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Journal of Biochemistry Jan 1989A cross-linked complex between plastocyanin and cytochrome f was prepared by incubation in the presence of a water soluble carbodiimide and its kinetic properties were...
A cross-linked complex between plastocyanin and cytochrome f was prepared by incubation in the presence of a water soluble carbodiimide and its kinetic properties were studied. The optical spectra, oxidation-reduction potentials and isoelectric pH of plastocyanin and cytochrome f did not change upon the formation of the cross-linked complex. Studies on the ionic strength effect on the electron transfer rate from cross-linked plastocyanin to ferricyanide indicated that the negative charge on the reaction site of plastocyanin was masked upon the cross-linking. It was also suggested that the sign of the net charge near the cytochrome f heme edge changed from positive to negative upon the cross-linking. On the other hand, electrostatic interactions between cross-linked plastocyanin and P700 seemed to be essentially the same as those in the case of native plastocyanin, although the rate of electron transfer from cross-linked plastocyanin to P700 was severely reduced. We also measured the intra-complex electron transfer from cytochrome f to plastocyanin. This suggested that the covalently cross-linked complex is a valid model of the electron transfer encounter complex. Based on these results, the reaction sites of plastocyanin with P700 and cytochrome f were discussed.
Topics: Carbodiimides; Chromatography, High Pressure Liquid; Cytochromes; Cytochromes f; Electrophoresis, Polyacrylamide Gel; Isoelectric Focusing; Kinetics; Oxidation-Reduction; Plant Proteins; Plants; Plastocyanin
PubMed: 2738049
DOI: 10.1093/oxfordjournals.jbchem.a122627 -
Journal of Biochemistry Oct 1980The protein-protein electron transfer reactions between cytochrome f and plastocyanin, both purified from Brassica komatsuna (Brassica rapa L. var. pervirdis Bailey),...
The protein-protein electron transfer reactions between cytochrome f and plastocyanin, both purified from Brassica komatsuna (Brassica rapa L. var. pervirdis Bailey), have been studied as a function of pH, ionic strength, and temperature. The second-order rate constant for the oxidation of ferrocytochrome f by plastocyanin was found to be k = 4.5 X 10(7) M-1 x S-1 at pH 7.0 mu 0.2 M, and 20 degrees C, with activation parameters delta H not equal to = 8.4 kcal/mol and delta S not equal to = 4.9 cal/mol x deg. Respective rate constant and activation parameters obtained for the reduction of ferricytochrome f by plastocyanin were k = 1.9 X 10(7) M-1 x S-1, delta H not equal to = 8.6 kcal/mol, and delta S not equal to = 3.9 cal/mol x deg. The high rate constants for these reactions and delta S not equal to = 4.9 cal/mol x deg. Respective rate constant and activation parameters obtained for the reduction of ferricytochrome f by plastocyanin were k = 1.9 X 10(7) M-1 x S-1, delta H not equal to = 8.6 kcal/mol, and delta S not equal to = 3.9 cal/mol x deg. The high rate constants for these reactions are attributable not to a low activation enthalpy but to a positive activation entropy term. The rate constants both for the oxidation and the reduction of cytochrome f by plastocyanin drastically decreased with increasing ionic strength, indicating the importance of electrostatic interactions. Divalent cations are more effective than monovalent cations in reducing the rates of these reactions. The rate constants for the oxidation of cytochrome f by plastocyanin are constant between pH 6.0 and 9.0 but decrease markedly above pH 9.0 and below pH 6.0. In the case of the reduction of cytochrome f by plastocyanin, an optimum pH around 7.0 was obtained and a biphasic feature was observed at alkaline pH. The results are discussed in relation to photosynthetic electron transport systems.
Topics: Brassica; Calorimetry; Cytochromes; Cytochromes f; Electron Transport; Hydrogen-Ion Concentration; Kinetics; Osmolar Concentration; Plant Proteins; Plants; Plastocyanin
PubMed: 7451412
DOI: 10.1093/oxfordjournals.jbchem.a133072 -
Journal of Biochemistry Feb 1992The green alga Pediastrum boryanum synthesizes alternatively two photosynthetic electron carrier proteins, plastocyanin and cytochrome c-553, depending on the copper...
The green alga Pediastrum boryanum synthesizes alternatively two photosynthetic electron carrier proteins, plastocyanin and cytochrome c-553, depending on the copper concentration of the medium. We studied the levels at which the syntheses of the two proteins are regulated. Plastocyanin and cytochrome c-553 were purified from P. boryanum NIES-301 cells, having apparent molecular weights of 14,600 and about 12,000, respectively. Western blotting with antisera raised against these proteins showed accumulation of (apo)plastocyanin and (apo)cytochrome c-553 in the cells grown with (2 microM) and without added CuSO4, respectively, but no accumulation of the precursor proteins in both cultures. The translatable mRNAs for the two proteins were examined by in vitro translation with total RNA and wheat germ extract followed by immunoprecipitation and SDS-PAGE. The 21-kDa polypeptide (preapoplastocyanin) was detected with anti-plastocyanin serum in copper-sufficient cells; the 23-kDa polypeptide (preapocytochrome c-553) with anti-cytochrome c-553 serum in copper-deficient cells. The translatable mRNA for preapoplastocyanin appeared in 1 h and (apo)plastocyanin in 2-3 h after the addition of 2 microM CuSO4 to the copper-deficient culture. The translatable mRNA for preapocytochrome c-553 disappeared within 4-5 h, while (apo)cytochrome c-553 disappeared more slowly. It is concluded that the syntheses of plastocyanin and cytochrome c-553 are regulated by copper at the pre-translational (i.e., transcriptional or post-transcriptional) level in P. boryanum NIES-301.
Topics: Blotting, Western; Cells, Cultured; Chlorophyta; Copper; Cytochrome c Group; Electrophoresis; Plastocyanin; Protein Biosynthesis; RNA, Messenger; Transcription, Genetic
PubMed: 1314811
DOI: 10.1093/oxfordjournals.jbchem.a123740 -
Journal of Molecular Biology Sep 1983The structure of poplar plastocyanin in the oxidized (CuII) state at pH 6.0 has been refined, using 1.6 A resolution counter data. The starting co-ordinates were...
The structure of poplar plastocyanin in the oxidized (CuII) state at pH 6.0 has been refined, using 1.6 A resolution counter data. The starting co-ordinates were obtained from the 2.7 A electron density map computed with phases derived by the multiple isomorphous replacement method. The model was refined successively by constrained real space, unrestrained reciprocal space, and restrained reciprocal space least-squares methods. The final residual R value is 0.17 for 8285 reflections (I greater than 2 sigma (I)). It is estimated that the root-mean-square standard deviation of the atomic positions is 0.1 A when averaged over all atoms, and 0.05 A for the Cu ligand atoms alone. The refined structure retains all the essential features of the 2.7 A model. The co-ordination geometry of the copper atom is confirmed as being distorted tetrahedral. The two Cu-N(His) bonds, 2.10 and 2.04 A, are within the range normally found in low molecular weight CuII complexes with Cu-N(imidazole) bonds. The Cu-S(Cys) bond, 2.13 A, is also normal, but the Cu-S(Met) bond, 2.90 A, is sufficiently long to raise important questions about its significance. The hydrogen-bonding and secondary structure can now be assigned confidently. Forty-four water molecules are included in the final model. Repetition of the refinement, using new data to 1.9 A resolution recorded from crystals at pH 4.2, has led to a residual R value of 0.16 for 6060 reflections (I greater than sigma (I)). There are few significant changes in the structure of poplar CuII-plastocyanin between pH 6.0 and pH 4.2. In particular, the geometry of the copper site is not affected. The observed changes in redox behaviour of plastocyanin at low pH are therefore unlikely to be connected with structural changes in the oxidized form of the protein. A number of features of the molecular structure appear to be directly related to the function of plastocyanin as an electron carrier in photosynthesis. Comparison between the known amino acid sequences of 67 plant plastocyanins reveals 52 conserved and 11 conservatively substituted residues in a total of 99. If three algal plastocyanin sequences are included in the comparison, there are still 26 conserved and 12 conservatively substituted residues. In many cases, the importance of these residues in determining the tertiary structure can be rationalized.
Topics: Amino Acid Sequence; Copper; Crystallography; Hydrogen Bonding; Hydrogen-Ion Concentration; Ligands; Models, Molecular; Oxidation-Reduction; Plant Proteins; Plastocyanin; Protein Conformation; Water
PubMed: 6620385
DOI: 10.1016/s0022-2836(83)80064-3 -
Physical Biology Jun 2006Most biological functions, including photosynthetic activity, are mediated by protein interactions. The proteins plastocyanin and cytochrome f are reaction partners in a...
Most biological functions, including photosynthetic activity, are mediated by protein interactions. The proteins plastocyanin and cytochrome f are reaction partners in a photosynthetic electron transport chain. We designed a 3D computer simulation model of diffusion and interaction of spinach plastocyanin and turnip cytochrome f in solution. It is the first step in simulating the electron transfer from cytochrome f to photosystem 1 in the lumen of thylakoid. The model is multiparticle and it can describe the interaction of several hundreds of proteins. In our model the interacting proteins are represented as rigid bodies with spatial fixed charges. Translational and rotational motion of proteins is the result of the effect of stochastic Brownian force and electrostatic force. The Poisson-Boltzmann formalism is used to determine the electrostatic potential field generated around the proteins. Using this model we studied the kinetic characteristics of plastocyanin-cytochrome f complex formation for plastocyanin mutants at pH 7 and a variety of ionic strength values.
Topics: Computer Simulation; Cytochromes f; Kinetics; Models, Chemical; Models, Molecular; Photosynthesis; Plastocyanin; Protein Binding; Protein Conformation; Static Electricity
PubMed: 16829698
DOI: 10.1088/1478-3975/3/2/004 -
European Journal of Biochemistry Mar 1990Spinach plastocyanin and horse heart cytochrome c have been shown, by monitoring the behaviour of the hyperfine-shifted heme resonances of Fe(III) cytochrome c on...
Spinach plastocyanin and horse heart cytochrome c have been shown, by monitoring the behaviour of the hyperfine-shifted heme resonances of Fe(III) cytochrome c on titration with Cu(II) plastocyanin, to form a 1:1 complex with a dissociation constant of 67 mM (D2O, pH* 7.0, 300 K). The interaction sites on the plastocyanin surface have been investigated in one- and two-dimensional NMR experiments involving competition for plastocyanin between cytochrome c and the paramagnetic cation Cr(NH3)(3+)6. The plastocyanin resonances which are paramagnetically broadened in the spectrum of plastocyanin alone are also broadened in the spectrum of the mixture of the two proteins. This shows that, on the NMR time scale, no plastocyanin residues are hidden from Cr(NH3)(3+)6 by complexation with cytochrome c. [It has been shown that Cr(NH3)(3+)6 does not disrupt formation of the complex between the two proteins.] It appears that initial complexation of cytochrome c takes place at the acidic east site of plastocyanin, and that the extensive negative electrostatic surface of plastocyanin accommodates the paramagnetic probe and cytochrome c simultaneously in a dynamic ternary complex. The location of the electron transfer site on plastocyanin is discussed.
Topics: Animals; Binding Sites; Copper; Cytochrome c Group; Ferric Compounds; Ferrous Compounds; Heme; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Conformation; Oxidation-Reduction; Plant Proteins; Plastocyanin; Surface Properties
PubMed: 2156702
DOI: 10.1111/j.1432-1033.1990.tb15418.x -
FEBS Letters Mar 1993Native plastocyanin from Synechocystis 6803 has been isolated and purified to electrophoretic homogeneity. The corresponding gene (petE) has been cloned and expressed in...
Native plastocyanin from Synechocystis 6803 has been isolated and purified to electrophoretic homogeneity. The corresponding gene (petE) has been cloned and expressed in E. coli, thus leading to a protein completely identical to plastocyanin purified from the cyanobacterial cells. The petE gene product is correctly processed in E. coli as deduced from the N-terminal amino acid sequences. These results, along with the identical physicochemical and kinetic properties of the two protein preparations, confirm that expression of petE in E. coli is an adequate tool to address the study of Synechocystis plastocyanin by site-directed mutagenesis.
Topics: Amino Acid Sequence; Base Sequence; Cyanobacteria; Escherichia coli; Isoelectric Point; Molecular Sequence Data; Molecular Weight; Oligodeoxyribonucleotides; Plastocyanin; Recombinant Proteins; Spectrum Analysis
PubMed: 8458419
DOI: 10.1016/0014-5793(93)80558-c -
Biochemistry Dec 1997The blue copper protein plastocyanin and the heme protein cytochrome c6 differ in composition and in structure but perform the same function in the photosynthetic... (Comparative Study)
Comparative Study
Comparison of the physiologically equivalent proteins cytochrome c6 and plastocyanin on the basis of their electrostatic potentials. Tryptophan 63 in cytochrome c6 may be isofunctional with tyrosine 83 in plastocyanin.
The blue copper protein plastocyanin and the heme protein cytochrome c6 differ in composition and in structure but perform the same function in the photosynthetic electron-transport chain. We compare these two proteins on the basis of their electrostatic potentials in order to understand the structural basis of their functional equivalence. In the first approach, we use a monopole-dipole approximation of the electrostatic potentials to superimpose the proteins. The resulting alignment suggests that Tyr51 in cytochrome c6 corresponds to Tyr83 in plastocyanin. But since Tyr51 is not conserved in all known cytochrome c6 sequences, a physiological role of this residue is questionable. In a more sophisticated approach, we applied the recently-developed Fame (flexible alignment of molecule ensembles) algorithm, in which molecules are superimposed by optimizing the similarity of their electrostatic potentials with respect to the relative orientation of the molecules. On the basis of the Fame alignments of plastocyanin and cytochrome c6, we analyze the docking and the electron-transfer reactions of these two proteins with its physiological reaction partner cytochrome f. We derive functional analogies for individual amino acids in possible electron-transfer paths in the interprotein redox reactions. We identify two surface patches in cytochrome c6 that may be involved in electron-transfer paths. The hydrophobic patch with the exposed heme edge in cytochrome c6 may be equivalent to the hydrophobic patch with His87 in plastocyanin, whereas Trp63 in cytochrome c6 may be equivalent to Tyr83 in plastocyanin. An aromatic amino acid is present at the position of Trp63 in all known cytochrome c6 sequences. The electronic coupling between the heme and the copper site on the one side and several potentially important amino acid residues on the other is analyzed by the Pathways method. We have proposed recently that Lys65 of cytochrome f and Tyr83 of plastocyanin form a cation-pi system, which may be involved in a two-step mechanism of the electron-transfer reaction between these two proteins from higher plants. Now we corroborate this proposal by analyzing available amino acid sequences.
Topics: Algorithms; Animals; Azurin; Bacterial Proteins; Chlamydomonas reinhardtii; Copper; Cytochromes; Cytochromes f; Electron Transport; Models, Molecular; Photosynthesis; Plant Proteins; Plastocyanin; Protein Structure, Secondary; Protein Structure, Tertiary; Pseudomonas aeruginosa; Structure-Activity Relationship; Tryptophan; Tyrosine
PubMed: 9405052
DOI: 10.1021/bi971241v -
Preparative Biochemistry 1975Homogeneous preparations of ferredoxin, plastocyanin, and chloroplast coupling factor (CF1) have been isolated from spinach by a combined procedure in which supernatants...
Homogeneous preparations of ferredoxin, plastocyanin, and chloroplast coupling factor (CF1) have been isolated from spinach by a combined procedure in which supernatants from preparation of chloroplasts are used for isolation of ferredoxin and the chloroplasts serve as the source of plastocyanin. The proteins were purified by DEAE-cellulose chromatography and gel filtration, after precipitation with acetone in the case of ferredoxin or release from membranes in the case of plastocyanin. The proteins obtained by this procedure are pure, as evidenced by absorption ratios (ferredoxin, A420/A276 = 0.47-0.48; plastocyanin, A278/A597 = 1.2) and by the fact that both proteins migrate as single bands on polyacrylamide gels in the presence of sodium dodecyl sulfate.
Topics: Chloroplasts; Chromatography, DEAE-Cellulose; Electrophoresis, Polyacrylamide Gel; Ferredoxins; Plant Proteins; Plants; Plastocyanin
PubMed: 1187556
DOI: 10.1080/00327487508061579 -
Archives of Biochemistry and Biophysics May 1992The electron transfer reactions between a lipid bilayer-modified gold electrode and oxidized spinach plastocyanin have been studied by cyclic voltammetry, using either...
The electron transfer reactions between a lipid bilayer-modified gold electrode and oxidized spinach plastocyanin have been studied by cyclic voltammetry, using either an electrically neutral phosphatidylcholine (PC) bilayer or a positively charged PC bilayer containing 40 mol% dimethyldioctadecylammonium chloride, at two ionic strengths of electrolyte (0.02 and 0.2 M NaClO4). Plastocyanin was found to interact strongly enough with the lipid membrane to support an efficient electron transfer reaction with the electrode. The interaction forces, and therefore the mode of diffusion of plastocyanin molecules to the electrode, which limits the electron transfer rate, could be controlled by the PC concentration. At low lipid concentrations (0-5 mg/ml), electrostatically attractive interactions between specific microelectroactive sites on the surface of the lipid membrane and plastocyanin molecules predominate, producing a radial mode of diffusion of the protein molecules to the electrode surface. On the other hand, at high lipid concentrations (greater than 5 mg/ml), interaction between plastocyanin and the lipid membrane occurs via hydrophobic forces, and a linear diffusion of protein molecules limits the electron transfer process. These observations support and extend other experimental and theoretical results which indicate two possible sites on the surface of the plastocyanin molecule, one hydrophobic and one negatively charged, which are able to participate in electron transfer reactions. We conclude that electrochemical measurements with the present system provide a new approach to the study of redox protein-membrane interactions.
Topics: Electrochemistry; Electrodes; Electron Transport; Lipid Bilayers; Oxidation-Reduction; Phosphatidylcholines; Plants; Plastocyanin; Protein Binding
PubMed: 1567192
DOI: 10.1016/0003-9861(92)90699-w