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At least 19 recordsLinked to original sources
PubMed1991

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.

Cytochrome c Group↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

High-resolution solution structure of reduced French bean plastocyanin and comparison with the crystal structure of poplar plastocyanin.

The three-dimensional solution structure of reduced (CuI) plastocyanin from French bean leaves has been determined by distance geometry and restrained molecular dynamics methods using constraints obtained from 1H n.m.r. (nuclear magnetic resonance) spectroscopy. A total of 1244 experimental constraints were used, including 1120 distance constraints, 103 dihedral angle constraints and 21 hydrogen bond constraints. Stereospecific assignments were made for 26 methylene groups and the methyls of 11 valines. Additional constraints on copper co-ordination were included in the restrained dynamics calculations. The structures are well defined with average atomic root-mean-square deviations from the mean of 0.45 A for all backbone heavy atoms and 1.08 A for side-chain heavy atoms. French bean plastocyanin adopts a beta-sandwich structure in solution that is similar to the X-ray structure of reduced poplar plastocyanin; the average atomic root-mean-square difference between 16 n.m.r. structures and the X-ray structure is 0.76 A for all backbone heavy atoms. The conformations of the side-chains that constitute the hydrophobic core of French bean plastocyanin are very well defined. Of 47 conserved residues that populate a single chi 1 angle in solution, 43 have the same rotamer in the X-ray structure. Many surface side-chains adopt highly preferred conformations in solution, although the 3J alpha beta coupling constants often indicate some degree of conformational averaging. Some surface side-chains are disordered in both the solution and crystal structures of plastocyanin. There is a striking correlation between measures of side-chain disorder in solution and side-chain temperature factors in the X-ray structure. Side-chains that form a distinctive acidic surface region, believed to be important in binding other electron transfer proteins, appear to be disordered. Fifty backbone amide protons form hydrogen bonds to carbonyls in more than 60% of the n.m.r. structures; 45 of these amide protons exchange slowly with solvent deuterons. Ten hydrogen bonds are formed between side-chain and backbone atoms, eight of which are correlated with decreased proton exchange. Of the 60 hydrogen bonds formed in French bean plastocyanin, 56 occur in the X-ray structure of the poplar protein; two of the missing hydrogen bonds are absent as a result of mutations. It appears that molecular dynamics refinement of highly constrained n.m.r. structures allows accurate prediction of the pattern of hydrogen bonding.

Amino Acid Sequence↗
PubMed1988

Interaction of plastocyanin with photosystem I: a chemical cross-linking study of the polypeptide that binds plastocyanin.

Plastocyanin has been covalently cross-linked to photosystem I (PSI) by using a water-soluble cross-linker, N-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linking reaction is light stimulated and results in the disappearance of a single 19-kDa subunit of PSI with the formation of a new protein-staining component of 31 kDa. The new product at 31 kDa reacts with both plastocyanin and 19-kDa subunit antibodies. Carboxyl group modified plastocyanin does not form a cross-linked product with PSI, implying that the negatively charged surface-exposed groups on plastocyanin are necessary to stabilize binding. These results demonstrate a specific interaction of plastocyanin with PSI and further implicate a specific protein to which plastocyanin binds to facilitate electron transfer to the P700 reaction center.

Chlorophyll↗
PubMed1997

Comparison of the physiologically equivalent proteins cytochrome c6 and plastocyanin on the basis of their electrostatic potentials. Tryptophan 63 in cytochrome c6 may be isofunctional with tyrosine 83 in plastocyanin.

The blue copper protein plastocyanin and the heme protein cytochrome c6 differ in composition and in structure but perform the same function in the photosynthetic electron-transport chain. We compare these two proteins on the basis of their electrostatic potentials in order to understand the structural basis of their functional equivalence. In the first approach, we use a monopole-dipole approximation of the electrostatic potentials to superimpose the proteins. The resulting alignment suggests that Tyr51 in cytochrome c6 corresponds to Tyr83 in plastocyanin. But since Tyr51 is not conserved in all known cytochrome c6 sequences, a physiological role of this residue is questionable. In a more sophisticated approach, we applied the recently-developed Fame (flexible alignment of molecule ensembles) algorithm, in which molecules are superimposed by optimizing the similarity of their electrostatic potentials with respect to the relative orientation of the molecules. On the basis of the Fame alignments of plastocyanin and cytochrome c6, we analyze the docking and the electron-transfer reactions of these two proteins with its physiological reaction partner cytochrome f. We derive functional analogies for individual amino acids in possible electron-transfer paths in the interprotein redox reactions. We identify two surface patches in cytochrome c6 that may be involved in electron-transfer paths. The hydrophobic patch with the exposed heme edge in cytochrome c6 may be equivalent to the hydrophobic patch with His87 in plastocyanin, whereas Trp63 in cytochrome c6 may be equivalent to Tyr83 in plastocyanin. An aromatic amino acid is present at the position of Trp63 in all known cytochrome c6 sequences. The electronic coupling between the heme and the copper site on the one side and several potentially important amino acid residues on the other is analyzed by the Pathways method. We have proposed recently that Lys65 of cytochrome f and Tyr83 of plastocyanin form a cation-pi system, which may be involved in a two-step mechanism of the electron-transfer reaction between these two proteins from higher plants. Now we corroborate this proposal by analyzing available amino acid sequences.

Algorithms↗
PubMed2006

Photo processes on self-associated cationic porphyrins and plastocyanin complexes 1. Ligation of plastocyanin tyrosine 83 onto metalloporphyrins and electron-transfer fluorescence quenching.

The spectroscopic properties of the self-associated complexes formed between the anionic surface docking site of spinach plastocyanin and the cationic metalloporphyrins, in which the tyrosine 83 (Y83) moiety is placed just below the docking site, tetrakis(N-methyl-4-pyridyl)porphyrin (Pd(II)TMPyP(4+) and Zn(II)TMPyP(4+)), have been studied and reported herein. The fluorescence quenching phenomenon of the self-assembled complex of Zn(II)TMPyP(4+)/plastocyanin has also been discovered. The observed red-shifting of the Soret and Q-bands of the UV-visible spectra, ca. 9 nm for Pd(II)TMPyP(4+)/plastocyanin and ca. 6 nm for the Zn(II)TMPyP(4+)/plastocyanin complexes, was explained in terms of exciton theory coupled with the Gouterman model. Thus, the hydroxyphenyl terminus of the Y83 residue of the self-associated plastocyanin/cationic porphyrin complexes was implicated in the charge-transfer ligation with the central metal atoms of these metalloporphyrins. Moreover, ground-state spectrometric-binding studies between Pd(II)TMPyP(4+) and the Y83 mutant plastocyanin (Y83F-PC) system proved that Y83 moiety of plastocyanin played a critical role in the formation of such ion-pair complexes. Difference absorption spectra and the Job's plots showed that the electrostatic attractions between the cationic porphyrins and the anionic patch of plastocyanin, bearing the nearby Y83 residue, led to the predominant formation of a self-associated 1:1 complex in the ground-state with significantly high binding constants (K = (8.0 +/- 1.1) x 10(5) M(-1) and (2.7 +/- 0.8) x 10(6) M(-1) for Pd(II)TMPyP(4+) and zinc variant, respectively) in low ionic strength buffer, 1 mM KCl and 1 mM phosphate buffer (pH 7.4). Molecular modeling calculations supported the formation of a 1:1 self-associated complex between the porphyrin and plastocyanin with an average distance of ca. 9 A between the centers of mass of the porphyrin and Y83 positioned just behind the anionic surface docking site on the protein surface. The photoexcited singlet state of Zn(II)TMPyP(4+) was quenched by the Y83 residue of the self-associated plastocyanin in a static mechanism as evidenced by steady-state and time-resolved fluorescence experiments. Even when all the porphyrin was complexed (more than 97%), significant residual fluorescence from the complex was observed such that the amplitude of quenching of the singlet state of uncomplexed species was enormously obscured.

Algorithms↗
PubMed1996

Light-induced charge separation between plastocyanin and the iron-sulfur clusters FA and FB in the complex of plastocyanin and photosystem I.

The light-induced electron transfer in a crosslinked complex between plastocyanin and photosystem I from spinach was studied by EPR at low temperature. Electron donation from reduced plastocyanin to P700+ was observed under illumination above a temperature of about 160 K, resulting in a second charge separation and an electron transfer from rereduced P700 to the terminal electron acceptors FA/FB. The charge-separated state Pc oxP700+ [FA/FB](2-) was found to be stable at 15 K. Implications of these results for the kinetic constants of the donation reaction and the backtransfer of electrons from reduced acceptors as well as for the structural models of the terminal acceptors are discussed.

Cross-Linking Reagents↗
PubMed1996

Enforced interaction of one molecule of plastocyanin with two molecules of cytochrome c and an electron-transfer reaction involving the hydrophobic patch on the plastocyanin surface.

Laser flash photolysis is used to study the photoinduced electron-transfer reaction cyt(III)//pc(II) + 3Zncyt --> cyt(III)//pc(I) + Zincyt+ at pH 7.0 and 25 degrees. In the covalent (symbol//) complex cyt(III)//pc(II) the acidic patch in cupriplastocyanin is directly cross-linked to the basic patch in ferricytochrome c. The triplet state of zinc cytochrome c reduces the pc(II) moiety, not the cyt(III) moiety, of the covalent complex. The reaction is strictly bimolecular in the entire range of ionic strength studied, from 1.25 mM to 1.00 M. The two reactants interact only transiently, in a collisional complex, and do not form a persistent complex cyt(III)//pc(II)/Zncyt. Because noncovalent (symbol/) association of three separate protein molecules is far less probable than association of the covalent complex and another protein molecule, we conclude that, without the aid of covalent cross-links, one molecule of plastocyanin will not form a ternary complex with two molecules of cytochrome c, cyt/pc/cyt. Dependence of the rate constant on ionic strength is analyzed in terms of van Leeuwen theory of electrostatic interactions, which recognizes the importance of dipole moments of the proteins. This analysis shows that 3Zncyt reacts with the hydrophobic patch in the pc(II) moiety of the covalent complex cyt(III)//pc(II). At high ionic strength, at which electrostatic interactions are practically abolished, the blue copper site is reduced with approximately equal rates via the hydrophobic patch in the pc(II) moiety of the complex and via the acidic patch in free pc(II). This is evidence that the two distinct patches on the plastocyanin surface are comparable in their intrinsic "conductivity" for electrons coming to the copper site. Positively charged and electroneutral redox partners tend to react at the acidic patch (although not necessarily at the initial docking site in this broad patch) for electrostatic, not electronic, reasons. Earlier theorectical studies disagreed about the relative electronic conductivities of the two patches. This experimental study corroborates very recent theoretical studies that found the two patches to be comparable in the efficiency of electron transfer.

Animals↗
PubMed2004

Brownian dynamics study of cytochrome f interactions with cytochrome c6 and plastocyanin in Chlamydomonas reinhardtii plastocyanin, and cytochrome c6 mutants.

Using Brownian dynamics simulations, all of the charged residues in Chlamydomonas reinhardtii cytochrome c(6) (cyt c(6)) and plastocyanin (PC) were mutated to alanine and their interactions with cytochrome f (cyt f) were modeled. Systematic mutation of charged residues on both PC and cyt c(6) confirmed that electrostatic interactions (at least in vitro) play an important role in bringing these proteins sufficiently close to cyt f to allow hydrophobic and van der Waals interactions to form the final electron transfer-active complex. The charged residue mutants on PC and cyt c(6) displayed similar inhibition classes. Our results indicate a difference between the two acidic clusters on PC. Mutations D44A and E43A of the lower cluster showed greater inhibition than do any of the mutations of the upper cluster residues. Replacement of acidic residues on cyt c(6) that correspond to the PC's lower cluster, particularly E70 and E69, was observed to be more inhibitory than those corresponding to the upper cluster. In PC residues D42, E43, D44, D53, D59, D61, and E85, and in cyt c(6) residues D2, E54, K57, D65, R66, E70, E71, and the heme had significant electrostatic contacts with cyt f charged residues. PC and cyt c(6) showed different binding sites and orientations on cyt f. As there are no experimental cyt c(6) mutation data available for algae, our results could serve as a good guide for future experimental work on this protein. The comparison between computational values and the available experimental data (for PC-cyt f interactions) showed overall good agreement, which supports the predictive power of Brownian dynamics simulations in mutagenesis studies.

Amino Acid Substitution↗
PubMed1996

Binding dynamics and electron transfer between plastocyanin and photosystem I.

The mechanism of the electron transfer from the soluble protein plastocyanin to the multiprotein complex of photosystem I from spinach has been studied in detail. The two kinetic components of P700+ reduction by plastocyanin after a laser flash, showing a constant half-life of 11 microseconds and a variable half-life of the second-order reaction, respectively, are used to monitor the electron transfer from bound and soluble plastocyanin. The effect of increasing concentration of reduced plastocyanin on both of these kinetic components and the competition by oxidized plastocyanin is used to estimate the individual dissociation constants of the complex between the proteins in each of its oxidized and reduced state. The dissociation constant of oxidized plastocyanin is about six times larger than that of 7 microM found for reduced plastocyanin and purified PSI. Consistent with this result the midpoint redox potential of plastocyanin bound to photosystem I either in equilibrium with soluble plastocyanin or after cross-linking to photosystem I is found to be 50-60 mV higher than that of soluble plastocyanin. It is concluded that the driving force of the intracomplex electron transfer is decreased in favor of an optimized turnover of photosystem I. Double-flash excitation shows that oxidized plastocyanin has to leave the complex after the electron transfer before a new reduced plastocyanin molecule can bind to photosystem I. This release of oxidized plastocyanin with a half-life of about 60 microseconds limits the turnover of photosystem I. All data are consistently described by a model including the formation of a complex at a single binding site of photosystem I. Differences in the rate and binding constants are discussed with respect to the structure and the electrostatic and hydrophobic interactions stabilizing the complex as well as their modification by the membrane environment in situ.

Algorithms↗
PubMed1987

The effect of ethylenediamine chemical modification of plastocyanin on the rate of cytochrome f oxidation and P-700+ reduction.

Chemical modification of plastocyanin was carried out using ethylenediamine plus a water-soluble carbodiimide, which has the effect of replacing a negatively charged carboxylate group with a positively charged amino group at pH 6-8. The conditions were adjusted to produce a series of singly and doubly modified forms of plastocyanin. Differences in charge configuration allowed separation of these forms on a Pharmacia fast protein liquid chromatograph using a Mono Q anion exchange column. These forms were used to study the interaction of plastocyanin with its reaction partner cytochrome f. The rate of cytochrome f oxidation was progressively inhibited upon incorporation of increasing numbers of ethylenediamine moieties indicating a positively charged binding site on cytochrome f. However, differential inhibition was obtained for the various singly modified forms allowing mapping of the binding site on plastocyanin. The greatest inhibition was found for forms modified at negatively charged residues Nos. 42-45 and Nos. 59-61 which comprise a negative patch surrounding Tyr-83. In contrast, the form modified at residue No. 68, on the opposite side of the globular plastocyanin molecule, showed the least inhibition. It can be concluded that the binding site for cytochrome f is located in the vicinity of residues Nos. 42-45 and Nos. 59-61. Modification of plastocyanin at residues Nos. 42-45 showed no effect on the rate of P-700+ reduction, suggesting that these residues are not involved in the binding of Photosystem I. However, an increase in the rate of P-700+ reduction was observed for plastocyanins modified at residue No. 68 or Nos. 59-61, which is consistent with the idea that the reaction domain of Photosystem I is negatively charged and Photosystem I binds at the top of the molecule and accepts electrons via His-87 in plastocyanin. These results raise the possibility that plastocyanin can bind both cytochrome f and Photosystem I simultaneously. The effect of ethylenediamine modification on the formal potential of plastocyanin was also examined. The formal potential of control plastocyanin was found to be +372 +/- 5 mV vs. normal hydrogen electrode at pH 7. All modified forms showed a positive shift in formal potential. Singly modified forms showed increases in formal potentials between +8 and +18 mV with the largest increases being observed for plastocyanins modified at residues Nos. 42-45 or Nos. 59-61.

Amino Acid Sequence↗
PubMed1981

Effect of carboxyl group modification on redox properties and electron donation capability of spinach plastocyanin.

Spinach plastocyanin was chemically modified by using a water-soluble carbodiimide to form an amide bond between a protein carboxyl group and one amino group of ethylenediamine. On the average, four plastocyanin carboxyl groups were replaced with positively charged amino groups. Modified plastocyanin facilitated high rates of electron donation to the oxidized P700 reaction center of photosystem I particles in the absence of cations. Control plastocyanin was totally inactive in the absence of divalent cations due to charge repulsion between the negatively charged plastocyanin and photosystem I proteins. The Km for the binding of modified plastocyanin to photosystem I particles was 2.1 uM compared to 36.5 uM for control plastocyanin in the presence of MgCl2. Therefore, chemical modification was mre effective than charge shielding by cations in facilitating the binding of plastocyanin to photosystem I. Chemical modification also increased the midpoint redox potential of plastocyanin from +380 to +420 mV, which indicated an alteration of the copper environment. Ethylenediamine was covalently attached to tryptic peptides from plastocyanin that contained amino acid residues 42-45, 59-61, and 68 which are highly conserved glutamic acid and aspartic acid residues. Chemically altering the charge on these residues changed the activity of plastocyanin which indicates that these amino acids are involved in the ionic regulation of the plastocyanin-PSI interaction. Because chemical modification also altered the environment of the chromophore, certain of these amino acids must be located near the copper site.

Carbodiimides↗
PubMed1996

Analysis of the 1H-NMR chemical shifts of Cu(I)-, Cu(II)- and Cd-substituted pea plastocyanin. Metal-dependent differences in the hydrogen-bond network around the copper site.

To compare cadmium-substituted plastocyanin with copper plastocyanin, the 1H-NMR spectra of CuI-, CuII- and Cd-plastocyanin from pea have been analyzed. Full assignments of the spectra of CuI- and Cd-plastocyanin indicate chemical shift differences up to 1 ppm. The affected protons are located in the four loops that surround the Cu site. The largest differences were found for protons in the hydrogen bond network which stabilizes this part of the protein. This suggests that the chemical shift differences are caused by very small but extensive structural changes in the network upon replacement of CuI by Cd. For CuII-plastocyanin the resonances of 72% of the protons observed in the CuI form have been identified. Protons within approximately 0.9 nm of the CuII were not observed due to fast paramagnetic relaxation. The protons between 0.9-1.7 nm from the CuII showed chemical shift differences up to 0.4 ppm compared to both CuI- and Cd-plastocyanin. These differences can be predicted assuming that they represent pseudocontact shifts. When corrected for the pseudocontact shift contribution, the CuII-plastocyanin chemical shifts were nearly all identical within error to those of the Cd form, but not of the CuI-plastocyanin, indicating that the CuII-plastocyanin structure, in as far as it can be observed, resembles Cd-rather than CuI-plastocyanin. In a single stretch of residues (64-69) chemical shift differences remained between all three forms after correction. The fact that pseudocontact shifts were observed for protons which were not broadened may be attributable to the weaker distance dependence of the pseudocontact shift effect compared to paramagnetic relaxation. This results in two shells around the Cu atom, an inner paramagnetic shell (0-0.9 nm), in which protons are not observed due to broadening, and an outer paramagnetic shell (0.9-1.7 nm), in which protons can be observed and show pseudocontact shifts. It is concluded that Cd-plastocyanin is a suitable redox-inactive substitute for Cu-plastocyanin.

Amino Acid Sequence↗
PubMed1993

The plastocyanin-deficient phenotype of Chlamydomonas reinhardtii Ac-208 results from a frame-shift mutation in the nuclear gene encoding preapoplastocyanin.

Ac-208 is a plastocyanin-deficient mutant of Chlamydomonas reinhardtii that contains only 2-3% of the wild-type level of plastocyanin-encoding mRNA and no detectable plastocyanin. Sequence analysis of the ac-208 plastocyanin-encoding gene reveals a single nucleotide insertion in the first exon compared with the wild-type gene; this alters the reading frame and results in a premature nonsense codon. We have introduced the genomic sequence encoding plastocyanin from a wild-type strain into ac-208 by cotransformation with a selectable marker encoding nitrate reductase. Of 22 nit+ transformants characterized, nine contained additional plastocyanin-encoding sequences (compared with untransformed cells) and each of these nine transformants was found to accumulate the protein. Transformants that do not contain newly introduced plastocyanin sequences retain the plastocyanin-deficient phenotype. The introduced plastocyanin-encoding sequences are stable during mitotic growth in liquid culture over a period of several months, as is expression from the introduced sequences. We suggest that the decreased steady state level of plastocyanin-encoding messages is a consequence of the frame-shift mutation in the structural gene. The ability to complement ac-208 with plastocyanin-encoding sequences will allow the introduction and analysis of in vitro mutagenized plastocyanin sequences in vivo in transgenic C. reinhardtii cells.

Amino Acid Sequence↗
PubMed1996

Voltammetry of Plastocyanin at a Graphite Electrode: Effects of Structure, Charge, and Electrolyte.

Comparative voltammetric studies on Anabaena variabilis plastocyanin (positively charged at neutral pH) and spinach and poplar plastocyanins (negatively charged at neutral pH) have been undertaken at an edge-plane graphite electrode as a function of ionic strength, pH, and Mg(2+) concentration at 3 degrees C. The aim was to provide a more detailed understanding of the influence of the electrode-protein (solution) interfacial characteristics, as well as the variation of the formal potential with both the nature of the plastocyanin species and the pH. As might be expected, some of the interfacial properties associated with the positive charge on A. variabilis plastocyanin are the opposite of those observed with the negatively charged plastocyanins. For example, the linear diffusion component of the mass transport process for A. variabilis plastocyanin under the conditions of cyclic voltammetry is decreased and the radial diffusion component is increased by the addition of Mg(2+), whereas the reverse occurs with poplar and spinach plastocyanins. The voltammetrically determined reversible potentials for A. variabilis plastocyanin are considerably less positive than those for spinach and poplar plastocyanins, in agreement with values calculated from chemically based redox studies. Ionic strength effects, as determined by addition of NaClO(4) over the concentration range 0.005-0.20 M, are negligible for all three proteins. The addition of Mg(2+) causes a significant shift in the reversible potential toward more positive values for spinach and poplar plastocyanin but only a small positive shift for A. variabilis plastocyanin. The difference is attributed to a specific binding effect. The addition of Mg(2+) also dramatically alters the pH dependence of the reversible potential, indicating that the equilibrium between the protonated and unprotonated forms of reduced plastocyanin is modified by binding of Mg(2+) to the protein. It is concluded that the effects of biologically relevant redox-inactive cations such as Mg(2+) or Ca(2+) have to be considered carefully in studies of the redox chemistry of metalloproteins.

Journal Article↗