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1.
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.  相似文献   

2.
Chemical modification of plastocyanin was carried out using 4-chloro-3,5-dinitrobenzoic acid, which has the effect of replacing positive charges on amino groups with negatively charged carboxyl groups. Four singly-modified forms were obtained which were separated using anion exchange FPLC. The four forms were modified at the N-terminal valine and at lysines 54, 71 and 77. The rates of reaction with mammalian cytochrome c were increased for all four modified plastocyanins. In contrast, the rates of reaction with cytochrome f were inhibited for the forms modified at residues 1, 54 and 77, whereas no effect was observed for the form modified at residue 71. Modification had no effect on either the midpoint redox potential or the reaction with K3Fe(CN)6. These results are consistent with a model in which charged residues on plastocyanin located at or near the binding site for cytochrome f recognize the positively-charged binding site on cytochrome f. In contrast, charged residues located at points on plastocyanin distant from the cytochrome f binding site recognize the net negative charge on the cytochrome f molecule. Based on these considerations, Glu-68 may be within the interaction sphere of cytochrome f, suggesting that cytochrome f may donate electrons to plastocyanin at either Tyr-83 or His-87.  相似文献   

3.
The plastocyanin binding domain of photosystem I.   总被引:2,自引:0,他引:2       下载免费PDF全文
The molecular recognition between plastocyanin and photosystem I was studied. Photosystem I and plastocyanin can be cross-linked to an active electron transfer complex. Immunoblots and mass spectrometric analysis of proteolytic peptides indicate that the two negative patches conserved in plant plastocyanins are cross-linked with lysine residues of a domain near the N-terminus of the PsaF subunit of photosystem I. Conversion of these negative to uncharged patches of plastocyanin by site-directed mutation D42N/E43Q/D44N/E45Q and E59Q/E60Q/D61N respectively, reveals the first patch to be essential for the electrostatic interaction in the electron transfer complex with photosystem I and the second one to lower the redox potential. The domain in PsaF, not found in cyanobacteria, is predicted to fold into two amphipathic alpha-helices. The interacting N-terminal helix lines up six lysines on one side which may guide a fast one-dimensional diffusion of plastocyanin and provide the electrostatic attraction at the attachment site, in addition to the hydrophobic interaction in the area where the electron is transferred to P700 in the reaction center of photosystem I. This two-step interaction is likely to increase the electron transfer rate by more than two orders of magnitude in plants as compared with cyanobacteria. Our data resolve the controversy about the function of PsaF.  相似文献   

4.
The role of electrostatic interactions in determining the rate of electron transfer between cytochrome f and plastocyanin has been examined in vitro with mutants of turnip cytochrome f and mutants of pea and spinach plastocyanins. Mutation of lysine residues Lys58, Lys65 and Lys187 of cytochrome f to neutral or acidic residues resulted in decreased binding constants and decreased rates of electron transfer to wild-type pea plastocyanin. Interaction of the cytochrome f mutant K187E with the pea plastocyanin mutant D51K gave a further decrease in electron transfer rate, indicating that a complementary charge pair at these positions could not compensate for the decreased overall charge on the proteins. Similar results were obtained with the interaction of the cytochrome f mutant K187E with single, double and triple mutants of residues in the acidic patches of spinach plastocyanin. These results suggest that the lysine residues of the basic patch on cytochrome f are predominantly involved in long-range electrostatic interactions with plastocyanin. However, analysis of the data using thermodynamic cycles provided evidence for the interaction of Lys187 of cytochrome f with Asp51, Asp42 and Glu43 of plastocyanin in the complex, in agreement with a structural model of a cytochrome f-plastocyanin complex determined by NMR.  相似文献   

5.
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.  相似文献   

6.
Two mutants of plastocyanin have been constructed by site-directed mutagenesis in spinach and pea to elucidate the binding and electron transfer properties between plastocyanin and spinach Photosystem 1. The conserved, surface-exposed Tyr-83 has been replaced by phenylalanine and leucine in plastocyanin from both species and the proteins have been expressed in Escherichia coli. The reaction mechanism of electron transfer from plastocyanins to photooxidized P700 in Photosystem 1 has been studied by laser-flash absorption spectroscopy. The experimental data were interpreted with a model involving a rate-limiting conformational change, preceding the intracomplex electron transfer. The pea proteins show an overall facilitated reaction with spinach Photosystem 1, compared to spinach plastocyanins. The changes are small but significant, indicating a more efficient electron transfer within the transient complex. In addition, for the spinach leucine mutant, the equilibrium within the plastocyanin-Photosystem 1 complex is more displaced towards the active conformation than for the corresponding wild-type. Absorption spectra, EPR and reduction potentials for the mutants are similar to those of the corresponding wild-type, although small shifts are observed in the spectra of the Tyr83Leu proteins. Based on these results, it is suggested that Photosystem 1 from spinach is capable of using both pea and spinach plastocyanin as an efficient electron donor and that the former even can stimulate the Photosystem 1 reduction. The origin of the stimulation is discussed in terms of differences in surface-exposed residues. Since the effects of the mutations are small, it can be concluded that electron transfer to Photosystem 1 does not occur via Tyr-83.Abbreviations cyt- cytochrome - IPTG- isopropyl--d-thiogalactopyranoside - P,P700- reaction-center chlorophyll - Pc- plastocyanin - PS 1- Photosystem 1 - SDS-PAGE- sodium dodecyl sulfate polyacrylamide gel electrophoresis - WT- wild-type  相似文献   

7.
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.  相似文献   

8.
A combination of site-directed mutagenesis and NMR chemical shift perturbation analysis of backbone and side-chain protons has been used to characterize the transient complex of the photosynthetic redox proteins plastocyanin and cytochrome f. To elucidate the importance of charged residues on complex formation, the complex of cytochrome f and E43Q/D44N or E59K/E60Q spinach plastocyanin double mutants was studied by full analysis of the (1)H chemical shifts by use of two-dimensional homonuclear NMR spectra. Both mutants show a significant overall decrease in chemical shift perturbations compared with wild-type plastocyanin, in agreement with a large decrease in binding affinity. Qualitatively, the E43Q/D44N mutant showed a similar interaction surface as wild-type plastocyanin. The interaction surface in the E59K/E60Q mutant was distinctly different from wild type. It is concluded that all four charged residues contribute to the affinity and that residues E59 and E60 have an additional role in fine tuning the orientation of the proteins in the complex.  相似文献   

9.
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)  相似文献   

10.
The reduction of plastocyanin by cytochromes c and f has been investigated with mutants of spinach plastocyanin in which individual, highly conserved surface residues have been modified. These include Leu-12 and Phe-35 in the 'northern' hydrophobic patch and Tyr-83 and Asp-42 in the 'eastern' acidic patch. The differences observed all involved binding rather than the intrinsic rates of electron transfer. The Glu-12 and Ala-12 mutants showed small but significant decreases in binding constant with cytochrome c, even though the cytochrome is not expected to make contact with the northern face of plastocyanin. These results, and small changes in the EPR parameters, suggested that these mutations cause small conformational changes in surface residues on the eastern face of plastocyanin, transmitted through the copper centre. In the case of cytochrome f, the Glu-12 and Ala-12 mutants also bound less strongly, but Leu12Asn showed a marked increase in binding constant, suggesting that cytochrome f can hydrogen bond directly to Asn-12 in the reaction complex. A surprising result was that the kinetics of reduction of Asp42Asn were not significantly different from wild type, despite the loss of a negative charge.  相似文献   

11.
The interaction between cytochrome f and its electron acceptor plastocyanin (PC) was studied. To address the question of which specific regions and which of the positively charged residues of cytochrome f are important for the interaction with the negatively charged residues of PC we have used two different experimental approaches. Cytochrome f was proteolytically cleaved and fragments that could bind to a PC-affinity column were isolated. The smallest of these fragments was analysed to give information on the minimum structural requirement for binding to PC. By this procedure, we identified a peptide of approx. 11 kDa, containing the heme binding site, and having an N-terminal sequence identical to that of the mature cytochrome f. This finding suggests that the first 90 amino acids of cytochrome f contain at least some of the residues interacting with PC. The second approach involved modification of Arg residues of cytochrome f with the specific chemical modifier, hydroxyphenylglyoxal (HPG). Cytochrome f modification was performed in the absence of PC to enable identification of residues that are protected from modification when PC is bound to cytochrome f. Two peptides containing Arg residues which are modified in the absence of PC, but are not modified when PC is present, were isolated. Sequence analysis of these two peptides revealed that Arg residues no. 88 and 154 of cytochrome f are the residues that are protected from modification when cytochrome f is bound to PC, suggesting a role for these residues in the binding of cytochrome f to PC.  相似文献   

12.
Jerry Brand  Anthony San Pietro 《BBA》1973,325(2):255-265
1. Chloroplast fragments from either Chlamydomonas reinhardi or spinach, which lack plastocyanin, or from Euglena gracilis depleted of cytochrome c552, require a large excess of exogenously added plastocyanin or cytochrome c552 to restore Photosystem I activity.2. In the presence of a small amount of polylysine, Photosystem I activity of chloroplast fragments is stimulated greatly by plastocyanin or cytochrome c552, and the reaction is saturated at a lower concentration of these proteins. Higher concentrations of polylysine inhibit Photosystem I activity; the inhibition is not reversed by plastocyanin or cytochrome c552.3. Salt protects chloroplast fragments from stimulation by polylysine plus plastocyanin or cytochrome c552, and also reverses this stimulation.4. The data suggest that polylysine, at low concentration, enhances binding of plastocyanin or cytochrome c552 to chloroplast membranes, thereby increasing the effective concentration at their site of function. The total inhibition of Photosystem I activity, independent of the presence of plastocyanin or cytochrome c552, at higher polylysine concentrations is similar probably to that observed previously in chloroplasts which retain their plastocyanin.  相似文献   

13.
Fifteen ancestral genotypes of United States soybean cultivars were screened for differences in photosynthetic electron transport capacity using isolated thylakoid membranes. Plants were grown in controlled environment chambers under high or low irradiance conditions. Thylakoid membranes were isolated from mature leaves. Photosynthetic electron transport was assayed as uncoupled Hill activity using 2,6-dichlorophenolindophenol (DCIP). Soybean electron transport activity was dependent on genotype and growth irradiance and ranged from 6 to 91 mmol DCIP reduced [mol chlorophyll]–1 s–1. Soybean plastocyanin pool size ranged from 0.1 to 1.3 mol plastocyanin [mol Photosystem I]–1. In contrast, barley and spinach electron transport activities were 140 and 170 mmol DCIP reduced [mol chlorophyll]–1 s–1, respectively, with plastocyanin pool sizes of 3 to 4 mol plastocyanin [mol Photosystem I]–1. No significant differences in the concentrations of Photosystem II, plastoquinone, cytochrome b6f complexes, or Photosystem I were observed. Thus, genetic differences in electron transport activity were correlated with plastocyanin pool size. The results suggested that plastocyanin pool size can vary significantly and may limit photosynthetic electron transport capacity in certain species such as soybean. Soybean plastocyanin consisted of two isoforms with apparent molecular masses of 14 and 11 kDa, whereas barley and spinach plastocyanins each consisted of single polypeptides of 8 and 12 kDa, respectively.Abbreviations DAP days after planting - DCIP 2,6-dichlorophenolindophenol - LiDS lithium dodecyl sulfate - PPFD photosynthetic photon flux density (mol photons m–2 s–1) - PS I Photosystem I - PS II Photosystem II - P700 reaction center of Photosystem I The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.  相似文献   

14.
Polylysine was found to increase the efficiency of electron donation from plastocyanin to P700+ in highly resolved Photosystem I subchloroplast particles. This increase in efficiency is due to a decrease in the Km for plastocyanin in the presence of polylysine and is similar to results obtained with divalent cations. Cytochrome f photooxidation is observed in the presence of plastocyanin and divalent cations but not in the presence of plastocyanin and polylysine. The results indicate that the binding of polylysine to plastocyanin prevents the reduction of plastocyanin by cytochrome f.  相似文献   

15.
The "Triton Subchloroplast Fraction I" or "TSF-I particles" can be further fractionated into a cytochrome fraction and a P-700-containing fraction essentially free of cytochromes. The cytochrome complex contains cytochromes f and b6 in approx. equimolar amounts, and, in addition, also plastocyanin and one iron-sulfur protein, all in the bound state. Bound plastocyanin was characterized by EPR spectroscopy. The EPR spectrum of the bound iron-sulfur protein resembles that previously detected in Phostosystem I particles under highly reducing conditions at lower than -560 mV. The redox potential of P-700 in the cytochrome-free high-P-700 particles was measured to be +468 mV; those of cytochromes f and b6 are +345 and -140 mV, respectively. Among the four components present in the complex, only cytochrome f can be coupled to a Photosystem I particle and undergoes photooxidation. This coupled photooxidation is totoally inhibited by KCN and only partially inhibited by HgCl2. The similarity of the complex containing cytochromes f and b6, plastocyanin, and an iron-sulfur protein to complexes III and IV of the mitochondrial respiratory redox chain and a possible involvement of the complex in cyclic photophosphorylation are noted and discussed.  相似文献   

16.
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.  相似文献   

17.
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.  相似文献   

18.
Electron transfer from plastocyanin to photosystem I.   总被引:9,自引:3,他引:6       下载免费PDF全文
Mutant plastocyanins with Leu at position 10, 90 or 83 (Gly, Ala and Tyr respectively in wildtype) were constructed by site-specific mutagenesis of the spinach gene, and expressed in transgenic potato plants under the control of the authentic plastocyanin promoter, as well as in Escherichia coli as truncated precursor intermediates carrying the C-terminal 22 amino acid residues of the transit peptide, i.e. the thylakoid-targeting domain that acts as a bacterial export signal. The identity of the purified plastocyanins was verified by matrix-assisted laser desorption/ionization mass spectrometry. The formation of a complex between authentic or mutant spinach plastocyanin and isolated photosystem I and the electron transfer has been studied from the biphasic reduction kinetics of P700+ after excitation with laser flashes. The formation of the complex was abolished by the bulky hydrophobic group of Leu at the respective position of G10 or A90 which are part of the conserved flat hydrophobic surface around the copper ligand H87. The rate of electron transfer decreased by both mutations to < 20% of that found with wildtype plastocyanin. We conclude that the conserved flat surface of plastocyanin represents one of two crucial structural elements for both the docking at photosystem I and the efficient electron transfer via H87 to P700+. The Y83L mutant exhibited faster electron transfer to P700+ than did authentic plastocyanin. This proves that Y83 is not involved in electron transfer to P700 and suggests that electron transfer from cytochrome f and to P700 follows different routes in the plastocyanin molecule. Plastocyanin (Y83L) expressed in either E. coli or potato exhibited different isoelectric points and binding constants to photosystem I indicative of differences in the folding of the protein. The structure of the binding site at photosystem I and the mechanism of electron transfer are discussed.  相似文献   

19.
David B. Knaff 《BBA》1973,292(1):186-192
Removal of plastocyanin from Photosystem I subchloroplast particles had no effect on the Photosystem I photooxidation of cytochrome f. Chloroplasts depleted of plastocyanin by sonication lost the ability to reduce cytochrome f in Photosystem II light. Addition of plastocyanin restored the photoreduction of cytochrome f. These results are consistent with a plastocyanin site on the reducing side of cytochrome f.  相似文献   

20.
This paper reports the results of research on the interaction between the cytochrome f of the active cytochrome b 6 f complex (incubated with Cd-, Zn-, and Ag-substituted plastocyanins) and Cu-plastocyanin. The presented studies show, that the metal derivatives of plastocyanin can have an influence on the photosynthetic electron transfer path: cytochrome b 6 f complex — photosystem I. The metal-substituted plastocyanins occupy the plastocyanin electron transfer site of the cytochrome f. The stopped-flow measurements show, that although the metal derivatives of plastocyanin do not influence the rate of cyt f- Pc electron transfer, creation of the non-electron-transfer complexes characterised by a strong binding between the cyt f and substituted plastocyanins and their slow release, dependent on the redox state of the substituted metal, results in the decrease of a turnover of the cytochrome complex. The research was done in the Department of Plant Biochemistry, Freiburg University, Sch?nzlestrasse 1, 79 104 Freiburg, Germany  相似文献   

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