Organization of Photosystem I Polypeptides (A Structural Interaction between the PsaD and PsaL Subunits)

The wild-type, PsaD-less, and PsaL-less strains of the cyanobacterium Synechocystis sp. PCC 6803 were used to study subunit interactions in photosystem I (PSI). When the membranes of a PsaD-less strain were solubilized with Triton X-100 and PSI was purified using ion-exchange chromatography and sucrose-gradient ultracentrifugation, the PsaL subunit was substantially removed from the core of PSI, whereas other subunits, such as PsaE and PsaF, were quantitatively retained during purification. When the wild-type PSI was exposed to increasing concentrations of NaI, the PsaE, PsaD, and PsaC subunits were gradually removed, whereas PsaF, PsaL, PsaK, and PsaJ resisted removal by up to 3 M NaI. The absence of PsaL enhanced the accessibility of PsaD to removal by NaI. Treatment of the wild-type PSI complexes with glutaraldehyde at 4[deg] C resulted in a 29-kD cross-linked product between PsaD and PsaL. The formation of such cross-linked species was independent of PSI concentrations, suggesting an intracomplex cross-linking between PsaD and PsaL. Taken together, these results demonstrate a structural interaction between PsaD and PsaL that plays a role in their association with the PSI core.     

The PsaC subunit of photosystem I provides an essential lysine residue for fast electron transfer to ferredoxin.

PsaC is the stromal subunit of photosystem I (PSI) which binds the two terminal electron acceptors FA and FB. This subunit resembles 2[4Fe-4S] bacterial ferredoxins but contains two additional sequences: an internal loop and a C-terminal extension. To gain new insights into the function of the internal loop, we used an in vivo degenerate oligonucleotide-directed mutagenesis approach for analysing this region in the green alga Chlamydomonas reinhardtii. Analysis of several psaC mutants affected in PSI function or assembly revealed that K35 is a main interaction site between PsaC and ferredoxin (Fd) and that it plays a key role in the electrostatic interaction between Fd and PSI. This is based upon the observation that the mutations K35T, K35D and K35E drastically affect electron transfer from PSI to Fd, as measured by flash-absorption spectroscopy, whereas the K35R change has no effect on Fd reduction. Chemical cross-linking experiments show that Fd interacts not only with PsaD and PsaE, but also with the PsaC subunit of PSI. Replacement of K35 by T, D, E or R abolishes Fd cross-linking to PsaC, and cross-linking to PsaD and PsaE is reduced in the K35T, K35D and K35E mutants. In contrast, replacement of any other lysine of PsaC does not alter the cross-linking pattern, thus indicating that K35 is an interaction site between PsaC and its redox partner Fd.     

Assembly of protein subunits within the stromal ridge of photosystem I. Structural changes between unbound and sequentially PS I-bound polypeptides and correlated changes of the magnetic properties of the terminal iron sulfur clusters

The X-ray structure of Photosystem I (PS I) from Synechococcus elongatus was recently solved at 2.5A resolution (PDB entry 1JB0). It provides a structural model for the stromal subunits PsaC, PsaD and PsaE, which comprise the "stromal ridge" of PS I. In a separate set of studies the three-dimensional solution structures of the unbound, recombinant PsaC (PDB entry 1K0T) and PsaE (PDB entries 1PSF, 1QP2 and 1GXI) subunits were solved by NMR. The PsaC subunit of PS I is a small (9.3 kDa) protein that harbors binding sites for two [4Fe-4S] clusters F(A) and F(B), which are the terminal electron acceptors in PS I. Comparison of the PsaC structure in solution with that in the X-ray structure of PS I reveals significant differences between them which are summarized and evaluated here. Changes in the magnetic properties of [4Fe-4S] centers F(A) and F(B) are related to changes in the protein structure of PsaC, and they are further influenced by the presence of PsaD. Based on experimental evidence, three assembly stages are analyzed: PsaC(free), PsaC(only), PsaC(PS I). Unbound, recombinant PsaD, studied by NMR, has only a few elements of secondary structure and no stable three-dimensional structure in solution. When PsaD is bound in PS I, it has a well-defined three-dimensional structure. For PsaE the three-dimensional structure is very similar in solution and in the PS I-bound form, with the exception of two loop regions. We suggest that the changes in the structures of PsaC and PsaD are caused by the sequential formation of multiple networks of contacts between the polypeptides of the stromal ridge and between those polypeptides and the PsaA/PsaB core polypeptides. The three-dimensional structure of the C(2)-symmetric F(X)-binding loops on PsaA and PsaB were also analyzed and found to be significantly different from the binding sites of other proteins that contain interpolypeptide [4Fe-4S] clusters. The aim of this work is to relate contact information to structural changes in the proteins and to propose a model for the assembly of the stromal ridge of PS I based on this analysis.     

An insight into the assembly and organization of photosystem I complex in the thylakoid membranes of the thermophilic cyanobacterium, Mastigocladus laminosus

The present study characterizes the assembly and organization of Photosystem I (PSI) complex, and its individual subunits into the thylakoid membranes of the thermophilic cyanobacterium, Mastigocladus laminosus. PSI is a multiprotein complex that contains peripheral as well as integral subunits. Hence, it serves as a suitable model system for understanding the formation and organization of membrane protein complexes. In the present study, two peripheral cytosol facing subunits of PSI, namely, PsaD and PsaE were overexpressed in E. coli and used for assembly studies. The gene encoding PsaK, an integral membrane spanning subunit of PSI, was cloned and the deduced amino acid sequence revealed PsaK to have two transmembrane alpha-helices. The characterization of the in vitro assembly of the peripheral subunits, PsaD and PsaE, as well as of the integral subunit, PsaK, was performed by incubating each subunit with thylakoids isolated from Mastigocladus laminosus. All three subunits studied were found to assemble into the thylakoids in a spontaneous mechanism, showing no requirement for cytosolic factors or NTP's (nucleotide 5'-triphosphate). Nevertheless, further characterization of the assembly of PsaK revealed its membrane integration to be most efficient at 55 degrees C. The associations and protein-protein interactions between different subunits within the assembled PSI complex were directly quantified by measurements performed using the BIACORE technology. The preliminary results indicated the existence of specific interaction between PsaD and PsaE, and revealed a very high binding affinity between PsaD and the PSI electron acceptor ferridoxin (Kd = 5.8 x 10(-11) M). PsaE has exhibited a much lower binding affinity for ferridoxin (Kd = 3.1 x 10(-5) M), thereby supporting the possibility of PsaE being one of the subunits responsible for the dissociation of ferridoxin from the PSI complex.     

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.     

Molecular interactions between photosystem I and ferredoxin: an integrated energy frustration and experimental model

The stromal domain (PsaC, PsaD, and PsaE) of photosystem I (PSI) reduces transiently bound ferredoxin (Fd) or flavodoxin. Experimental structures exist for all of these protein partners individually, but no experimental structure of the PSI/Fd or PSI/flavodoxin complexes is presently available. Molecular models of Fd docked onto the stromal domain of the cyanobacterial PSI site are constructed here utilizing X‐ray and NMR structures of PSI and Fd, respectively. Predictions of potential protein‐protein interaction regions are based on experimental site‐directed mutagenesis and cross‐linking studies to guide rigid body docking calculations of Fd into PSI, complemented by energy landscape theory to bring together regions of high energetic frustration on each of the interacting proteins. The results identify two regions of high localized frustration on the surface of Fd that contain negatively charged Asp and Glu residues. This study predicts that these regions interact predominantly with regions of high localized frustration on the PsaC, PsaD, and PsaE chains of PSI, which include several residues predicted by previous experimental studies. Copyright © 2014 John Wiley & Sons, Ltd.     

Isolation of thylakoid membrane complexes from rice by a new double-strips BN/SDS-PAGE and bioinformatics prediction of stromal ridge subunits interaction

Thylakoid membrane complexes of rice (Oryza sativa L.) play crucial roles in growth and crop production. Understanding of protein interactions within the complex would provide new insights into photosynthesis. Here, a new "Double-Strips BN/SDS-PAGE" method was employed to separate thylakoid membrane complexes in order to increase the protein abundance on 2D-gels and to facilitate the identification of hydrophobic transmembrane proteins. A total of 58 protein spots could be observed and subunit constitution of these complexes exhibited on 2D-gels. The generality of this new approach was confirmed using thylakoid membrane from spinach (Spinacia oleracea) and pumpkin (Cucurita spp). Furthermore, the proteins separated from rice thylakoid membrane were identified by the mass spectrometry (MS). The stromal ridge proteins PsaD and PsaE were identified both in the holo- and core- PSI complexes of rice. Using molecular dynamics simulation to explore the recognition mechanism of these subunits, we showed that salt bridge interactions between residues R19 of PsaC and E168 of PasD as well as R75 of PsaC and E91 of PsaD played important roles in the stability of the complex. This stromal ridge subunits interaction was also supported by the subsequent analysis of the binding free energy, the intramolecular distances and the intramolecular energy.     

Structural analysis of the photosystem I supercomplex of cyanobacteria induced by iron deficiency

Here we describe the three-dimensional structure of the newly discovered CP43'-photosystem I (PSI) supercomplex of cyanobacteria calculated by single-particle analysis of images obtained by electron cryomicroscopy (cryo-EM). This large membrane protein complex has a molecular mass of approximately 2 MDa and is found in cyanobacteria when grown in iron deficient media. It is composed of a reaction center trimer surrounded by 18 subunits of the chlorophyll a binding CP43'protein, encoded by the isiA gene, which increases the light harvesting capacity of PSI by approximately 70%. By modeling higher-resolution structural data obtained from X-ray crystallography into the three-dimensional (3D) cryo-EM map, we have been able to gain a better understanding of the structure and functional properties of this supermolecular complex. We have identified three separate clusters of chlorophyll molecules at the periphery of the PSI core which may aid energy transfer from the CP43' antenna ring to the reaction center. Moreover, it is shown that despite the replacement of ferredoxin with flavodoxin as an electron acceptor under iron stress conditions, the 3D map has density to accommodate the extrinsic proteins, PsaC, PsaD, and PsaE. The presence of these three proteins was also confirmed by immunoblotting.     

Absence of PsaC subunit allows assembly of photosystem I core but prevents the binding of PsaD and PsaE in Synechocystis sp. PCC6803

In photosystem I (PSI) of oxygenic photosynthetic organisms the psaC polypeptide, encoded by the psaC gene, provides the ligands for two [4Fe-4S] clusters, F_A and F_B. Unlike other cyanobacteria, two different psaC genes have been reported in the cyanobacterium Synechocystis 6803, one (copy 1) with a deduced amino acid sequence identical to that of tobacco and another (copy 2) with a deduced amino acid sequence similar to those reported for other cyanobacteria. Insertion of a gene encoding kanamycin resistance into copy 2 resulted in a photosynthesis-deficient strain, CDK25, lacking the PsaC, PsaD and PsaE polypeptides in isolated thylakoid membranes, while the PsaA/PsaB and PsaF subunits were found. Growth of the mutant cells was indistinguishable from that of wild-type cells under light-activated heterotrophic growth (LAHG). A reversible P700⁺ signal was detected by EPR spectroscopy in the isolated thylakoids during illumination at low temperature. Under these conditions, the EPR signals attributed to F_A and F_B were absent in the mutant strain, but a reversible F_x signal was present with broad resonances at g=2.079, 1.903, and 1.784. Addition of PsaC and PsaD proteins to the thylakoids gave rise to resonances at g=2.046, 1.936, 1.922, and 1.880; these values are characteristic of an interaction-type spectrum of F_A ^- and F_B ^-. In room-temperature optical spectroscopic analysis, addition of PsaC and PsaD to the thylakoids also restored a 30 ms kinetic transient which is characteristic of the P700⁺ [F_A/F_B]^- backreaction. Expression of copy 1 was not detected in cells grown under LAHG and under mixotrophic conditions. These results demonstrate that copy 2 encodes the PsaC polypeptide in PSI in Synechocystis 6803, while copy 1 is not involved in PSI; that the PsaC polypeptide is necessary for stable assembly of PsaD and PsaE into PSI complex in vivo; and that PsaC, PsaD and PsaE are not needed for assembly of PsaA-PsaB dimer and electron transport from P700 to F_x.     

Biochemical and molecular characterization of photosystem I deficiency in the NCS6 mitochondrial mutant of maize

Interorganellar signaling interactions are poorly understood. The maize non-chromosomal stripe (NCS) mutants provide models to study the requirement of mitochondrial function for chloroplast biogenesis and photosynthesis. Previous work suggested that the NCS6 mitochondrial mutation, a cytochrome oxidase subunit 2 (cox2) deletion, is associated with a malfunction of Photosystem I (PSI) in defective chloroplasts of mutant leaf sectors (Gu et al., 1993). We have now quantified the reductions of photosynthetic rates and PSI activity in the NCS6 defective stripes. Major reductions of the plastid-coded PsaC and nucleus-coded PsaD and PsaE PSI subunits and of their corresponding mRNAs are seen in mutant sectors; however, although thepsaA/B mRNA is greatly reduced, levels of PsaA and PsaB (the core proteins of PSI) are only slightly decreased. Levels of the PsaL subunit and its mRNA appear to be unchanged. Tested subunits of other thylakoid membrane complexes – PSII, Cyt b₆/f, and ATP synthase, have minor (or no) reductions within mutant sectors. The results suggest that specific signaling pathways sense the dysfunction of the mitochondrial electron transport chain and respond to down-regulate particular PSI mRNAs, leading to decreased PSI accumulation in the chloroplast. The reductions of both nucleus and plastid encoded components indicate that complex interorganellar signaling pathways may be involved.     

Unique constitution of photosystem I with a novel subunit in the cyanobacterium Gloeobacter violaceus PCC 7421

Constitution of the photosystem I complex isolated from the cyanobacterium Gloeobacter violaceus PCC 7421 was investigated by tricine-urea-SDS-PAGE, followed by peptide mass fingerprinting or N-terminal sequencing. Eight subunits (PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaL and PsaM) were identified as predicted from the genome sequence. A novel subunit (PsaZ) was discovered, but PsaI, PsaJ, PsaK and PsaX were absent. PsaB has a C-terminal extension with 155 amino acids in addition to the conserved region and this domain is similar to the peptidoglycan-binding domain. These results suggest that PS I complexes of G. violaceus have unique structural properties.     

Identification of surface-exposed domains on the reducing side of photosystem I.

Photosystem I (PSI) is a multisubunit enzyme that catalyzes the light-driven oxidation of plastocyanin or cytochrome c6 and the concomitant photoreduction of ferredoxin or flavodoxin. To identify the surface-exposed domains in PSI of the cyanobacterium Synechocystis sp. PCC 6803, we mapped the regions in PsaE, PsaD, and PsaF that are accessible to proteases and N-hydroxysuccinimidobiotin (NHS-biotin). Upon exposure of PSI complexes to a low concentration of endoproteinase glutamic acid (Glu)-C, PsaE was cleaved to 7.1- and 6.6-kD N-terminal fragments without significant cleavage of other subunits. Glu63 and Glu67, located near the C terminus of PsaE, were the most likely cleavage sites. At higher protease concentrations, the PsaE fragments were further cleaved and an N-terminal 9.8-kD PsaD fragment accumulated, demonstrating the accessibility of Glu residue(s) in the C-terminal domain of PsaD to the protease. Besides these major, primary cleavage products, several secondary cleavage sites on PsaD, PsaE, and PsaF were also identified. PsaF resisted proteolysis when PsaD and PsaE were intact. Glu88 and Glu124 of PsaF became susceptible to endoproteinase Glu-C upon extensive cleavage of PsaD and PsaE. Modification of PSI proteins with NHS-biotin and subsequent cleavage by endoproteinase Glu-C or thermolysin showed that the intact PsaE and PsaD, but not their major degradation products lacking C-terminal domains, were heavily biotinylated. Therefore, lysine-74 at the C terminus of PsaE was accessible for biotinylation. Similarly, lysine-107, or lysine-118, or both in PsaD could be modified by NHS-biotin.     

Polypeptide composition of the Photosystem I complex and the Photosystem I core protein from Synechococcus sp. PCC 6301.

The polypeptide composition of the Photosystem I complex from Synechococcus sp. PCC 6301 was determined by sodium-dodecyl sulfate polyacrylamide gel electrophoresis and N-terminal amino acid sequencing. The PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaK and PsaL proteins, as well as three polypeptides with apparent masses less than 8 kDa and small amounts of the 12.6 kDa GlnB (PII) protein, wee present in the Photosystem I complex. No proteins homologous to the PsaG and PsaH subunits of eukaryotic Photosystem I complexes were detected. When the Photosystem I complex was treated with 6.8 M urea and ultrafiltered using a 100 kDa cutoff membrane, the resulting Photosystem I core protein was found to be depleted of the PsaC, PsaD and PsaE proteins. The filtrate contained the missing proteins, along with five proteolytically-cleaved polypeptides with apparent masses of less than 16 kDa and with N-termini identical to that of the PsaD protein. The PsaF and PsaL proteins, along with the three less than 8 kDa polypeptides, were not released from the Photosystem I complex to any significant extent, but low-abundance polypeptides with N-termini identical to those of PsaF and PsaL were found in the filtrate with apparent masses slightly smaller than those found in the native Photosystem I complex. When the filtrate was incubated with FeCl3, Na2S and beta-mercaptoethanol in the presence of the isolated Photosystem I core protein, the PsaC, PsaD and PsaE proteins were rebound to reconstitute a Photosystem I complex functional in light-induced electron flow from P700 to FA/FB. In the absence of the iron-sulfur reconstitution agents, there was little rebinding of the PsaC, psaD or PsaE proteins to the Photosystem I core protein. No binding of the truncated PsaD polypeptides occurred, either in the presence or absence of the iron-sulfur reagents. The reconstitution of the FA/FB iron-sulfur clusters thus appears to be a necessary precondition for rebinding of the PsaC, psaD and psaE proteins to the Photosystem I core protein.     

Solution structure of the unbound, oxidized Photosystem I subunit PsaC, containing [4Fe-4S] clusters FA and FB: a conformational change occurs upon binding to Photosystem I

This work presents the three-dimensional NMR solution structure of recombinant, oxidized, unbound PsaC from Synechococcus sp. PCC 7002. Constraints are derived from homo- and heteronuclear one-, two- and three-dimensional (1)H and (15)N NMR data. Significant differences are outlined between the unbound PsaC structure presented here and the available X-ray structure of bound PsaC as an integral part of the whole cyanobacterial PS I complex. These differences mainly concern the arrangement of the N- and C-termini with respect to the [4Fe-4S] core domain. In the NMR solution structure of PsaC the C-terminal region assumes a disordered helical conformation, and is clearly different from the extended coil conformation, which is one of the structural elements required to anchor PsaC to the PS I core heterodimer. In solution the N-terminus of PsaC is in contact with the pre-C-terminal region but slides in between the latter and the iron-sulfur core region of the protein. Together, these features result in a concerted movement of the N-terminus and pre-C-terminal region away from the F(A) binding site, accompanied by a bending of the N-terminus. In comparison, the same terminal regions are positioned much closer to F(A) and take up an anti-parallel beta-sheet arrangement in PsaC bound to PS I. The conformational changes between bound and unbound PsaC correlate with the differences reported earlier for the EPR spectra of reduced F(A) and F(B) in bound versus unbound PsaC. The observed different structural features in solution are highly relevant for unraveling the stepwise assembly process of the stromal PsaC, PsaD and PsaE subunits to the PS I core heterodimer. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0321-3.     

The PsaD subunit of photosystem I. Mutations in the basic domain reduce the level of PsaD in the membranes.

The PsaD subunit of photosystem I (PSI) is a peripheral protein that provides a docking site for ferredoxin and interacts with the PsaB, PsaC, and PsaL subunits of PSI. We used site-directed mutagenesis to determine the function of a basic region in PsaD of the cyanobacterium Synechocystis sp. PCC 6803. We generated five mutant strains in which one or more charged residues were altered. Western blotting showed that replacement of lysine (Lys)-74 with glutamine or glutamic acid led to a substantial decrease in the level of PsaD in the membranes. The mutant PSI complexes showed reduced NADP+ photoreduction activity mediated by ferredoxin; the decrease in activity correlated with the reduced level of PsaD. Using protein synthesis inhibitors we showed that the degradation rates of the mutant and wild-type PsaD were similar, indicating a defect in the assembly of the mutant protein. Treatment of the mutant PSI complexes with a different concentration of NaI showed that the mutations decreased affinity between PsaD and the transmembrane components of PSI. With glutaraldehyde, the mutant and wild-type PsaD proteins could be cross-linked with PsaC, but the PsaD-PsaL cross-linked product was reduced drastically when arginine-72, Lys-74, and Lys-76 were mutated simultaneously. These studies demonstrate that the basic residues in the central region of PsaD, especially Lys-74, are crucial in the assembly of PsaD into the PSI complex.     

Electrostatic influence of PsaC protein binding to the PsaA/PsaB heterodimer in photosystem I

The absence of the PsaC subunit in the photosystem I (PSI) complex (native PSI complex) by mutagenesis or chemical manipulation yields a PSI core (P700-F(X) core) that also lacks subunits PsaD and PsaE and the two iron-sulfur clusters F(A) and F(B), which constitute an integral part of PsaC. In this P700-F(X) core, the redox potentials (E(m)) of the two quinones A(1A/B) and the iron-sulfur cluster F(X) as well as the corresponding protonation patterns are investigated by evaluating the electrostatic energies from the solution of the linearized Poisson-Boltzmann equation. The B-side specific Asp-B558 changes its protonation state significantly upon isolating the P700-F(X) core, being mainly protonated in the native PSI complex but ionized in the P700-F(X) core. In the P700-F(X) core, E(m)(A(1A/B)) remains practically unchanged, whereas E(m)(F(X)) is upshifted by 42 mV. With these calculated E(m) values, the electron transfer rate from A(1) to F(X) in the P700-F(X) core is estimated to be slightly faster on the A(1A) side than that of the wild type, which is consistent with kinetic measurements.     

Organization of photosystem I polypeptides examined by chemical cross-linking.

Photosystem I from the cyanobacterium Synechocystis sp. PCC 6803 was examined using the chemical cross-linkers glutaraldehyde and N-ethyl-1-3-[3-(dimethylamino)propyl]carbodiimide to investigate the organization of the polypeptide subunits. Thylakoid membranes and photosystem I, which was isolated by Triton X-100 fractionation, were treated with cross-linking reagents and were resolved using a Tricine/urea low-molecular-weight resolution gel system. Subunit-specific antibodies and western blotting analysis were used to identify the components of cross-linked species. These analyses identified glutaraldehyde-dependent cross-linking products composed of small amounts of PsaD and PsaC, PsaC and PsaE, and PsaE and PsaF. The novel cross-link between PsaE and PsaF was also observed following treatment with N-ethyl-1-3-[3-(dimethylamino)propyl]carbodiimide. These cross-linking results suggest a structural interaction between PsaE and PsaF and predict a transmembrane topology for PsaF.     

Control of electron transport in photosystem I by the iron-sulfur cluster FX in response to intra- and intersubunit interactions

Photosystem I (PS I) is a transmembranal multisubunit complex that mediates light-induced electron transfer from plactocyanine to ferredoxin. The electron transfer proceeds from an excited chlorophyll a dimer (P700) through a chlorophyll a (A0), a phylloquinone (A1), and a [4Fe-4S] iron-sulfur cluster FX, all located on the core subunits PsaA and PsaB, to iron-sulfur clusters FA and FB, located on subunit PsaC. Earlier, it was attempted to determine the function of FX in the absence of FA/B mainly by chemical dissociation of subunit PsaC. However, not all PsaC subunits could be removed from the PS I preparations by this procedure without partially damaging FX. We therefore removed subunit PsaC by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp. PCC 6803. Cells could not grow under photosynthetic conditions when subunit PsaC was deleted, yet the PsaC-deficient mutant cells grew under heterotrophic conditions and assembled the core subunits of PS I in which light-induced electron transfer from P700 to A1 occurred. The photoreduction of FX was largely inhibited, as seen from direct measurement of the extent of electron transfer from A1 to FX. From the crystal structure it can be seen that the removal of subunits PsaC, PsaD, and PsaE in the PsaC-deficient mutant resulted in the braking of salt bridges between these subunits and PsaB and PsaA and the formation of a net of two negative surface charges on PsaA/B. The potential induced on FX by these surface charges is proposed to inhibit electron transport from the quinone. In the complete PS I complex, replacement of a cysteine ligand of FX by serine in site-directed mutation C565S/D566E in subunit PsaB caused an approximately 10-fold slow down of electron transfer from the quinone to FX without much affecting the extent of this electron transfer compared with wild type. Based on these and other results, we propose that FX might have a major role in controlling electron transfer through PS I.     

Decreased stability of photosystem I in dgd1 mutant of Arabidopsis thaliana

The dgd1 mutant of Arabidopsis thaliana provides us with a powerful tool for revealing the specific role of digalactosyldiacylglycerol (DGDG) in photosynthesis. Blue-native polyacrylamide gel electrophoresis analysis revealed that photosystem I (PSI) subunits are assembled into a PSI complex, and that a PSI subcomplex lacking stroma side subunits was also present. PSI subunits in the dgd1 mutant were decreased to a similar level compared with that in the wild type (WT) Arabidopsis. Further experiments showed that PSI subunits in the stroma side, PsaD and PsaE, in the dgd1 mutant were more susceptible to removal by chaotropic agents than those in the WT plant, indicating that the stability of PsaD and PsaE is impaired in the dgd1 mutant. These results provide evidence that DGDG is important for the stability of the PSI complex.     

Genetic analysis of the Photosystem I subunits from the red alga, Galdieria sulphuraria

Currently, there are very little data available regarding the photosynthetic apparatus of red algae. We have analyzed the genes for Photosystem I in the recently sequenced genome of the red alga Galdieria sulphuraria. All subunits that are conserved between plants and cyanobacteria were unambiguously identified in the Galdieria genome: PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaI, PsaJ, PsaK and PsaL. From the plant specific subunits, PsaN and PsaO were identified but the sequence homology was much lower than for the subunits that are present in plants and cyanobacteria. The subunit PsaX, which is specific for thermophilic cyanobacteria, is not present in the Galdieria genome, whereas PsaM is a plastid-encoded protein as in other red algae. The sequences of the core subunits of PSI were further analyzed by mapping of the conserved areas in the crystal structures of cyanobacterial and plant PSI. The structural comparison shows that PSI from the red alga Galdieria may represent a common ancestral structure at the interface between cyanobacterial and plant PSI. Some subunits have a “zwitter” structure that contains structural elements that show similarities with either plant or cyanobacterial PSI. The structure of PsaL, which is responsible for the trimerization of PSI in cyanobacteria, lacks a short helix and the Ca²⁺ binding site, which are essential for trimer formation indicating that the Galdieria PSI is a monomer. However the sequence homology to plant PsaL is low and lacks strong conservation of the interaction sites with PsaH. Furthermore, the sites for interaction of plant PSI with the LHCI complex are not well conserved between plants and Galdieria, which may indicate that Galdieria may contain a PSI that is evolutionarily much more ancient than PSI from green algae, plants and the current cyanobacteria.