-
Biochimica Et Biophysica Acta.... Jul 2019Small diffusible redox proteins play a ubiquitous role in bioenergetic systems, facilitating electron transfer (ET) between membrane bound complexes. Sustaining high ET...
Small diffusible redox proteins play a ubiquitous role in bioenergetic systems, facilitating electron transfer (ET) between membrane bound complexes. Sustaining high ET turnover rates requires that the association between extrinsic and membrane-bound partners is highly specific, yet also sufficiently weak to promote rapid post-ET separation. In oxygenic photosynthesis the small soluble electron carrier protein plastocyanin (Pc) shuttles electrons between the membrane integral cytochrome bf (cytbf) and photosystem I (PSI) complexes. Here we use peak-force quantitative nanomechanical mapping (PF-QNM) atomic force microscopy (AFM) to quantify the dynamic forces involved in transient interactions between cognate ET partners. An AFM probe functionalised with Pc molecules is brought into contact with cytbf complexes, immobilised on a planar silicon surface. PF-QNM interrogates the unbinding force of the cytbf-Pc interactions at the single molecule level with picoNewton force resolution and on a time scale comparable to the ET time in vivo (ca. 120 μs). Using this approach, we show that although the unbinding force remains unchanged the interaction frequency increases over five-fold when Pc and cytbf are in opposite redox states, so complementary charges on the cytbf and Pc cofactors likely contribute to the electrostatic forces that initiate formation of the ET complex. These results suggest that formation of the docking interface is under redox state control, which lowers the probability of unproductive encounters between Pc and cytbf molecules in the same redox state, ensuring the efficiency and directionality of this central reaction in the 'Z-scheme' of photosynthetic ET.
Topics: Cytochrome b6f Complex; Electron Transport; Oxidation-Reduction; Photosynthesis; Plastocyanin; Protein Binding; Protein Conformation; Single-Cell Analysis; Spinacia oleracea
PubMed: 31247170
DOI: 10.1016/j.bbabio.2019.06.013 -
Plant Molecular Biology Jan 1995Plastocyanin from the thermophilic cyanobacterium Phormidium laminosum has been purified, a partial amino acid sequence obtained and the gene cloned and sequenced. The... (Comparative Study)
Comparative Study
Plastocyanin from the thermophilic cyanobacterium Phormidium laminosum has been purified, a partial amino acid sequence obtained and the gene cloned and sequenced. The derived amino acid sequence indicates that the plastocyanin protein is initially synthesized with an N-terminal leader sequence of 34 amino acids to direct it across the thylakoid membrane. The leader sequence consists of a positively charged N-terminal region, a hydrophobic region and a cleavage site, which are characteristic both of higher-plant chloroplast thylakoid transfer domains and of bacterial leader peptides. The petE gene and flanking regions have been cloned in Escherichia coli, and the plastocyanin protein is expressed and directed to the periplasmic space, with concomitant processing to the mature form. Targeting to the periplasm and processing of the plastocyanin protein in E. coli appears to be dependent on components of the Sec apparatus, since the unprocessed precursor accumulates in the cytoplasm of a secA mutant. Expression of plastocyanin in E. coli is copper-inducible and apparently controlled at the level of transcription, leading to the conclusion that copper-regulated promoters exist in the regions flanking the gene and are recognized in a heterologous system. Possible implications for gene expression and protein targeting in the cyanobacterium are discussed.
Topics: Adenosine Triphosphatases; Amino Acid Sequence; Bacterial Proteins; Base Sequence; Cloning, Molecular; Copper; Cyanobacteria; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genes, Bacterial; Membrane Transport Proteins; Molecular Sequence Data; Plastocyanin; Recombinant Proteins; SEC Translocation Channels; SecA Proteins; Sequence Analysis; Sequence Homology, Amino Acid
PubMed: 7865788
DOI: 10.1007/BF00019189 -
The Journal of Biological Chemistry Jan 1986The crystal structure of Hg(II)-plastocyanin has been determined and refined at a resolution of 1.9 A. The crystals were prepared by soaking crystals of... (Comparative Study)
Comparative Study
The crystal structure of Hg(II)-plastocyanin has been determined and refined at a resolution of 1.9 A. The crystals were prepared by soaking crystals of Cu(II)-plastocyanin from poplar leaves (Populus nigra var. italica) in a solution of a mercuric salt. Replacement of the Cu(II) atom in plastocyanin by Hg(II) causes only minor changes in the geometry of the metal site, and there are few significant changes elsewhere in the molecule. It is concluded that, as in the case of the native protein, the geometry of the metal site is determined by the polypeptide. The weak metal-S(methionine) bond found in Cu(II)-plastocyanin remains weak in Hg(II)-plastocyanin. The "flip" of a proline side chain close to the metal site from a C gamma-exo conformation in Cu(II)-plastocyanin to a C gamma-endo conformation in Hg(II)-plastocyanin suggests that this region of the molecule is particularly flexible. Crystallographic evidence for the close similarity of the Hg(II)- and Cu(II)-plastocyanin structures was originally obtained from electron density difference maps at 2.5-A resolution. The refinement of the structure was begun with a set of atomic coordinates taken from the structure of Cu(II)-plastocyanin. A Hg(II) atom was substituted for the Cu(II) atom, and the side chains of 6 residues in the vicinity of the metal site were omitted. Three series of stereochemically restrained least-squares refinement calculations were interspersed with two stages of model adjustment followed by phase extension. Fifty-nine water molecules were located. The final structure has a crystallographic residual R = 0.16.
Topics: Copper; Crystallization; Mercury; Models, Molecular; Plant Proteins; Plastocyanin; Stereoisomerism; Trees
PubMed: 3941073
DOI: 10.2210/pdb3pcy/pdb -
Acta Crystallographica. Section D,... Feb 1999The crystal structure of the 'blue' copper protein plastocyanin from the cyanobacterium Phormidium laminosum has been solved and refined using 2.8 A X--ray data. P....
The crystal structure of the 'blue' copper protein plastocyanin from the cyanobacterium Phormidium laminosum has been solved and refined using 2.8 A X--ray data. P. laminosum plastocyanin crystallizes in space group P43212 with unit-cell dimensions a = 86.57, c = 91.47 A and with three protein molecules per asymmetric unit. The final residual R is 19.9%. The structure was solved using molecular replacement with a search model based on the crystal structure of a close homologue, Anabaena variabilis plastocyanin (66% sequence identity). The molecule of P. laminosum plastocyanin has 105 amino-acid residues. The single Cu atom is coordinated by the same residues - two histidines, a cysteine and a methionine - as in other plastocyanins. In the crystal structure, the three molecules of the asymmetric unit are related by a non-crystallographic threefold axis. A Zn atom lies between each pair of neighbouring molecules in this ensemble, being coordinated by a surface histidine residue of one molecule and by two aspartates of the other.
Topics: Amino Acid Sequence; Crystallography, X-Ray; Cyanobacteria; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Plastocyanin; Protein Conformation; Sequence Homology, Amino Acid; Zinc
PubMed: 10089349
DOI: 10.1107/s0907444998012074 -
Journal of Biochemistry Aug 1989Plastocyanin was purified from a multicellular, marine green alga, Ulva arasakii, by conventional methods to homogeneity. The oxidized plastocyanin showed absorption...
Plastocyanin was purified from a multicellular, marine green alga, Ulva arasakii, by conventional methods to homogeneity. The oxidized plastocyanin showed absorption maxima at 252, 276.8, 460, 595.3, and 775 nm, and shoulders at 259, 265, 269, and 282.5 nm; the ratio A276.8/A595.3 was 1.5. The midpoint redox potential was determined to be 0.356 V at pH 7.0 with a ferri- and ferrocyanide system. The molecular weight was estimated to be 10,200 and 11,000 by SDS-PAGE and by gel filtration, respectively. U. arasakii also has a small amount of cytochrome c6, like Enteromorpha prolifera. The amino acid sequence of U. arasakii plastocyanin was determined by Edman degradation and by carboxypeptidase digestion of the plastocyanin, six tryptic peptides, and five staphylococcal protease peptides. The plastocyanin contained 98 amino acid residues, giving a molecular weight of 10,236 including one copper atom. The complete sequence is as follows: AQIVKLGGDDGALAFVPSKISVAAGEAIEFVNNAGFPHNIVFDEDAVPAGVDADAISYDDYLNSKGETV VRKLSTPGVY G VYCEPHAGAGMKMTITVQ. The sequence of U. arasakii plastocyanin is closet to that of the E. prolifera protein (85% homology). A phylogenetic tree of five algal and two higher plant plastocyanins was constructed by comparing the amino acid differences. The branching order is considered to be as follows: a blue-green alga, unicellular green algae, multicellular green algae, and higher plants.
Topics: Amino Acid Sequence; Amino Acids; Apoproteins; Chlorophyll; Chlorophyta; Cyanobacteria; Cytochromes; Cytochromes f; Hydrolysis; Methylation; Molecular Sequence Data; Peptide Fragments; Peptides; Plant Proteins; Plants; Plastocyanin; Terminology as Topic
PubMed: 2509442
DOI: 10.1093/oxfordjournals.jbchem.a122845 -
Biochimica Et Biophysica Acta Feb 1974(1) An electron paramagnetic resonance (EPR) signal was observed at g = 2.05 in the low temperature spectra of intact cells of green, red and blue-green algae and of...
(1) An electron paramagnetic resonance (EPR) signal was observed at g = 2.05 in the low temperature spectra of intact cells of green, red and blue-green algae and of spinach chloroplasts. The g-value and the shape of the signal were similar to that of purified, soluble plastocyanin. (2) The amount of the copper protein, determined from the EPR signal height, was estimated to be nearly the same in all the studied organisms on the basis of the concentration of chlorophyll. Furthermore, it was found that the amount of the copper protein, determined from the EPR signal height in spinach chloroplasts corresponds with that of plastocyanin as determined chemically by Katoh, S., Suga, I., Shiratori, I. and Takamiya, A. (1961) Arch. Biochem. Biophys. 94, 136-141. (3) Experiments with far-red and red illumination show that the site of the copper protein in vivo is in the electron transport pathway between Photosystems 1 and 2. Plastocyanin is not oxidized by illumination at 77 degrees K, indicating that no electron transfer occurs between the primary electron donor of Photosystem 1, P700, and plastocyanin at that temperature. Furthermore, the experiments suggest that in the intact cells of the studied algae, plastocyanin is not only reduced by Photosystem 2 but also by cyclic electron transport around Photosystem 1.
Topics: Algal Proteins; Chlorella; Chloroplasts; Electron Spin Resonance Spectroscopy; Oxidation-Reduction; Plant Proteins; Plastocyanin
PubMed: 19400039
DOI: 10.1016/0005-2728(74)90011-5 -
FEBS Letters Sep 2000A study by two-dimensional electrophoresis showed that the soluble, lumenal fraction of Arabidopsis thaliana thylakoids can be resolved into 300 protein spots. After...
A study by two-dimensional electrophoresis showed that the soluble, lumenal fraction of Arabidopsis thaliana thylakoids can be resolved into 300 protein spots. After subtraction of low-intensity spots and accounting for low-level stromal contamination, the number of more abundant, lumenal proteins was estimated to be between 30 and 60. Two of these proteins have been identified: a novel plastocyanin that also was the predominant component of the total plastocyanin pool, and a putative ascorbate peroxidase. Import studies showed that these proteins are routed to the thylakoid lumen by the Sec- and delta pH-dependent translocation pathways, respectively. In addition, novel isoforms of PsbO and PsbQ were identified.
Topics: Amino Acid Sequence; Arabidopsis; Ascorbate Peroxidases; Cell Fractionation; Chloroplasts; Molecular Sequence Data; Peroxidases; Plastocyanin; Proteome; Sequence Homology, Amino Acid; Thylakoids
PubMed: 11034343
DOI: 10.1016/s0014-5793(00)01890-1 -
Journal of Biochemistry Oct 1994Plastocyanin is a copper protein that functions as an electron carrier in the thylakoid lumen of the chloroplast. To characterize the transit peptide of plastocyanin and...
Plastocyanin is a copper protein that functions as an electron carrier in the thylakoid lumen of the chloroplast. To characterize the transit peptide of plastocyanin and develop expression systems for it in Escherichia coli, three kinds of expression vectors which encode different size precursor plastocyanin molecules were constructed. Their expression, processing, and copper-binding activity have been examined. When the full-length cDNA encoding the precursor plastocyanin from Silene pratensis was expressed in E. coli, a large amount of precursor plastocyanin accumulated in insoluble aggregates. Its accumulation level was increased by the addition of copper ions. About six percent of precursor plastocyanin molecules were transported into the periplasmic space and processed to the mature protein. On the other hand, expression of the intermediate size cDNA, which contains the hydrophobic domain and basic amino acid of C-terminal transit peptide, caused exclusive translocation to the periplasmic space and correct processing to the mature size. The addition of copper ions increased the holo-protein content, but did not change the polypeptide content of mature plastocyanin, indicating that translocation and processing are independent of the incorporation of copper ions. The mature plastocyanin content corresponds to 8% (w/w) of the total E. coli protein content (123 mg per liter of culture). The purified mature holo-protein showed almost the same spectroscopic and kinetic properties as those of purified spinach plastocyanin. Expression of the cDNA encoding the mature polypeptide and two preceeding amino acid residues caused the accumulation of only a small amount of plastocyanin.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Amino Acid Sequence; Base Sequence; Binding Sites; Copper; Escherichia coli; Gene Expression; Genetic Vectors; Molecular Sequence Data; Plasmids; Plastocyanin; Protein Precursors; Protein Processing, Post-Translational; Recombinant Proteins
PubMed: 7883757
DOI: 10.1093/oxfordjournals.jbchem.a124602 -
Metallomics : Integrated Biometal... Feb 2011Cupredoxins are small proteins that contain type I copper centers, which are ubiquitous in nature. They function as electron transfer shuttles between proteins. This... (Review)
Review
Cupredoxins are small proteins that contain type I copper centers, which are ubiquitous in nature. They function as electron transfer shuttles between proteins. This review of the structure and properties of native cupredoxins, and those modified by site-directed mutagenesis, illustrates how these proteins may have evolved to specifically bind copper, develop recognition sites for specific redox partners, tune redox potential for a particular function, and allow for efficient electron transfer through the protein matrix. This is relevant to the general understanding of the roles of metals in energy metabolism, respiration and photosynthesis.
Topics: Azurin; Binding Sites; Copper; Models, Molecular; Plastocyanin
PubMed: 21258692
DOI: 10.1039/c0mt00061b -
European Journal of Biochemistry Nov 1996To compare cadmium-substituted plastocyanin with copper plastocyanin, the 1H-NMR spectra of CuI-, CuII- and Cd-plastocyanin from pea have been analyzed. Full assignments...
Analysis of the 1H-NMR chemical shifts of Cu(I)-, Cu(II)- and Cd-substituted pea plastocyanin. Metal-dependent differences in the hydrogen-bond network around the copper site.
To compare cadmium-substituted plastocyanin with copper plastocyanin, the 1H-NMR spectra of CuI-, CuII- and Cd-plastocyanin from pea have been analyzed. Full assignments of the spectra of CuI- and Cd-plastocyanin indicate chemical shift differences up to 1 ppm. The affected protons are located in the four loops that surround the Cu site. The largest differences were found for protons in the hydrogen bond network which stabilizes this part of the protein. This suggests that the chemical shift differences are caused by very small but extensive structural changes in the network upon replacement of CuI by Cd. For CuII-plastocyanin the resonances of 72% of the protons observed in the CuI form have been identified. Protons within approximately 0.9 nm of the CuII were not observed due to fast paramagnetic relaxation. The protons between 0.9-1.7 nm from the CuII showed chemical shift differences up to 0.4 ppm compared to both CuI- and Cd-plastocyanin. These differences can be predicted assuming that they represent pseudocontact shifts. When corrected for the pseudocontact shift contribution, the CuII-plastocyanin chemical shifts were nearly all identical within error to those of the Cd form, but not of the CuI-plastocyanin, indicating that the CuII-plastocyanin structure, in as far as it can be observed, resembles Cd-rather than CuI-plastocyanin. In a single stretch of residues (64-69) chemical shift differences remained between all three forms after correction. The fact that pseudocontact shifts were observed for protons which were not broadened may be attributable to the weaker distance dependence of the pseudocontact shift effect compared to paramagnetic relaxation. This results in two shells around the Cu atom, an inner paramagnetic shell (0-0.9 nm), in which protons are not observed due to broadening, and an outer paramagnetic shell (0.9-1.7 nm), in which protons can be observed and show pseudocontact shifts. It is concluded that Cd-plastocyanin is a suitable redox-inactive substitute for Cu-plastocyanin.
Topics: Amino Acid Sequence; Cadmium; Copper; Electron Spin Resonance Spectroscopy; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Pisum sativum; Plastocyanin
PubMed: 8954163
DOI: 10.1111/j.1432-1033.1996.0132r.x