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Rafael Picorel - One of the best experts on this subject based on the ideXlab platform.
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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 <5 a small reversible change at 672.5 nm is measured that correlates with a reversible change at 542 nm, indicating that inactive Pheophytin a will absorb at this wavelength.
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pigment content of d1 d2 cytochrome b559 reaction center preparations after removal of cp47 contamination an immunological study
Biochemistry, 1995Co-Authors: Jose Javier Pueyo, Michael Seibert, Esmeralda Moliner, Rafael PicorelAbstract:Isolated D1 -D2-cytochrome b559 photosystem I1 reaction center preparations with pigment stoichiometry higher than 4 chlorophylls per 2 Pheophytins can be contaminated with CP47 proximal antenna complex. Reaction centers prepared by a modification of the Nanba-Satoh procedure and containing about 6 chlorophylls per 2 Pheophytins showed immuno-cross-reactivity when probed with a monoclonal antibody raised against the CP47 polypeptide. Furthermore, they could be fractionated successfully by Superose- 12 sieve chromatography into two different populations. The first few fractions off the column contained a more definitive 435 nm shoulder corresponding to increased chlorophyll content, and showed strong immuno-cross-reactivity with the CP47 antibody. The peak fractions off the column displayed a less prominent 435 nm shoulder, and did not cross-react with the antibody. Moreover, when a 6-chlorophyll preparation was mixed with Sepharose beads coupled to CP47 antibody, the eluted material corresponded to a preparation of about 4 chlorophylls per 2 Pheophytins and did not show any cross- reaction with the antibody against CP47. The amount of CP47 protein in the 6-chlorophyll preparation as quantitated using Coomassie Blue staining or from gel blots was sufficient to account for most of the extra 2 chlorophylls. We conclude that D 1 -D2-cytochrome b559 preparations containing more than 4 chlorophylls per 2 Pheophytins can be contaminated with small amounts of CP47-D1 -D2-Cyt b559 complex and that native photosystem I1 reaction centers contain 4 core chlorophylls per 2 Pheophytins.
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spectroscopic characterization of two forms of the d1 d2 cytochrome b559 complex from sugar beet
Photochemistry and Photobiology, 1993Co-Authors: Guillermo Montoya, Rafael Cases, Inmaculada Yruela, Rafael PicorelAbstract:Two D1-D2-cytochrome b559 complex forms, called RCIIa and RCIIb, with different pigment stoichi-ometry were characterized using absorption and surface-enhanced resonance Raman scattering spectroscopy and spectral gaussian deconvolution. Electronic absorption spectra of the RCIIb at 277 K showed significant differences compared to RCIIa, i.e. a strong decrease in the absorbance due to carotenoid and chlorophyll for the same amount of Pheophytin. A reduced carotenoid and chlorophyll content in RCIIb was also observed in the surface-enhanced resonance Raman scattering spectra. Spectral deconvolution elicited three main absorption bands at 680, 672 and 669–670 nm, which were ascribed to P680, Pheophytin and accessory chlorophyll, respectively. In addition, a minor component around 667 nm was observed in the RCIIb, most probably due to some reaction center inactivation. Calculation of the relative area under the gaussians together with pigment stoichiometry data suggest that the 680, 672 and 669–670 nm components contain, respectively, two chlorophylls, two Pheophytins and four chlorophylls for the RCIIa, and two chlorophylls, two Pheophytins and two chlorophylls for the RCIIb.
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precise location of the cu ii inhibitory binding site in higher plant and bacterial photosynthetic reaction centers as probed by light induced absorption changes
Journal of Biological Chemistry, 1993Co-Authors: Inmaculada Yruela, Miguel Alfonso, Guillermo Montoya, Ortiz I De Zarate, Rafael PicorelAbstract:Light-dependent absorption change at 325 nm, ascribed to QA activity, was strongly reduced in the presence of Cu(II) in oxygen-evolving core complex. This change was much less affected in the presence of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), indicating that the Cu(II)-binding site is different from that of the DCMU and that Cu(II) blocks QA reduction. Cu(II) did not eliminate the absorption change at 545 nm, ascribed to Pheophytin reduction, in Na2S2O4-treated oxygen-evolving core and D1-D2-cytochrome b559 complexes. This indicates that Cu(II) does not affect the electron transport between P680 and Pheophytin. Moreover, the activity of the bacterial reaction center probed by the absorption change at 790 nm was inhibited by Cu(II), but the signal at 530 nm, associated to the reduction of bacterioPheophytin in Na2S2O4-treated reaction center, was not inhibited. We conclude that Cu(II) impaired the photosynthetic electron transport between Pheophytin and QA in both higher plants and photosynthetic bacteria. Cu(II) would bind to an amino acid(s) highly conserved in non-oxygenic and oxygenic reaction centers, which is(are) necessary for the electron transfer between Pheophytin and QA. Based on the atomic structure of the bacterial reaction center several schemes of possible Cu(II) binding are shown.
Lutz A Eichacker - One of the best experts on this subject based on the ideXlab platform.
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light harvesting chlorophyll a b binding protein stably inserts into etioplast membranes supplemented with zn Pheophytin a b
Journal of Biological Chemistry, 1997Co-Authors: Andrea Kuttkat, Lutz A Eichacker, Ingrid Edhofer, Harald PaulsenAbstract:Abstract Light-harvesting chlorophylla/b-binding protein, LHCP, or its precursor, pLHCP, cannot be stably inserted into barley etioplast membranes in vitro. However, when these etioplast membranes are supplemented with the chlorophyll analogs Zn-Pheophytin a/b, synthesizedin situ from Zn-pheophorbide a/b and digeranyl pyrophosphate, pLHCP is inserted into a protease-resistant state. This proves that chlorophyll is the only component lacking in etioplast membranes that is necessary for stable LHCP insertion. Synthesis of Zn-Pheophytin b alone promotes insertion of LHCP in vitro into a protease-resistant state, whereas synthesis of Zn-Pheophytin a alone does not. Insertion of pLHCP into etioplast membranes can also be stimulated by adding chlorophyll a and chlorophyll b to the membranes, albeit at a significantly lower efficiency as compared with Zn-Pheophytin a/b synthesized in situ. When pLHCP is inserted into chlorophyll- or Zn-Pheophytin-supplemented etioplast membranes and then assayed with protease, only the protease digestion product indicative of the monomeric major light-harvesting chlorophyll a/b complex (LHCII) is found but not the one indicating trimeric complexes. In this respect, chlorophyll- or Zn-Pheophytin-supplemented etioplast membranes resemble thylakoid membranes at an early greening stage: pLHCP inserted into plastid membranes from greening barley is assembled into trimeric LHCII only after more than 1 h of greening.
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stabilization of chlorophyll a binding apoproteins p700 cp47 cp43 d2 and d1 by chlorophyll a or zn Pheophytin a
Journal of Biological Chemistry, 1996Co-Authors: Lutz A Eichacker, Michael Helfrich, Bernd MullerAbstract:Abstract Stabilization of chlorophyll a-binding apoproteins P700, CP47, CP43, D2, and D1 against proteolytic degradation has been investigated through in vitro synthesis of chlorophyll a or Zn-Pheophytin a in intact etioplasts from barley. Stabilization of the apoproteins was dependent on the concentration of chlorophyll a or Zn-Pheophytin a. Zn-Pheophytin a was superior to chlorophyll a with respect to the concentration of pigment required for an equal yield of the stabilized chlorophyll a protein CP47, CP43, and P700 and for the total yield of chlorophyll a proteins. Zn-Pheophytin a was most efficient for stabilizing CP47 and, at an increased concentration, efficient for stabilizing CP43, P700, and D1. Stabilization of apoproteins was highest after de novo synthesis of 90-300 pmol of Zn-Pheophytin a or of about 400-600 pmol of chlorophyll a/4.2 × 107 etioplasts. The yield of stabilized chlorophyll proteins decreased at higher concentrations of Zn-Pheophytin a, but was unaffected by higher concentrations of chlorophyll a.
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stabilization of the chlorophyll binding apoproteins p700 cp47 cp43 d2 and d1 by synthesis of zn Pheophytin a in intact etioplasts from barley
FEBS Letters, 1996Co-Authors: Lutz A Eichacker, Bernd Muller, Michael HelfrichAbstract:Chlorophyll a was compared with Zn-Pheophytin a for stabilization of chlorophyll binding apoproteins, P700, CP47, CP43, D2, and D1, in intact etioplasts from barley (Hordeum vulgare L.). Intact etioplasts were shown to effectively translate the chlorophyll apoproteins, to take up and esterify the exogenously added substrates, chlorophyllide a and Zn-pheophorbide a, with geranylgeraniolpyrophosphate. For stabilization of P700, CP47, D2, and D1, the product, Zn-Pheophytin a, was shown to substitute for chlorophyll a. Stabilization of CP43 was selectively increased in the presence of Zn-Pheophytin a. The degree of stabilization was shown to depend on the amount of newly synthesized Zn-Pheophytin a and on the central atom of the chlorophyll molecule.
Michael Helfrich - One of the best experts on this subject based on the ideXlab platform.
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stabilization of chlorophyll a binding apoproteins p700 cp47 cp43 d2 and d1 by chlorophyll a or zn Pheophytin a
Journal of Biological Chemistry, 1996Co-Authors: Lutz A Eichacker, Michael Helfrich, Bernd MullerAbstract:Abstract Stabilization of chlorophyll a-binding apoproteins P700, CP47, CP43, D2, and D1 against proteolytic degradation has been investigated through in vitro synthesis of chlorophyll a or Zn-Pheophytin a in intact etioplasts from barley. Stabilization of the apoproteins was dependent on the concentration of chlorophyll a or Zn-Pheophytin a. Zn-Pheophytin a was superior to chlorophyll a with respect to the concentration of pigment required for an equal yield of the stabilized chlorophyll a protein CP47, CP43, and P700 and for the total yield of chlorophyll a proteins. Zn-Pheophytin a was most efficient for stabilizing CP47 and, at an increased concentration, efficient for stabilizing CP43, P700, and D1. Stabilization of apoproteins was highest after de novo synthesis of 90-300 pmol of Zn-Pheophytin a or of about 400-600 pmol of chlorophyll a/4.2 × 107 etioplasts. The yield of stabilized chlorophyll proteins decreased at higher concentrations of Zn-Pheophytin a, but was unaffected by higher concentrations of chlorophyll a.
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stabilization of the chlorophyll binding apoproteins p700 cp47 cp43 d2 and d1 by synthesis of zn Pheophytin a in intact etioplasts from barley
FEBS Letters, 1996Co-Authors: Lutz A Eichacker, Bernd Muller, Michael HelfrichAbstract:Chlorophyll a was compared with Zn-Pheophytin a for stabilization of chlorophyll binding apoproteins, P700, CP47, CP43, D2, and D1, in intact etioplasts from barley (Hordeum vulgare L.). Intact etioplasts were shown to effectively translate the chlorophyll apoproteins, to take up and esterify the exogenously added substrates, chlorophyllide a and Zn-pheophorbide a, with geranylgeraniolpyrophosphate. For stabilization of P700, CP47, D2, and D1, the product, Zn-Pheophytin a, was shown to substitute for chlorophyll a. Stabilization of CP43 was selectively increased in the presence of Zn-Pheophytin a. The degree of stabilization was shown to depend on the amount of newly synthesized Zn-Pheophytin a and on the central atom of the chlorophyll molecule.
Bernd Muller - One of the best experts on this subject based on the ideXlab platform.
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stabilization of chlorophyll a binding apoproteins p700 cp47 cp43 d2 and d1 by chlorophyll a or zn Pheophytin a
Journal of Biological Chemistry, 1996Co-Authors: Lutz A Eichacker, Michael Helfrich, Bernd MullerAbstract:Abstract Stabilization of chlorophyll a-binding apoproteins P700, CP47, CP43, D2, and D1 against proteolytic degradation has been investigated through in vitro synthesis of chlorophyll a or Zn-Pheophytin a in intact etioplasts from barley. Stabilization of the apoproteins was dependent on the concentration of chlorophyll a or Zn-Pheophytin a. Zn-Pheophytin a was superior to chlorophyll a with respect to the concentration of pigment required for an equal yield of the stabilized chlorophyll a protein CP47, CP43, and P700 and for the total yield of chlorophyll a proteins. Zn-Pheophytin a was most efficient for stabilizing CP47 and, at an increased concentration, efficient for stabilizing CP43, P700, and D1. Stabilization of apoproteins was highest after de novo synthesis of 90-300 pmol of Zn-Pheophytin a or of about 400-600 pmol of chlorophyll a/4.2 × 107 etioplasts. The yield of stabilized chlorophyll proteins decreased at higher concentrations of Zn-Pheophytin a, but was unaffected by higher concentrations of chlorophyll a.
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stabilization of the chlorophyll binding apoproteins p700 cp47 cp43 d2 and d1 by synthesis of zn Pheophytin a in intact etioplasts from barley
FEBS Letters, 1996Co-Authors: Lutz A Eichacker, Bernd Muller, Michael HelfrichAbstract:Chlorophyll a was compared with Zn-Pheophytin a for stabilization of chlorophyll binding apoproteins, P700, CP47, CP43, D2, and D1, in intact etioplasts from barley (Hordeum vulgare L.). Intact etioplasts were shown to effectively translate the chlorophyll apoproteins, to take up and esterify the exogenously added substrates, chlorophyllide a and Zn-pheophorbide a, with geranylgeraniolpyrophosphate. For stabilization of P700, CP47, D2, and D1, the product, Zn-Pheophytin a, was shown to substitute for chlorophyll a. Stabilization of CP43 was selectively increased in the presence of Zn-Pheophytin a. The degree of stabilization was shown to depend on the amount of newly synthesized Zn-Pheophytin a and on the central atom of the chlorophyll molecule.
Inmaculada Yruela - One of the best experts on this subject based on the ideXlab platform.
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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 <5 a small reversible change at 672.5 nm is measured that correlates with a reversible change at 542 nm, indicating that inactive Pheophytin a will absorb at this wavelength.
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spectroscopic characterization of two forms of the d1 d2 cytochrome b559 complex from sugar beet
Photochemistry and Photobiology, 1993Co-Authors: Guillermo Montoya, Rafael Cases, Inmaculada Yruela, Rafael PicorelAbstract:Two D1-D2-cytochrome b559 complex forms, called RCIIa and RCIIb, with different pigment stoichi-ometry were characterized using absorption and surface-enhanced resonance Raman scattering spectroscopy and spectral gaussian deconvolution. Electronic absorption spectra of the RCIIb at 277 K showed significant differences compared to RCIIa, i.e. a strong decrease in the absorbance due to carotenoid and chlorophyll for the same amount of Pheophytin. A reduced carotenoid and chlorophyll content in RCIIb was also observed in the surface-enhanced resonance Raman scattering spectra. Spectral deconvolution elicited three main absorption bands at 680, 672 and 669–670 nm, which were ascribed to P680, Pheophytin and accessory chlorophyll, respectively. In addition, a minor component around 667 nm was observed in the RCIIb, most probably due to some reaction center inactivation. Calculation of the relative area under the gaussians together with pigment stoichiometry data suggest that the 680, 672 and 669–670 nm components contain, respectively, two chlorophylls, two Pheophytins and four chlorophylls for the RCIIa, and two chlorophylls, two Pheophytins and two chlorophylls for the RCIIb.
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precise location of the cu ii inhibitory binding site in higher plant and bacterial photosynthetic reaction centers as probed by light induced absorption changes
Journal of Biological Chemistry, 1993Co-Authors: Inmaculada Yruela, Miguel Alfonso, Guillermo Montoya, Ortiz I De Zarate, Rafael PicorelAbstract:Light-dependent absorption change at 325 nm, ascribed to QA activity, was strongly reduced in the presence of Cu(II) in oxygen-evolving core complex. This change was much less affected in the presence of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), indicating that the Cu(II)-binding site is different from that of the DCMU and that Cu(II) blocks QA reduction. Cu(II) did not eliminate the absorption change at 545 nm, ascribed to Pheophytin reduction, in Na2S2O4-treated oxygen-evolving core and D1-D2-cytochrome b559 complexes. This indicates that Cu(II) does not affect the electron transport between P680 and Pheophytin. Moreover, the activity of the bacterial reaction center probed by the absorption change at 790 nm was inhibited by Cu(II), but the signal at 530 nm, associated to the reduction of bacterioPheophytin in Na2S2O4-treated reaction center, was not inhibited. We conclude that Cu(II) impaired the photosynthetic electron transport between Pheophytin and QA in both higher plants and photosynthetic bacteria. Cu(II) would bind to an amino acid(s) highly conserved in non-oxygenic and oxygenic reaction centers, which is(are) necessary for the electron transfer between Pheophytin and QA. Based on the atomic structure of the bacterial reaction center several schemes of possible Cu(II) binding are shown.