The structure and unusual pH dependence of plastocyanin from the fern Dryopteris crassirhizoma. The protonation of an active site histidine is hindered by pi-pi interactions

Spectroscopic properties, amino acid sequence, electron transfer kinetics, and crystal structures of the oxidized (at 1.7 A resolution) and reduced form (at 1.8 A resolution) of a novel plastocyanin from the fern Dryopteris crassirhizoma are presented. Kinetic studies show that the reduced form of Dryopteris plastocyanin remains redox-active at low pH, under conditions where the oxidation of the reduced form of other plastocyanins is inhibited by the protonation of a solvent-exposed active site residue, His87 (equivalent to His90 in Dryopteris plastocyanin). The x-ray crystal structure analysis of Dryopteris plastocyanin reveals pi-pi stacking between Phe12 and His90, suggesting that the active site is uniquely protected against inactivation. Like higher plant plastocyanins, Dryopteris plastocyanin has an acidic patch, but this patch is located closer to the solvent-exposed active site His residue, and the total number of acidic residues is smaller. In the reactions of Dryopteris plastocyanin with inorganic redox reagents, the acidic patch (the "remote" site) and the hydrophobic patch surrounding His90 (the "adjacent" site) are equally efficient for electron transfer. These results indicate the significance of the lack of protonation at the active site of Dryopteris plastocyanin, the equivalence of the two electron transfer sites in this protein, and a possibility of obtaining a novel insight into the photosynthetic electron transfer system of the first vascular plant fern, including its molecular evolutionary aspects. This is the first report on the characterization of plastocyanin and the first three-dimensional protein structure from fern plant.     

Unusual properties of plastocyanin from the fern Dryopteris crassirhizoma.

The effect of pH on the (1)H NMR spectrum, reduction potential, and self-exchange rate constant of the novel plastocyanin (PCu) from the fern plant Dryopteris crassirhizoma has been studied. The results are compared with those for the higher-plant PCu from parsley. In the (1)H NMR spectrum of D. crassirhizoma PCu(I), there is no sign that either of the His ligands is protonated at pH* down to 5.4. The reduction potentials of D. crassirhizoma and parsley PCu are 382 and 379 mV, respectively, at pH 7.4. When the pH value is decreased, the reduction potential of parsley PCu is seen to increase quite dramatically, consistent with protonation at His87 in PCu(I). A pK(a) of 5.8 is obtained from the electrochemistry data, consistent with a value of 5.6 determined by NMR. The reduction potential of D. crassirhizoma PCu exhibits a much less pronounced dependence on pH. The self-exchange rate constant of D. crassirhizomaPCu(I) is 3.4 x 10(3) M(-1) s(-1) at pH* 7.9. This is the smallest self-exchange rate constant reported to date for a PCu and can be rationalized by considering the altered distribution of charged residues on the surface of the D. crassirhizoma protein compared to the charge distributions of other higher-plant PCus. The self-exchange rate constant increases to 9 x 10(3) M(-1) s(-1) at pH* 5.4, consistent with enhanced protein-protein association at lower pH*, and the absence of His87 protonation in D. crassirhizoma PCu(I) in the accessible pH range.     

Role of electrostatics in the interaction between cytochrome f and plastocyanin of the cyanobacterium Phormidium laminosum

The role of charged residues on the surface of plastocyanin from the cyanobacterium Phormidium laminosum in the reaction with soluble cytochrome f in vitro was studied using site-directed mutagenesis. The charge on each of five residues on the eastern face of plastocyanin was neutralized and/or inverted, and the effect of the mutation on midpoint potentials was determined. The dependence of the overall rate constant of reaction, k(2), on ionic strength was investigated using stopped-flow spectrophotometry. Removing negative charges (D44A or D45A) accelerated the reaction and increased the dependence on ionic strength, whereas removing positive charges slowed it down. Two mutations (K46A, K53A) each almost completely abolished any influence of ionic strength on k(2), and three mutations (R93A, R93Q, R93E) each converted electrostatic attraction into repulsion. At low ionic strength, wild type and all mutants showed an inhibition which might be due to changes in the interaction radius as a consequence of ionic strength dependence of the Debye length or to effects on the rate constant of electron transfer, k(et). The study shows that the electrostatics of the interaction between plastocyanin and cytochrome f of P. laminosum in vitro are not optimized for k(2). Whereas electrostatics are the major contributor to k(2) in plants [Kannt, A., et al. (1996) Biochim. Biophys. Acta 1277, 115-126], this role is taken by nonpolar interactions in the cyanobacterium, leading to a remarkably high rate at infinite ionic strength (3.2 x 10(7) M(-1) s(-1)).     

A comparative flash-photolysis study of electron transfer from pea and spinach plastocyanins to spinach Photosystem 1. A reaction involving a rate-limiting conformational change

Two mutants of plastocyanin have been constructed by site-directed mutagenesis in spinach and pea to elucidate the binding and electron transfer properties between plastocyanin and spinach Photosystem 1. The conserved, surface-exposed Tyr-83 has been replaced by phenylalanine and leucine in plastocyanin from both species and the proteins have been expressed in Escherichia coli. The reaction mechanism of electron transfer from plastocyanins to photooxidized P700 in Photosystem 1 has been studied by laser-flash absorption spectroscopy. The experimental data were interpreted with a model involving a rate-limiting conformational change, preceding the intracomplex electron transfer. The pea proteins show an overall facilitated reaction with spinach Photosystem 1, compared to spinach plastocyanins. The changes are small but significant, indicating a more efficient electron transfer within the transient complex. In addition, for the spinach leucine mutant, the equilibrium within the plastocyanin-Photosystem 1 complex is more displaced towards the active conformation than for the corresponding wild-type. Absorption spectra, EPR and reduction potentials for the mutants are similar to those of the corresponding wild-type, although small shifts are observed in the spectra of the Tyr83Leu proteins. Based on these results, it is suggested that Photosystem 1 from spinach is capable of using both pea and spinach plastocyanin as an efficient electron donor and that the former even can stimulate the Photosystem 1 reduction. The origin of the stimulation is discussed in terms of differences in surface-exposed residues. Since the effects of the mutations are small, it can be concluded that electron transfer to Photosystem 1 does not occur via Tyr-83.Abbreviations cyt- cytochrome - IPTG- isopropyl--d-thiogalactopyranoside - P,P700- reaction-center chlorophyll - Pc- plastocyanin - PS 1- Photosystem 1 - SDS-PAGE- sodium dodecyl sulfate polyacrylamide gel electrophoresis - WT- wild-type     

Prokaryote-eukaryote relationship and the amino acid sequence of plastocyanin from Anabaena variabilis.

The amino acid sequence of plastocyanin from the prokaryotic blue-green alga Anabaena variabilis was determined. The protein consists of a single polypeptide chain of 105 residues. The amino acid sequence of the plastocyanin was compared with that of the eukaryotic green alga Chlorella fusca and with those of higher-plant plastocyanins. The considerable similarity between the prokaryotic and eukaryotic plastocyanins is discussed. Detailed evidence for the sequence of the protein has been deposited as Supplementary Publication SUP 50051 (13 pages) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem J. (1975) 145, 5.     

The inhibition of photosynthetic electron transport in spinach chloroplasts by low osmolarity.

The reversible inhibition, by low osmolarity, of the rate of electron transport through photosystem 1 has been investigated in spinach chloroplasts. By use of different electron donor systems to photosystem 1, inhibitors of plastocyanin, and by measurement of the extent of photooxidation of the photosystem 1 reaction center P700, the inhibition site has been localized on the electron donor side of this photosystem. From comparison of the influence of impermeant and permeant salts on the electron transport rate, and from the effect of ionic strength on the oxidation of externally added plastocyanin by subchloroplast preparations, it is concluded that low ionic strength within the thylakoids inhibits the photooxidation of endogenous plastocyanin by P700. The results are taken as evidence that plastocyanin is oxidized by P700 at the internal (lumen) side of the osmotic barrier in the thylakoid membrane.     

Role of electrostatics in the interaction between plastocyanin and photosystem I of the cyanobacterium Phormidium laminosum.

The interactions between photosystem I and five charge mutants of plastocyanin from the cyanobacterium Phormidium laminosum were investigated in vitro. The dependence of the overall rate constant of reaction, k2, on ionic strength was investigated using laser flash photolysis. The rate constant of the wild-type reaction increased with ionic strength, indicating repulsion between the reaction partners. Removing a negative charge on plastocyanin (D44A) accelerated the reaction and made it independent of ionic strength; removing a positive charge adjacent to D44 (K53A) had little effect. Neutralizing and inverting the charge on R93 slowed the reaction down and increased the repulsion. Specific effects of MgCl2 were observed for mutants K53A, R93Q and R93E. Thermodynamic analysis of the transition state revealed positive activation entropies, suggesting partial desolvation of the interface in the transition state. In comparison with plants, plastocyanin and photosystem I of Phormidium laminosum react slowly at low ionic strength, whereas the two systems have similar rates in the range of physiological salt concentrations. We conclude that in P. laminosum, in contrast with plants in vitro, hydrophobic interactions are more important than electrostatics for the reactions of plastocyanin, both with photosystem I (this paper) and with cytochrome f[Schlarb-Ridley, B.G., Bendall, D.S. & Howe, C.J. (2002) Biochemistry41, 3279-3285]. We discuss the implications of this conclusion for the divergent evolution of cyanobacterial and plant plastocyanins.     

The electron-transfer site of spinach plastocyanin

Two sites for electron transfer have been proposed for plastocyanin: one near the copper ion and the other close to the acid patch formed by residues 42-45. Calculations of electrostatic properties of spinach plastocyanin and ionic strength dependences of electron-transfer reactions of this protein have been used to distinguish between these two sites. Calculations show that the electric potential field of spinach plastocyanin is highly asymmetric and that the protein has a dipole moment of 360 D. The negative end of the dipole axis emerges between the negative patches formed by residues 42-45, which is though to be the cation binding site, and residues 59-61. The angles between the dipole vector and vectors from the center of mass to the copper ion and to the acid patch are 90 degrees and 30 degrees, respectively. The angle between the dipole vector and a line from the center of mass to the site of electron transfer is evaluated from the ionic strength dependence of electron-transfer rates at pH 7.8 with the help of equations developed by Van Leeuwen et al. [van Leeuwen, J.W., Mofers, F.J.M., & Veerman, E.C.I. (1981) Biochim. Biophys. Acta 635, 434] and Van Leeuwen [van Leeuwen, J.W. (1983) Biochim. Biophys. Acta 743, 408]. The angles found are 85 degrees, 110 degrees, and 75 +/- 15 degrees for reactions with tris(1,10-phenanthroline)cobalt(III), hexacyanoferrate(III), and ferrocytochrome c, respectively. The electric potential field calculations suggest that the hexacyanoferrate(III) interaction angle corresponds to a unique site of minimum repulsion at the hydrophobic region of the protein surface, close to the copper ion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of an acidic plastocyanin from Microcystis aeruginosa

Plastocyanin and cytochrome c-553 are two functionally equivalent electron carriers in the photosynthetic chain of cyanobacteria. Microcystis aeruginosa, a unicellular cyanobacterium which grows well at a high pH (8.6) and which was not known to possess plastocyanin, has been studied for its ability to synthesize plastocyanin in culture media with and without Cu. In the absence of Cu, an acidic cytochrome c-553 alone was isolated. With the inclusion of 2 microM Cu, cytochrome c-553 synthesis was partially suppressed and an acidic plastocyanin was isolated. A newly developed procedure, using high concentrations of ammonium sulfate to fractionate water-soluble proteins on Sephacryl S-200 was successfully used to isolate and concentrate the plastocyanin, thus allowing it to be further purified to homogeneity. This protein has an isoelectric point of 4.8 which is similar to the pI value reported for other acidic plastocyanins from higher plants and green algae. Its N-terminal sequence of the first 15 amino acids has been determined; 9 of these amino acids are identical to those in the sequence of the basic plastocyanin from Anabaena variabilis.     

Ionic strength and pH dependence of the reaction between plastocyanin and Photosystem 1. Evidence of a rate-limiting conformational change

The electron-transfer reaction between spinach wild-type plastocyanin (Pc(WT)) two site-directed mutants, Pc(Thr79His) and Pc(Lys81His), and spinach Photosystem 1 particles, has been studied as a function of protein concentration, ionic strength and pH by using laser-flash absorption spectroscopy. The kinetic data are interpreted using the simplest possible three-step model, involving a rate-limiting conformational change preceding intracomplex electron transfer. The three proteins show similar concentration, pH and ionic strength dependencies. The effects of ionic strength and pH on the reaction indicate a strong influence of complementary charges on complex formation and stabilization. Studies with apoprotein support the opinion that the hydrophobic patch is critical for an productive interaction with the reaction center of Photosystem 1. Together with earlier site-directed mutagenesis studies, the absence of a detectable Photosystem 1 reaction in the presence of reduced azurin, stellacyanin, cytochrome c and cytochrome c₅₅₁, demonstrates the existence of a high level of specificity in the protein-protein interface in the formation of an efficient electron-transfer complex.     

pH-dependent redox activity and fluxionality of the copper site in amicyanin from Thiobacillus yersutus as studied by 300- and 600- MHz 1H NMR

The kinetics of the deuteronation of one of the copper ligand histidines of the reduced Type I blue-copper protein amicyanin from Thiobacillus versutus was studied as a function of temperature by 300- and 600- MHz 1H NMR. The NMR data were analyzed with the help of a three site exchange model. Deuteron exchange between the histidine ligand and the solution appears to be catalyzed by phosphate. After deuteronation the histidine can occur in two conformations. The electron self-exchange rate of amicyanin was determined as a function of temperature and ionic strength. At 298 K, pD = 8.6, I = 0.05 M, the ese rate amounts to 1.3 x 10(5) M-1 S-1. The activation parameters amount to delta H not equal to = (52 +/- 3) kJ/mol and delta S not equal to = (26 +/- 9) J/mol.K. The dependence of the ese rate on ionic strength is small. The deuteronated amicyanin appears to be redox-inactive. The experimental findings clearly distinguish amicyanin from other classes of blue-copper proteins like the azurins and the pseudo-azurins.     

Spectroscopic characterization of plastocyanin from hornwort

Plastocyanin isolated from an aquatic higher plant, Ceratophyllum demersum L. (hornwort), has been characterized by electronic absorption, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopies. The visible absorption, CD, and EPR spectra of hornwort plastocyanin indicated a complete similarity of blue copper center to those of terrestrial higher plants and algae. However, the ultraviolet absorption spectrum of hornwort plastocyanin exhibited a lower tyrosine (Tyr) and a higher phenylalanine (Phe) content of the protein comparing with other plastocyanins. The ratio of Phe/Tyr residues was estimated to be 9 by amino acid analysis. The hornwort plastocyanin resembles in amino acid composition terrestrial higher plant plastocyanins rather than alga plastocyanins but is peculiar in the content of Phe and Tyr residues.     

Electron transfer from the Rieske iron-sulfur protein (ISP) to cytochrome f in vitro. Is a guided trajectory of the ISP necessary for competent docking?

The time course of electron transfer in vitro between soluble domains of the Rieske iron-sulfur protein (ISP) and cytochrome f subunits of the cytochrome b(6)f complex of oxygenic photosynthesis was measured by stopped-flow mixing. The domains were derived from Chlamydomonas reinhardtii and expressed in Escherichia coli. The expressed 142-residue soluble ISP apoprotein was reconstituted with the [2Fe-2S] cluster. The second-order rate constant, k(2)((ISP-f)) = 1.5 x 10(6) m(-1) s(-1), for ISP to cytochrome f electron transfer was <10(-2) of the rate constant at low ionic strength, k(2)((f-PC))(> 200 x 10(6) m(-1) s(-1)), for the reduction of plastocyanin by cytochrome f, and approximately 1/30 of k(2)((f-PC)) at the ionic strength estimated for the thylakoid interior. In contrast to k(2)((f-PC)), k(2)((ISP-f)) was independent of pH and ionic strength, implying no significant role of electrostatic interactions. Effective pK values of 6.2 and 8.3, respectively, of oxidized and reduced ISP were derived from the pH dependence of the amplitude of cytochrome f reduction. The first-order rate constant, k(1)((ISP-f)), predicted from k(2)((ISP-f)) is approximately 10 and approximately 150 times smaller than the millisecond and microsecond phases of cytochrome f reduction observed in vivo. It is proposed that in the absence of electrostatic guidance, a productive docking geometry for fast electron transfer is imposed by the guided trajectory of the ISP extrinsic domain. The requirement of a specific electrically neutral docking configuration for ISP electron transfer is consistent with structure data for the related cytochrome bc(1) complex.     

Electrostatic and steric control of electron self-exchange in cytochromes c, c551, and b5.

The ionic strength dependence of the electron self-exchange rate constants of cytochromes c, c551, and b5 has been analyzed in terms of a monopole-dipole formalism (van Leeuwen, J.W. 1983. Biochim. Biophys. Acta. 743:408-421). The dipole moments of the reduced and oxidized forms of Ps. aeruginosa cytochrome c551 are 190 and 210 D, respectively (calculated from the crystal structure). The projections of these on the vector from the center of mass through the exposed heme edge are 120 and 150 D. For cytochrome b5, the dipole moments calculated from the crystal structure are 500 and 460 D for the reduced and oxidized protein; the projections of these dipole moments through the exposed heme edge are -330 and -280 D. A fit of the ionic strength dependence of the electron self-exchange rate constants gives -280 (reduced) and -250 (oxidized) D for the center of mass to heme edge vector. The self-exchange rate constants extrapolated to infinite ionic strength of cytochrome c, c551, and b5 are 5.1 x 10(5), 2 x 10(7), and 3.7 x 10(5) M-1 s-1, respectively. The extension of the monopole-dipole approach to other cytochrome-cytochrome electron transfer reactions is discussed. The control of electron transfer by the size and shape of the protein is investigated using a model which accounts for the distance of the heme from each of the surface atoms of the protein. These calculations indicate that the difference between the electrostatically corrected self-exchange rate constants of cytochromes c and c551 is due only in part to the different sizes and heme exposures of the two proteins.     

The role of individual lysine residues in the basic patch on turnip cytochrome f for electrostatic interactions with plastocyanin in vitro.

The role of electrostatic interactions in determining the rate of electron transfer between cytochrome f and plastocyanin has been examined in vitro with mutants of turnip cytochrome f and mutants of pea and spinach plastocyanins. Mutation of lysine residues Lys58, Lys65 and Lys187 of cytochrome f to neutral or acidic residues resulted in decreased binding constants and decreased rates of electron transfer to wild-type pea plastocyanin. Interaction of the cytochrome f mutant K187E with the pea plastocyanin mutant D51K gave a further decrease in electron transfer rate, indicating that a complementary charge pair at these positions could not compensate for the decreased overall charge on the proteins. Similar results were obtained with the interaction of the cytochrome f mutant K187E with single, double and triple mutants of residues in the acidic patches of spinach plastocyanin. These results suggest that the lysine residues of the basic patch on cytochrome f are predominantly involved in long-range electrostatic interactions with plastocyanin. However, analysis of the data using thermodynamic cycles provided evidence for the interaction of Lys187 of cytochrome f with Asp51, Asp42 and Glu43 of plastocyanin in the complex, in agreement with a structural model of a cytochrome f-plastocyanin complex determined by NMR.     

Electron transfer from plastocyanin to photosystem I.

Mutant plastocyanins with Leu at position 10, 90 or 83 (Gly, Ala and Tyr respectively in wildtype) were constructed by site-specific mutagenesis of the spinach gene, and expressed in transgenic potato plants under the control of the authentic plastocyanin promoter, as well as in Escherichia coli as truncated precursor intermediates carrying the C-terminal 22 amino acid residues of the transit peptide, i.e. the thylakoid-targeting domain that acts as a bacterial export signal. The identity of the purified plastocyanins was verified by matrix-assisted laser desorption/ionization mass spectrometry. The formation of a complex between authentic or mutant spinach plastocyanin and isolated photosystem I and the electron transfer has been studied from the biphasic reduction kinetics of P700+ after excitation with laser flashes. The formation of the complex was abolished by the bulky hydrophobic group of Leu at the respective position of G10 or A90 which are part of the conserved flat hydrophobic surface around the copper ligand H87. The rate of electron transfer decreased by both mutations to < 20% of that found with wildtype plastocyanin. We conclude that the conserved flat surface of plastocyanin represents one of two crucial structural elements for both the docking at photosystem I and the efficient electron transfer via H87 to P700+. The Y83L mutant exhibited faster electron transfer to P700+ than did authentic plastocyanin. This proves that Y83 is not involved in electron transfer to P700 and suggests that electron transfer from cytochrome f and to P700 follows different routes in the plastocyanin molecule. Plastocyanin (Y83L) expressed in either E. coli or potato exhibited different isoelectric points and binding constants to photosystem I indicative of differences in the folding of the protein. The structure of the binding site at photosystem I and the mechanism of electron transfer are discussed.     

Hydrolysis of proteins using dipeptidyl aminopeptidases: analysis of the N-terminal portion of spinach plastocyanin

The exopeptidases dipeptidyl aminopeptidases I and IV were used to hydrolyze the N-terminal portion of spinach plastocyanin to dipeptides. The enzymes were used individually as well as in a mixture and the dipeptides were analyzed by combined gas chromatography-mass spectrometry. Data are presented for native plastocyanin and the S-methylated protein. Of the 98 residues which make up this protein, the first 44 were released in the form of 22 dipeptides by the combined action of DAP I and DAP IV. These dipeptides were aligned by homology to other plastocyanins of known sequence. The results demonstrate the versatility of the two enzymes in hydrolyzing proteins to obtain information on their primary sequence.     

Computer simulation of interaction of photosystem 1 with plastocyanin and ferredoxin

We designed 3D multiparticle computer models to simulate diffusion and interactions of spinach plastocyanin and ferredoxin with plant photosystem 1 in a solution. Using these models we studied kinetic characteristics of plastocyanin-photosystem 1 and ferredoxin-photosystem 1 complex formation at a variety of ionic strength values. The computer multiparticle models demonstrate non-monotonic dependences of complex formation rates on the ionic strength as the result of long-range electrostatic interactions. Our calculations show that the decrease in the association second-order rate constant at low values of the ionic strength is caused by the protein pairs spending more time in “wrong” orientations which do not satisfy the docking conditions and so do not form the final complex capable of the electron transfer.     

Immunological comparisons of higher plant plastocyanins

Antisera were prepared in rabbits to purified plastocyanins of Spinacia oleracea and Urtica dioica. Using the method of micro-complement fixation, the immunological cross-reactivity of these antisera with plastocyanins from 37 species of plants was determined. Cross-reactivity between antisera to spinach plastocyanin and 11 plastocyanins from other plant species showed a positive correlation with distance on an ancestral amino acid sequence affinity tree constructed by the method of Dayhoff and Eck [1]. The importance of serological data as a supplement to amino acid sequence data in evolutionary studies is discussed.