Purification of plastocyanin and cytochrome c6 from plants, green algae, and cyanobacteria.

Plastocyanin and cytochrome c6 are widely distributed over the oxygen-evolving photosynthetic organisms. The two proteins are functionally equivalent, but strongly differ in their global electrostatic charge. In fact, they are acidic in eukaryotes, but either neutral or basic in cyanobacteria. Such a difference in their electrostatic features is a critical factor in designing the purification procedure, which must thus be modified and adapted accordingly. This chapter reports the methods for producing (including cell cultures), isolating, and purifying plastocyanin and cytochrome c6--which greatly differ in their isoelectric point--from a number of eukaryotic and prokaryotic organisms.

Isolation and identification of poplar isoplastocyanins.

An improved four-stage isolation and purification procedure for preparing poplar isoplastocyanins is described in detail. Absorbance (UV-VIS) spectroscopy and isoelectric focusing (IEF) are used to determine the protein purity and identity. The present procedure increases twice the total plastocyanin (PC) yield. Four PC isoform fractions are consecutively isolated at the third chromatographic step: oxidized PCa(II) and PCb(II) and reduced PCb(I) and PCa(I). PCa(II) and PCb(II) obtained at the fourth chromatographic step are highly purified PC isoforms which show the purity index (p.i.) A278/A597 < or = 0.85. Isoelectric points (pl values) of the PC isoforms are found to be at pH 3.92 +/- 0.04 for PCa and at pH 3.85 +/- 0.02 for PCb. The results of appropriate biological experiments that include the highly purified poplar PC isoforms could give answers to the questions about the physiological significance of PC dimorphism for photosynthesis.

Some physicochemical peculiarities of poplar plastocyanins a and b.

The redox potentials of poplar plastocyanins a and b (PCa, PCb) were determined by spectrophotometric titrations of their reduced forms with [Fe(CN)6]3-. It was found that the two isoforms have the following millimolar extinction coefficients epsilon597 equilibrium constants Keq of one-electron exchange with [Fe(CN)6]4-/[Fe(CN)6]3-, and standard electron potentials E0: PCa: epsilon597 = (4.72 +/- 0.08) mM(-1) cm(-1), Keq = 0.133 +/- 0.009, E0' = (354 +/- 11) mV; PCb: epsilon597 = (5.23 +/- 0.16) mM(-1) cm(-1), Keq = 0.175 +/- 0.010, E0' = (363 +/- 12) mV. The pH dependence of the redox potential of PCb was studied too. It was found, that the value of E0' for PCb is constant in the pH range 6.5-9.5, but decreases in the range 4.8-6.5. On the whole, the dependence resembles that of PC from some well-known plant species, including poplar PCa. The changes of E0' in the pH-dependent region for poplar PCb, however, are smaller and are 13 mV per pH unit, whereas in the other well-known plant species the changes are about 50-60 mV per pH unit. It has been assumed that the weaker pH dependence of EO' of PCb accounts for some structural differences between PCa and PCb.

Competitive inhibition of electron donation to photosystem 1 by metal-substituted plastocyanin.

The electron transfer from wild-type spinach plastocyanin (Pc) to photosystem 1 has been studied by flash-induced absorption changes at 830 nm. The decay kinetics of photo-oxidized P700 are drastically slower in the presence of Ag(I)-substituted Pc, while addition of Zn(II)-substituted Pc has a weaker effect. The metal-substituted forms of Pc act as competitive inhibitors of the reaction between normal, Cu-containing, Pc and P700. The inhibition constants obtained from an analysis of the kinetic data were 30 and 410 microM for Ag(I)- and Zn(II)-substituted Pc, respectively. When the Gly8Asp mutant form of Pc was used instead of the wild-type form, the corresponding values were found to be 77 and 442 microM. If the Ag- and Zn-derivatives can be considered as structural mimics of reduced and oxidized CuPc, respectively, our results imply that there is a redox-induced decrease in the affinity between Pc and photosystem 1 that follows the electron donation to P700. Our data also imply that the Gly8Asp mutation can diminish the magnitude of this change. The findings reported here are consistent with a reaction mechanism where the electron transfer in the complex between Pc and photosystem 1 is assumed to be reversible.

Dynamics in the transient complex of plastocyanin-cytochrome f from Prochlorothrix hollandica.

The nature of transient protein complexes can range from a highly dynamic ensemble of orientations to a single well-defined state. This represents variation in the equilibrium between the encounter and final, functional state. The transient complex between plastocyanin (Pc) and cytochrome f (cytf) of the cyanobacterium Prochlorothrix hollandica was characterized by NMR spectroscopy. Intermolecular pseudocontact shifts and chemical shift perturbations were used as restraints in docking calculations to determine the structure of the wild-type Pc-cytf complex. The orientation of Pc is similar to orientations found in Pc-cytf complexes from other sources. Electrostatics seems to play a modest role in complex formation. A large variability in the ensemble of lowest energy structures indicates a dynamic nature of the complex. Two unusual hydrophobic patch residues in Pc have been mutated to the residues found in other plastocyanins (Y12G/P14L). The binding constants are similar for the complexes of cytf with wild-type Pc and mutant Pc, but the chemical shift perturbations are smaller for the complex with mutant Pc. Docking calculations for the Y12G/P14L Pc-cytf complex did not produce a converged ensemble of structures. Simulations of the dynamics were performed using the observed averaged NMR parameters as input. The results indicate a surprisingly large amplitude of mobility of Y12G/P14L Pc within the complex. It is concluded that the double mutation shifts the complex further from the well-defined toward the encounter state.

[Purification of plastocynin from Ulva pertusa by column chromatography and analysis of its N-terminal amino acid sequence].

Protein plastocyanin from a green alga, Ulva pertusa, has been purified. Samples were homogenized in 0.02 mol/L phosphate buffer (pH 7.2) and then centrifuged to remove debris and subjected to ammonium sulfate fractionation (40%-80% saturation). Ion exchange column chromatography with DEAE-Sepharose Fast Flow and gel filtration column chromatography with Sephadex G-75 were then employed for further purification of plastocyanin. Three peaks, A, B and C, were eluted with 0.01 mol/L phosphate buffer, containing a NaCl linear gradient from 0 to 1.0 mol/L at the flow rate of 32 mL/h through DEAE-Sepharose chromatography. The protein fractions containing the plastocyanin were then purified further with Sephardex G-75 column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophores (SDS-PAGE) is analysis indicates that the protein was purified to homogeneity and its relative molecular mass is 10,000. N-terminal amino acid sequence was used to identify the protein. The protein was transblotted to PVDF membrane and N-terminal amino acid sequence was performed via Edman degradation with an automated amino acid sequencer. The 20 N-terminal amino acid residues are AAIVKLGPDDGSLAFVPSKI, which share 85% homology with the 20 N-terminal amino acid sequence of U. prolifera and U. arasakii, and share 90% homology with the ones of U. pertusa formerly reported.

Novel plastocyanin containing phenylalanine-83 from the gymnosperm Ginkgo biloba.

Plastocyanin was purified from the gymnosperm Ginkgo biloba L., and its complete amino acid sequence was determined. The protein was shown to contain Phe-83 instead of Tyr-83 conserved in other land plant plastocyanins. This is the first report of the characterization and complete amino acid sequence of a gymnosperm plastocyanin.

Purification of plastocyanin and cytochrome c6 from plants, green algae, and cyanobacteria.

Plastocyanin and cytochrome c6 are widely distributed over the oxygen-evolving photosynthetic organisms. The two proteins are functionally equivalent, but strongly differ in their global electrostatic charge. In fact, they are acidic in eukaryotes, but either neutral or basic in cyanobacteria. The difference in their electrostatic features is a critical factor in designing the purification procedure, which must be modified and adapted accordingly. This chapter reports the methods for producing (including cell cultures), isolating and purifying plastocyanin and cytochrome c6-which greatly differ in their isoelectric point-from a number of eukaryotic and prokaryotic organisms.

PAA1, a P-type ATPase of Arabidopsis, functions in copper transport in chloroplasts.

Copper (Cu) is an essential trace element with important roles as a cofactor in many plant functions, including photosynthesis. However, free Cu ions can cause toxicity, necessitating precise Cu delivery systems. Relatively little is known about Cu transport in plant cells, and no components of the Cu transport machinery in chloroplasts have been identified previously. Cu transport into chloroplasts provides the cofactor for the stromal enzyme copper/zinc superoxide dismutase (Cu/ZnSOD) and for the thylakoid lumen protein plastocyanin, which functions in photosynthetic electron transport from the cytochrome b(6)f complex to photosystem I. Here, we characterized six Arabidopsis mutants that are defective in the PAA1 gene, which encodes a member of the metal-transporting P-type ATPase family with a functional N-terminal chloroplast transit peptide. paa1 mutants exhibited a high-chlorophyll-fluorescence phenotype as a result of an impairment of photosynthetic electron transport that could be ascribed to decreased levels of holoplastocyanin. The paa1-1 mutant had a lower chloroplast Cu content, despite having wild-type levels in leaves. The electron transport defect of paa1 mutants was evident on medium containing <1 micro M Cu, but it was suppressed by the addition of 10 micro M Cu. Chloroplastic Cu/ZnSOD activity also was reduced in paa1 mutants, suggesting that PAA1 mediates Cu transfer across the plastid envelope. Thus, PAA1 is a critical component of a Cu transport system in chloroplasts responsible for cofactor delivery to plastocyanin and Cu/ZnSOD.

Kinetic and cross-linking studies on the interactions of negative patch mutant plastocyanin from Silene pratensis with photosystem I complexes from cyanobacteria, green algae, and plants.

The site-directed mutants of negative patches on silene plastocyanin (PC) were used to investigate the change of interactions between photosystem I (PSI) and PC during the course of evolution from cyanobacteria to plants. The net charges of two highly conserved negative patches (#42-45 and #59-61) on silene PC were systematically modified from -4 to +1. PSI complexes from cucumber and Chlamydomonas reinhardtii were efficient electron acceptors for silene PC. The increase of net charge on the negative patch (#42-45) of silene PC decreased the reduction rates of PSI from cucumber and Chlamydomonas, while the modification of the other negative patch (#59-61) had no effect. Though the addition of MgCl2 decreased the reduction rate of cucumber PSI, the decrease was severely diminished in the case of Chlamydomonas PSI, and the reduction rate increased with increasing concentration of MgCl2 when the net charge of the negative patch (#42-45) was modified to +1. The PSI complexes from Anabaena variabilis and Synechosystis sp. PCC 6803 were inefficient electron acceptors for silene PC and their rates were almost independent of the net charge of the negative patches, as well as the ionic strength of the reaction mixtures. Silene PC specifically cross-linked to the PsaF subunit of PSI complexes from cucumber, Chlamydomonas, Anabaena, and Synechosystis sp. PCC 6803. Modification of the negative patch (#42-45) inhibited the formation of cross-linked adducts in all the cases examined, whereas modification of the other negative patch (#59-61) had essentially no effect. Based on these results, the changes of electrostatic interactions between PC and PSI during the course of evolution from cyanobacteria to plants are discussed.

Expression of Anabaena PCC 7937 plastocyanin in Synechococcus PCC 7942 enhances photosynthetic electron transfer and alters the electron distribution between photosystem I and cytochrome-c oxidase.

The petE gene encoding plastocyanin precursor protein from the cyanobacterium Anabaena PCC 7937 was introduced in the cyanobacterial host strain Synechococcus PCC 7942. The host normally only uses cytochrome c553 as Photosystem I (PS I) donor. The heterologous gene was efficiently expressed using the inducible Escherichia coli trc promoter. Accumulation of plastocyanin protein depended on the presence of Cu2+. The protein was accurately targeted to the thylakoid lumen, from which it could be isolated in the mature form. Redox difference spectroscopy proved the presence of a Cu2+ ion in the holoenzyme. Isolated heterologous plastocyanin was functional in reconstitution of in vitro electron transfer to PS I. The presence of Anabaena plastocyanin in Synechococcus thylakoid membranes increased PS I electron transfer rate 2.5 times. Analysis of P700 redox and PS II fluorescence transients in vivo showed a faster electron transfer through PS I because of enhanced electron supply in the presence of plastocyanin. In addition, the distribution of electrons between photosynthetic and respiratory electron transfer changed. Plastocyanin preferentially donates electrons to PS I rather than to the respiratory cytochrome-c oxidase complex and is not functionally equivalent to cytochrome c553.

Chloroplast protein import. Chloroplast envelopes and thylakoids have different abilities to unfold proteins.

Proteins have to be at least partially unfolded upon passage through the biological membranes. Previous studies with a dihydrofolate reductase fusion protein containing a chloroplast transit peptide showed that stabilization of the tertiary structure of the fusion protein by binding of a ligand, methotrexate, failed to block its translocation across the envelopes, suggesting that chloroplast envelopes have strong activity to unfold proteins [America, T., Hageman, J., Guéra, A., Rook, F., Archer, K., Keegstra, K. & Weisbeek, P. (1994) Plant Mol. Biol. 24, 283-294]. In the present study, we have analyzed in vitro translocation of a fusion protein consisting of the entire plastocyanin precursor and dihydrofolate reductase across the chloroplast envelope membranes and the thylakoid membrane. In the presence of methotrexate, the fusion protein was imported into the stroma but its translocation across the thylakoid membrane was blocked. The fusion protein that bound to the envelope became susceptible to digestion by thermolysin. These results suggest that, while the envelope membranes can unfold the methotrexate-bound fusion protein to allow its passage, the thylakoid membrane cannot unfold the fusion protein that has re-bound to methotrexate in the stroma.

Periplasmic fractionation of Escherichia coli yields recombinant plastocyanin despite the absence of a signal sequence.

Poplar plastocyanin has been expressed in E. coli from a synthetic gene cloned into the T7 expression system. Despite the absence of a signal sequence, large quantities of the recombinant protein were readily obtained by procedures typically used to isolate proteins from the bacterial periplasm. Several different fractionation methods were equally successful. The presence of plastocyanin in these fractions does not reflect wholesale leakage of intracellular proteins, since neither beta-galactosidase activity nor the bulk of Escherichia coli proteins were released by the fractionation. The identity of the overexpressed protein was unequivocally proven to be poplar plastocyanin by N-terminal amino acid sequence analysis and by spectroscopic characterization of the purified blue copper protein.

High-resolution solution structure of reduced parsley plastocyanin.

A high-resolution three-dimensional solution structure of parsley plastocyanin has been determined using 1H-NMR-derived data. An ensemble of 30 conformers has been calculated, exhibiting an atomic root mean square distribution about the mean coordinate positions of 0.37 +/- 0.03 A for backbone atoms and 0.75 +/- 0.04 A for all heavy atoms. (These values exclude residues 8-10 which are disordered.) The global fold of parsley plastocyanin is closely similar to those of other plastocyanins which have been structurally characterized by X-ray diffraction and NMR methods. However, deletion of residues at positions 57 and 58 of the consensus plastocyanin sequence causes elimination of a turn found in most higher plant plastocyanins. This turn is located in an acidic patch binding site, which consists of two clusters of acidic residues at positions 42-45 and 59-61. These residues surround the side chain of Tyr 83, which has been shown to be involved in binding of and electron transfer from cytochrome f, one of plastocyanin's physiological partners. The acidic recognition site is further disrupted in parsley plastocyanin by nonconservative substitution of two charged residues at positions 59 and 60. The NMR-derived structures show that E53, E85, and E95 compensate for these substitutions and give parsley plastocyanin an acidic recognition site of similar extent to that of other higher plant plastocyanins.(ABSTRACT TRUNCATED AT 250 WORDS)

Twin plastocyanin dimorphism in tobacco.

Two iso-plastocyanin fractions, oxidized b-plastocyanin, PCb(II) and reduced a-plastocyanin, PCa(I), have been isolated from whole tobacco leaves by conventional chromatography on DEAE-cellulose. The isoelectric points of PCa and PCb at 10 degrees C were found to be 3.99 and 3.97, respectively. When the primary structures were analysed, a microheterogeneity within both PCa and PCb was observed. By appropriate peptide arrangements the amino-acid sequences of two PCa (PCa' and PCa") and two PCb (PCb' and PCb") have been differentiated. All four sequences contain 99 amino-acid residues. PCa' and PCa" differ in one position, where Ser-58 in PCa' is replaced by Pro in PCa".PCb' and PCb" differ in three positions, where Gly-65, Thr-81 and Ala-85 in PCb' are replaced by Ala, Ser and Ser in PCb", respectively. PCa (PCa'/PCa") generally differs from PCb (PCb'/PCb") in three positions, where Val-52, Glu-61 and Tyr-62 in PCa'/PCa" are replaced by Ala, Asp and Leu in PCb'/PCb", respectively. Fluorescence spectra of oxidized tobacco PCa and PCb have been characterized with an emission-maximum position at around 340 nm. The presence of one extra tyrosyl (Tyr-62) in PCa results in a weak increase of the maximal intensity in conjunction with a slight blue-shift of the maximum position.

The 1.5-A crystal structure of plastocyanin from the green alga Chlamydomonas reinhardtii.

The crystal structure of plastocyanin from the green alga Chlamydomonas reinhardtii has been determined at 1.5-A resolution with a crystallographic R factor of 16.8%. Plastocyanin is a small (98 amino acids), blue copper-binding protein that catalyzes the transfer of electrons in oxygenic photosynthesis from cytochrome f in the quinol oxidase complex to P700+ in photosystem I. Chlamydomonas reinhardtii plastocyanin is an eight-stranded, antiparallel beta-barrel with a single copper atom coordinated in quasitetrahedral geometry by two imidazole nitrogens (from His-37 and His-87), a cysteine sulfur (from Cys-84), and a methionine sulfur (from Met-92). The molecule contains a region of negative charge surrounding Tyr-83 (the putative distant site of electron transfer) and an exclusively hydrophobic region surrounding His-87; these regions are thought to be involved in the recognition of reaction partners for the purpose of directing electron transfer. Chlamydomonas reinhardtii plastocyanin is similar to the other plastocyanins of known structure, particularly the green algal plastocyanins from Enteromorpha prolifera and Scenedesmus obliquus. A potential "through-bond" path of electron transfer has been identified in the protein that involves the side chain of Tyr-83, the main-chain atoms between residues 83 and 84, the side chain of Cys-84, the copper atom, and the side chain of His-87.

Synechocystis 6803 plastocyanin isolated from both the cyanobacterium and E. coli transformed cells are identical.

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.

Subunit composition of Photosystem I complex that catalyzes light-dependent transfer of electrons from plastocyanin to ferredoxin.

The PSI core complex prepared from cucumber cotyledons, which contains 80 chlorophylls per reaction center (P700) and eight polypeptides with apparent molecular masses of 65/63, 20, 19.5, 18.5, 17.5, 7.6, and 5.8 kDa, has been shown to catalyze the light-dependent transfer of electrons from plastocyanin to ferredoxin. The "native" PSI complex, which contains more than fifteen polypeptides and 120 chlorophylls per P700, did not show higher activity. Any attempt to deplete subunit(s) of the core complex decreased its activity. These results suggest that in addition to light-harvesting chlorophyll a/b protein complexes, several genes of psaA-psaK, which have been proposed as components of PSI complex, are not involved in the activity of PSI complex. It was also found that the amount of 18.5-kDa polypeptide in the PSI complex affects the activity: when this polypeptide was largely depleted, the complex was almost inactive. The inactivation was due to inhibition of electron transfer from plastocyanin to photooxidized P700. Chemical cross-linking and N-terminal amino acid sequencing experiments indicated that the 18.5-kDa polypeptide is the plastocyanin-docking protein and the psaF gene product. The function of the psaF gene product was discussed.

Reconstitution of mature plastocyanin from precursor apo-plastocyanin expressed in Escherichia coli.

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.

Expression of spinach plastocyanin in E. coli.

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.