| 1. |
- Sigfridsson, Kalle, et al.
(författare)
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Electron transfer in ruthenium-modified spinach plastocyanin mutants
- 1998
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Ingår i: Archives of Biochemistry and Biophysics. - : Academic Press. - 0003-9861 .- 1096-0384. ; 351:2, s. 197-206
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Tidskriftsartikel (refereegranskat)abstract
- Four site-directed mutants of spinach plastocyanin, Pc(Leu12His), Pc(Leu15His), Pc(Thr79His), and Pc(Lys81His), have been modified by covalent attachment of a photoactive [Ru(bpy)2(im)]2+ complex at the surface-exposed histidine residues. The Pc-Ru complexes were characterized with optical absorption, CD, and EPR spectroscopy and their spectra were found to be similar to the unmodified proteins except in the case of the Pc(Leu12His) mutant which lost the Cu ion irreversibly during the Ru modification. Electron transfer (ET) within the other Pc-Ru complexes was studied with time-resolved optical spectroscopy, using an external-quencher approach. The fully reduced [Cu(I), Ru(II)] proteins were photoexcited and subsequently oxidized by an external quencher, [Ru(NH3)6]Cl3, forming the [Cu(I), Ru(III)] proteins. This was followed by an internal ET from Cu(I) to Ru(III). The rates of the internal ET reactions exhibit an exponential dependence on metal-to-metal separation, with a decay factor of 1.1 A-1. From a temperature-dependence study of the Ru-modified Pc(Lys81His) protein, a reorganization energy for the Cu-to-Ru ET reaction of 1.2 eV was determined. In this analysis it was found necessary to include an appreciable temperature dependence in the driving force of the ET reaction.
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| 2. |
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| 3. |
- Ejdebäck, Mikael, 1969-, et al.
(författare)
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Effects of codon usage and vector-host combinations on the expression of spinach plastocyanin in Escherichia coli
- 1997
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Ingår i: Protein Expression and Purification. - : Elsevier. - 1046-5928 .- 1096-0279. ; 11:1, s. 17-25
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Tidskriftsartikel (refereegranskat)abstract
- Spinach plastocyanin has been expressed in Escherichia coli and exported to the periplasmic space. The effects of codon usage, expression system, growth length, and temperature on expression levels in LB medium were investigated. A stretch of codons, rare in E. coli, was identified and replaced with highly expressed codons, increasing the yield by at least 20%. Plastocyanin was more efficiently expressed under the T7 promoter than under the lac promoter. Maximum yields were obtained at 37 degrees C when growing the cells for 16 h after induction. The optimized expression system produced 38 mg holoprotein per liter culture. In this system it was also possible to express plastocyanin in minimal medium, at a yield of 10 mg per liter. N-terminal sequencing and mass spectrometry showed that plastocyanin was correctly processed. The expressed plastocyanin was purified to homogeneity, as shown by an A278/A597 ratio of 1.0, and together with amino acid analysis and the determination of oxidized and total copper contents, both the absorption coefficients for epsilon 278 and for epsilon 597 were determined to be 4700 M-1 cm-1.
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| 4. |
- Ejdebäck, Mikael, 1969-
(författare)
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Studies on spinach plastocyanin and mutants : Expression in Escherichia coli, folding and function
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Photosynthesis is a process in which photons from sunlight excite chlorophylls in the thylakoid membranes of plants, algae and cyanobacteria. The photo-oxidised reaction centre chlorophyll P700 is re-reduced by an electron transferred from the soluble, blue copper protein plastocyanin. The oxidised plastocyanin dissociates and binds to the cytochrome b6f complex, where it accepts an electron and a new redox cycle can begin. Plastocyanin has three regions of importance for the interaction with its redox partners, a hydrophobic patch and two acidic (negatively charged) patches. Electrostatic interactions between opposite charges are important for the association and the specificity and stability of the formed complexes.In this work the interactions with photosystem 1 and cytochrome c have been studied using mutants of plastocyanin. The mutations introduced in the small acidic patch and position 88 of plastocyanin had small effects on the binding to photosystem 1 as compared to the weak binding reported for mutants in the large acidic patch. The affinity was increased by the Glu60Gln, Glu60Lys and Asp61Lys mutations and a more efficient electron transfer was observed for the Gln88Lys mutation. The association between Pc mutated in the small acidic patch and cytochrome c was weakened and the rearrangement hindered by lysines in positions 59 and 60.The development of an efficient expression system for spinach plastocyanin in the bacterium Escherichia coli made it possible to produce sufficient amounts of isotopically labelled plastocyanin for NMR experiments. This technique was used for solving the structure of the complex between plastocyanin and cytochrome f. The hydrophobic patch on plastocyanin binds to an area close to the heme on cytochrome f. Electrostatic interactions between opposite charges on the two proteins are also important. The short distance from the heme to the copper ligand His87 suggests an electron transfer from the heme via Tyr1 or Phe4 on cytochrome f.The involvement of specific amino-acid residues in copper binding or folding of plastocyanin has also been examined by site-directed mutagenesis. The copper-binding histidines have been replaced by other amino acids, but no blue protein could be produced. The stability of the different redox forms of copper plastocyanin as well as the zinc protein has also been determined by guanidinium-induced unfolding.
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| 5. |
- Ivković-Jensen, Maja M., et al.
(författare)
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Comparing the rates and the activation parameters for the forward reaction between the triplet state of zinc cytochrome c and cupriplastocyanin and the back reaction between the zinc cytochrome c cation radical and cuproplastocyanin
- 1999
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 38:5, s. 1589-1597
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Tidskriftsartikel (refereegranskat)abstract
- This is a comparative study of the photoinduced (so-called forward) electron-transfer reaction 3Zncyt/pc(II) --> Zncyt+/pc(I), between the triplet state of zinc cytochrome c (3Zncyt) and cupriplastocyanin [pc(II)], and the thermal (so-called back) electron-transfer reaction Zncyt+/pc(I) --> Zncyt/pc(II), between the cation (radical) of zinc cytochrome c (Zncyt+) and cuproplastocyanin [pc(I)], which follows it. Both reactions occur between associated (docked) reactants, and the respective unimolecular rate constants are kF and kB. Our previous studies showed that the forward reaction is gated by a rearrangement of the diprotein complex. Now we examine the back reaction and complare the two. We study the effects of temperature (in the range 273.3-302.9 K) and viscosity (in the range 1.00-17.4 cP) on the rate constants and determine enthalpies (DeltaH), entropies (DeltaS), and free energies (DeltaG) of activation. We compare wild-type spinach plastocyanin, the single mutants Tyr83Leu and Glu59Lys, and the double mutant Glu59Lys/Glu60Gln. The rate constant kB for wild-type spinach plastocyanin and its mutants markedly depends on viscosity, an indication that the back reaction is also gated. The activation parameters DeltaH and DeltaS show that the forward and back reactions have similar mechanisms, involving a rearrangement of the diprotein complex from the initial binding configuration to the reactive configuration. The rearrangements of the complexes 3Zncyt/pc(II) and Zncyt+/pc(I) that gate their respective reactions are similar but not identical. Since the back reaction of all plastocyanin variants is faster than the forward reaction, the difference in free energy between the docking and the reactive configuration is smaller for the back reaction than for the forward reaction. This difference is explained by the change in the electrostatic potential on the plastocyanin surface as Cu(II) is reduced to Cu(I). It is the smaller DeltaH that makes DeltaG smaller for the back reaction than for the forward reaction.
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| 6. |
- Ivković-Jensen, Maja M., et al.
(författare)
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Effects of single and double mutations in plastocyanin on the rate constant and activation parameters for the rearrangement gating the electron-transfer reaction between the triplet state of zinc cytochrome c and cupriplastocyanin
- 1998
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 37:26, s. 9557-9569
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Tidskriftsartikel (refereegranskat)abstract
- The unimolecular rate constant for the photoinduced electron-transfer reaction 3Zncyt/pc(II) --> Zncyt+/pc(I) within the electrostatic complex of zinc cytochrome c and spinach cupriplastocyanin is kF. We report the effects on kF of the following factors, all at pH 7.0: 12 single mutations on the plastocyanin surface (Leu12Asn, Leu12Glu, Leu12Lys, Asp42Asn, Asp42Lys, Glu43Asn, Glu59Gln, Glu59Lys, Glu60Gln, Glu60Lys, Gln88Glu, and Gln88Lys), the double mutation Glu59Lys/Glu60Gln, temperature (in the range 273.3-302.9 K), and solution viscosity (in the range 1. 00-116.0 cP) at 283.2 and 293.2 K. We also report the effects of the plastocyanin mutations on the association constant (Ka) and the corresponding free energy of association (DeltaGa) with zinc cytochrome c at 298.2 K. Dependence of kF on temperature yielded the activation parameters DeltaH, DeltaS, and DeltaG. Dependence of kF on solution viscosity yielded the protein friction and confirmed the DeltaG values determined from the temperature dependence. The aforementioned intracomplex reaction is not a simple electron-transfer reaction because donor-acceptor electronic coupling (HAB) and reorganizational energy (lambda), obtained by fitting of the temperature dependence of kF to the Marcus equation, deviate from the expectations based on precedents and because kF greatly depends on viscosity. This last dependence and the fact that certain mutations affect Ka but not kF are two lines of evidence against the mechanism in which the electron-transfer step is coupled with the faster, but thermodynamically unfavorable, rearrangement step. The electron-transfer reaction is gated by the slower, and thus rate determining, structural rearrangement of the diprotein complex; the rate constant kF corresponds to this rearrangement. Isokinetic correlation of DeltaH and DeltaS parameters and Coulombic energies of the various configurations of the Zncyt/pc(II) complex consistently show that the rearrangement is a facile configurational fluctuation of the associated proteins, qualitatively the same process regardless of the mutations in plastocyanin. Correlation of kF with the orientation of the cupriplastocyanin dipole moment indicates that the reactive configuration of the diprotein complex involves the area near the residue 59, between the upper acidic cluster and the hydrophobic patch. Kinetic effects and noneffects of plastocyanin mutations show that the rearrangement from the initial (docking) configuration, which involves both acidic clusters, to the reactive configuration does not involve the lower acidic cluster and the hydrophobic patch but involves the upper acidic cluster and the area near the residue 88.
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| 7. |
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| 8. |
- Olesen, Kenneth, et al.
(författare)
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Electron transfer to photosystem 1 from spinach plastocyanin mutated in the small acidic patch : ionic strength dependence of kinetics and comparison of mechanistic models
- 1999
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 38:50, s. 16695-16705
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Tidskriftsartikel (refereegranskat)abstract
- A set of plastocyanin (Pc) mutants, probing the small acidic patch (Glu59, Glu60, and Asp61) and a nearby residue, Gln88, has been constructed to provide further insight into the electron transfer process between Pc and photosystem 1. The negatively charged residues were changed into their neutral counterparts or to a positive lysine. All mutant proteins exhibited electron transfer kinetics qualitatively similar to those of the wild type protein over a wide range of Pc concentrations. The kinetics were slightly faster for the Gln88Lys mutant, while they were significantly slower for the Glu59Lys mutant. The data were analyzed with two different models: one involving a conformational change of the Pc-photosystem 1 complex that precedes the electron transfer step (assumed to be irreversible) [Bottin, H., and Mathis, P. (1985) Biochemistry 24, 6453-6460] and another where no conformational change occurs, the electron transfer step is reversible, and dissociation of products is explicitly taken into account [Drepper, F., Hippler, M., Nitschke, W., and Haehnel, W. (1996) Biochemistry 35, 1282-1295]. Both models can account for the observed kinetics in the limits of low and high Pc concentrations. To discriminate between the models, the effects of added magnesium ions on the kinetics were investigated. At a high Pc concentration (0.7 mM), the ionic strength dependence was found to be consistent with the model involving a conformational change but not with the model where the electron transfer is reversible. One residue in the small acidic patch, Glu60, seems to be responsible for the major part of the ionic strength dependence of the kinetics.
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| 9. |
- Ubbink, Marcellus, et al.
(författare)
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The structure of the complex of plastocyanin and cytochrome f, determined by paramagnetic NMR and restrained rigid-body molecular dynamics
- 1998
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Ingår i: Structure. - : Elsevier. - 0969-2126 .- 1878-4186. ; 6:3, s. 323-335
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
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