Reconstitution of barley photosystem I reveals that the N-terminus of the PSI-D subunit is essential for tight binding of PSI-C

Removal of the peripheral subunits PSI-C, -D and -E from the photosystem I (PSI) complex of barley requires a urea treatment much harsher than required to remove the similar subunits from cyanobacterial PSI. The resulting PSI barley core was reconstituted by addition of the E. coli expressed subunits PSI-C and -D, and PSI-E isolated from barley. Western blotting, flash photolysis and NADP⁺ photoreduction measurements demonstrated complete and specific removal of the three subunits from the core and efficient reconstitution of the complex after addition of PSI-C, -D and -E. Flash photolysis reveals that PSI-D is essential for binding of functional PSI-C to the PSI core. An N-terminally truncated barley PSI-D lacking 24 amino acid residues and thus being without the N-terminal extension characteristic for higher plant PSI-D proteins reconstitutes the PSI core to 50% of the level obtained with intact PSI-D as demonstrated by flash photolysis and NADP⁺ photoreduction measurements. Cyanobacterial PSI-D is functionally equivalent to truncated barley PSI-D with respect to its activity to reconstitute the PSI core. This shows that the N-terminal extension of plant PSI-D plays a key role in binding PSI-C to the core. The plant-specific N-terminus of PSI-D is hypothesized to execute its function through interaction with a plant-specific PSI subunit, possibly PSI-H. An anchoring function of the N-terminus of PSI-D would also explain the harsh treatment needed to obtain a plant PSI core. PSI-E is important for efficient NADP⁺ reduction but does not influence electron transfer to iron-sulphur centres A/B nor binding of PSI-C. The enhancing effect of PSI-E on NADP⁺ reduction is independent of the presence of the N-terminus of PSI-D.     

Evidence for the involvement of PSI-E subunit in the reduction of ferredoxin by photosystem I.

Of the stroma-accessible proteins of photosystem I (PSI) from Synechocystis sp. PCC 6803, the PSI-C, PSI-D and PSI-E subunits have already been characterized, and the corresponding genes isolated. PCR amplification and cassette mutagenesis were used in this work to delete the psaE gene. PSI particles were isolated from this mutant, which lacks subunit PSI-E, and the direct photoreduction of ferredoxin was investigated by flash absorption spectroscopy. The second order rate constant for reduction of ferredoxin by wild type PSI was estimated to be approximately 10(9) M-1s-1. Relative to the wild type, PSI lacking PSI-E exhibited a rate of ferredoxin reduction decreased by a factor of at least 25. After reassociation of the purified PSI-E polypeptide, the original rate of electron transfer was recovered. When a similar reconstitution was performed with a PSI-E polypeptide from spinach, an intermediate rate of reduction was observed. Membrane labeling of the native PSI with fluorescein isothiocyanate allowed the isolation of a fluorescent PSI-E subunit. Peptide analysis showed that some residues following the N-terminal sequence were labeled and thus probably accessible to the stroma, whereas both N- and C-terminal ends were probably buried in the photosystem I complex. Site-directed mutagenesis based on these observations confirmed that important changes in either of the two terminal sequences of the polypeptide impaired its correct integration in PSI, leading to phenotypes identical to the deleted mutant. Less drastic modifications in the predicted stroma exposed sequences did not impair PSI-E integration, and the ferredoxin photoreduction was not significantly affected. All these results lead us to propose a structural role for PSI-E in the correct organization of the site involved in ferredoxin photoreduction.     

Nearest-neighbor analysis of higher-plant photosystem I holocomplex.

Photosystem 1 (PSI) preparations from barley (Hordeum vulgare) and spinach (Spinacia oleracea) were subjected to chemical cross-linking using the cleavable homobifunctional cross-linkers dithiobis(succinimidylpropionate) and 3,3'-dithiobis(sulfosuccinimidyl-propionate). The overall pattern of cross-linked products was analyzed by the simple but powerful technique of diagonal electrophoresis, in which the disulfide bond in the cross-linker was cleaved between the first and second dimensions of the gel, and immunoblotting. A large number of cross-linked products were identified. Together with preexisting data on the structure of PSI, it was deduced that the subunits PSI-D, PSI-H, PSI-I, and PSI-L occupy one side of the complex, whereas PSI-E, PSI-F, and PSI-J occupy the other. PSI-K and PSI-G appear to be adjacent to Lhca3 and Lhca2, respectively, and not close to the other small subunits. Experiments with isolated light-harvesting complex I preparations indicate that the subunits are organized as dimers, which seem to associate to the PSI-A/PSI-B proteins independent of each other. We suggest which PSI subunit corresponds to each membrane-spanning helix in the cyanobacterial PSI structure, and present a model for higher-plant PSI.     

Protein-protein and protein-function relationships in Arabidopsis photosystem I: cluster analysis of PSI polypeptide levels and photosynthetic parameters in PSI mutants

In flowering plants, photosystem I (PSI) mediates electron transport across the thylakoid membrane and contains at least 14 proteins. The availability of co-suppression and/or mutant lines deficient for individual PSI polypeptides in Arabidopsis thaliana allows one to assign functions to PSI subunits. We have performed cluster analysis on an extensive set of data on PSI polypeptide levels in ten different PSI mutants. This type of analysis serves to group proteins that exhibit similar changes in amount in different genotypes, and also identifies genotypes which show similar PSI compositions. The interdependence of levels of PSI-C, -D and -E, of -H and -L, and of Lhca2 and 3, which was previously proposed based on the study of single genotypes or on cross-linking experiments, was confirmed by our analyses. In addition, the levels of the lumenal subunits F and N are found to be interdependent. The incorporation of photosynthetic parameters into the cluster analysis revealed that the level of photosynthetic state transitions correlates with the abundance of PSI-H in all 8 genotypes tested, supporting the hypothesis that PSI-H serves as a docking site for LHCII during state transitions.     

Molecular aspects of photosystem I

Photosystem I (PSI) in higher plants consists of 17 polypeptide subunits. Cofactors are chlorophyll a and b , β-carotene, phylloquinone and iron-sulfur clusters. Eight subunits are specific for higher plants while the remaining ones are also present in cyanobacteria. Two 80-kDa subunits (PSI-A and -B) constitute the major part of PSI and bind most of the pigments and electron donors and acceptors. The 9-kDa PSI-C carries the remaining electron acceptors which are [4Fe-4S] iron sulfur clusters. PSI-D, -E and -H have importance for integrity and function at the stromal face of PSI while PSI-F has importance for function at the lumenal face. PSI-N is localized at the lumenal side, but its function is unknown. Four subunits are light-harvesting chlorophyll a/b -binding proteins. The remaining subunits are integral membrane proteins with poorly understood function. Subunit interactions have been studied in reconstitution experiments and by cross-linking studies. Based on these data, it is concluded that iron-sulfur cluster F_B is proximal to F_X and that F_A is the terminal acceptor in PSI. Similarities between PSI and the reaction center from green sulfur bacteria are discussed.     

Inhibition of electron flow around photosystem I in chloroplasts of Cd-treated maize plants is due to Cd-induced iron deficiency

Photosystem I activity of chloroplasts isolated from 21 days old maize seedlings ( Zea mays L. cv. Hidosil) cultivated in a nutrient solution containing different concentrations of Cd (10,20,30μM) was investigated. Cd markedly decreased ferredoxin(Fd)-dependent NADP⁺ photoreduction, while it had no effect on electron transport from 2. 6-dichlorophenolindophenol to methyl viologen, indicating that the metal interferred with electron transport on the reducing side of photosystem I. The decrease in electron transport correlated with a low Fd content, which in turn was correlated with a low Fe concentration, suggesting Cd-induced Fe deficiency. In in vitro experiments direct Cd inhibition of Fd-dependent NADP⁺ photoreduction required much higher Cd concentrations than those observed in Cd-treated plants.     

Sulfide-induced oxygen uptake by isolated spinach chloroplasts catalyzed by photosynthetic electron transport

In addition to an inhibitory effect on the photoreduction of NADP⁺ by isolated spinach chloroplasts ( Spinacea oleracea L. cv. Melody Hybrid), sulfide initiated oxygen uptake by chloroplasts upon illumination, both in presence and absence of an electron acceptor. Sulfide-induced oxygen uptake was sensitive to DCMU demonstrating the involvement of photosynthetic electron transport. Addition of superoxide dismutase to the chloroplast suspension prevented the sulfide-induced oxygen uptake, which indicated that sulfide may be oxidized by the chloroplast, its oxidation being initiated by superoxide formed upon illumination (at the reducing side of PSI). Tris-induced inhibition of NADP⁺ photo-reduction could not be abolished by sulfide, which indicated that sulfide could not act as an electron donor for PSI.     

Cosuppression of photosystem I subunit PSI-H in Arabidopsis thaliana. Efficient electron transfer and stability of photosystem I is dependent upon the PSI-H subunit

PSI-H is an intrinsic membrane protein of 10 kDa that is a subunit of photosystem I (PSI). PSI-H is one of the three PSI subunits found only in eukaryotes. The function of PSI-H was characterized in Arabidopsis plants transformed with a psaH cDNA in sense orientation. Cosuppressed plants containing less than 3% PSI-H are smaller than wild type when grown on sterile media but are similar to wild type under optimal conditions. PSI complexes lacking PSI-H contain 50% PSI-L, whereas other PSI subunits accumulate in wild type amounts. PSI devoid of PSI-H has only 61% NADP+ photoreduction activity compared with wild type and is highly unstable in the presence of urea as determined from flash-induced absorbance changes at 834 nm. Our data show that PSI-H is required for stable accumulation of PSI and efficient electron transfer in the complex. The plants lacking PSI-H compensate for the less efficient PSI with a 15% increase in the P700/chlorophyll ratio, and this compensation is sufficient to prevent overreduction of the plastoquinone pool as evidenced by normal photochemical quenching of fluorescence. Nonphotochemical quenching is approximately 60% of the wild type value, suggesting that the proton gradient across the thylakoid membrane is decreased in the absence of PSI-H.     

Arabidopsis thaliana plants lacking the PSI-D subunit of photosystem I suffer severe photoinhibition,have unstable photosystem I complexes,and altered redox homeostasis in the chloroplast stroma

The PSI-D subunit of photosystem I is a hydrophilic subunit of about 18 kDa, which is exposed to the stroma and has an important function in the docking of ferredoxin to photosystem I. We have used an antisense approach to obtain Arabidopsis thaliana plants with only 5-60% of PSI-D. No plants were recovered completely lacking PSI-D, suggesting that PSI-D is essential for a functional PSI in plants. Plants with reduced amounts of PSI-D showed a similar decrease in all other subunits of PSI including the light harvesting complex, suggesting that in the absence of PSI-D, PSI cannot be properly assembled and becomes degraded. Plants with reduced amounts of PSI-D became light-stressed even in low light although they exhibited high non-photochemical quenching (NPQ). The high NPQ was generated by upregulating the level of violaxanthin de-epoxidase and PsbS, which are both essential components of NPQ. Interestingly, the lack of PSI-D affected the redox state of thioredoxin. During the normal light cycle thioredoxin became increasingly oxidized, which was observed as decreasing malate dehydrogenase activity over a 4-h light period. This result shows that photosynthesis was close to normal the first 15 min, but after 2-4 h photoinhibition dominated as the stroma progressively became less reduced. The change in the thiol disulfide redox state might be fatal for the PSI-D-less plants, because reduction of thioredoxin is one of the main switches for the initiation of CO2 assimilation and photoprotection upon light exposure.     

Characterization of a redox active cross-linked complex between cyanobacterial photosystem I and soluble ferredoxin.

A covalent stoichiometric complex between photosystem I (PSI) and ferredoxin from the cyanobacterium Synechocystis sp. PCC 6803 was generated by chemical cross-linking. The photoreduction of ferredoxin, studied by laser flash absorption spectroscopy between 460 and 600 nm, is a fast process in 60% of the covalent complexes, which exhibit spectral and kinetic properties very similar to those observed with the free partners. Two major phases with t(1/2) <1 micros and approximately 10-14 micros are observed at two different pH values (5.8 and 8.0). The remaining complexes do not undergo fast ferredoxin reduction and 20-25% of the complexes are still able to reduce free ferredoxin or flavodoxin efficiently, thus indicating that ferredoxin is not bound properly in this proportion of covalent complexes. The docking site of ferredoxin on PSI was determined by electron microscopy in combination with image analysis. Ferredoxin binds to the cytoplasmic side of PSI, with its mass center 77 angstroms distant from the center of the trimer and in close contact with a ridge formed by the subunits PsaC, PsaD and PsaE. This docking site corresponds to a close proximity between the [2Fe- 2S] center of ferredoxin and the terminal [4Fe-4S] acceptor FII of PSI and is very similar in position to the docking site of flavodoxin, an alternative electron acceptor of PSI.     

Structural and functional properties of the cyanobacterial photosystem I complex

Photosystem I (PSI) complexes have been isolated from two cyanobacterial strains, Synechococcus sp. PCC 7002 and 6301. These complexes contain six to seven low molecular mass subunits in addition to the two high molecular mass subunits previously shown to bind the primary reaction center components. Chemical cross-linking of ferredoxin to the complex identified a 17.5-kDa subunit as the ferredoxin-binding protein in the Synechococcus sp. PCC 6301-PSI complex. The amino acid sequence of this subunit, deduced from the DNA sequence of the gene, confirmed its identity as the psaD gene product. A 17-kDa subunit cross-links to the electron donor, cytochrome c-553, in a manner analogous to the cross-linking of plastocyanin to the higher plant PSI complex. Using antibodies raised against the spinach psaC gene product (a 9-kDa subunit which binds Fe-S centers A and B), we identified an analogous protein in the cyanobacterial PSI complex.     

Ferredoxin Cross-Links to a 22 kD Subunit of Photosystem I

We have used a cross-linking approach to study the interaction of ferredoxin (Fd) with photosystem I (PSI). The cross-linking reagent N-ethyl-3-(3-dimethylaminopropyl) carbodiimide was found to cross-link spinach Fd to a 22 kilodalton subunit of PSI in both isolated spinach (Spinacia oleracea) PSI complexes and spinach thylakoid membranes. The product had an apparent molecular weight of 38 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was identified as a cross-linked product using specific antibodies to Fd and the 22 kilodalton subunit. In both a native PSI complex (200 Chl/P700) and a PSI core complex (100 Chl/P700), a second cross-linked product at 36 kilodaltons was seen. The latter cross-reacted with an antibody to Fd but did not cross-react with antibodies directed against the 24.3, 22, 19, 17.3 or 8.5 kilodalton, or psaC subunits of PSI. Its composition remains to be determined. In thylakoids only the 38 kilodalton product was observed along with a cross-linked complex of Fd and Fd:NADP⁺ reductase.     

The ferredoxin docking site of photosystem I

The reaction center of photosystem I (PSI) reduces soluble ferredoxin on the stromal side of the photosynthetic membranes of cyanobacteria and chloroplasts. The X-ray structure of PSI from the cyanobacterium Synechococcus elongatus has been recently established at a 2.5 A resolution [Nature 411 (2001) 909]. The kinetics of ferredoxin photoreduction has been studied in recent years in many mutants of the stromal subunits PsaC, PsaD and PsaE of PSI. We discuss the ferredoxin docking site of PSI using the X-ray structure and the effects brought by the PSI mutations to the ferredoxin affinity.     

Purification and membrane topology of PSI-D and PSI-E, two subunits of the photosystem I reaction center.

Structural studies have been conducted on polypeptides PSI-D and PSI-E, which are extrinsic but firmly bound to the photosystem I reaction center. These subunits are predicted to be involved in the correct interaction with soluble electron acceptor(s), like ferredoxin. We designed an original method to extract both polypeptides directly from thylakoid membranes and to purify them: a stepwise extraction with NaSCN followed by size fractionation and reverse-phase HPLC. Investigation of the in situ topology of PSI-D and PSI-E was undertaken using monoclonal antibody binding, controlled proteolysis, peptide sequencing and electron microscopy. The precise identification of numerous proteolytic sites indicates that the entire N-terminal regions of PSI-E (up to Glu15) and PSI-D (up to Lys15) are exposed to the medium. Partial mapping of the exposed epitopes was possible using purified fragments of each polypeptide. In the case of PSI-E, this mapping confirmed the accessibility of the N-terminal part, and suggested the need for another exposed sequence, probably located after Met39 in the second half of the protein. For PSI-D, this mapping revealed that the sequence between Met74 and Met140, including the most basic amino acid clusters, is also partly accessible. These experiments provide the first detailed informations, although still partial, on the topology of these polypeptides. They give a preliminary basis for hypotheses concerning the sites of interaction with the soluble counterparts.     

Single and double knockouts of the genes for photosystem I subunits G,K, and H of Arabidopsis. Effects on photosystem I composition,photosynthetic electron flow,and state transitions

Photosystem I (PSI) of higher plants contains 18 subunits. Using Arabidopsis En insertion lines, we have isolated knockout alleles of the genes psaG, psaH2, and psaK, which code for PSI-G, -H, and -K. In the mutants psak-1 and psag-1.4, complete loss of PSI-K and -G, respectively, was confirmed, whereas the residual H level in psah2-1.4 is due to a second gene encoding PSI-H, psaH1. Double mutants, lacking PSI-G, and also -K, or a fraction of -H, together with the three single mutants were characterized for their growth phenotypes and PSI polypeptide composition. In general, the loss of each subunit has secondary, in some cases additive, effects on the abundance of other PSI polypeptides, such as D, E, H, L, N, and the light-harvesting complex I proteins Lhca2 and 3. In the G-less mutant psag-1.4, the variation in PSI composition suggests that PSI-G stabilizes the PSI-core. Levels of light-harvesting complex I proteins in plants, which lack simultaneously PSI-G and -K, indicate that PSI subunits other than G and K can also bind Lhca2 and 3. In the same single and double mutants, psag-1.4, psak-1, psah2-1.4, psag-1.4/psah2-1.4, and psag-1.4/psak-1 photosynthetic electron flow and excitation energy quenching were analyzed to address the roles of the various subunits in P700 reduction (mediated by PSI-F and -N) and oxidation (PSI-E), and state transitions (PSI-H). Based on the results, we also suggest for PSI-K a role in state transitions.     

Light-harvesting complex II binds to several small subunits of photosystem I

Mobile light-harvesting complex II (LHCII) is implicated in the regulation of excitation energy distribution between Photosystem I (PSI) and Photosystem II (PSII) during state transitions. To investigate how LHCII interacts with PSI during state transitions, PSI was isolated from Arabidopsis thaliana plants treated with PSII or PSI light. The PSI preparations were made using digitonin. Chemical cross-linking using dithio-bis(succinimidylpropionate) followed by diagonal electrophoresis and immunoblotting showed that the docking site of LHCII (Lhcb1) on PSI is comprised of the PSI-H, -L, and -I subunits. This was confirmed by the lack of energy transfer from LHCII to PSI in the digitonin-PSI isolated from plants lacking PSI-H and -L. Digitonin-PSI was purified further to obtain an LHCII.PSI complex, and two to three times more LHCII was associated with PSI in the wild type in State 2 than in State 1. Lhcb1 was also associated with PSI from plants lacking PSI-K, but PSI from PSI-H, -L, or -O mutants contained only about 30% of Lhcb1 compared with the wild type. Surprisingly, a significant fraction of the LHCII bound to PSI in State 2 was not phosphorylated. Cross-linking prior to sucrose gradient purification resulted in copurification of phosphorylated LHCII in the wild type, but not with PSI from the PSI-H, -L, and -O mutants. The data suggest that migration of LHCII during state transitions cannot be explained sufficiently by different affinity of phosphorylated and unphosphorylated LHCII for PSI but is likely to involve structural changes in thylakoid organization.     

Purification and characterization of ferredoxin-NADP+ reductase from the green alga Chlorella fusca

Ferredoxin-NADP⁺ reductase (FNR, EC I.18.1.2) from the green algae Chlorella fusca Shihira et Kraus 211–15, was purified to homogeneity. The molecular mass was 36.8 kDa as determined by SDS-polyacrylamide gel electrophoresis. The enzyme exhibits the typical spectrum of a flavoprotein with an absorption maximum at 459 nm and an A_273/459 ratio of 7.2. It contains one mol of FAD per mol of protein and the calculated extinction coefficient is 9.8 m M cm⁻¹. Four different forms of the purified enzyme were detected by isoelectric focusing (pI between 5.4 and 5.9), even when protease inhibitors were used during the first steps of the purification. Kinetic parameters were determined for several FNR-catalyzed reactions. NADP⁺ photoreduction gave comparable rates when either ferredoxin or flavodoxin was used.     

Down-regulation of the PSI-F subunit of photosystem I (PSI) in Arabidopsis thaliana. The PSI-F subunit is essential for photoautotrophic growth and contributes to antenna function

The PSI-F subunit of photosystem I is a transmembrane protein with a large lumenal domain. The role of PSI-F was investigated in Arabidopsis plants transformed with an antisense construct of the psaF cDNA. Several plant lines with reduced amounts of the PSI-F subunit were generated. Many of the transgenic plants died, apparently because they were unable to survive without the PSI-F subunit. Plants with 5% of PSI-F were capable of photoautotrophic growth but were much smaller than wild-type plants. The plants suffered severely under normal growth conditions but recovered somewhat in the dark indicating chronic photoinhibition. Photosystem I lacking PSI-F was less stable, and the stromal subunits PSI-C, PSI-D, and PSI-E were present in lower amounts than in wild type. The lack of PSI-F resulted in an inability of light-harvesting complex I-730 to transfer energy to the P700 reaction center. In thylakoids deficient in PSI-F, the steady state NADP(+) reduction rate was only 10% of the wild-type levels indicating a lower efficiency in oxidation of plastocyanin. Surprisingly, the lack of PSI-F also gave rise to disorganization of the thylakoids. The strict arrangement in grana and stroma lamellae was lost, and instead a network of elongated and distorted grana was observed.