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Journal of Photochemistry and... May 2004This paper reports the results of the research on the interaction between the highly active cytochrome b(6)f complex and plastocyanin, both isolated from the same source...
This paper reports the results of the research on the interaction between the highly active cytochrome b(6)f complex and plastocyanin, both isolated from the same source - spinachia oleracea plants. An equilibrium constant K between the cytochrome f of the cytochrome b(6)f complex and plastocyanin has been estimated by two independent spectroscopic techniques: steady-state absorption spectroscopy and stopped-flow. The second-order rate constants k2 for forward and backward electron transfer between cytochrome f and plastocyanin have been found between 1.4-2 x 10(7) and 8-10 x 10(6) M(-1)s(-1), respectively, giving the value of an equilibrium constant of about 2+/-0.4 or a difference in redox potential between plastocyanin and cytochrome f of cytochrome b(6)f complex of ca. 17 mV. The value of K=1.7+/-0.3 has been estimated from steady-state experiments in which the initial and final concentrations of participating components after mixing have been estimated via differential spectra analysis or spectra deconvolution. We propose a method of evaluation of the final plastocyanin concentration after the electron transfer reaction between cytochrome bf complex and plastocyanin that overcomes the interference by the strong chlorophyll absorption in the spectral region where oxidised plastocyanin has its low extinction absorption band. The data from both experiments, in the system devoid of quinol being the electron donor to cytochrome b(6), suggest that in case of electron transfer from cytochrome f to plastocyanin electron transfer can either bypass cytochrome f or the Rieske iron-sulfur protein can be reduced prior to its movement to the quinol binding site of cytochrome b(6). The role of the Rieske protein in forward and backward electron transfer reactions is discussed.
Topics: Cytochrome b6f Complex; Electron Transport; Plant Leaves; Plastocyanin; Spectrum Analysis; Spinacia oleracea
PubMed: 15157909
DOI: 10.1016/j.jphotobiol.2004.03.007 -
FEBS Letters Dec 1990An expression vector designed for overexpression of plastocyanin in the periplasmic space of E. coli has been developed. The vector contains the signal peptide sequence...
An expression vector designed for overexpression of plastocyanin in the periplasmic space of E. coli has been developed. The vector contains the signal peptide sequence of Pseudomonas aeruginosa azurin and the mature sequence of spinach plastocyanin. The precursor is efficiently translocated to the periplasmic space and correctly processed to mature plastocyanin. No detectable amount of plastocyanin was present in the cytoplasmic or in the membrane fraction. A large scale preparation of the recombinant plastocyanin in a 20 litre fermentor yielded approximately 30 mg of pure plastocyanin. The recombinant protein obtained from E. coli shows CD, EPR and optical properties identical to plastocyanin isolated from spinach.
Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Electron Spin Resonance Spectroscopy; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Genetic Vectors; Molecular Sequence Data; Molecular Weight; Plants; Plasmids; Plastocyanin; Recombinant Proteins
PubMed: 2176166
DOI: 10.1016/0014-5793(90)80517-m -
Journal of Biochemistry Jan 1962
Topics: Plastocyanin; Proteins; Spinacia oleracea; Vegetables
PubMed: 14454304
DOI: 10.1093/oxfordjournals.jbchem.a127497 -
The Journal of Biological Chemistry Mar 1991In higher plants, the chloroplastic protein plastocyanin is synthesized as a transit peptide-containing precursor by cytosolic ribosomes and posttranslationally...
In higher plants, the chloroplastic protein plastocyanin is synthesized as a transit peptide-containing precursor by cytosolic ribosomes and posttranslationally transported to the thylakoid lumen. En route to the lumen, a plastocyanin precursor is first imported into chloroplasts and then further directed across the thylakoid membrane by a second distinct transport event. A partially processed form of plastocyanin is observed in the stroma during import experiments using intact chloroplasts and has been proposed to be the translocation substrate for the second step (Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365-375). To further characterize this second step, we have reconstituted thylakoid transport in a system containing in vitro-synthesized precursor proteins and isolated thylakoid membranes. This system was specific for lumenal proteins since stromal proteins lacking the appropriate targeting information did not accumulate in the thylakoid lumen. Plastocyanin precursor was taken up by isolated thylakoids, proteolytically processed to mature size, and converted to holo form. Translocation was temperature-dependent and was stimulated by millimolar levels of ATP but did not strictly require the addition of stromal factors. We have examined the substrate requirements of thylakoid translocation by testing the ability of different processed forms of plastocyanin to transport in the in vitro system. Interestingly, only the full-length plastocyanin precursor, not the partially processed intermediate form, was competent for transport in this in vitro system.
Topics: Adenosine Triphosphate; Biological Transport; Cell Wall; Chelating Agents; Electrophoresis, Polyacrylamide Gel; Plastocyanin; Protein Precursors; Temperature
PubMed: 2005123
DOI: No ID Found -
Plant & Cell Physiology May 2024The function of ascorbate peroxidase-related (APX-R) proteins, present in all green photosynthetic eukaryotes, remains unclear. This study focuses on APX-R from...
The function of ascorbate peroxidase-related (APX-R) proteins, present in all green photosynthetic eukaryotes, remains unclear. This study focuses on APX-R from Chlamydomonas reinhardtii, namely, ascorbate peroxidase 2 (APX2). We showed that apx2 mutants exhibited a faster oxidation of the photosystem I primary electron donor, P700, upon sudden light increase and a slower re-reduction rate compared to the wild type, pointing to a limitation of plastocyanin. Spectroscopic, proteomic and immunoblot analyses confirmed that the phenotype was a result of lower levels of plastocyanin in the apx2 mutants. The redox state of P700 did not differ between wild type and apx2 mutants when the loss of function in plastocyanin was nutritionally complemented by growing apx2 mutants under copper deficiency. In this case, cytochrome c6 functionally replaces plastocyanin, confirming that lower levels of plastocyanin were the primary defect caused by the absence of APX2. Overall, the results presented here shed light on an unexpected regulation of plastocyanin level under copper-replete conditions, induced by APX2 in Chlamydomonas.
Topics: Plastocyanin; Ascorbate Peroxidases; Chlamydomonas reinhardtii; Mutation; Copper; Oxidation-Reduction; Photosystem I Protein Complex; Plant Proteins; Cytochromes c6; Proteomics; Light
PubMed: 38591346
DOI: 10.1093/pcp/pcae019 -
The Journal of Biological Chemistry Sep 1992In certain cyanobacteria and algae, cytochrome c553 or plastocyanin can serve to carry electrons from the cytochrome bf complex to photosystem I. The availability of...
In certain cyanobacteria and algae, cytochrome c553 or plastocyanin can serve to carry electrons from the cytochrome bf complex to photosystem I. The availability of copper in the growth medium regulates which protein is present. To investigate copper induced control of gene expression we isolated these proteins from the cyanobacterium Synechocystis 6803. Using immunodetection and optical spectroscopy, the steady state levels of cytochrome c553 and plastocyanin were measured in cells grown at different copper concentrations. The results show that in cells grown in 20-30 nM copper, cytochrome c553 was present, whereas plastocyanin was not detected. The opposite behavior was observed in cells grown in the presence of 1 microM copper; plastocyanin was present, whereas cytochrome c553 could not be detected. Both proteins were present in cells grown in 0.3 microM copper. Northern analysis of total RNA, probed with a gene fragment for cytochrome c553 or the plastocyanin gene, showed that cells grown in the presence of 20-30 nM copper have message for cytochrome c553, but not for plastocyanin, whereas cells grown in 1 microM copper have message for plastocyanin, but not for cytochrome c553. These results demonstrate that copper regulates expression of both of the genes encoding cytochrome c553 and plastocyanin prior to translation in Synechocystis 6803.
Topics: Copper; Cyanobacteria; Cytochrome c Group; Gene Expression Regulation; Light; Plastocyanin; RNA, Messenger
PubMed: 1326543
DOI: No ID Found -
The EMBO Journal Mar 1994Mutant 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... (Comparative Study)
Comparative Study
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.
Topics: Amino Acid Sequence; Electron Transport; Escherichia coli; Genes, Plant; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Photosynthetic Reaction Center Complex Proteins; Photosystem I Protein Complex; Plants, Genetically Modified; Plastocyanin; Protein Conformation; Recombinant Proteins; Solanum tuberosum; Vegetables
PubMed: 8131737
DOI: 10.1002/j.1460-2075.1994.tb06351.x -
The Journal of Biological Chemistry Jun 2000The interaction between plastocyanin and the intact cytochrome bf complex, both from spinach, has been studied by stopped-flow kinetics with mutant plastocyanin to...
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 cytochrome f. 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 bf complex. 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.
Topics: Amino Acid Substitution; Arginine; Cytochrome b Group; Cytochrome b6f Complex; Kinetics; Leucine; Models, Molecular; Mutagenesis, Site-Directed; Oxidation-Reduction; Plastocyanin; Protein Conformation; Recombinant Proteins; Spinacia oleracea; Tyrosine
PubMed: 10837495
DOI: 10.1074/jbc.275.23.17590 -
Biochimica Et Biophysica Acta 2005The orientation of poplar plastocyanin in the complex with turnip cytochrome f has been determined by rigid-body calculations using restraints from paramagnetic NMR...
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 pK(a) values. The results are interpreted in a two-step model for complex formation.
Topics: Brassica napus; Cytochromes f; Hydrogen-Ion Concentration; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Osmolar Concentration; Plant Proteins; Plastocyanin; Protein Binding; Protein Conformation; Time Factors; Trees
PubMed: 15863096
DOI: 10.1016/j.bbabio.2004.12.002 -
Reconstitution of mature plastocyanin from precursor apo-plastocyanin expressed in Escherichia coli.Biochimica Et Biophysica Acta Jun 1991The precursor plastocyanin from Silene pratensis (white campion) has been expressed in Escherichia coli. The precursor protein was accumulated in insoluble aggregates...
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.
Topics: Amino Acid Sequence; Apoproteins; Blotting, Western; Copper; Cytochromes; Cytochromes f; Electron Transport; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Gene Expression Regulation, Bacterial; Genes, Bacterial; Kinetics; Molecular Sequence Data; Oxidation-Reduction; Photochemistry; Plants; Plasmids; Plastocyanin; Protein Precursors
PubMed: 2049372
DOI: 10.1016/s0005-2728(05)80226-9