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.     

NMR and homology modeling studies of copper(II)-halocyanin from Natronobacterium pharaonis bacteria

Halocyanin from the haloalkaliphilic archaean Natronobacterium pharaonis is a peripheral membrane type 1 blue copper protein with a single polypeptide chain of 163 amino acid residues. Halocyanin participates as putative electron carrier protein associated to an electron acceptor role for a terminal oxidase and has the lowest redox potential value reported to date for a BCP. NMR studies and homology modeling calculations were performed to evaluate the electronic properties of Cu(II)-halocyanin from Natronobacterium pharaonis. The copper coordination site properties of Cu(II)-halocyanin are discussed. The ¹H NMR spectra, isotropic chemical shifts and relaxation times for halocyanin are compared with those of other BCPs such as azurin, amicyanin, plastocyanin and stellacyanin. The wild-type Cu(II)-halocyanin presents almost the same ¹H NMR spectra in comparison with Cu(II)-plastocyanin as expected from a similar coordination symmetry. However, minor differences were found. In order to get some insight on these differences, a computational model for Cu(II)-halocyanin from N. pharaonis was built. Model is based on sequential homology of halocyanin with two different families of proteins: plastocyanins and pseudoazurins. Homology modeling was performed using two different structural templates and copper ion was added for further refinement of the coordination site. Proposed structure was in good agreement with NMR experimental information and is the first three-dimensional model reported to date of an halocyanin. Small differences were found in the copper coordination site with respect to other BCP with known structure. This work is also an interesting example of expertise-driven homology modeling across different protein families.     

The structural homology of amicyanin from Thiobacillus versutus to plant plastocyanins

The complete amino acid sequence of the blue copper protein amicyanin of Thiobacillus versutus, induced when the bacterium is grown on methylamine, has been determined as follows: QDKITVTSEKPVAAADVPADAVVVGIEKMKYLTPEVTIKAGETVYWVNGEVMPHNVA FKKGIVGEDAFRGEMMTKDQAYAITFNEAGSYDYFCTPHPFMRGKVIVE. The four copper ligand residues in this 106-residue-containing polypeptide chain are His54, Cys93, His96, and Met99. The Thiobacillus amicyanin is 52% similar to the amicyanin of Pseudomonas AM1, the only other copper protein known with the same spacing between the second histidine ligand and the methionine ligand. T. versutus amicyanin contains no cysteine bridge and is more closely related to the plant copper protein plastocyanin than to the bacterial copper protein azurin. Alignment of the two known amicyanin sequences with the consensus sequence of the plastocyanins and comparison with the known three-dimensional structure of poplar leaves plastocyanin reveals that the bacterial proteins have the same overall structure with two beta-sheets packed face to face. The major structural differences between the amicyanins and the plastocyanins appear to be located in two of the five loops that connect the six identified beta-strands of the amicyanins. The first of these two loops, connecting strands F and G, contains a ligand histidine and must have a different conformation from the same loop in the plastocyanins because it is shorter by two amino acids. Further differences occur in the loop connecting the strands D and E. This loop contains only 17 residues in amicyanin whereas the corresponding loop of plastocyanin contains 25 residues. Despite these differences the amicyanins appear much closer related to the plastocyanins than to the azurins. The present findings demonstrate that the occurrence of blue copper proteins with clearly plastocyanin-like features is not restricted to photosynthetic redox chains.     

Spectrochemical studies on the blue copper protein azurin from Alcaligenes denitrificans

Spectroscopic and electrochemical studies, incorporating electronic spectra, electron paramagnetic resonance (EPR) spectra, resonance Raman (RR) spectra, and measurements of the redox potential, have been carried out on the blue copper protein azurin, from Alcaligenes denitrificans. These data are correlated with the refined crystal structure of this azurin and with corresponding data for other blue copper proteins. The electronic spectrum, characterized by an intense (epsilon = 5100 M-1 cm-1) charge-transfer band at 619 nm, the EPR spectral parameters (g perpendicular = 2.059, g parallel of = 2.255, A parallel of = 60 X 10(-4) cm-1), and the resonance Raman spectrum are similar to those obtained from other azurins and from plastocyanins. Both the electronic spectrum and the EPR spectrum are unchanged over the pH range 4-10.5, but major changes occur above pH 12 and below pH 3.5. A small reversible change occurs at pH approximately 11.4. In the RR spectrum the Cu-S stretching mode is shown to contribute to all of the five principal RR peaks. Deuterium substitution produces shifts in at least seven of the peaks; these shifts may be attributable, at least in part, to the NH...S hydrogen bond to the copper-ligated Cys-112. Measurements of the redox potential, using spectroelectrochemical methods, over the temperature range 4.8-40.0 degrees C, give values for delta H0' and delta S0' of -55.6 kJ mol-1 and -97.0 J K-1 mol-1, respectively. The redox potential of A. denitrificans azurin at pH 7.0, Eo', is 276 mV. These data are interpreted in terms of a copper site, in azurin, comprising three strong bonds, in an approximately trigonal plane, from Cys-112, His-46, and His-117 and much longer axial approaches from Met-121 and the peptide carbonyl oxygen of Gly-45. Spectral differences within the azurin family and between azurin and plastocyanin are attributed to differences in the strengths of these axial interactions. Likewise, the distinctly lower Eo values for azurins, as compared with plastocyanins, are related to the more copper(II)-like site in azurin [with a weaker Cu-S(Met) interaction and a Cu-O interaction not found in plastocyanin]. On the other hand, the relative constancy of the EPR parameters between azurin and plastocyanin suggests they are not strongly influenced by weakly interacting axial groups.     

Characterization of Plastocyanin Isolated From Brazilian Elodea

Plastocyanin obtained from an aquatic higher plant, Brazilianelodea, was characterized by electronic absorption, CD, andEPR spectroscopy. The blue copper center of Brazilian elodeaplastocyanin is electronically and geometrically similar tothat of terrestrial higher-plant (cucumber, spinach, poplar,etc.) and algal plastocyanins as well as that of another aquatichigher-plant (hornwort) plastocyanin. The midpoint redox potentialof the copper in the plastocyanin was estimated to be +364 mVat pH 7.2, which is in the range of midpoint potentials from+347 to +395 mV reported for plastocyanins. The ratio of Phe/Tyrresidue was determined to be 3 by amino acid analysis. Valuesof 2–3.5 have been commonly observed in many higher-plantplastocyanins; a conspicuous exception to this is hornwort plastocyaninwith a Phe/Tyr value of 9. (Received January 16, 1986; Accepted April 18, 1987)     

The unique proline of the Prochlorothrix hollandica plastocyanin hydrophobic patch impairs electron transfer to photosystem I.

A number of surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in amino acids located in the amino-terminal hydrophobic patch of the copper protein, which presents a variant structure as compared with other plastocyanins. The single mutants Y12G, Y12F, Y12W, P14L, and double mutant Y12G/P14L have been produced. Their reactivity toward photosystem I has been analyzed by laser flash absorption spectroscopy. Plots of the observed rate constant with all mutants versus plastocyanin concentration show a saturation profile similar to that with wild-type plastocyanin, thus suggesting the formation of a plastocyanin-photosystem I transient complex. The mutations do not induce relevant changes in the equilibrium constant for complex formation but induce significant variations in the electron transfer rate constant, mainly with the two mutants at proline 14. Additionally, molecular dynamics calculations indicate that mutations at position 14 yield small changes in the geometry of the copper center. The comparative kinetic analysis of the reactivity of plastocyanin mutants toward photosystem I from different organisms (plants and cyanobacteria) reveals that reversion of the unique proline of Prochlorothrix plastocyanin to the conserved leucine of all other plastocyanins at this position enhances the reactivity of the Prochlorothrix protein.     

Formation of silver plastocyanin in Scenedesmus

Silver ions up to 5 microM do not affect growth of the green microalga Scenedesmus acutus. They induce formation of protein species precipitable by an antibody specific against plastocyanin. The metal is incorporated into a part of the induced protein in competition with copper. Bismuth, lead and molybdenum had no effect. The amount of both silver- and copper-containing plastocyanins so formed apparently regulates concurrently inhibition of soluble plastidic cytochrome c-553. The silver-copper competition for the build-up of blue plastocyanin can be shown with intact cells, not with isolated algal plastocyanin.     

Managing the solvent water polarization to obtain improved NMR spectra of large molecular structures

In large molecular structures, the magnetization of all hydrogen atoms in the solute is strongly coupled to the water magnetization through chemical exchange between solvent water and labile protons of macromolecular components, and through dipole–dipole interactions and the associated “spin diffusion” due to slow molecular tumbling. In NMR experiments with such systems, the extent of the water polarization is thus of utmost importance. This paper presents a formalism that describes the propagation of the water polarization during the course of different NMR experiments, and then compares the results of model calculations for optimized water polarization with experimental data. It thus demonstrates that NMR spectra of large molecular structures can be improved with the use of paramagnetic spin relaxation agents which selectively enhance the relaxation of water protons, so that a substantial gain in signal-to-noise can be achieved. The presently proposed use of a relaxation agent can also replace the water flip-back pulses when working with structures larger than about 30 kDa. This may be a valid alternative in situations where flip-back pulses are difficult to introduce into the overall experimental scheme, or where they would interfere with other requirements of the NMR experiment.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-3070-8     

Study of electrostatic potential surface distribution of wild-type plastocyanin Synechocystis solution structure determined by homonuclear NMR

Plastocyanin is a small (approximately 10 kDa), type I blue copper protein that works as an electron donor to photosystem I from cytochrome f in both chloroplast systems and in some strains of cyanobacteria. Comparative studies of the kinetic mechanisms of plastocyanins in different organisms show that the electron transfer from photosystem I happens by simple collision in cyanobacteria but through a intermediate transition complex in green algae and superior plants. Previous work has proved that this effect cannot be explained by structural variations across the different plastocyanins but it can be explained by differences in the electrostatic potential distribution at the protein surface. In that case, minor conformational errors at the amino acid side chain level may imply an important effect in the electrostatic potential distribution calculation. In this work we present a high resolution study of side chain conformation by homonuclear NMR for the reduced wild-type plastocyanin Synechocystis using intensity ratios for 2D-NOESY and 2D-H,H-TOCSY cross peaks at different mixing times. We also present the corresponding comparison with different plastocyanin structures and the effect in the electrostatic potential distribution at the protein surface. We discuss the importance of indirect J-coupling information from TOCSY-type experiments as complement for intraresidue distances derived from NOESY experiments in the determination of side chain orientation and stereo-specific assignments.     

Protein global fold determination using site-directed spin and isotope labeling

We describe a simple experimental approach for the rapid determination of protein global folds. This strategy utilizes site-directed spin labeling (SDSL) in combination with isotope enrichment to determine long-range distance restraints between amide protons and the unpaired electron of a nitroxide spin label using the paramagnetic effect on relaxation rates. The precision and accuracy of calculating a protein global fold from only paramagnetic effects have been demonstrated on barnase, a well-characterized protein. Two monocysteine derivatives of barnase, (H102C) and (H102A/Q15C), were 15N enriched, and the paramagnetic nitroxide spin label, MTSSL, attached to the single Cys residue of each. Measurement of amide 1H longitudinal relaxation times, in both the oxidized and reduced states, allowed the determination of the paramagnetic contribution to the relaxation processes. Correlation times were obtained from the frequency dependence of these relaxation processes at 800, 600, and 500 MHz. Distances in the range of 8 to 35 A were calculated from the magnitude of the paramagnetic contribution to the relaxation processes and individual amide 1H correlation times. Distance restraints from the nitroxide spin to amide protons were used as restraints in structure calculations. Using nitroxide to amide 1H distances as long-range restraints and known secondary structure restraints, barnase global folds were calculated having backbone RMSDs <3 A from the crystal structure. This approach makes it possible to rapidly obtain the overall topology of a protein using a limited number of paramagnetic distance restraints.     

Protein strain in blue copper proteins studied by free energy perturbations.

Free energy perturbations have been performed on two blue copper proteins, plastocyanin and nitrite reductase. By changing the copper coordination geometry, force constants, and charges, we have estimated the maximum energy with which the proteins may distort the copper coordination sphere. By comparing this energy with the quantum chemical energy cost for the same perturbation on the isolated copper complex, various hypotheses about protein strain have been tested. The calculations show that the protein can only modify the copper-methionine bond length by a modest amount of energy-<5 kJ/mol-and they lend no support to the suggestion that the quite appreciable difference in the copper coordination geometry encountered in the two proteins is a result of the proteins enforcing different Cu-methionine bond lengths. On the contrary, this bond is very flexible, and neither the geometry nor the electronic structure change appreciably when the bond length is changed. Moreover, the proteins are rather indifferent to the length of this bond. Instead, the Cu(II) coordination geometries in the two proteins represent two distinct minima on the potential surface of the copper ligand sphere, characterized by different electronic structures, a tetragonal, mainly sigma-bonded, structure in nitrite reductase and a trigonal, pi-bonded, structure in plastocyanin. In vacuum, the structures have almost the same energy, and they are stabilized in the proteins by a combination of geometric and electrostatic interactions. Plastocyanin favors the bond lengths and electrostatics of the trigonal structure, whereas in nitrite reductase, the angles are the main discriminating factor. Proteins 1999;36:157-174.     

A simple protocol to study blue copper proteins by NMR.

In the case of oxidized plastocyanin from Synechocystis sp. PCC6803, an NMR approach based on classical two and three dimensional experiments for sequential assignment leaves unobserved 14 out of 98 amino acids. A protocol which simply makes use of tailored versions of 2D HSQC and 3D CBCA(CO)NH and CBCANH leads to the identification of nine of the above 14 residues. The proposed protocol differs from previous approaches in that it does not involve the use of unconventional experiments designed specifically for paramagnetic systems, and does not exploit the occurrence of a corresponding diamagnetic species in chemical exchange with the blue copper form. This protocol is expected to extend the popularity of NMR in the structural studies of copper (II) proteins, allowing researchers to increase the amount of information available via NMR on the neighborhood of a paramagnetic center without requiring a specific expertise in the field. The resulting 3D spectra are standard spectra that can be handled by any standard software for protein NMR data analysis.     

Prediction of the secondary and tertiary structure of plastocyanin.

The amino acid sequences of nine plastocyanins were examined using four published methods for the prediction of secondary structure in proteins. The results of the four methods were combined in such a way as to maximize agreement, and the position of alpha helices, beta sheets, and beta turns in plastocyanin was predicted. From this result and other information, such as the position of conserved residues and the requirements for coordination of copper, a preliminary model for the mainchain folding of the molecule was presented.     

Plastocyanin-cytochrome f interactions: the influence of hydrophobic patch mutations studied by NMR spectroscopy

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.     

Complete amino acid sequence of plastocyanin from a green alga, Enteromorpha prolifera

The complete amino acid sequence of the plastocyanin from the green alga Enteromorpha prolifera has been determined by Edman degradation of the intact molecule and fragments produced by enzymatic cleavage of the polypeptide chain with chymotrypsin, Staphylococcus aureus protease, proline-specific endopeptidase, Lys-C endopeptidase and trypsin. The molecule consists of 98 amino acid residues with a calculated relative molecular mass of 10103. The amino acid sequence of E. prolifera plastocyanin shows a high degree of homology with those plastocyanins from other algae and higher plants. In particular, the four residues which are copper ligands in other plastocyanins and in the bacterial electron transport protein azurin (two histidines, one cysteine and one methionine) are conserved. Five out of the six acidic amino acid side-chains which create an 'acidic patch' on the surface of plastocyanin from Populus nigra var. italica [Colman, P. M. et al. (1978) Nature (Lond.) 272, 319-324] are conserved in the amino acid sequence of E. prolifera plastocyanin.     

The amino acid sequence of plastocyanin from Lactuca sativa (lettuce)

The amino acid sequence of plastocyanin from lettuce (Lactuca sativa L.) was determined by using a Beckman 890C automatic sequencer and by dansyl-phenylisothiocyanate analysis of peptides obtained by enzymic digestion of purified CNBr fragments. The protein consists of a single polypeptide chain of 99 residues, and shows close homology with other higher plant plastocyanins. The data are discussed in relation to the possible residues involved in the binding of copper in plastocyanin.     

A Brownian dynamics study of the interaction of Phormidium cytochrome f with various cyanobacterial plastocyanins

Brownian dynamics simulations were used to study the role of electrostatic forces in the interactions of cytochrome f from the cyanobacterium Phormidium laminosum with various cyanobacterial plastocyanins. Both the net charge on the plastocyanin molecule and the charge configuration around H92 (H87 in higher plants) are important in determining the interactions. Those plastocyanins (PCs) with a net charge more negative than -2.0, including those from Synechococcus sp. PCC7942, Synechocystis sp. 6803, and P. laminosum showed very little complex formation. On the other hand, complex formation for those with a net charge more positive than -2.0 (including Nostoc sp. PCC7119 and Prochlorothrix hollandica) as well as Nostoc plastocyanin mutants showed a linear dependence of complex formation upon the net charge on the plastocyanin molecule. Mutation of charged residues on the surface of the PC molecules also affected complex formation. Simulations involving plastocyanin mutants K35A, R93A, and K11A (when present) showed inhibition of complex formation. In contrast, D10A and E17A mutants showed an increase in complex formation. All of these residues surround the H92 (H87 in higher plant plastocyanins) ligand to the copper. An examination of the closest electrostatic contacts shows that these residues interact with D63, E123, R157, D188, and the heme on Phormidium cytochrome f. In the complexes formed, the long axis of the PC molecule lies perpendicular to the long axis of cytochrome f. There is considerable heterogeneity in the orientation of plastocyanin in the complexes formed.     

The transient complex of poplar plastocyanin with cytochrome f: effects of ionic strength and pH

The orientation of poplar plastocyanin in the complex with turnip cytochrome f has been determined by rigid-body calculations using restraints from paramagnetic NMR measurements. The results show that poplar plastocyanin interacts with cytochrome f with the hydrophobic patch of plastocyanin close to the heme region on cytochrome f and via electrostatic interactions between the charged patches on both proteins. Plastocyanin is tilted relative to the orientation reported for spinach plastocyanin, resulting in a longer distance between iron and copper (13.9 A). With increasing ionic strength, from 0.01 to 0.11 M, all observed chemical-shift changes decrease uniformly, supporting the idea that electrostatic forces contribute to complex formation. There is no indication for a rearrangement of the transient complex in this ionic strength range, contrary to what had been proposed earlier on the basis of kinetic data. By decreasing the pH from pH 7.7 to pH 5.5, the complex is destabilized. This may be attributed to the protonation of the conserved acidic patches or the copper ligand His87 in poplar plastocyanin, which are shown to have similar pK(a) values. The results are interpreted in a two-step model for complex formation.     

Spectroscopic (FTIR,FT-Raman,NMR and UV) and molecular structure investigations of 1,5-diphenylpenta-2,4-dien-1-one: a combined experimental and theoretical approach

The title molecule 1,5-diphenylpenta-2,4-dien-1-one (cinnamylideneacetophenone, CA) has been synthesised and characterised by FTIR, FT-Raman, NMR and UV–vis spectral analyses. The possible stable conformers of the CA molecule were searched by potential energy surface scan at B3LYP level of theory. The molecular geometry from X-ray determination of the CA molecule in the ground state has been compared using the density functional theory (DFT) with 6-31G(d,p) basis set. The harmonic vibrational modes, the corresponding wavenumbers and IR and Raman intensities of most stable conformer were calculated by the DFT method. The assignments of the fundamentals were proposed on the basis of total energy distribution calculations. The calculated ¹³C and ¹H NMR chemical shifts using gauge including atomic orbitals approach are in good agreement with the observed chemical shifts. The molecular stability and bond strength have been investigated by applying natural bond orbital analysis. Using the time-dependent DFT method, the electronic absorption spectrum of the title compound has been predicted and the electronic transitions within the molecule have been interpreted. The molecular electrostatic potential map was used for predicting possible hydrogen and oxygen bonding sites in the CA molecule.     

Backbone dynamics of plastocyanin in both oxidation states. Solution structure of the reduced form and comparison with the oxidized state

A model-free analysis based on (15)N R(1), (15)N R(2), and (15)N-(1)H nuclear Overhauser effects was performed on reduced (diamagnetic) and oxidized (paramagnetic) forms of plastocyanin from Synechocystis sp. PCC6803. The protein backbone is rigid, displaying a small degree of mobility in the sub-nanosecond time scale. The loops surrounding the copper ion, involved in physiological electron transfer, feature a higher extent of flexibility in the longer time scale in both redox states, as measured from D(2)O exchange of amide protons and from NH-H(2)O saturation transfer experiments. In contrast to the situation for other electron transfer proteins, no significant difference in the dynamic properties is found between the two redox forms. A solution structure was also determined for the reduced plastocyanin and compared with the solution structure of the oxidized form in order to assess possible structural changes related to the copper ion redox state. Within the attained resolution, the structure of the reduced plastocyanin is indistinguishable from that of the oxidized form, even though small chemical shift differences are observed. The present characterization provides information on both the structural and dynamic behavior of blue copper proteins in solution that is useful to understand further the role(s) of protein dynamics in electron transfer processes.