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Miguel A. De La Rosa - One of the best experts on this subject based on the ideXlab platform.
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Key Role of the Local Hydrophobicity in the East Patch of Plastocyanins on Their Thermal Stability and Redox Properties
'American Chemical Society (ACS)', 2018Co-Authors: Olloqui-sariego, José Luis, Miguel A. De La Rosa, Frutos-beltrán Estrella, Díaz-moreno Irene, Calvente J.j., Andreu Rafael, Díaz-quintana AntonioAbstract:Understanding the molecular basis of the thermal stability and functionality of redox proteins has important practical applications. Here, we show a distinct thermal dependence of the spectroscopic and electrochemical properties of two Plastocyanins from the thermophilic cyanobacterium Phormidium laminosum and their mesophilic counterpart from Synechocystis sp. PCC 6803, despite the similarity of their molecular structures. To explore the origin of these differences, we have mimicked the local hydrophobicity in the east patch of the thermophilic protein by replacing a valine of the mesophilic Plastocyanin by isoleucine. Interestingly, the resulting mutant approaches the thermal stability, redox thermodynamics, and dynamic coupling of the flexible site motions of the thermophilic protein, indicating the existence of a close connection between the hydrophobic packing of the east patch region of Plastocyanin and the functional control and stability of the oxidized and reduced forms of the protein.Peer Reviewe
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voltammetric study of the adsorbed thermophilic Plastocyanin from phormidium laminosum up to 90 c
Electrochemistry Communications, 2012Co-Authors: Jose Luis Olloquisariego, Antonio Diazquintana, Miguel A. De La Rosa, Estrella Frutosbeltran, Emilio Roldan, Juan Jose Calvente, Rafael AndreuAbstract:Abstract Redox thermodynamics and kinetics of Plastocyanin from Phormidium laminosum , and of azurin from Pseudonomas aeruginosa , have been investigated as a function of temperature by protein film voltammetry. To this purpose, both proteins have been physisorbed on a pyrolytic graphite edge electrode. A pronounced negative shift of the Plastocyanin standard potential, compared to a slight shift in the case of azurin, has been found upon increasing the temperature. Hence, significant conformational and/or solvation changes accompany the redox conversion of Plastocyanin. Lower electron transfer rate constants (by c.a. one order of magnitude) and higher activation enthalpies have been found for Plastocyanin as compared to azurin. The voltammetric response of azurin vanishes irreversibly at temperatures close to 60 °C, whereas the redox properties of Plastocyanin remain unaltered, except for some loss of electroactive protein, after heating the electrode at temperatures as high as 90 °C.
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Modulation of copper site properties by remote residues determines the stability of Plastocyanins.
FEBS letters, 2010Co-Authors: Francisco J. Muñoz-lópez, Miguel A. De La Rosa, Estrella Frutos Beltrán, Sofía Díaz-moreno, Irene Díaz-moreno, Gloria Subías, Antonio Díaz-quintanaAbstract:The metal cofactor determines the thermal stability in cupredoxins, but how the redox state of copper modulates their melting points remains unknown. The metal coordination environment is highly conserved in cyanobacterial Plastocyanins. However, the oxidised form is more stable than the reduced one in thermophilic Phormidium, but the opposite occurs in mesophilic Synechocystis. We have performed neutral amino-acid substitutions at loops of Phormidium Plastocyanin far from the copper site. Notably, mutation P49G/G50P confers a redox-dependent thermal stability similar to that of the mesophilic Plastocyanin. Moreover, X-ray absorption spectroscopy reveals that P49G/G50P mutation makes the electron density distribution at the oxidised copper site shift towards that of Synechocystis Plastocyanin.
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structure of the complex between Plastocyanin and cytochrome f from the cyanobacterium nostoc sp pcc 7119 as determined by paramagnetic nmr the balance between electrostatic and hydrophobic interactions within the transient complex determines the rel
Journal of Biological Chemistry, 2005Co-Authors: Irene Diazmoreno, Antonio Diazquintana, Miguel A. De La Rosa, Marcellus UbbinkAbstract:Abstract The complex between cytochrome f and Plastocyanin from the cyanobacterium Nostoc has been characterized by NMR spectroscopy. The binding constant is 16 mm–1, and the lifetime of the complex is much less than 10 ms. Intermolecular pseudo-contact shifts observed for the Plastocyanin amide nuclei, caused by the heme iron, as well as the chemical-shift perturbation data were used as the sole experimental restraints to determine the orientation of Plastocyanin relative to cytochrome f with a precision of 1.3 A. The data show that the hydrophobic patch surrounding tyrosine 1 in cytochrome f docks the hydrophobic patch of Plastocyanin. Charge complementarities are found between the rims of the respective recognition sites of cytochrome f and Plastocyanin. Significant differences in the relative orientation of both proteins are found between this complex and those previously reported for plants and Phormidium, indicating that electrostatic and hydrophobic interactions are balanced differently in these complexes.
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different modes of interaction in cyanobacterial complexes of Plastocyanin and cytochrome f
Biochemistry, 2005Co-Authors: Irene Diazmoreno, Peter B Crowley, Antonio Diazquintana, Miguel A. De La Rosa, Marcellus UbbinkAbstract:The highly efficient electron-transfer chain in photosynthesis demonstrates a remarkable variation among organisms in the type of interactions between the soluble electron-transfer protein Plastocyanin and it partner cytochrome f. The complex from the cyanobacterium Nostoc sp. PCC 7119 was studied using nuclear magnetic resonance spectroscopy and compared to that of the cyanobacterium Phormidium laminosum. In both systems, the main site of interaction on Plastocyanin is the hydrophobic patch. However, the interaction in the Nostoc complex is highly dependent on electrostatics, contrary to that of Phormidium, resulting in a binding constant that is an order of magnitude larger at low ionic strength for the Nostoc complex. Studies of the mixed complexes show that these differences in interactions are mainly attributable to the surface properties of the Plastocyanins.
Marcellus Ubbink - One of the best experts on this subject based on the ideXlab platform.
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dynamics in the transient complex of Plastocyanin cytochrome f from prochlorothrix hollandica
Journal of the American Chemical Society, 2008Co-Authors: Rinske Hulsker, George S Bullerjahn, Maria V Baranova, Marcellus UbbinkAbstract:The nature of transient protein complexes can range from a highly dynamic ensemble of orientations to a single well-defined state. This represents variation in the equilibrium between the encounter and final, functional state. The transient complex between Plastocyanin (Pc) and cytochrome f (cytf) of the cyanobacterium Prochlorothrix hollandica was characterized by NMR spectroscopy. Intermolecular pseudocontact shifts and chemical shift perturbations were used as restraints in docking calculations to determine the structure of the wild-type Pc−cytf complex. The orientation of Pc is similar to orientations found in Pc−cytf complexes from other sources. Electrostatics seems to play a modest role in complex formation. A large variability in the ensemble of lowest energy structures indicates a dynamic nature of the complex. Two unusual hydrophobic patch residues in Pc have been mutated to the residues found in other Plastocyanins (Y12G/P14L). The binding constants are similar for the complexes of cytf with wi...
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dynamics in the transient complex of Plastocyanin cytochrome f from prochlorothrix hollandica
Journal of the American Chemical Society, 2008Co-Authors: Rinske Hulsker, George S Bullerjahn, Maria V Baranova, Marcellus UbbinkAbstract:The nature of transient protein complexes can range from a highly dynamic ensemble of orientations to a single well-defined state. This represents variation in the equilibrium between the encounter and final, functional state. The transient complex between Plastocyanin (Pc) and cytochrome f (cytf) of the cyanobacterium Prochlorothrix hollandica was characterized by NMR spectroscopy. Intermolecular pseudocontact shifts and chemical shift perturbations were used as restraints in docking calculations to determine the structure of the wild-type Pc−cytf complex. The orientation of Pc is similar to orientations found in Pc−cytf complexes from other sources. Electrostatics seems to play a modest role in complex formation. A large variability in the ensemble of lowest energy structures indicates a dynamic nature of the complex. Two unusual hydrophobic patch residues in Pc have been mutated to the residues found in other Plastocyanins (Y12G/P14L). The binding constants are similar for the complexes of cytf with wi...
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Handbook of Metalloproteins - Cytochrome f/Plastocyanin Complex
Handbook of Metalloproteins, 2006Co-Authors: Marcellus UbbinkAbstract:In the photosynthetic electron transfer chain in plants, cytochrome f, a subunit of the cytochrome b6f complex, acts as electron donor for Plastocyanin. Plastocyanin is a small soluble protein, which shuttles electrons to photosystem 1. The structure of the short-lived and weak complex of the soluble domain of cytochrome f and Plastocyanin has been determined using nuclear magnetic resonance. It shows that the heme iron of cytochrome f and the copper ion of Plastocyanin are in close proximity, enabling fast electron transfer. The copper ligand His87 is located close to the iron ligand Tyr1, suggesting that electron transfer occurs via the hydrophobic patch of Plastocyanin. Electrostatic interactions are also important in the complex. These findings are discussed in the light of studies of the electron transfer kinetics. 3D Structure Keywords: electron transfer; cytochrome f; Plastocyanin; photosynthesis; paramagnetic; NMR
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structure of the complex between Plastocyanin and cytochrome f from the cyanobacterium nostoc sp pcc 7119 as determined by paramagnetic nmr the balance between electrostatic and hydrophobic interactions within the transient complex determines the rel
Journal of Biological Chemistry, 2005Co-Authors: Irene Diazmoreno, Antonio Diazquintana, Miguel A. De La Rosa, Marcellus UbbinkAbstract:Abstract The complex between cytochrome f and Plastocyanin from the cyanobacterium Nostoc has been characterized by NMR spectroscopy. The binding constant is 16 mm–1, and the lifetime of the complex is much less than 10 ms. Intermolecular pseudo-contact shifts observed for the Plastocyanin amide nuclei, caused by the heme iron, as well as the chemical-shift perturbation data were used as the sole experimental restraints to determine the orientation of Plastocyanin relative to cytochrome f with a precision of 1.3 A. The data show that the hydrophobic patch surrounding tyrosine 1 in cytochrome f docks the hydrophobic patch of Plastocyanin. Charge complementarities are found between the rims of the respective recognition sites of cytochrome f and Plastocyanin. Significant differences in the relative orientation of both proteins are found between this complex and those previously reported for plants and Phormidium, indicating that electrostatic and hydrophobic interactions are balanced differently in these complexes.
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the transient complex of poplar Plastocyanin with cytochrome f effects of ionic strength and ph
Biochimica et Biophysica Acta, 2005Co-Authors: Christian Lange, Irene Diazmoreno, Tobias Cornvik, Marcellus UbbinkAbstract:Abstract The orientation of poplar Plastocyanin in the complex with turnip cytochrome f has been determined by rigid-body calculations using restraints from paramagnetic NMR measurements. The results show that poplar Plastocyanin interacts with cytochrome f with the hydrophobic patch of Plastocyanin close to the heme region on cytochrome f and via electrostatic interactions between the charged patches on both proteins. Plastocyanin is tilted relative to the orientation reported for spinach Plastocyanin, resulting in a longer distance between iron and copper (13.9 A). With increasing ionic strength, from 0.01 to 0.11 M, all observed chemical-shift changes decrease uniformly, supporting the idea that electrostatic forces contribute to complex formation. There is no indication for a rearrangement of the transient complex in this ionic strength range, contrary to what had been proposed earlier on the basis of kinetic data. By decreasing the pH from pH 7.7 to pH 5.5, the complex is destabilized. This may be attributed to the protonation of the conserved acidic patches or the copper ligand His87 in poplar Plastocyanin, which are shown to have similar p K a values. The results are interpreted in a two-step model for complex formation.
Antonio Díaz-quintana - One of the best experts on this subject based on the ideXlab platform.
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Modulation of copper site properties by remote residues determines the stability of Plastocyanins.
FEBS letters, 2010Co-Authors: Francisco J. Muñoz-lópez, Miguel A. De La Rosa, Estrella Frutos Beltrán, Sofía Díaz-moreno, Irene Díaz-moreno, Gloria Subías, Antonio Díaz-quintanaAbstract:The metal cofactor determines the thermal stability in cupredoxins, but how the redox state of copper modulates their melting points remains unknown. The metal coordination environment is highly conserved in cyanobacterial Plastocyanins. However, the oxidised form is more stable than the reduced one in thermophilic Phormidium, but the opposite occurs in mesophilic Synechocystis. We have performed neutral amino-acid substitutions at loops of Phormidium Plastocyanin far from the copper site. Notably, mutation P49G/G50P confers a redox-dependent thermal stability similar to that of the mesophilic Plastocyanin. Moreover, X-ray absorption spectroscopy reveals that P49G/G50P mutation makes the electron density distribution at the oxidised copper site shift towards that of Synechocystis Plastocyanin.
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Mutations in both leucine 12 and lysine 33 in Plastocyanin from Synechocystis sp. PCC 6803 induce drastic changes in the hydrophobic interactions with Photosystem I
Photosynthesis Research, 2002Co-Authors: Antonio Díaz-quintana, Manuel Hervas, Berta De La Cerda, José A. Navarro, Miguel A. De La RosaAbstract:The role played by the residues Leu12 and Lys33 – which are both located at the north hydrophobic patch of Plastocyanin – in the interaction of the copper protein with Photosystem I from the cyanobacterium Synechocystis sp. PCC 6803 has been investigated by site-directed mutagenesis. A thermodynamic analysis of PS I reduction by wild-type and mutant Plastocyanins has been performed by laser-flash absorption spectroscopy. In all cases, the electron transfer is impaired by mutations, which induce drastic changes in the apparent activation entropy of the overall reaction. Substitution of Leu12 by alanine specifically affects the hydrophobic interactions with PS I, whereas replacement of Lys33 by glutamate not only induces local electrostatic changes, but also alters the hydrophobic interactions with the photosystem. The thermodynamic analysis of the reactivity of K33E mutant towards PS I reveals that the effect of the mutation can be reversed by addition of magnesium cations, which probably bind at a place close to Glu33. The electrostatic surface potential does thus modulate the hydrophobic interactions with PS I by altering the solvent accessibility of some surface residues.
Peter Weisbeek - One of the best experts on this subject based on the ideXlab platform.
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Silene Plastocyanin is fully functional in transgenic tobacco
Plant Science, 2003Co-Authors: Douwe De Boer, Annemiek Wilmink, Alice Lever, Fons Cremers, Paul J J Hooykaas, Peter WeisbeekAbstract:Abstract Transgenic tobacco plants, expressing the Silene pratensis (white campion) gene for the precursor of Plastocyanin, were analysed with respect to the functionality of the gene product. The gene was found to be expressed in all tissues that were examined due to the strong and constitutive cauliflower mosaic virus 35S promoter. In green tissue the Silene protein was transported into chloroplasts and routed to the chloroplasts lumen, where it was found processed to its mature size. In non-photosynthetic tissue the protein is transported into chromoplasts or leucoplasts. In plants grown in tissue culture the amount of endogenous tobacco Plastocyanin was found to be reduced significantly, but the Silene Plastocyanin was clearly detectable. When plants were dependent on photosynthesis for growth, due to depletion of sucrose from the medium, still only Silene Plastocyanin was present. This strongly suggests that the Silene protein can take over photosynthesis in transgenic tobacco when the endogenous Plastocyanin is not present. Silene Plastocyanin is considered to be fully functional, since both its transport to the chloroplast and its function in photosynthesis were retained in transgenic tobacco plants.
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Reconstitution of mature Plastocyanin from precursor apo-Plastocyanin expressed in Escherichia coli.
Biochimica et Biophysica Acta, 1991Co-Authors: Takashi Hibino, A. Douwe De Boer, Peter Weisbeek, Teruhiro TakabeAbstract:The precursor Plastocyanin from Silene pratensis (white campion) has been expressed in Escherichia coli. The precursor protein was accumulated in insoluble aggregates and partially purified as an apo-protein. The purified precursor apo-Plastocyanin was processed to the mature apo-Plastocyanin by chloroplast extracts. N-terminal amino-acid sequencing indicated that the processed protein was identical to the N-terminal amino-acid residues of mature Plastocyanin that was deduced from the nucleotide sequence. The copper could be incorporated into the apo-Plastocyanin of mature size in vitro, but could not into the precursor apo-Plastocyanin under the same conditions. Absorption spectra and reduction potential of the reconstituted mature Plastocyanin were indistinguishable from those of the purified spinach Plastocyanin. The electron transfer activities of the reconstituted Plastocyanin with both the Photosystem I reaction center (P700) and cytochrome f were almost the same as those of the purified spinach Plastocyanin.
Reinhold G Herrmann - One of the best experts on this subject based on the ideXlab platform.
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dynamic interaction of Plastocyanin with the cytochrome bf complex
Journal of Biological Chemistry, 2000Co-Authors: Jurgen Illerhaus, Lothar Altschmied, Reinhold G Herrmann, Jan Reichert, Wolfgang HaehnelAbstract:Abstract The interaction between Plastocyanin and the intact cytochrome bf complex, both from spinach, has been studied by stopped-flow kinetics with mutant Plastocyanin to elucidate the site of electron transfer and the docking regions of the molecule. Mutation of Tyr-83 to Arg or Leu provides no evidence for a second electron transfer path via Tyr-83 of Plastocyanin, which has been proposed to be the site of electron transfer from cytochromef. The data found with mutations of acidic residues indicate that both conserved negative patches are essential for the binding of Plastocyanin to the intact cytochrome bfcomplex. Replacing Ala-90 and Gly-10 at the flat hydrophobic surface of Plastocyanin by larger residues slowed down and accelerated, respectively, the rate of electron transfer as compared with wild-type Plastocyanin. These opposing effects reveal that the hydrophobic region around the electron transfer site at His-87 is divided up into two regions, of which only that with Ala-90 contributes to the attachment to the cytochrome bf complex. These binding sites of Plastocyanin are substantially different from those interacting with photosystem I. It appears that each of the two binding regions of Plastocyanin is split into halves, which are used in different combinations in the molecular recognition at the two membrane complexes.
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the Plastocyanin binding domain of photosystem i
The EMBO Journal, 1996Co-Authors: Michael Hippler, Reinhold G Herrmann, M Sutter, Lothar Altschmied, U Schroer, J. Reichert, Wolfgang HaehnelAbstract:Abstract The molecular recognition between Plastocyanin and photosystem I was studied. Photosystem I and Plastocyanin can be cross-linked to an active electron transfer complex. Immunoblots and mass spectrometric analysis of proteolytic peptides indicate that the two negative patches conserved in plant Plastocyanins are cross-linked with lysine residues of a domain near the N-terminus of the PsaF subunit of photosystem I. Conversion of these negative to uncharged patches of Plastocyanin by site-directed mutation D42N/E43Q/D44N/E45Q and E59Q/E60Q/D61N respectively, reveals the first patch to be essential for the electrostatic interaction in the electron transfer complex with photosystem I and the second one to lower the redox potential. The domain in PsaF, not found in cyanobacteria, is predicted to fold into two amphipathic alpha-helices. The interacting N-terminal helix lines up six lysines on one side which may guide a fast one-dimensional diffusion of Plastocyanin and provide the electrostatic attraction at the attachment site, in addition to the hydrophobic interaction in the area where the electron is transferred to P700 in the reaction center of photosystem I. This two-step interaction is likely to increase the electron transfer rate by more than two orders of magnitude in plants as compared with cyanobacteria. Our data resolve the controversy about the function of PsaF.
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electron transfer from Plastocyanin to photosystem i
The EMBO Journal, 1994Co-Authors: Wolfgang Haehnel, Ralf Bernd Klosgen, B Stahl, Dagmar Michl, B Huvermann, Michael Karas, T. Jansen, Kermit Gause, Reinhold G HerrmannAbstract:Abstract Mutant Plastocyanins with Leu at position 10, 90 or 83 (Gly, Ala and Tyr respectively in wildtype) were constructed by site-specific mutagenesis of the spinach gene, and expressed in transgenic potato plants under the control of the authentic Plastocyanin promoter, as well as in Escherichia coli as truncated precursor intermediates carrying the C-terminal 22 amino acid residues of the transit peptide, i.e. the thylakoid-targeting domain that acts as a bacterial export signal. The identity of the purified Plastocyanins was verified by matrix-assisted laser desorption/ionization mass spectrometry. The formation of a complex between authentic or mutant spinach Plastocyanin and isolated photosystem I and the electron transfer has been studied from the biphasic reduction kinetics of P700+ after excitation with laser flashes. The formation of the complex was abolished by the bulky hydrophobic group of Leu at the respective position of G10 or A90 which are part of the conserved flat hydrophobic surface around the copper ligand H87. The rate of electron transfer decreased by both mutations to < 20% of that found with wildtype Plastocyanin. We conclude that the conserved flat surface of Plastocyanin represents one of two crucial structural elements for both the docking at photosystem I and the efficient electron transfer via H87 to P700+. The Y83L mutant exhibited faster electron transfer to P700+ than did authentic Plastocyanin. This proves that Y83 is not involved in electron transfer to P700 and suggests that electron transfer from cytochrome f and to P700 follows different routes in the Plastocyanin molecule. Plastocyanin (Y83L) expressed in either E. coli or potato exhibited different isoelectric points and binding constants to photosystem I indicative of differences in the folding of the protein. The structure of the binding site at photosystem I and the mechanism of electron transfer are discussed.