by a low reduction potential (=<0 V). It was also demonstrated that DDQ treatment damages the oxygen evolving complex, leading to inhibition of oxygen evolution, decrease of the S2-state EPR multiline signal and release of Mn2+. In parallel, studies of model systems containing iron(III) protoporphyrin IX chloride (FeIIIPor), which is a good model compound for the Cyt B559 prosthetic group, were performed by using optical and EPR spectroscopy. The interaction of FeIIIPor with imidazole (Im) in weakly polar solvent results in formation of bis-imidazole coordinated heme iron (FeIIIPor Im2) which mimic the bis-histidine axial ligation of Cyt B559. The reaction of DDQ with the LS FeIIIPor Im2 complex leads to its transformation into the HS state (g@?=5.95, g@?=2.00). It was shown that the spin conversion occurs due to the donor-acceptor interaction of coordinated imidazole with this high-potential quinone causing the displacement of imidazole from the axial position. The similar mechanism of DDQ-induced spin change is assumed to be valid for the native membrane Cyt B559 in PSII centers. (Less)
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electron transfer from Cytochrome B559 and tyrosined to the s2 and s3 states of the water oxidizing complex in photosystem ii
Principles and Practice of Constraint Programming, 2003Co-Authors: Yashar Feyziyev, Bart J Van Rotterdam, Gabor Bernat, Stenbjorn StyringAbstract:We have investigated the electron transfer from reduced tyrosine YD (YDred) and Cytochrome B559 to the S2 and S3 states of the water oxidizing complex (WOC) in Photosystem II. The EPR signal of oxidized cyt B559, the S2 state multiline EPR signal and the EPR signal from YD@? were measured to follow the electron transfer to the S2 and S3 states at 245 and 275 K. The majority of the S2 centers was reduced directly from YDred but at 245 K we observed oxidation of cyt B559 in about 20% of the centers. Incubation of the YDredS3 state resulted in biphasic changes of the S2 multiline signal. The signal first increased due to reduction of the S3 state. Thereafter, the signal decreased due to decay of the S2 state. In contrast, the YD@? signal increased with a monophasic kinetics at both temperatures. Again, we observed oxidation of cyt B559 in about 20% of the PSII centers at 245 K. This oxidation correlated with the decay of the S2 state. The complex changes can be explained by the conversion of YDredS3 centers (present initially) to YD@?S1 centers, via the intermediate YD@?S2 state. The early increase of the S2 state multiline signal involves electron transfer from YDred to the S3 state resulting in formation of YD@?S2. This state is reduced by cyt B559 resulting in a single exponential oxidation of cyt B559. Taken together, these results indicate that the electron donor to S2 is cyt B559 while cyt B559 is unable to compete with YDred in the reduction of the S3 state in the pre-reduced samples. We also followed the decay of the S2 and S3 states and the oxidation of cyt B559 in samples where YD was oxidized from the start. In this case cyt B559 was able to reduce both the S2 and the S3 states suggesting that different pathways exist for the electron transfer from cyt B559 to the WOC. The activation energies for the YDredS2->YD@?S1 and YDredS3->YD@?S2 transformations are 0.57 and 0.67 eV, respectively, and the reason for these large activation energies is discussed. (Less)
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the role of Cytochrome B559 and tyrosined in protection against photoinhibition during in vivo photoactivation of photosystem ii
Biochimica et Biophysica Acta, 1999Co-Authors: Ann Magnuson, Maria Rova, Fikret Mamedov, Perolof Fredriksson, Stenbjorn StyringAbstract:The role of Cytochrome B559 and tyrosineD in protection against photoinhibition during in vivo photoactivation of Photosystem II
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changes in the oxidation state of Cytochrome B559 and tyrosine d during in vivo photoactivation of photosystem ii
11th Photosynthesis Congress, 1998Co-Authors: Ann Magnusson, Maria Rova, Fikret Mamedov, Perolof Fredriksson, Stenbjorn StyringAbstract:Changes in the oxidation state of Cytochrome B559 and tyrosine-D during in vivo photoactivation of photosystem II
James Barber - One of the best experts on this subject based on the ideXlab platform.
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Localisation of the PsbH subunit in photosystem II: a new approach using labelling of his-tags with a Ni2+-NTA gold cluster and single particle analysis
Journal of Molecular Biology, 2001Co-Authors: Claudia Büchel, Edward P. Morris, Elena V. Orlova, James BarberAbstract:Photosystem II core dimers were isolated from the green alga Chlamydomonas reinhardtii by Ni2+-affinity chromatography exploiting a 6 × His tag located at the N terminus of the PsbH protein. This protein is predicted to have a single transmembrane helix. In order to identify the location of PsbH within the photosystem II complex, the His-tagged core dimers were labelled using a Ni2+-NTA gold cluster and subjected to electron microscopy and image analysis. This new method enabled us to identify the location of the labelled His tag by statistical analysis of electron micrographs of the gold-labelled photosystem II complex. Comparison of these data with electron and X-ray crystallographic analysis of photosystem II indicates that the N terminus of PsbH is close to the two transmembrane helices of Cytochrome B559. Our analysis suggests that this approach is a powerful method to locate specific proteins within multisubunit complexes like photosystem II when crystallographic analysis is of insufficient resolution to directly identify amino acid side-chains. Moreover, it can be combined with cross-linking studies, and here we demonstrate that PsbH is a near neighbour of PsbX, which is consistent with the latter subunit being located close to the α and β-subunits of Cytochrome B559. However, cross-linking between PsbH and PsbW was not detected despite the fact that the latter cross-linked with the α-subunit of Cytochrome B559.
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substantial deletions in the de loop of the photosystem ii d1 protein do not prevent its turnover or cross linking with the α subunit of Cytochrome B559 a study using synechocystis sp pcc 6803 mutants
Journal of Plant Physiology, 1999Co-Authors: Roberto Barbato, Paula Mulo, Elena Bergo, Donatella Carbonera, Pirkko Maenpaa, Giorgio M Gracometti, James BarberAbstract:Summary Light-induced damage of photosystem II brings about the specific degradation of the reaction centre D1-protein. Under similar conditions, cross-linking occurs between this protein and the α-subunit of Cytochrome B559, giving rise to a 41-kDa adduct. In order to understand whether there is any relationship between the formation of the 41-kDa adduct and the D1-protein degradation, three deletion mutants of Synechocystis sp. PCC 6803 have been employed. The three mutants have deletions in the DE loop of the D1-protein, Δ(G240-V249), Δ(R225-F239) and Δ(R225-V249), which incorporates the ‹PEST-like› region and the FGQEEET motif. These regions have been implicated in the degradation and turnover of the D1 protein, and also in the formation of the 41-kDa adduct. Using a proteolytic digestion assay we show that the deletions induce conformational changes in the putative helical region of the DE-loop. However, the deletion mutants mantain their abilities to degrade and turnover the D1 protein, and also to generate the 41-kDa adduct, reinforcing the idea of a correlation between the two phenomena.
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Purification and Determination of Intact Molecular Mass by Electrospray Ionization Mass Spectrometry of the Photosystem II Reaction Center Subunits
Journal of Biological Chemistry, 1997Co-Authors: Jyoti Sharma, James Barber, Maria Panico, Howard R. MorrisAbstract:: A reverse phase high pressure liquid chromatography purification system for the rapid separation of photosystem II reaction center proteins free of salts and detergents is described. This procedure results in the isolation of the three small subunits: alpha- and beta-subunits of Cytochrome B559 and PsbI protein, with near base-line resolution between each peak, although the D1 and D2 proteins were partially deconvoluted. The molecular masses obtained by electrospray ionization mass spectrometry for the purified beta-subunit of Cytochrome B559, alpha-subunit of Cytochrome B559, and the PsbI protein, 4,394.8 +/- 0.4, 9,283.7 +/- 0.8, and 4,209.5 +/- 0.4 Da, respectively, are in excellent agreement with values obtained from previous characterization studies (Sharma, J., Panico, M., Barber, J., and Morris, H. R. (1997) J. Biol. Chem. 272, 3935-3943). Direct electrospray analysis of the D1 and D2 proteins suggests that these components exist in heterogeneous forms. The molecular mass ascribed to a predominant form of the D1 protein, 38, 040.9 +/- 6.5 Da, and the D2 protein, 39,456.1 +/- 7.7, are also in agreement with those expected for the mature nonphosphorylated states of these subunits.
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Characterization of the Low Molecular Weight Photosystem II Reaction Center Subunits and Their Light-induced Modifications by Mass Spectrometry
Journal of Biological Chemistry, 1997Co-Authors: Jyoti Sharma, James Barber, Maria Panico, Howard R. MorrisAbstract:Abstract A sensitive and simple reverse phase HPLC purification scheme was developed for the rapid separation of the small protein subunits from photosystem II reaction center preparations. The precise molecular masses of the α- and β-subunits of Cytochrome B559 and the psbI gene product from pea plants, found to be 4394.6 ± 0.6, 9283.6 ± 0.7, and 4209.5 ± 0.5 Da, respectively, were then successfully determined for the first time by electrospray- and fast atom bombardment-mass spectrometry. Discrepancies between the molecular weights assigned and those calculated from the respective DNA sequences were observed for α- and β-subunits of Cytochrome B559. Currently, the nucleotide sequence of the psbI gene product from pea plants is not available. Application of novel mapping and sequencing strategies has assured the elucidation of full primary structures of all of the purified subunits. The modifications identified here include the post-translational processing of the initiating methionine on both subunits of Cytochrome B559, NH2-terminal acetylation and an mRNA editing site at residue 26 (Ser → Phe) on the β-subunit, and retention of the NH2-terminal formyl-Met on the psbI gene product. In addition, specific oxidation of a single amino acid residue was identified on the psbI gene product and the β-subunit purified from light-treated reaction center preparations. Overall, these studies provide the first detailed primary structural characterization of the small subunits of the reaction center complex and their associated light-induced modifications.
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characterization of the light induced cross linking of the alpha subunit of Cytochrome B559 and the d1 protein in isolated photosystem ii reaction centers
Journal of Biological Chemistry, 1995Co-Authors: Roberto Barbato, Giulia Friso, Markella Ponticos, James BarberAbstract:Abstract Illumination of the isolated reaction center of photosystem II generates a protein of 41 kDa molecular mass. Using immunoblotting, it is confirmed that the protein is an adduct of the D1 protein and the α-subunit of Cytochrome B559. Its formation seems to be photochemically induced, being independent of temperature between 4 and 20°C and unaffected by a mixture of protease inhibitors. The maximum levels are detected when the pH is in the region 6.5-8.5 and when illumination intensities are moderate. Although higher light intensities induce a higher rate of formation, the accumulation of elevated levels of the 41-kDa protein does not occur due to light-induced degradation. This degradation is also unaffected by the presence of protease inhibitors. Proteolytic mapping and N-terminal sequencing indicates that the cross-linking process involves the N-terminal serine of the α-subunit of Cytochrome B559 and D1 residues in the 239-244 FGQEEE motif close to the QB binding site. In conclusion, the results indicate that the N terminus of the α-subunit is exposed on the stromal side of photosystem II in such a way as to undergo light-induced cross-linking in the QB region of the D1 protein. They also suggest that the 41-kDa adduct may be an intermediate before the light-induced cleavage of the D1 protein in the FGQEEE region.
