Localization of the reaction side of plastocyanin from immunological and kinetic studies with inside-out thylakoid vesicles

(1) The effect of four active antisera against plastocyanin on Photosystem I-driven electron transport and phosphorylation was investigated in spinach chloroplasts. Partial inhibition of electron transport and stimulation of plastocyanin-dependent phosphorylation were sometimes observed after adding amounts of antibodies which were in large excess and not related to the plastocyanin content of the chloroplasts. This indicates effects of the antibodies on the membrane. (2) The antibodies against plastocyanin neither directly nor indirectly agglutinated unbroken chloroplast membranes. (3) The plastocyanin content of right-side-out and inside-out thylakoid vesicles isolated by aqueous polymer two-phase partition from chloroplasts disrupted by Yeda press treatment was determined by quantitative rocket electroimmunodiffusion. Right-side-out vesicles retained about 25%, inside-out vesicles none of the original amount of plastocyanin. (4) The effect of externally added plastocyanin on the reduction of P-700 was studied by monitoring the absorbance changes at 703 nm after a long flash. In inside-out vesicles P-700 was reduced by the added plastocyanin but not in right-side-out vesicles and class II chloroplasts. These results provide strong evidence for a function of plastocyanin at the internal side of the thylakoid membrane.     

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.     

Plastocyanin conformation. The effect of nitrotyrosine modification and pH

Plastocyanin isolated from several species including spinach, poplar, and lettuce showed conformational changes both upon reduction and upon lowering the pH as determined by near-ultraviolet absorption and fluorescence measurements. The fluorescence excitation maximum was at 278 nm for all species of plastocyanin measured. In the case of spinach, the emission maximum was at 310–312 nm, similar to a tyrosine residue in solution. The fluorescence intensity increased 22% upon reduction of plastocyanin at pH 7.0. In poplar plastocyanin, the emission maximum was shifted to 335 nm and increased only 10% upon reduction. The 335 nm emission peak observed in poplar plastocyanin is attributed to Tyr 80 which is hydrogen bonded to a carbonyl group on the protein backbone. Tyr 83 was also shown to undergo fluorescence changes upon reduction since the redox state-dependent fluorescence changes decreased for a nitrotyrosine (nitrotyrosine-plastocyanin) derivative of this residue. These results show that the east face of the molecule, which contains both Tyr 80 and 83 as well as a possible binding site [1,2], undergoes conformational changes upon reduction. These conformational changes may be involved in promoting smooth electron transport between plastocyanin and its reaction partners. Both the absorption and fluorescence were found to be pH dependent. The quantum yield for fluorescence increased sharply below pH 6 for both oxidized and reduced spinach plastocyanin. This may be related to the appearance of a redox-inactive form of reduced plastocyanin [3]. The conformational changes observed at low pH may provide a mechanism for control of electron transport by the proton gradient. Low concentrations of CaCl₂ (10 mM) had no effect on plastocyanin fluorescence. However, addition of 2.7 M (NH₄)₂SO₄ eliminated the redox-dependent fluorescence changes.     

Electron transfer reactions of chemically modified plastocyanin with P700 and cytochrome f. Importance of local charges

Chemically modified spinach plastocyanin, in which negatively charged carboxyl residues are replaced with positively charged amino residues, has been prepared. Four distinct species of chemically modified plastocyanin, having 1 to 4 mol of modified carboxyl residue per mol of plastocyanin, could be separated by ion-exchange chromatography on DEAE-Sephacel. The rate of electron transfer from reduced cytochrome f to oxidized singly substituted plastocyanin was 30% of that of the native unmodified plastocyanin, and the reaction rate decreased further with increasing number of modified carboxyl residues. These results indicate the importance of electrostatic interactions between the negative charges on plastocyanin and the positive charges on cytochrome f in this reaction. Since the overall net charge of cytochrome f is negative at neutral pH, the positive charges on cytochrome f involved in the reaction should be localized ones. On the other hand, the rates of electron transfer from reduced singly and doubly substituted plastocyanin to photooxidized P700 in the P700-chlorophyll alpha protein complex were similar to that of native plastocyanin, which suggests that these carboxyl residues have only a minor role in the electron transfer to P700. Although divalent cation is essential for the electron transfer from native plastocyanin to P700 at neutral pH, the triply substituted plastocyanin could donate electrons to P700 even without MgCl2, and the rate of this reaction reached the maximum at a low concentration of MgCl2 (less than 2.5 mM). The modification of four carboxyl residues per plastocyanin molecule activated this reaction to the maximum level without MgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the intermolecular complex between plastocyanin and cytochrome f from spinach

In oxygenic photosynthesis, plastocyanin shuttles electrons between the membrane-bound complexes cytochrome b6f and photosystem I. The homologous complex between cytochrome f and plastocyanin, both from spinach, is the object of this study. The solution structure of the reduced spinach plastocyanin was determined using high field NMR spectroscopy, whereas the model structure of oxidized cytochrome f was obtained by homology modeling calculations and molecular dynamics. The model structure of the intermolecular complex was calculated using the program AUTODOCK, taking into account biological information obtained from mutagenesis experiments. The best electron transfer pathway from the heme group of cytochrome f to the copper ion of plastocyanin was calculated using the program HARLEM, obtaining a coupling decay value of 1.8 x 10(-4). Possible mechanisms of interaction and electron transfer between plastocyanin and cytochrome f were discussed considering the possible formation of a supercomplex that associates one cytochrome b6f, one photosystem I, and one plastocyanin.     

Multiple sites of DCIP reduction by sonicated Oat chloroplasts: Role of plastocyanin

1. Oat chloroplasts, in the presence of 0.02 M methylamine, reduce 2,6 dichlorophenolindophenol (DCIP) at a rate of 350–500 μmoles/mg chl per h, in saturating light. Brief sonication for approx. 1 min lowers the rate to approx. 50 μmoles/mg chl per h; longer sonication does not reduce activity further. During brief sonication, plastocyanin is lost from the chloroplasts. When plastocyanin is added back to sonicated fragments, DCIP reduction is approximately doubled to 100 μmoles/mg chl per h.

2. When oxidized plastocyanin is added, a transient is observed when light is first turned on: this is due to a reduction of the plastocyanin before DCIP reduction begins. When reduced plastocyanin is added, a different transient occurs: this is due to a fast photoreduction of DCIP by the plastocyanin and is followed by the slower steady state reduction of DCIP by water. When light is turned off before complete reduction of DCIP, a transient reduction of oxidized plastocyanin by reduced DCIP is seen. Insensitivity of these transients to 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and the greater effectiveness of 710 nm light, along with the known capacity of plastocyanin to mediate electron transfer to System I, prove that an intrinsically fast reduction of DCIP occurs at a site close to the primary photoreduced product of System I.

3. After brief sonication and washing, no residual plastocyanin was detected in chloroplast fragments, and the rate of the slow DCIP reduction (about 50μmoles/mg chl per h) sustained by such fragments was essentially identical to that maintained by fragments of mutants lacking System I activity. Following et al.⁹, the simplest explanation for this slow DCIP reduction is that is occurs at a site close to System II and the system I is not involved.

4. A very slow transient reduction of DCIP occurs after extinguishing light; this presumably involves another reduction site close to System II, as suggested by ⁹.     

EFFECT OF PLASTOCYANIN ON PHOTOPHOSPHORYLATION IN ISOLATED CHLOROPLASTS

The effect of addition of plastocyanin on photophosphorylationin isolated chloroplasts was studied in comparison with itseffect on photoreduction. Catalytic amounts of added plastocyaninstimulated the HILL reaction with NADP as oxidant, but the photophosphorylationcoupled to this reaction was not influenced by the additionof plastocyanin. The addition of NH₄+ or the complete phosphorylatingsystem did not affect the plastocyanin-induced increase in rateof the photoreduction. The rate of photoreduction of NADP, FMN,or indigo carmine in the presence of DPIP and ascorbate wasmuch accelerated by added plastocyanin, but these reactionswere not coupled to ATP formation. As reported earlier, isolated chloroplasts can utilize plastocyaninas oxidant in the HILL reaction. This reaction was found tobe accompanied by ATP formation, but the efficiency in thiscase was somewhat lower than in photophosphorylation coupledto the HILL reaction with the usual oxidants. The possible mechanism of these reactions is described brieflyand a scheme for reactions of plastocyanin in the electron transferin chloroplasts is presented. (Received July 20, 1965; )     

Preliminary crystallographic data for plastocyanins from an alga (Enteromorpha prolifera) and from cucumber (Cucumis sativus)

The plastocyanins from a green alga (Enteromorpha prolifera) and cucumber (Cucumis sativus) have been crystallized. Crystal data are as follows: E. prolifera plastocyanin, space group I4, a = b = 53.9 A, c = 59.4 A, Z = 8; C. sativus plastocyanin, space group P4(1) (or P4(3) ), a = b = 66.7 A, c = 46.0 A, Z = 8. Accordingly, the asymmetric units of the crystals contain one and two molecules, respectively.     

Cytochrome f and plastocyanin kinetics in Chlorella pyrenoidosa. II. Reduction kinetics and electric field increase in the 10 ms range

On dark-adapted Chlorella, after one flash, plastocyanin (PC) undergoes reduction with a half-time of 7 ms. After 4 or 5 flashes, the reduction of PC+ in the 10 ms range is suppressed, and the level of oxidized plastocyanin increases during the next few flashes before reaching a stationary value. Cytochrome f exhibits approximately the same pattern. The reduction of PC+ and cytochrome f+ in the 10 ms range is correlated with an increase of the electrice field named phase b (Joliot, P. and Delosme, R., Biochim. Biophys. Acta 357 (1974) 267-284). Both need the presence of a compound R' in the reduced state. A dark electron transfer involving a carrier of electrons across the membrane, a proton carrier, R' as terminal reducant, PC+ and cytochrome f+ as terminal oxidants, would account for this field generation. Cooperation between the electron transfer chains is implied at the level of plastocyanin oxidation. An equilibrium constant of about 2 is observed between cytochrome f and plastocyanin before 1 ms and after 500 ms after the photochemical reactions. We observe that cytochrome f and plastocyanin are not connected from 1 to 100 ms after a photochemical reaction. The equilibrium constant between plastocyanin and P-700 remains large [20] under these conditions.     

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.     

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.     

Electrostatic analysis and Brownian dynamics simulation of the association of plastocyanin and cytochrome f.

The oxidation of cytochrome f by the soluble cupredoxin plastocyanin is a central reaction in the photosynthetic electron transfer chain of all oxygenic organisms. Here, two different computational approaches are used to gain new insights into the role of molecular recognition and protein-protein association processes in this redox reaction. First, a comparative analysis of the computed molecular electrostatic potentials of seven single and multiple point mutants of spinach plastocyanin (D42N, E43K, E43N, E43Q/D44N, E59K/E60Q, E59K/E60Q/E43N, Q88E) and the wt protein was carried out. The experimentally determined relative rates (k(2)) for the set of plastocyanin mutants are found to correlate well (r(2) = 0.90 - 0.97) with the computed measure of the similarity of the plastocyanin electrostatic potentials. Second, the effects on the plastocyanin/cytochrome f association rate of these mutations in the plastocyanin "eastern site" were evaluated by simulating the association of the wild type and mutant plastocyanins with cytochrome f by Brownian dynamics. Good agreement between the computed and experimental relative rates (k(2)) (r(2) = 0.89 - 0.92) was achieved for the plastocyanin mutants. The results obtained by applying both computational techniques provide support for the fundamental role of the acidic residues at the plastocyanin eastern site in the association with cytochrome f and in the overall electron-transfer process.     

Effects of pH on the kinetics of redox reactions in and around the cytochromebf complex in an isolated system

Rate-coefficients describing the electron transfer reactions between P700 and plastocyanin, between cytochromef in cytochromebf complexes and plastocyanin, and between decyl plastoquinol and cytochromebf complexes were determined as a function of pH in the range 4–10 from flash-induced absorbancy changes at four wavelengths. The reactions between P700 and plastocyanin, and between cytochromef and plastocyanin were optimised when there was electrostatic interaction between ionised acidic groups in plastocyanin with a pK_a of 4.3–4.7 and ionised basic constituents in P700 (assumed to be in the PSI-F subunit) and in cytochromef, with a pK_b of 8.9–9.4. The basic groups are thought to be lysine rather than arginine. This mechanism agrees with that inferred from effects of ionic strength changes on rate-coefficients. The relation between the second-order rate-coefficient for decyl plastoquinol oxidation by thebf complex and pH was characterised by a pK_a of 6.1. This is interpreted as showing that the anion radical form of that quinol, which has a pK_a of 6, and which becomes progressively protonated when pH is changed from 7 to 5, is essential to reduce cytochromeb-563 (low potential) during quinol oxidation. Above pH 9, permanent effects were observed on this rate-coefficient, which were absent in the reactions between P700, plastocyanin and cytochromef.     

On the functional organization of electron transport from plastoquinone to Photosystem I

Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl₂, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700⁺ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700⁺ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b₆ - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700⁺. The P-700 kinetics indicate an electron transfer from the cytochrome b₆ - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.     

Direct electrochemistry of spinach plastocyanin at a lipid bilayer-modified electrode: cyclic voltammetry as a probe of membrane-protein interactions.

The electron transfer reactions between a lipid bilayer-modified gold electrode and oxidized spinach plastocyanin have been studied by cyclic voltammetry, using either an electrically neutral phosphatidylcholine (PC) bilayer or a positively charged PC bilayer containing 40 mol% dimethyldioctadecylammonium chloride, at two ionic strengths of electrolyte (0.02 and 0.2 M NaClO4). Plastocyanin was found to interact strongly enough with the lipid membrane to support an efficient electron transfer reaction with the electrode. The interaction forces, and therefore the mode of diffusion of plastocyanin molecules to the electrode, which limits the electron transfer rate, could be controlled by the PC concentration. At low lipid concentrations (0-5 mg/ml), electrostatically attractive interactions between specific microelectroactive sites on the surface of the lipid membrane and plastocyanin molecules predominate, producing a radial mode of diffusion of the protein molecules to the electrode surface. On the other hand, at high lipid concentrations (greater than 5 mg/ml), interaction between plastocyanin and the lipid membrane occurs via hydrophobic forces, and a linear diffusion of protein molecules limits the electron transfer process. These observations support and extend other experimental and theoretical results which indicate two possible sites on the surface of the plastocyanin molecule, one hydrophobic and one negatively charged, which are able to participate in electron transfer reactions. We conclude that electrochemical measurements with the present system provide a new approach to the study of redox protein-membrane interactions.     

Copper(I)-incorporation into spinach apoplastocyanin

Spinach plastocyanin was converted into the apoprotein. CuSO₄ and oxidized Cu(II)- thionein reacted with the apoprotein to Cu(II) plastocyanin. Cu(I) transfer from Cu(I)0-thionein was only 15%. The structural analogue of the copper thiolate chromophore [Cu(I)(thiourea)₃]Cl as well as [Cu(CH₃CN)₄]ClO₄ successfully formed the Cu(I)- holoprotein. Characteristic circular dichroism bands at θ₂₈₄ (?5300 deg·cm²·dmol^?1 and θ₃₁₀ (+3300 deg·cm²·dmol^?1) were seen. Upon oxidation with ferricyanide and dialysis against phosphate buffer the correct Cu(II) binding into the active centre of Cu(II) plastocyanin was confirmed by EPR-measurements. The use of [Cu(I)(thiourea)₃] Cl as a convenient Cu(I) source for reconstitution studies on copper proteins is highly recommended.     

Kinetics of electron transfer between plastocyanin and the soluble CuA domain of cyanobacterial cytochrome c oxidase

It has been shown that efficient functioning of photosynthesis and respiration in the cyanobacterium Synechocystis PCC 6803 requires the presence of either cytochrome c6 or plastocyanin. In order to check whether the blue copper protein plastocyanin can act as electron donor to cytochrome c oxidase, we investigated the intermolecular electron transfer kinetics between plastocyanin and the soluble CuA domain (i.e. the donor binding and electron entry site) of subunit II of the aa3-type cytochrome c oxidase from Synechocystis. Both copper proteins were expressed heterologously in Escherichia coli. The forward and the reverse electron transfer reactions were studied yielding apparent bimolecular rate constants of (5.1+/-0.2) x 10(4) M(-1) s(-1) and (8.5+/-0.4) x 10(5) M(-1) s(-1), respectively (20 mM phosphate buffer, pH 7). This corresponds to an apparent equilibrium constant of 0.06 in the physiological direction (reduction of CuA), which is similar to Keq values calculated for the reaction between c-type cytochromes and the soluble fragments of other CuA domains. The potential physiological role of plastocyanin in cyanobacterial respiration is discussed.     

Inhibition of Plastocyanin to P700+ Electron Transfer in Chlamydomonas reinhardtii by Hyperosmotic Stress

Oxygen electrode and fluorescence studies demonstrate that linear electron transport in the freshwater alga Chlamydomonas reinhardtii can be completely abolished by abrupt hyperosmotic shock. We show that the most likely primary site of inhibition of electron transfer by hyperosmotic shock is a blockage of electron transfer between plastocyanin (PC) or cytochrome c(6) and P(700). The effects on this reaction were reversible upon dilution of the osmolytes and the stability of plastocyanin or photosystem (PS) I was unaffected. Electron micrographs of osmotically shocked cells showed a significant decrease in the thylakoid lumen volume. Comparison of estimated lumenal width with the x-ray structures of plastocyanin and PS I suggest that lumenal space contracts during HOS so as to hinder the movement of docking to PS I of plastocyanin or cytochrome c(6).     

The purification of cytochrome f and plastocyanin using affinity chromatography

Both plastocyanin and cytochrome f were purified using a combination of affinity chromatography together with established methods. Plastocyanin was partially purified using the method of Davis and San Pietro (Anal. Biochem. 95 (1979) 254-259), after which it was further purified using a column of cytochrome c covalently attached to Sepharose 4B. The affinity column was prepared using the method of Godinot and Gautheron (Methods Enzymol. 54 (1979) 112-114). The final purity index ratio (A278/A597) was less than 1.2, which is equal to that obtained using the more expensive FPLC procedure (Anderson, G.P., Sanderson, D.G., Lee, C.H., Durell, S., Anderson, L.B. and Gross, E.L. (1987) Biochim. Biophys. Acta 894, issue 3). Cytochrome f was partially purified using a modification of the method of Matazaki et al. (Plant Cell. Physiol. 16 (1975) 237-246) and bound to an affinity column of plastocyanin covalently attached to Sepharose 4B. Cytochrome f purified using this procedure had a purity index ratio (A554.5/A277) of 1.2. Both proteins are tyrosine proteins containing no tryptophan residues. After the affinity chromatography step, the fluorescence emission spectrum of either plastocyanin or cytochrome f was typical of a tyrosine protein free from tryptophan contamination.