Role of acidic amino acid residues of PsaD subunit on limiting the affinity of photosystem I for ferredoxin.

The PsaD subunit of photosystem I is one of the central polypeptides for the interaction with ferredoxin, its acidic electron acceptor. In the cyanobacterium Synechocystis 6803, this role is partly performed by a sequence extending approximately from histidine 97 to arginine 119, close to the C-terminus. In the present work, acidic amino acids D100, E105, and E109 are shown to moderate the affinity of Photosystem I for ferredoxin. Most single replacements of these residues by neutral amino acids increased the affinity for ferredoxin, resulting in a dissociation constant as low as 0.015 microM for the E105Q mutant (wild-type K(D) = 0.4 microM). This is the first report on the limitation of photosystem I affinity for ferredoxin due to acidic amino acids from PsaD subunit. It highlights the occurrence of a negative control on the binding during the formation of transient complexes between electron carriers.

Multiple functions for the C terminus of the PsaD subunit in the cyanobacterial photosystem I complex.

PsaD subunit of Synechocystis sp PCC 6803 photosystem I (PSI) plays a critical role in the stability of the complex and is part of the docking site for ferredoxin (Fd). In the present study we describe major physiological and biochemical effects resulting from mutations in the accessible C-terminal end of the protein. Four basic residues were mutated: R111, K117, K131, and K135, and a large 36-amino acid deletion was generated at the C terminus. PSI from R111C mutant has a 5-fold decreased affinity for Fd, comparable with the effect of the C terminus deletion, and NADP+ is photoreduced with a 2-fold decreased rate, without consequence on cell growth. The K117A mutation has no effect on the affinity for Fd, but decreases the stability of PsaE subunit, a loss of stability also observed in R111C and the deletion mutants. The double mutation K131A/K135A does not change Fd binding and reduction, but decreases the overall stability of PSI and impairs the cell growth at temperatures above 30 degrees C. Three mutants, R111C, K117A, and the C-terminal deleted exhibit a higher content of the trimeric form of PSI, in apparent relation to the removal of solvent accessible positive charges. Various regions in the C terminus of cyanobacterial PsaD thus are involved in Fd strong binding, PSI stability, and accumulation of trimeric PSI.

Importance of the region including aspartates 57 and 60 of ferredoxin on the electron transfer complex with photosystem I in the cyanobacterium Synechocystis sp. PCC 6803.

Ferredoxin reduction by photosystem I has been studied by flash-absorption spectroscopy. Aspartate residues 20, 57, and 60 of ferredoxin were changed to alanine, cysteine, arginine, or lysine. On the one hand, electron transfer from photosystem I to all mutated ferredoxins still occurs on a microsecond time scale, with half-times of ferredoxin reduction mostly conserved compared to wild-type ferredoxin. On the other hand, the total amplitude of the fast first-order reduction varies largely when residues 57 or 60 are modified, in apparent relation to the charge modification (neutralized or inverted). Substituting these two residues for lysine or arginine induce strong effects on ferredoxin binding (up to sixfold increase in K(D)), whereas the same substitution on aspartate 20, a spatially related residue, results in moderate effects (maximum twofold increase in K(D)). In addition, double mutations to arginine or lysine were performed on both aspartates 57 and 60. The mutated proteins have a 15- to 20-fold increased K(D) and show strong modifications in the amplitudes of the fast reduction kinetics. These results indicate that the acidic area of ferredoxin including aspartates 57 and 60, located opposite to the C-terminus, is crucial for high affinity interactions with photosystem I.

Essential role of a single arginine of photosystem I in stabilizing the electron transfer complex with ferredoxin.

PsaE is one of the photosystem I subunits involved in ferredoxin binding. The central role of arginine 39 of this 8-kDa peripheral polypeptide has been established by a series of mutations. The neutral substitution R39Q leads to a 250-fold increase of the dissociation constant K(d) of the photosystem I-ferredoxin complex, as large as the increase induced by PsaE deletion. At pH 8.0, this K(d) value strongly depends on the charge of the residue substituting Arg-39: 0.22 microM for wild type, 1.5 microM for R39K, 56 microM for R39Q, and more than 100 microM for R39D. The consequences of arginine 39 substitution for the titratable histidine were analyzed as a function of pH. The K(d) value of R39H is increased 140 times at pH 8.0 but only 5 times at pH 5.8, which is assigned to the protonation of histidine at low pH. In the mutant R39Q, the association rate of ferredoxin was decreased 3-fold compared with wild type, whereas an 80-fold increase is calculated for the dissociation rate. We propose that a major contribution of PsaE is to provide a prominent positive charge at position 39 for controlling the electrostatic interaction and lifetime of the complex with ferredoxin.

Ferredoxin reduction by photosystem I from Synechocystis sp. PCC 6803: toward an understanding of the respective roles of subunits PsaD and PsaE in ferredoxin binding.

The process of ferredoxin reduction by photosystem I has been extensively investigated by flash-absorption spectroscopy in psaD and psaE deleted mutants from Synechocystis sp. PCC 6803. In both mutants, the dissociation constant for the photosystem I/ferredoxin complex at pH 8 is considerably increased as compared to the wild type: approximately 25- and 100-fold increases are found for PsaD-less and PsaE-less photosystem I, respectively. However, at high ferredoxin concentrations, submicrosecond and microsecond kinetics of electron transfer similar to that observed in the wild type are present in both mutants. The presence of these fast kinetic components indicates that the relative positions of ferredoxin and of the terminal photosystem I acceptor are not significantly disturbed by the absence of either PsaD or PsaE. The second-order rate constant of ferredoxin reduction is lowered 10- and 2-fold for PsaD-less and PsaE-less photosystem I, respectively. Assuming a simple binding equilibrium between photosystem I and ferredoxin, PsaD appears to be important for the guiding of ferredoxin to its binding site (main effect on the association rate) whereas PsaE seems to control the photosystem I/ferredoxin complex lifetime (main effect on the dissociation rate). The properties of electron transfer from photosystem I to ferredoxin were also studied at pH 5. 8. In the psaE deleted mutant as in the wild type, the change of pH from 8 to 5.8 induces a 10-fold increase in affinity of ferredoxin for photosystem I. In the absence of PsaD, this pH effect is not observed, in favor of this subunit being mostly responsible for the low pH increased affinity.

Monoacylation of ribonuclease A enables its transport across an in vitro model of the blood-brain barrier.

A major challenge in correcting disorders affecting the central nervous system is to induce blood-brain barrier (BBB) crossing of exogenous biological compounds such as proteins or specific nucleic acid sequences. Fatty acids, due to their high membrane affinity and low toxicity, are good potential candidates to promote this barrier crossing when covalently bound to proteins. In this paper, we report that regiospecific monoacylation of ribonuclease A (RNase A) enables its transport across an in vitro model of the BBB. Myristoylated, palmitoylated and stearoylated RNases A were prepared using reversed micelles as microreactors. All the purified acylated RNases A kept their original enzymatic activity. A single fatty acid moiety was linked to RNase A through the alpha-amino group of its N-terminal lysine as shown by powerful analytical techniques. The ability of monoacylated RNases A to cross an in vitro model of the BBB is strictly dependent on the acyl chain length, which must be at least 16 carbon atoms long.

The PsaE subunit is required for complex formation between photosystem I and flavodoxin from the cyanobacterium Synechocystis sp. PCC 6803.

The photoreduction of the oxidized and the semiquinone form of flavodoxin by photosystem I particles (PSI) from the wild type and a psaE deletion strain from the cyanobacterium Synechocystis sp. PCC 6803 was analyzed by flash-absorption spectroscopy to investigate a possible involvement of the PsaE subunit in this photoreduction process. The kinetics of the reduction of oxidized flavodoxin display a single-exponential component for both PSI preparations. Limiting electron transfer rates kobs of approximately 500 and approximately 900 s -1 are deduced for the wild type and PSI from the psaE-less mutant, respectively, indicating that the PsaE subunit is not important for this photoreduction process. In the case of wild-type PSI, the reduction of flavodoxin semiquinone is a biphasic process, displaying a fast first-order phase with a t1/2 of approximately 13 micro(s) which is then followed by a slower, concentration-dependent phase, for which a second-order rate constant k2 of 2.2 x 10(8) M-1 cm-1 is calculated. In contrast, photoreduction of the semiquinone by PSI from the psaE-less mutant is monoexponential, displaying only one second-order component with a second-order rate constant similar to those observed for wild-type PSI (k2 = 1.5 x 10(8) M-1 cm-1). The fast first-order component which is interpreted as an electron transfer process within a preformed complex between flavodoxin semiquinone and PSI is almost completely absent in the reduction of flavodoxin by the PsaE-less PSI. A similar loss of the fast phase is also observed for the photoreduction of flavodoxin semiquinone by PSI from a Synechococcus elongatus psaE-less mutant. Upon reconstitution of isolated PsaE to the PsaE-less PSI in vitro, approximately 80% of the fast first-order kinetic component is recovered, indicating that PsaE is required for high-affinity binding of the flavodoxin semiquinone to PSI. In addition, chemical cross-linking assays show that flavodoxin can no longer be cross-linked to PSI in detectable amounts when PsaE is missing on the reaction center. Taken together, these experiments indicate that the PsaE subunit is required for complex formation between PSI and flavodoxin but is not required for an efficient forward electron transfer from photosystem I to both forms of flavodoxin.

Structural organization of the major subunits in cyanobacterial photosystem 1. Localization of subunits PsaC, -D, -E, -F, and -J.

Based on an improved isolation procedure using perfusion chromatography, trimeric Photosystem 1 (PS1) complexes have been isolated from various deletion mutants of the mesophilic cyanobacterium Synechocystis PCC 6803. These mutants are only deficient in the deleted subunits, which was carefully checked by high resolution gel electrophoresis in combination with immunoblotting. These highly purified and well characterized PS1 particles were then examined by electron microscopy, followed by computer-aided image processing with single particle averaging techniques as described earlier (Kruip, J., Boekema, E. J., Bald, D., Boonstra, A. F., and Rögner, M. (1993) J. Biol. Chem. 268, 23353-23360). This precise methodological approach allowed a confident localization of the PS1 subunits PsaC, -D, -E, -F, and -J; it also shows shape and size of these subunits once integrated in the PS1 complex. Subunits PsaC, -D, and -E form a ridge on the stromal site, with PsaE toward the edge of each monomer within the trimer and PsaD extending toward the trimeric center, leaving PsaC in between. PsaF (near PsaE) and PsaJ are close together on the outer edge of each monomer; their proximity is also supported by chemical cross-linking, using the zero-length cross-linker EDC. This localization of PsaF contradicts the position suggested by the published low resolution x-ray analysis and shows for the first time the existence of at least one transmembrane alpha-helix for PsaF. A topographic three-dimensional map has been drawn from this set of results showing the location of the major PS1 subunits (besides PsaA and PsaB). These data also led to the assignment of electron density in the recent medium resolution x-ray structure for PS1 (Krauss, N., Schubert, W.-D., Klukas, O., Fromme, P., Witt, H. T., Saenger, W. (1996) Nat. Struct. Biol. 3, 965-973).

Mutagenesis of photosystem I in the region of the ferredoxin cross-linking site: modifications of positively charged amino acids.

The psaD gene isolated from the cyanobacterium Synechocystis sp. PCC 6803 has been mutated in the region encoding a cross-linking site for ferredoxin. A glucose tolerant strain of Synechocystis 6803 was first deleted for psaD, and the resulting PS-I was characterised by EPR and flash absorption spectroscopy. The major modification related to the absence of the PsaD subunit is the disappearance of the first order reduction of ferredoxin which is replaced by a second order reaction. Reconstitution of the deleted PS-I with the purified PsaD polypeptide restored 80% of the fast photoreduction of ferredoxin. The deletion of PsaD has no apparent effect on the main biochemical features of the resulting depleted PS-I complex, with the exception of minor modifications to the FA/FB centers. The deleted strain was transformed by a series of psaD genes mutated at three conserved residues, all located close to the ferredoxin cross-linking site. The resulting photosystem I complexes were extensively studied by flash absorption spectroscopy. Unexpectedly, the change of Lys 106 involved in the cross-linking of ferredoxin for an uncharged amino acid has almost no effect (mutation K106A). However, the functional consequences of more drastic substitutions of either Lys 106 or Arg 111 indicate a role for these two basic amino acids in the binding and submicrosecond reduction of ferredoxin. Various mutations of the unique His at position 97 show that this amino acid is involved in the increased affinity of PS-I for ferredoxin when the pH is lowered. This histidine could be central in regulating in vivo the rate of ferredoxin reduction as a precise sensor of the local proton concentration.

Identification of the amino acids involved in the functional interaction between photosystem I and ferredoxin from Synechocystis sp. PCC 6803 by chemical cross-linking.

Ferredoxin isolated from the cyanobacterium Synechocystis sp. PCC 6803 has been chemically cross-linked to purified photosystem I from the same organism. The reaction was catalyzed by N-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of N-hydroxysulfosuccinimide. A short reaction time and neutral pH values can be used in the presence of the two reagents, ensuring the integrity of both of the proteins and the iron-sulfur cluster of the ferredoxin. The only covalent complex detected comprised ferredoxin and the photo-system I (PSI)-D subunit, as identified by antibodies probing after electrophoresis. Electron paramagnetic resonance measurements of this covalent complex have shown that the cross-linked ferredoxin was entirely photoreducible by photosystem I and that the molar ratio of ferredoxin to PSI was close to 1. Extensive sequencing of the peptides obtained after proteolysis of the purified cross-linked product led to the identification of a covalent bond between glutamic acid 93 of ferredoxin and lysine 106 of the PSI-D subunit.

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.

Ferredoxin and flavodoxin from the cyanobacterium Synechocystis sp PCC 6803.

The unicellular cyanobacterium Synechocystis sp PCC 6803 is capable of synthesizing two different Photosystem-I electron acceptors, ferredoxin and flavodoxin. Under normal growth conditions a [2Fe-2S] ferredoxin was recovered and purified to homogeneity. The complete amino-acid sequence of this protein was established. The isoelectric point (pI = 3.48), midpoint redox potential (Em = -0.412 V) and stability under denaturing conditions were also determined. This ferredoxin exhibits an unusual electrophoretic behavior, resulting in a very low apparent molecular mass between 2 and 3.5 kDa, even in the presence of high concentrations of urea. However, a molecular mass of 10,232 Da (apo-ferredoxin) is calculated from the sequence. Free thiol assays indicate the presence of a disulfide bridge in this protein. A small amount of ferredoxin was also found in another fraction during the purification procedure. The amino-acid sequence and properties of this minor ferredoxin were similar to those of the major ferredoxin. However, its solubility in ammonium sulfate and its reactivity with antibodies directed against spinach ferredoxin were different. Traces of flavodoxin were also recovered from the same fraction. The amount of flavodoxin was dramatically increased under iron-deficient growth conditions. An acidic isoelectric point was measured (pI = 3.76), close to that of ferredoxin. The midpoint redox potentials of flavodoxin are Em1 = -0.433 V and Em2 = -0.238 V at pH 7.8. Sequence comparison based on the 42 N-terminal amino acids indicates that Synechocystis 6803 flavodoxin most likely belongs to the long-chain class, despite an apparent molecular mass of 15 kDa determined by SDS-PAGE.

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.

Amino acid sequence of photosystem I subunit IV from the cyanobacterium Synechocystis PCC 6803.

We describe here the complete amino acid sequence of photosystem I subunit IV from Synechocystis 6803. The molecular mass of 8.0 kDa is lower than in higher plants and Chlamydomonas, due to the lack of a characteristic, proline-rich, N-terminal sequence. The remaining sequence exhibits a good conservation, with a hydrophilic and strongly basic N-terminal head followed by two hydrophobic domains. There is no possibility of classical membrane-spanning alpha helices. This component is likely to be one of the most stroma accessible subunits of photosystem I.

Cloning and sequencing of spinach cDNA clones encoding the 20 kDa PS I polypeptide.

Apart from the 8 kDa subunit, which is of chloroplast origin, most of the small polypeptides of the PS I reaction center from higher plants are encoded in nuclear genes. We describe here the first nucleotide sequence of a nuclear component of this photosystem, the precursor of the 20 kDa protein. The deduced sequence of the large transit peptide (55-60 amino acids) is rich in serine/threonine residues and has a net positive charge, which are classical features of these precursors. The sequence itself is mainly hydrophilic, with no possibility of classical membrane-spanning alpha-helices; it exhibits an interesting stretch of five basic amino acids in close vicinity: Thr-Arg-Leu-Arg-Ser-Lys-Tyr-Lys-Ile-Lys-Tyr.

Screening of monoclonal antibodies using antigens labeled with acetylcholinesterase: application to the peripheral proteins of photosystem 1.

An original immunoenzymatic screening method, based on the use of antigens labeled with the stable enzyme acetylcholinesterase (AChE, EC 3.1.1.7), is described. The high turnover of this enzyme results in a very sensitive detection of antibodies. In this method, monoclonal antibodies from the supernatants of hybridoma cultures are immobilized on a solid phase coated with anti-mouse immunoglobulins and react simultaneously with the appropriate antigen labeled with biotin molecules. In a second step, biotinylated acetylcholinesterase is in turn associated to the system via avidin interactions and subsequently detected by a colorimetric assay. The method appears more sensitive and easier to use than either the corresponding radioimmunological test using a 125I-iodinated antigen or the same type of enzymatic immunoassay performed with biotinylated horseradish peroxidase instead of biotinylated AChE. The combined use of microtiter plates, solid-phase separation, and colorimetric detection allows a high level of automation of the method which makes it very efficient to process a large number of samples. This technique has been successfully applied to the screening of monoclonal antibodies directed against peripheral proteins of the photosystem 1 (PS1) membrane complex in photosynthesis. A complete set of antibodies recognizing these PS1 components was selected. The same technique was also tested in competition immunoassays and appears to be a very precise and useful tool for quantifying PS1 polypeptides in different biological extracts, including sodium dodecyl sulfate-denatured membranes. This can be of special interest for studying the biogenesis of membrane complexes.

Protein identification of purified particles isolated from spinach thylakoids by deoxycholate electrophoresis.

Three thylakoid complexes were isolated by deoxycholate preparative electrophoresis. The protein composition of each fraction was analyzed by SDS analytical electrophoresis. No protein of the PS 1 enriched fraction (fraction 1) was found in the PS 2 enriched fraction (fraction 2) and inversely. The antenna complex (fraction 3) did not have any contamination by proteins of fraction 1 or fraction 2. Fraction 1 was mainly composed of the CP1, the reaction center complex of the PS1, and by low molecular weight proteins, previously found in other PS 1 preparations. Tentative assignments of these proteins are presented; among them are iron sulfur proteins. After analytical SDS electrophoresis of fraction 2, the reaction center complex was dissociated. Nevertheless three proteins of 50 kD, 42 kD and 35 kD were assigned to this complex. Fraction 2 contained also the three cytochromes of the thylakoid membranes: cyt f, cyt b6, cyt b559. Fraction 3 was exclusively composed of one protein pigment complex, CP2.

Effect of various denaturing treatments on the structure and activity of a purified CP1 complex.

Spinach CP1 complex, purified as previously described [16], was submitted to various dissociating treatments. Chaotropic agents, like urea and thiocyanate salts, remained without effect on the structure and photooxidation of the complex, just SDS at very high concentrations was able to dissociate the chlorophyll from the polypeptides and to abolish the photoreaction. Proteolytic enzymes have no more action on the apparent structure and activity of CP1, but some of them do cleave the large polypeptides (65 kD) into smaller ones, as observed after pigments dissociation. This last result might be an important step in the search for a smaller active P700 protein complex.

Contribution to the structural characterization of eucaryotic PSI reaction centre-I. Critical analysis of the polypeptidic composition of different P700 enriched fractions.

In thylakoid membranes, several peptides of high MW† are present which may interfere with the study of CP1's components. Modifying Cleveland's technique [7] for limited proteolysis, we have characterized the polypeptides found in the 60 kD region. Some may result from incomplete washing of the CF1 while others come from the CP1; indeed, this chlorophyll protein complex, which has a higher MW (above 100 kD), very often undergoes a dissociation into smaller components of about 60 KD MW.Analysis of the protein content of different preparations commonly used to obtain PSI reaction centre enriched fractions has been performed. The α and ß subunits of CF1 are among the main contaminants of most of these preparations. A further purification step is described which can be applied to all these preparations, but numerous peptides are still present in the active fractions. It is most unlikely that all these polypeptides are required for the primary photochemical event, and this emphasizes the necessity to find a new simple method to purify PSI reaction centres.

Contribution to the structural characterization of eucaryotic PSI reaction centre - II. Characterization of a highly purified photoactive SDS-CP1 complex.

Under precise conditions, SDS PAGE† allows purification of a photoactive P700-chla-protein complex from eucaryotic cells. The yield of P700 recovery is close to 100%. A total protein content equivalent to about 140 kD for one mole of P700 has been estimated by chemical analysis, and electrophoresis revealed the presence of two peptidic chains with MWs close to 65 kD. Photochemical and structural properties of this complex are given and compared with those of other complexes previously isolated.