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.     

Structure comparison between oxidized and reduced plastocyanin from a fern, Dryopteris crassirhizoma.

The X-ray crystal structures of oxidized and reduced plastocyanin obtained from the fern Dryopteris crassirhizoma have been determined at 1.7 and 1.8 A resolution, respectively. The fern plastocyanin is unique in the longer main chain composed of 102 amino acid residues and in the unusual pH dependence due to the pi-pi stacking interaction around the copper site [Kohzuma, T., et al. (1999) J. Biol. Chem. 274, 11817-11823]. Here we report the structural comparison between the fern plastocyanin and other plastocyanins from cyanobacteria, green algae, and other higher plants, together with the structural changes of fern plastocyanin upon reduction. Glu59 hydrogen bonds to the OH of Tyr83, which is thought to be a possible conduit for electrons, in the oxidized state. However, it moves away from Tyr83 upon reduction like poplar plastocyanin.     

Experimental and numerical study of the poplar plastocyanin isoforms using Tyr as a probe for electrostatic similarity and dissimilarity

A detailed study of the tyrosine spectral characteristics was carried out in a broad range of pHs for both isoforms of plastocyanin from poplar. It was found that Tyr 80 is always protonated while Tyr 83 can form a tirosinate at high pHs. The pK(a) of Tyr 83 is practically identical in plastocyanin a and b, but the quenching of its spectrum is different in the isoforms. This provides insights that the acidic patches surrounding Tyr 83 have different electrostatic properties in plastocyanin a and b. The protonation states and the electrostatic interactions were numerically modeled on the existing plastocyanin a structure and on a homology model of plastocyanin b. The results of numerical calculations agree with the experimental findings and identify several differences in the titration behavior of the acidic patches. The difference of the tyrosine quenching pH profiles of the isoforms is rationalized by the differences in the calculated pK(a)'s of amino acids in the neighboring acidic clusters.     

Analysis of the near-ultraviolet absorption and circular dichroic spectra of parsley plastocyanin for the effects of pH and copper center conformation changes

The absorption and circular dichroic (CD) spectra of parsley plastocyanin (PC) were measured in order to determine the effects of changes in primary amino acid sequence on both the copper center and protein components of the PC molecule. The near-ultraviolet (uv) absorption and CD spectra of parsley PC were found to be qualitatively similar to those of spinach, poplar, and lettuce PC, except for the near-uv CD spectrum of the reduced form at low pH (ca. pH 5.0). The CD spectrum of reduced parsley PC in the 250-265 nm wavelength region changes from positive to negative ellipticity upon reduction of pH, and is characterized by a pKa value of 5.7. This pKa value is the same as that for the protonation of the histidine 87 copper ligand, observed by NMR, and the change in conformation of the copper center. Similar processes are believed to occur in the other PC species at lower pH values. Thus, the pH-dependent perturbations of the near-uv CD spectra of reduced PC are interpreted as due to transitions in the reduced copper center. The increase in the near-uv absorption spectrum of reduced PC can be divided into pH-independent and pH-dependent portions. The pH-independent portion resembles the absorption spectrum of tetrahedral Cu(I) metallothionein, suggesting the presence of Cu(I)-Cys 84 and/or Cu(I)-Met 92 charge transfer transitions in the near-uv absorption spectra of reduced PC. The pH dependence of the absorption spectrum changes and the pH difference absorption spectrum indicate that tyrosine residues may contribute to at least a part of the pH-dependent portion of the absorption increase of reduced PC.     

Crystal structure determinations of oxidized and reduced plastocyanin from the cyanobacterium Synechococcus sp. PCC 7942.

The crystal structures of oxidized and reduced plastocyanins from Synechococcus sp. PCC 7942 have been determined at 1.9 and 1.8 A resolution, respectively, at pH 5.0. The protein consists of only 91 amino acid residues, the smallest number known for a plastocyanin, and apparently lacks the mostly conserved acidic patch that is believed to be important for recognition with electron-transfer partners. The protein has two acidic residues, Glu42 and Glu85, around Tyr83, which is thought to be a possible conduit for electrons, but these are neutralized by Arg88 and Lys58. Residue Arg88 interacts with Tyr83 through a pi-pi interaction in which the guanidinium group of the former completely overlaps the aromatic ring of the tyrosine. Reduction of the protein at pH 5.0 causes a lengthening of one Cu-N(His) bond by 0.36 A, despite the small rms deviation of 0.08 A calculated for the backbone atoms. Moreover, significant conformational changes of Arg88 and Lys58, along with the movement of a water molecule adjacent to the OH group of Tyr83, were observed on reduction; the guanidinium group of Arg88 rotates by more than 11 degrees, and the water molecule moves by 0.42 A. The changes around the copper site and the alterations around Tyr83 may be linked to the reduction of the copper.     

Modeling of the electrostatic potential field of plastocyanin

The DelPhi computer program is used to calculate the electrostatic potential field of the photosynthetic electron transport protein plastocyanin. Knowledge of the potential field is important for understanding the mechanisms by which plastocyanin interacts with other charged reagents. The program uses a macroscopic, continuum approach in which the protein and solvent are assigned different dielectric constants, the crystal structure of the protein defines the dielectric boundary, and the ionic strength of the solvent is taken into account. The potential field is determined by numerically solving the Poisson-Boltzmann equation. The field surrounding plastocyanin is characterized by a region of positive potential over the copper center active site, and a region of negative potential over the adjacent association site containing tyrosine 83. The shape and magnitude of the potential field shows a strong dependence on the ionic strength and pH of the solvent. The program is able to accurately predict the effect of the copper center oxidation state on the pKa of a tetranitromethane derivative of tyrosine 83 using an intrinsic protein dielectric constant of 2 to 4. Evidence is also presented that the glutamate 68 side chain is exposed to the solvent to a greater extent in the solution structure of plastocyanin than in the crystal structure.     

Cytochrome f: Structure,function and biosynthesis

Cytochrome f is an intrinsic membrane component of the cytochrome bf complex, transferring electrons from the Rieske FeS protein to plastocyanin in the thylakoid lumen. The protein is held in the thylakoid membrane by a single transmembrane span located near its C-terminus with a globular hydrophilic domain extending into the lumen. The globular domain of the turnip protein has recently been crystallised, offering the prospect of a detailed three-dimensional structure. Reaction with plastocyanin involves localised positive charges on cytochrome f interacting with the acidic patch on plastocyanin and electron transfer via the surface-exposed tyrosine residue (Tyr83) of plastocyanin. Apocytochrome f is encoded in the chloroplast genome and is synthesised with an N-terminal presequence which targets the protein to the thylakoid membrane. The synthesis of cytochrome f is coordinated with the synthesis of the other subunits of the cytochrome bf complex.     

The interaction of nitrotyrosine-83 plastocyanin with cytochromes f and c: pH dependence and the effect of an additional negative charge on plastocyanin.

Spinach plastocyanin was selectively modified using tetranitromethane which incorporates a nitro group ortho to the hydroxyl group of tyrosine 83 (Anderson, G.P., Draheim, J.E. and Gross, E.L. (1985) Biochim. Biophys. Acta 810, 123-131). This tyrosine residue has been postulated to be part of the cytochrome f binding site on plastocyanin. Since the hydroxyl moiety of nitrotyrosine 83 is deprotonated above its pK of 8.3, it provides a useful modification for studying the effect of an extra negative charge on the interaction of plastocyanin with cytochrome f. No effect on cytochrome f oxidation was observed at pH 7 under conditions in which the hydroxyl moiety is protonated. However, the rate of cytochrome f oxidation increased at pH values greater than 8, reaching a maximum at pH 8.6 and decreasing at still higher pH values. The increase was half-maximal at pH 8.3 which is the pK for the hydroxyl moiety on nitrotyrosine 83. In contrast, the rate of cytochrome f oxidation for control plastocyanin was independent of pH from pH 7 to 8.6. These results show that increasing the negative charge on plastocyanin at Tyr-83 increases the ability to react with cytochrome f, supporting the hypothesis that cytochrome f interacts with plastocyanin at this location. In contrast, the reaction of Ntyr-83 plastocyanin with mammalian cytochrome c was independent of pH, suggesting that its mode of interaction with plastocyanin is different from that of cytochrome f. A comparison of the effects of Ntyr-83 modification of plastocyanin with the carboxyl- and amino-group modifications reported previously suggests that plastocyanin binds to cytochrome f in such a way that electrons could be donated to plastocyanin at either of its two binding sites.     

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)     

Correlation of proton release and electrochromic shifts of the optical spectrum due to oxidation of tyrosine in reaction centers from Rhodobacter sphaeroides

Reaction centers from the Y(L167) mutant of Rhodobacter sphaeroides, containing a highly oxidizing bacteriochlorophyll dimer and a tyrosine residue substituted at Phe L167, were compared to reaction centers from the Y(M) mutant, with a tyrosine at M164, and a quadruple mutant containing a highly oxidizing dimer but no nearby tyrosine residue. Distinctive features in the light-induced optical and EPR spectra showed that the oxidized bacteriochlorophyll dimer was reduced by Tyr L167 in the Y(L167) mutant, resulting in a tyrosyl radical, as has been found for Tyr M164 in the Y(M) mutant. In the Y(L167) mutant, the net proton uptake after formation of the tyrosyl radical and the reduced primary quinone ranged from +0.1 to +0.3 H(+)/reaction center between pH 6 and pH 10, with a dependence that is similar to the quadruple mutant but different than the large proton release observed in the Y(M) mutant. In the light-induced absorption spectrum in the 700-1000 nm region, the Y(L167) mutant exhibited unique changes that can be assigned as arising primarily from an approximately 30 nm blue shift of the dimer absorption band. The optical signals in the Y(L167) mutant were pH dependent, with a pK(a) value of approximately 8.7, indicating that the tyrosyl radical is stabilized at high pH. The results are modeled by assuming that the phenolic proton of Tyr L167 is trapped in the protein after oxidation of the tyrosine, resulting in electrostatic interactions with the tetrapyrroles and nearby residues.     

pH dependent conformational changes and electrostatic effects in plastocyanin

Reduction of plastocyanin (PC) caused a change in the electric field at the surface of the molecule which resulted in a 0.3 pH unit increase in the pK_a of a nitrated derivative of Tyr 83. This change in electrical potential could alter the affinity for cytochrome f which is known to bind at this site. Conversely, properties of the copper center, including the pH dependence of the reduction potential, are regulated by the charge on the surface of the molecule. Both the reduction potential and conformation (as measured by near-UV circular dichroic spectra) were pH dependent. Thus the conformation and electrostatic behavior of PC are dependent on oxidiation state, pH and surface charge, raising the possibility that its redox activity is controlled by the pH gradient.     

Kinetic studies on the oxidation of cytochrome b 5 Phe35 mutants with cytochrome c, plastocyanin and inorganic complexes

To illustrate the functions of the aromatic residue Phe35 of cytochrome b(5) and to give further insight into the roles of the Phe35-containing hydrophobic patch and/or aromatic channel of cytochrome b(5), we studied electron transfer reactions of cytochrome b(5) and its Phe35Tyr and Phe35Leu variants with cytochrome c, with the wild-type and Tyr83Phe and Tyr83Leu variants of plastocyanin, and with the inorganic complexes [Fe(EDTA)](-), [Fe(CDTA)](-) and [Ru(NH(3))(6)](3+). The changes at Phe35 of cytochrome b(5) and Tyr83 of plastocyanin do not affect the second-order rate constants for the electron transfer reactions. These results show that the invariant aromatic residues and aromatic patch/channel are not essential for electron transfer in these systems.     

The surface-exposed tyrosine residue Tyr83 of pea plastocyanin is involved in both binding and electron transfer reactions with cytochrome f.

Site-directed mutants of the pea plastocyanin gene in which the codon for the surface-exposed Tyr83 has been changed to codons for Phe83 and Leu83 have been expressed in transgenic tobacco plants. The mutant proteins have been purified to homogeneity and their conformations shown not to differ significantly from the wild-type plastocyanin by 1H-NMR and CD. Overall rate constants for electron transfer (k2) from cytochrome f to plastocyanin have been measured by stopped-flow spectrophotometry and rate constants for binding (ka) and association constants (KA) have been measured from the enhanced Soret absorption of cytochrome f on binding plastocyanin. These measurements allow the calculation of the intrinsic rate of electron transfer in the binary complex. An 8-fold decrease in the overall rate of electron transfer to the Phe83 mutant is due entirely to a decreased association constant for cytochrome f, whereas the 40-fold decrease in the overall rate of electron transfer to the Leu83 mutant is due to weaker binding and a lower intrinsic rate of electron transfer. This indicates that Tyr83 is involved in binding to cytochrome f and forms part of the main route of electron transfer.     

Conformational changes in plastocyanin

The visible and near-uv absorption and circular dichroic spectra were determined for spinach and poplar plastocyanin under a variety of conditions. The visible spectra showed that the copper center was invariant to changes in species, chemical modification with ethylenediamine, and addition of high concentrations of salt [2.7 M (NH4)2SO4]. In contrast, the near-uv spectra were sensitive to these conditions. Reduction of plastocyanin also altered its near-uv absorption and circular dichroic spectra. It is unlikely that these spectral changes were due to charge transfer bands since the near-uv CD spectrum of apo-plastocyanin was almost identical to that of reduced plastocyanin. There were no corresponding changes in the far-uv spectra which monitor protein secondary structure. The most likely explanation is that the protein has a flexible tertiary conformation. Conformational changes may be important in regulating electron transport. If plastocyanin is a mobile electron carrier, differential binding of the oxidized and reduced forms of plastocyanin to its reaction partners cytochrome f and P700 could facilitate electron transport.     

Exploring the function of Tyr83 in bacteriorhodopsin: features of the Y83F and Y83N mutants.

Tyrosine-83, a residue which is conserved in all halobacterial retinal proteins, is located at the extracellular side in helix C of bacteriorhodopsin. Structural studies indicate that its hydroxyl group is hydrogen bonded to Trp189 and possibly to Glu194, a residue which is part of the proton release complex (PRC) in bacteriorhodopsin. To elucidate the role of Tyr83 in proton transport, we studied the Y83F and Y83N mutants. The Y83F mutation causes an 11 nm blue shift of the absorption spectrum and decreases the size of the absorption changes seen upon dark adaptation. The light-induced fast proton release, which accompanies formation of the M intermediate, is observed only at pH above 7 in Y83F. The pK(a) of the PRC in M is elevated in Y83F to about 7.3 (compared to 5.8 in WT). The rate of the recovery of the initial state (the rate of the O --> BR transition) and light-induced proton release at pH below 7 is very slow in Y83F (ca. 30 ms at pH 6). The amount of the O intermediate is decreased in Y83F despite the longer lifetime of O. The Y83N mutant shows a similar phenotype in respect to proton release. As in Y83F, the recovery of the initial state is slowed several fold in Y83N. The O intermediate is not seen in this mutant. The data indicate that the PRC is functional in Y83F and Y83N but its pK(a) in M is increased by about 1.5 pK units compared to the WT. This suggests that Tyr83 is not the main source for the proton released upon M formation in the WT; however, Tyr83 is involved in the proton release affecting the pK(a) of the PRC in M and the rate of proton transport from Asp85 to PRC during the O --> bR transition. Both the Y83F and the Y83N mutations lead to a greatly decreased functionality of the pigment at high pH because most of the pigment is converted into the inactive P480 species, with a pK(a) 8-9.     

Deciphering the structural role of histidine 83 for heme binding in hemophore HasA

Heme carrier HasA has a unique type of histidine/tyrosine heme iron ligation in which the iron ion is in a thermally driven two spin states equilibrium. We recently suggested that the H-bonding between Tyr75 and the invariantly conserved residue His83 modulates the strength of the iron-Tyr75 bond. To unravel the role of His83, we characterize the iron ligation and the electronic properties of both wild type and H83A mutant by a variety of spectroscopic techniques. Although His83 in wild type modulates the strength of the Tyr-iron bond, its removal causes detachment of the tyrosine ligand, thus giving rise to a series of pH-dependent equilibria among species with different axial ligation. The five coordinated species detected at physiological pH may represent a possible intermediate of the heme transfer mechanism to the receptor.     

Iodinated derivatives of the hormone avian pancreatic polypeptide

Reaction of avian pancreatic polypeptide with an iodine monochloride reagent at both pH 4 and pH 7.5 results in the differential modification of the four tyrosine residues in this peptide hormone. A total of 19 distinct iodinated derivatives were isolated by reverse-phase high-performance liquid chromatography, and their sites of iodination were characterized by both tryptic mapping and leucine aminopeptidase techniques coupled with HPLC. The pH 4 reaction produced 16 derivatives which, overall, represented substantial iodination at each tyrosine residue, whereas the pH 7.5 reaction was more directed, producing only 7 derivatives. Iodination at the C-terminal tyrosineamide 36 predominated at both pH values, and diiodo-Tyr 36 was found in the majority of the pH 7.5 derivatives. The relative of the four tyrosine residues with ICl were as follows: at pH 7.5, Tyr 36 much greater than Tyr 21 much greater than Tyr 27 greater than Tyr 7; at pH 4, Tyr 36 greater than Tyr 27 greater than Tyr 7 greater than Tyr 21.     

Trapped tyrosyl radical populations in modified reaction centers from Rhodobacter sphaeroides

The photosynthetic reaction center from the purple bacterium Rhodobacter sphaeroides has been modified such that the bacteriochlorophyll dimer, when it becomes oxidized after light excitation, is capable of oxidizing tyrosine residues. One factor in this ability is a high oxidation-reduction midpoint potential for the dimer, although the location and protein environment of the tyrosine residue appear to be critical as well. These factors were tested in a series of mutants, each of which contains changes, at residues L131, M160, M197, and M210, that give rise to a bacteriochlorophyll dimer with a midpoint potential of at least 800 mV. The protein environment was altered near tyrosine residues that are either present in the wild type or introduced by mutagenesis, focusing on residues that could act as acceptors for the phenolic proton of the tyrosine upon oxidation. These mutations include Ser M190 to His, which is near Tyr L162, the combination of His M193 to Tyr and Arg M164 to His, which adds a Tyr-His pair, and the combinations of Arg L135 to Tyr with Tyr L164 to His, Arg L135 to Tyr with Tyr L144 to Glu, and Arg L135 to Tyr with Tyr L164 to Phe. Radicals were produced in the mutants by using light to initiate electron transfer. The radicals were trapped by freezing the samples, and the relative populations of the oxidized dimer and tyrosyl radicals were determined by analysis of low-temperature electron paramagnetic resonance spectra. The mutants all showed evidence of tyrosyl radical formation at high pH, and the extent of radical formation at Tyr L135 with pH differed depending on the identity of L144 and L164. The results show that tyrosine residues within approximately 10 A of the dimer can become oxidized when provided with a suitable protein environment.     

Redox-induced conformational changes in plastocyanin: an infrared study.

The conformational changes associated with the redox transition of plastocyanin (PC) were investigated by absorption and reaction-induced infrared spectroscopy. In addition to spectral features readily ascribed to beta and turn protein secondary structures, the amide I band shows a major component band at 1647 cm(-1) in both redox states of the protein. The sensitivity of this component to deuteration and increasing temperature suggests that PC adopts an unusual secondary structure in solution, which differs from those described for other type I copper proteins, such as azurin and halocyanin. The conformations of oxidized and reduced PC are different, as evidenced (1) by analysis of their amide I band contour and the electrochemically induced oxidized-minus-reduced difference spectrum and (2) by their different thermal stability. The redox-induced difference spectrum exhibits a number of difference bands within the conformationally sensitive amide I band that could be assigned to peptide C=O modes, in light of their small shift upon deuteration, and to signals attributable to side chain vibrational modes of Tyr residues. Lowering the pH to 4.8 induces destabilization of both redox states of the protein, more pronounced for reduced PC, without significantly affecting their secondary structure. Besides the conformational differences obtained at neutral pH, the oxidized-minus-reduced difference spectrum shows two broad and strong negative bands at 1405 and 1571 cm(-1), assigned to COO(-) vibrations, and a broad positive band at 1710 cm(-1), attributed to the C=O vibration of a COOH group(s). These bands are indicative of a protonation of (an) Asp or Glu side chain(s) upon plastocyanin oxidation at acidic pH.     

Crystal structure of spinach plastocyanin at 1.7 A resolution.

The crystal structure of plastocyanin from spinach has been determined using molecular replacement, with the structure of plastocyanin from poplar as a search model. Successful crystallization was facilitated by site-directed mutagenesis in which residue Gly8 was substituted with Asp. The region around residue 8 was believed to be too mobile for the wild-type protein to form crystals despite extensive screening. The current structure represents the oxidized plastocyanin, copper (II), at low pH (approximately 4.4). In contrast to the similarity in the core region as compared to its poplar counterpart, the structure shows some significant differences in loop regions. The most notable is the large shift of the 59-61 loop where the largest shift is 3.0 A for the C(alpha) atom of Glu59. This results in different patterns of electrostatic potential around the acidic patches for the two proteins.