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The Journal of Biological Chemistry Mar 2003Three surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in methionine 33, located...
Three surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in methionine 33, located in the hydrophobic patch of the copper protein, and in arginine 86 and proline 53, both located in the eastern hydrophilic area. The reactivity toward photosystem I of single mutants M33N, P53A, P53E, R86Q, R86E, and the double mutant M33N/P14L has been studied by laser flash absorption spectroscopy. All the mutations yield increased reactivity of plastocyanin toward photosystem I as compared with wild type plastocyanin, thus indicating that in Prochlorothrix electron donation to photosystem I is not optimized. The most drastic increases in the intracomplex electron transfer rate are obtained with mutants in methionine 33, whereas replacing arginine 86 only modestly affects the plastocyanin-photosystem I equilibrium constant for complex formation. Mutations at position 53 also promote major changes in the association of plastocyanin with photosystem I, yielding a change from a mechanism involving complex formation to a simpler collisional interaction. Molecular dynamics calculations indicate that mutations at position 33 promote changes in the H-bond network around the copper center. The comparative kinetic analysis of the reactivity of Prochlorothrix plastocyanin mutants toward photosystem I from other cyanobacteria reveals that mutations M33N, P53A, and P53E result in enhanced general reactivity.
Topics: Base Sequence; DNA Primers; Hydrogen Bonding; Models, Molecular; Mutagenesis, Site-Directed; Nuclear Magnetic Resonance, Biomolecular; Photosynthetic Reaction Center Complex Proteins; Plastocyanin; Prochlorothrix
PubMed: 12509429
DOI: 10.1074/jbc.M211913200 -
Acta Crystallographica. Section D,... Sep 2006Plastocyanin from the cyanobacterium Anabaena variabilis was heterologously produced in Escherichia coli and purified. Plate-like crystals were obtained by...
Plastocyanin from the cyanobacterium Anabaena variabilis was heterologously produced in Escherichia coli and purified. Plate-like crystals were obtained by crystallization in 1.15 M trisodium citrate and 7.67 mM sodium borate buffer pH 8.5. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.85, b = 45.81, c = 63.41 Angstrom. The structure of the oxidized protein was solved to a resolution of 1.6 Angstrom using plastocyanin from Phormidium laminosum as a search model. Two molecules were found in the asymmetric unit. The electrostatic surface of the basic protein showed a large population of positively charged residues in the northern site, whereas the eastern site lacked the two strongly negatively charged patches. The copper ion was found to be relatively mobile and there were two distinct conformations of His61.
Topics: Amino Acid Sequence; Anabaena; Borates; Citrates; Crystallization; Crystallography, X-Ray; Electrons; Escherichia coli; Hydrogen-Ion Concentration; Molecular Conformation; Molecular Sequence Data; Plastocyanin; Protein Conformation; Protein Structure, Secondary; Sequence Homology, Amino Acid; Static Electricity
PubMed: 16929103
DOI: 10.1107/S0907444906023638 -
Biochemical and Biophysical Research... Jan 1985A complex between chlorophyll a and plastocyanin has been prepared by dialysis of mixtures of chlorophyll in Triton X-100 micellar solution with the protein. The complex...
A complex between chlorophyll a and plastocyanin has been prepared by dialysis of mixtures of chlorophyll in Triton X-100 micellar solution with the protein. The complex appears to contain no more than one chlorophyll per plastocyanin molecule, and is non-fluorescent, whether the protein is in the oxidized or reduced state. The lack of fluorescence suggests that the chlorophyll is adsorbed very close to the Cu center.
Topics: Chlorophyll; Chlorophyll A; Octoxynol; Plant Proteins; Plastocyanin; Polyethylene Glycols; Spectrophotometry
PubMed: 3970712
DOI: 10.1016/0006-291x(85)90649-7 -
Physical Chemistry Chemical Physics :... Sep 2010The electronic absorption, electronic circular dichroism and X-ray absorption spectroscopy of the blue copper protein plastocyanin is studied with density functional...
The electronic absorption, electronic circular dichroism and X-ray absorption spectroscopy of the blue copper protein plastocyanin is studied with density functional theory, time-dependent density functional theory and multireference configuration interaction in conjunction with classical molecular dynamics simulations. A strong correlation is observed between the excitation energy of the intense ligand to metal charge transfer band and the copper-cysteine sulfur bond length. The results suggest that the copper-cysteine sulfur bond length in the crystal structure of plastocyanin is too short and should be closer to the corresponding bond lengths in related blue copper proteins. Averaging over many structural conformations is required to reproduce the major features of the experimental circular dichroism spectra. A correlation between the rotational strength of the ligand to metal charge transfer band and the distortion of the copper atom from the plane of the cysteine sulfur and histidine nitrogen atoms is found. X-ray absorption calculations show a smaller sulfur p orbital character in the singly occupied molecular orbital of cucumber basic protein compared to plastocyanin.
Topics: Catalytic Domain; Circular Dichroism; Copper; Crystallography, X-Ray; Molecular Dynamics Simulation; Plastocyanin; Sulfur; X-Ray Absorption Spectroscopy
PubMed: 20532328
DOI: 10.1039/c001805h -
Structure (London, England : 1993) Mar 1998The reduction of plastocyanin by cytochrome f is part of the chain of photosynthetic electron transfer reactions that links photosystems II and I. The reaction is rapid...
BACKGROUND
The reduction of plastocyanin by cytochrome f is part of the chain of photosynthetic electron transfer reactions that links photosystems II and I. The reaction is rapid and is influenced by charged residues on both proteins. Previously determined structures show that the plastocyanin copper and cytochrome f haem redox centres are some distance apart from the relevant charged sidechains, and until now it was unclear how a transient electrostatic complex can be formed that brings the redox centres sufficiently close for a rapid reaction.
RESULTS
A new approach was used to determine the structure of the transient complex between cytochrome f and plastocyanin. Diamagnetic chemical shift changes and intermolecular pseudocontact shifts in the NMR spectrum of plastocyanin were used as input in restrained rigid-body molecular dynamics calculations. An ensemble of ten structures was obtained, in which the root mean square deviation of the plastocyanin position relative to cytochrome f is 1.0 A. Electrostatic interaction is maintained at the same time as the hydrophobic side of plastocyanin makes close contact with the haem area, thus providing a short electron transfer pathway (Fe-Cu distance 10.9 A) via residues Tyr1 or Phe4 (cytochrome f) and the copper ligand His87 (plastocyanin).
CONCLUSIONS
The combined use of diamagnetic and paramagnetic chemical shift changes makes it possible to obtain detailed information about the structure of a transient complex of redox proteins. The structure suggests that the electrostatic interactions 'guide' the partners into a position that is optimal for electron transfer, and which may be stabilised by short-range interactions.
Topics: Binding Sites; Cytochromes; Cytochromes f; Electron Transport; Magnetic Resonance Spectroscopy; Models, Molecular; Plastocyanin; Protein Binding; Protein Conformation
PubMed: 9551554
DOI: 10.1016/s0969-2126(98)00035-5 -
Biochemistry Dec 2002Transient complex formation between plastocyanin from Prochlorothrix hollandica and cytochrome f from Phormidium laminosum was investigated using nuclear magnetic...
Transient complex formation between plastocyanin from Prochlorothrix hollandica and cytochrome f from Phormidium laminosum was investigated using nuclear magnetic resonance (NMR) spectroscopy. Binding curves derived from NMR titrations at 10 mM ionic strength reveal a 1:1 stoichiometry and a binding constant of 6 (+/-2) x 10(3) M(-1) for complex formation, 1 order of magnitude larger than that for the physiological plastocyanin-cytochrome f complex from Ph. laminosum. Chemical-shift perturbation mapping indicates that the hydrophobic patch of plastocyanin is involved in the complex interface. When the unusual hydrophobic patch residues of P. hollandica plastocyanin were reverted to the conserved residues found in most other plastocyanins (Y12G/P14L), the binding constant for the interaction with cytochrome f was unaffected. However, the chemical shift perturbation map was considerably different, and the size of the average perturbation decreased by 40%. The complexes of both the wild-type and double mutant plastocyanin with cytochrome f were sensitive to ionic strength, contrary to the physiological complex. The possible implications of these findings for the mechanism of transient complex formation are discussed.
Topics: Binding Sites; Conserved Sequence; Cyanobacteria; Cytochromes; Cytochromes f; Glycine; Histidine; Hydrophobic and Hydrophilic Interactions; Leucine; Macromolecular Substances; Mutagenesis, Site-Directed; Nuclear Magnetic Resonance, Biomolecular; Plastocyanin; Prochlorothrix; Proline; Protons; Tyrosine
PubMed: 12501198
DOI: 10.1021/bi026349b -
Archives of Biochemistry and Biophysics Nov 1992The thermal stability of plastocyanin (PC) was determined as a function of oxidation state of the copper center and the presence of oxidants, reductants, oxygen, and... (Comparative Study)
Comparative Study
The thermal stability of plastocyanin (PC) was determined as a function of oxidation state of the copper center and the presence of oxidants, reductants, oxygen, and EDTA. It was found that the copper center and its ligands play a crucial role in maintaining the stability of PC. Thermal denaturation was monitored by using far-uv circular dichroism (CD) spectra to monitor changes in secondary structure, the near-uv CD ellipticity at 280 nm to monitor changes in tertiary structure, and the absorbance at 597 nm and the 255-nm CD transition to monitor changes in the copper center. Reduced PC (Tm = 71 degrees C) was found to be more stable than the oxidized form (Tm = 61 degrees C). The Tm was increased by addition of reductants, removal of oxygen, or addition of EDTA. Two distinct denatured forms (designated D1 and D2) were separated by anion exchange fast protein liquid chromatography. Neither form contained a native copper center. Form D2 retained the characteristic 280-nm CD band but showed an altered far-uv CD spectrum. Its formation was inhibited by the addition of reductants or the removal of oxygen. It could be refolded to form native, Cu-PC upon incubation with copper plus a reductant such as dithionite. These results suggest that its formation involves the reversible oxidation of a group on the PC molecule, possibly a ligand to the copper such as Cys 84 or Met 92. Form D1 occurred in the presence of ferricyanide or at high temperatures in the presence of oxygen. EDTA inhibited its formation. Form D1 lost the 280-nm CD transition and its far-uv CD spectrum was altered. No renaturation was observed suggesting that Form D1 is the product of an irreversible oxidation step possibly involving a histidine ligand to the copper. Forms D1 and D2 are not interconvertible and represent the endpoints of two different denaturation pathways.
Topics: Anaerobiosis; Circular Dichroism; Drug Stability; Edetic Acid; Hot Temperature; Oxidants; Oxidation-Reduction; Plants; Plastocyanin; Protein Conformation; Protein Denaturation; Spectrophotometry; Thermodynamics
PubMed: 1416972
DOI: 10.1016/0003-9861(92)90429-z -
Indian Journal of Biochemistry &... Oct 2008Two total plastocyanin (PC) fractions - loosely bound (lPC) and strongly bound (sPC) were extracted (84% and 16%, respectively) from the homogenate of Scenedesmus acutus...
Two total plastocyanin (PC) fractions - loosely bound (lPC) and strongly bound (sPC) were extracted (84% and 16%, respectively) from the homogenate of Scenedesmus acutus MT8. Two-fold isolation-purification procedure including DE-52 chromatography separated IPC into a smaller oxidized [IPC (II)] and a larger reduced [IPC(I)] fractions, in contrast to sPC, where sPC(ll) greatly dominated over sPC(I). Analytical isoelectric focusing (IEF) separated IPC(II) into two main fractions only in the presence of 8 M urea, implying microheterogeneity. Preparative IEF in immobiline pH-gradient of 3.2-4.1 separated IPC(II) into two blue fractions - a more alkaline IPC(II) and a more acidic IPC"(II), which were probably stereoisomers. Their UV-Vis spectra exhibited rarely observed tryptophane (291.5 nm) and some differences at 270 and 287 nm. The exact molecular masses of apo-/holo-lPC (10131 Da/10194 Da) were determined by mass spectrometry. The number of -SH groups was determined from the mass difference between alkylated with 4-vinylpyridine (4-VP) and non-alkylated protein. Additionally, a simple procedure for simultaneous separation of both primary structure and stereoisomers of PC was developed.
Topics: Chromatography, Ion Exchange; Isoelectric Focusing; Plastocyanin; Scenedesmus; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Ultraviolet
PubMed: 19069842
DOI: No ID Found -
Molecular Plant Mar 2009Two homologous plastocyanin isoforms are encoded by the genes PETE1 and PETE2 in the nuclear genome of Arabidopsis thaliana. The PETE2 transcript is expressed at...
Two homologous plastocyanin isoforms are encoded by the genes PETE1 and PETE2 in the nuclear genome of Arabidopsis thaliana. The PETE2 transcript is expressed at considerably higher levels and the PETE2 protein is the more abundant isoform. Null mutations in the PETE genes resulted in plants, designated pete1 and pete2, with decreased plastocyanin contents. However, despite reducing plastocyanin levels by over approximately 90%, a pete2 null mutation on its own affects rates of photosynthesis and growth only slightly, whereas pete1 knockout plants, with about 60-80% of the wild-type plastocyanin level, did not show any alteration. Hence, plastocyanin concentration is not limiting for photosynthetic electron flow under optimal growth conditions, perhaps implying other possible physiological roles for the protein. Indeed, plastocyanin has been proposed previously to cooperate with cytochrome c(6A) (Cyt c(6A)) in thylakoid redox reactions, but we find no evidence for a physical interaction between the two proteins, using interaction assays in yeast. We observed homodimerization of Cyt c(6A) in yeast interaction assays, but also Cyt c(6A) homodimers failed to interact with plastocyanin. Moreover, phenotypic analysis of atc6-1 pete1 and atc6-1 pete2 double mutants, each lacking Cyt c(6A) and one of the two plastocyanin-encoding genes, failed to reveal any genetic interaction. Overexpression of either PETE1 or PETE2 in the pete1 pete2 double knockout mutant background results in essentially wild-type photosynthetic performance, excluding the possibility that the two plastocyanin isoforms could have distinct functions in thylakoid electron flow.
Topics: Amino Acid Sequence; Arabidopsis; Electron Transport; Molecular Sequence Data; Mutation; Oxidation-Reduction; Photosynthesis; Plastocyanin; Protein Isoforms; Sequence Homology, Amino Acid; Thylakoids
PubMed: 19825610
DOI: 10.1093/mp/ssn041 -
Physical Review. E, Statistical... Sep 2000The dynamical behavior of water around plastocyanin has been investigated in a wide temperature range by molecular dynamics simulation. The mean square displacements of...
The dynamical behavior of water around plastocyanin has been investigated in a wide temperature range by molecular dynamics simulation. The mean square displacements of water oxygen atoms show, at long times, a t(alpha) trend for all temperatures. Below 150 K, alpha is constant and equal to 1; at higher temperatures it drops to a value significantly smaller than 1, and thereafter decreases with increasing temperature. The occurrence of such an anomalous diffusion matches the onset of the dynamical transition observed in the protein. The intermediate scattering function of water is characterized, at high temperature, by a stretched exponential decay evolving, at low temperature, toward a two step relaxation behavior, which becomes more evident on increasing the exchanged wave vector q. Both the mean square displacements and the intermediate scattering functions show, beyond the ballistic regime, a plateau, which progressively extends for longer times as long as the temperature is lowered, such behavior reflecting trapping of water molecules within a cage formed by the nearest neighbors. At low temperature, a low frequency broad inelastic peak is observed in the dynamical structure factor of hydration water; such an excess of vibrational modes being reminiscent of the boson peak, characteristic of disordered, amorphous systems. All these features, which are typical of complex systems, can be traced back to the glassy character of the hydration water and suggest a dynamical coupling occurring at the macromolecule-solvent interface.
Topics: Computer Simulation; Glass; Kinetics; Macromolecular Substances; Motion; Plastocyanin; Temperature; Vibration; Water
PubMed: 11088920
DOI: 10.1103/physreve.62.3991