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PubMed1997

Strains of Synechocystis sp. PCC 6803 with altered PsaC. II. EPR and optical spectroscopic properties of FA and FB in aspartate, serine, and alanine replacements of cysteines 14 and 51.

A psaC deletion mutant of the unicellular cyanobacterium Synechocystis sp. PCC 6803 was utilized to incorporate site-specific amino acid substitutions in the cysteine residues that ligate the FA and FB iron-sulfur clusters in Photosystem I (PS I). Cysteines 14 and 51 of PsaC were changed to aspartic acid (C14DPsaC, C51DPsaC, C14D/C51DPsaC), serine (C14SPsaC, C51SPsaC), and alanine (C14APsaC, C51APsaC), and the properties of FA and FB were characterized by electron paramagnetic resonance spectroscopy and time-resolved optical spectroscopy. The C14DPsaC-PS I and C14SPsaC-PS I complexes showed high levels of photoreduction of FA with g values of 2.045, 1. 944, and 1.852 after illumination at 15 K, but there was no evidence of reduced FB in the g = 2 region. The C51DPsaC-PS I and C51SPsaC-PS I complexes showed low levels of photoreduction of FB with g values of 2.067, 1.931, and 1.881 after illumination at 15 K, but there was no evidence of reduced FA in the g = 2 region. The presence of FB was inferred in C14DPsaC-PS I and C14SPsaC-PS I, and the presence of FA was inferred in C51DPsaC-PS I and C51SPsaC-PS I by magnetic interaction in the photoaccumulated spectra and by the equal spin concentration of the irreversible P700(+) cation generated by illumination at 77 K. Flash-induced optical absorbance changes at 298 K in the presence of a fast electron donor indicate that two electron acceptors function after FX in the four mutant PS I complexes at room temperature. These data suggest that a mixed-ligand [4Fe-4S] cluster is present in the mutant sites of C14X-PS I and C51X-PS I (where X = D or S), but that the proposed spin state of S = 3/2 renders the resonances undetectable in the g = 2 region. The C14APsaC-PS I, C51APsaC-PS I and C14D/C51DPsaC-PS I complexes show only the photoreduction of FX, consistent with the absence of PsaC. These results show that only those PsaC proteins that contain two [4Fe-4S] clusters are capable of assembling onto PS I cores in vivo.

Amino Acids↗
PubMed1999

Site-directed mutagenesis of the PsaC subunit of photosystem I. F(b) is the cluster interacting with soluble ferredoxin.

The two [4Fe-4S] clusters F(A) and F(B) are the terminal electron acceptors of photosystem I (PSI) that are bound by the stromal subunit PsaC. Soluble ferredoxin (Fd) binds to PSI via electrostatic interactions and is reduced by the outermost iron-sulfur cluster of PsaC. We have generated six site-directed mutants of the green alga Chlamydomonas reinhardtii in which residues located close to the iron-sulfur clusters of PsaC are changed. The acidic residues Asp(9) and Glu(46), which are located one residue upstream of the first cysteine liganding cluster F(B) and F(A), respectively, were changed to a neutral or a basic amino acid. Although Fd reduction is not affected by the E46Q and E46K mutations, a slight increase of Fd affinity (from 1.3- to 2-fold) was observed by flash absorption spectroscopy for the D9N and D9K mutant PSI complexes. In the FA(2) triple mutant (V49I/K52T/R53Q), modification of residues located next to the F(A) cluster leads to partial destabilization of the PSI complex. The electron paramagnetic resonance properties of cluster F(A) are affected, and a 3-fold decrease of Fd affinity is observed. The introduction of positively charged residues close to the F(B) cluster in the FB(1) triple mutant (I12V/T15K/Q16R) results in a 60-fold increase of Fd affinity as measured by flash absorption spectroscopy and a larger amount of PsaC-Fd cross-linking product. The first-order kinetics are similar to wild type kinetics (two phases with t((1)/(2)) of <1 and approximately 4.5 microseconds) for all mutants except FB(1), where Fd reduction is almost monophasic with t((1)/(2)) < 1 microseconds. These data indicate that F(B) is the cluster interacting with Fd and therefore the outermost iron-sulfur cluster of PSI.

Animals↗
PubMed1996

Active photosynthesis in cyanobacterial mutants with directed modifications in the ligands for two iron-sulfur clusters on the PsaC protein of photosystem I.

The PsaC protein of the Photosystem I (PSI) complex in thylakoid membranes coordinates two [4Fe-4S] clusters, FA and FB. Although it is known that PsaC participates in electron transfer to ferredoxin, the pathway of electrons through this protein is unknown. To elucidate the roles of FA and FB, we created two site-directed mutant strains of the cyanobacterium Anabaena variabilis ATCC 29413. In one mutant, cysteine 13, a ligand for FB was replaced by an aspartic acid (C13D); in the other mutant, cysteine 50, a ligand for FA was modified similarly (C50D). Low-temperature electron paramagnetic resonance studies demonstrated that the C50D mutant has a normal FB center and a modified FA center. In contrast, the C13D strain has normal FA, but failed to reveal any signal from FB. Room-temperature optical studies showed that C13D has only one functional electron acceptor in PsaC, whereas two such acceptors are functional in the C50D and wild-type strains. Although both mutants grow under photoautotrophic conditions, the rate of PSI-mediated electron transfer in C13D under low light levels is about half that of C50D or wild type. These data show that (i) FB is not essential for the assembly of the PsaC protein in PSI and (ii) FB is not absolutely required for electron transfer from the PSI reaction center to ferredoxin.

Amino Acid Sequence↗
PubMed2025

The critical role of PSAC channel in malaria parasite survival is driven home by phenotypic screening under relevant nutrient levels.

Spreading resistance to front-line treatments necessitate the search for new classes of antimalarials. Limitations of standard screening conditions lead us to develop an assay using culture media that more closely reflects nutrient levels in human serum to reveal new therapeutically relevant parasite pathways. Our approach was validated by testing 22k compounds followed by a full 750k compound screen and identified 29 chemotypes with higher activity in nutrient restricted media that were further characterized. Through a combination of chemo-genomics and innovative photocatalytic proximity labeling proteomics, we identified the target of two compounds as the CLAG3 component of the plasmodial surface anion channel (PSAC). Strikingly, every one of the other 29 chemotypes selected was also found to block PSAC activity, highlighting the importance of this nutrient channel for parasite survival under physiological conditions. The effect of PSAC inhibitors in the in vivo humanized mouse model was confirmed.

Animals↗
PubMed1994

The PsaC protein is necessary for the stable association of the PsaD, PsaE, and PsaL proteins in the photosystem I complex: analysis of a cyanobacterial mutant strain.

The PsaC protein binds two 4Fe-4S centers, FA and FB, in the photosystem I (PSI) protein complex. In the T398 strain of Anabaena variabilis ATCC 29413, the psaC gene encoding this protein has been insertionally inactivated by the introduction of a neomycin resistance gene cartridge in the coding region. Photosystem I complex was purified through native gel electrophoresis of beta-dodecyl maltoside solubilized thylakoid membranes from wild-type and T398 strains of Anabaena 29413. The PSI complex from T398 strain retained functionally active P700, the reaction center chlorophylls. Interestingly, purified PSI complex from T398 cells lacked the PsaD, PsaE, and PsaL polypeptides. Western analysis with polyclonal antibodies raised against these proteins indicated that the two stromal exposed polypeptides, PsaD and PsaE, are absent in isolated thylakoid membranes from T398 cells. The PsaL polypeptide could be detected at a level comparable to that in wild-type thylakoid membranes, although it is absent in the PSI preparation from the mutant. These observations suggest that the PsaC protein is essential for the stable association of PsaD and PsaE, two hydrophilic, extrinsic polypeptides. Moreover, PsaL, a hydrophobic protein is loosely associated with PSI and is lost during the isolation of the PSI complex.

Anabaena↗
PubMed1998

PsaC subunit of photosystem I is oriented with iron-sulfur cluster F(B) as the immediate electron donor to ferredoxin and flavodoxin.

The PsaC subunit of photosystem I (PS I) binds two [4Fe-4S] clusters, F(A) and F(B), functioning as electron carriers between F(X) and soluble ferredoxin. To resolve the issue whether F(A) or F(B) is proximal to F(X), we used single-turnover flashes to promote step-by-step electron transfer between electron carriers in control (both F(A) and F(B) present) and HgCl2-treated (F(B)-less) PS I complexes from Synechococcus sp. PCC 6301 and analyzed the kinetics of P700+ reduction by monitoring the absorbance changes at 832 nm in the presence of a fast electron donor (phenazine methosulfate (PMS)). In control PS I complexes exogenously added ferredoxin, or flavodoxin could be photoreduced on each flash, thus allowing P700+ to be reduced from PMS. In F(B)-less complexes, both in the presence and in the absence of ferredoxin or flavodoxin, P700+ was reduced from PMS only on the first flash and was reduced from F(X)- on the following flashes, indicating lack of electron transfer to ferredoxin or flavodoxin. In the F(B)-less complexes, a normal level of P700 photooxidation was detected accompanied by a high yield of charge recombination between P700+ and F(A)- in the presence of a slow donor, 2,6-dichlorophenol-indophenol. This recombination remained the only pathway of F(A)- reoxidation in the presence of added ferredoxin, consistent with the lack of forward electron transfer. F(A)- could be reoxidized by methyl viologen in F(B)-less PS I complexes, although at a concentration two orders of magnitude higher than is required in wild-type PS I complexes, thus implying the presence of a diffusion barrier. The inhibition of electron transfer to ferredoxin and flavodoxin was completely reversed after reconstituting the F(B) cluster. Using rate versus distance estimates for electron transfer rates from F(X) to ferredoxin for two possible orientations of PsaC, we conclude that the kinetic data are best compatible with PsaC being oriented with F(A) as the cluster proximal to F(X) and F(B) as the distal cluster that donates electrons to ferredoxin.

Biophysical Phenomena↗
PubMed1995

Modified ligands to FA and FB in photosystem I. I. Structural constraints for the formation of iron-sulfur clusters in free and rebound PsaC.

Cysteines 14, 21, 34, 51, or 58 in PsaC of photosystem I (PS I) were replaced with aspartic acid (C21D and C58D), serine (C14S, C34S, and C51S), and alanine (C14A, C34A, and C51A). When free in solution, the C34S and C34A holoproteins contained two S = 1/2 ground state [4Fe-4S] clusters; all other mutant proteins contained [3Fe-4S] clusters and [4Fe-4S] clusters; in addition, there was evidence in C14S, C51S, C14A, and C51A for high spin (S = 3/2) [4Fe-4S] clusters, presumably in the modified site. These findings are consistent with the assignment of C14, C21, C51, and C58, but not C34, as ligands to FA and FB. The [4Fe-4S] clusters in the unmodified sites in C14S, C51S, C14A, and C51A remained highly electronegative, with Em values ranging from -495 to -575 mV. The [3Fe-4S] clusters in the modified sites were driven 400 to 450 mV more oxidizing than the native [4Fe-4S] clusters, with Em values ranging from -98 mV to -171 mV. A C14D/C51D double mutant contains [3Fe-4S] and S = 1/2 [4Fe-4S] clusters, showing that the 3Cys.1Asp motif is also able to accommodate a low spin cubane. When C34S, C34A, C14S, C51S, C14A, and C51A were rebound to P700-FX cores, electron transfer to FA/FB was regained, but functional reconstitution has not yet been achieved for C21D, C58D, or C14D/C51D. These data imply that PsaC requires two iron-sulfur clusters to refold, one of which must be a cubane. Since two [4Fe-4S] clusters are found in all reconstituted PS I complexes, the presence of two cubanes in free PsaC may be a necessary precondition for binding to P700-FX cores.

Amino Acid Sequence↗
PubMed1995

Modified ligands to FA and FB in photosystem I. II. Characterization of a mixed ligand [4Fe-4S] cluster in the C51D mutant of PsaC upon rebinding to P700-Fx cores.

A Photosystem I (PS I) complex reconstituted with PsaC-C51D (aspartate in lieu of cysteine in position 51) shows light-induced EPR signals with g values, line widths, and photoreduction behavior characteristic of FB. Contrary to an earlier report, a [3Fe-4S] cluster was not located in the reconstituted PS I complex. Instead, a second set of resonances with g values of 2.044, 1.942, and 1.853 becomes EPR-visible when the C51D-PS I complex is measured at 4.2 K. This fast relaxing center, termed FA' is likely to represent a [4Fe-4S] cluster in the mixed ligand (3Cys.1Asp) site. Redox studies show that the Em of FA' and FB are -630 mV and -575 mV, respectively. Room temperature optical studies support the presence of two functioning electron acceptors subsequent to Fx, and NADP+ photoreduction rates mediated by ferredoxin and flavodoxin are nearly equivalent to the wild type. In addition to [3Fe-4S] clusters and S = 1/2 ground state [4Fe-4S] clusters, the free PsaC-C51D protein shows resonances near g = 5.5, which may represent a population of high spin (S = 3/2) [4Fe-4S] clusters in the mixed ligand FA' site. Similar to the C14D-PS I mutant complex, it is proposed that the P700-Fx core selectively rebinds those free PsaC-C51D proteins that contain two [4Fe-4S] clusters. These studies show that primary photochemistry and electron transfer rates in PS I are relatively unaffected by the presence of a highly reducing, mixed ligand cluster in the FA' site.

Aspartic Acid↗
PubMed1997

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).

Bacterial Proteins↗
PubMed1992

Identification of a second psaC gene in the cyanobacterium Synechocystis sp. PCC6803.

The psaC gene encodes the 9 kDa protein subunit of photosystem I that carries the iron-sulfur centers FA and FB. The paper describes the sequence and transcription analysis of a second psaC gene (psaC2) from Synechocystis sp. PCC6803. ThepsaC2 gene is transcribed into an abundant monocistronic mRNA. The deduced amino acid sequence of PSI-C2 is very similar to the protein sequences from two other cyanobacteria (Synechococcus sp. PCC7002 and Nostoc sp. PCC8009). In contrast, the recently isolated psaC1 gene is not transcribed and the PSI-C1 amino acid sequence shows the highest similarity to the proteins from higher plants.

Amino Acid Sequence↗
PubMed1992

Site-directed conversion of a cysteine to aspartate leads to the assembly of a [3Fe-4S] cluster in PsaC of photosystem I. The photoreduction of FA is independent of FB.

The terminal electron acceptors FA and FB exist as two [4Fe-4S] clusters located on the 8.9-kDa PsaC protein in photosystem I. We have used site-directed mutagenesis to produce a complementary pair of mutant PsaC proteins in which specific cysteine ligands to the [4Fe-4S] clusters were changed to aspartic acid residues. The mutant proteins, denoted C14D and C51D, were overproduced in Escherichia coli; the iron-sulfur clusters were inserted in vitro; and the reconstituted proteins were rebound to the P700-FX core of Synechococcus sp. PCC 6301 in the presence of the PsaD protein. In complexes reconstituted with C51D a rhombic ESR spectrum with g-values of 2.063, 1.934, and 1.879 in the reduced state identifies the intact [4Fe-4S] cluster as FB, while an intense axial spectrum with g-values of 2.020 and 1.997 in the oxidized state identifies the altered cluster in the aspartate site as a [3Fe-4S] cluster. The [3Fe-4S] cluster corresponding to FA can be reduced chemically with dithionite and photochemically by illumination at room temperature but is not reduced by illumination at 15 K. With reconstituted C14D a rhombic ESR spectrum with g-values of 2.043, 1.942, and 1.853 in the reduced state identified the unaltered [4Fe-4S] cluster as FA, while a complex spectrum with a gz-value of 2.194 and an asymmetric gx,y set of resonances between 2.092 and 1.999 indicates an altered cluster of unknown identity in the site containing the aspartate ligand. The ESR signals arising from the altered cluster corresponding to FB are not diminished by illumination at either room temperature or 15 K.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartic Acid↗
PubMed1997

Strains of synechocystis sp. PCC 6803 with altered PsaC. I. Mutations incorporated in the cysteine ligands of the two [4Fe-4S] clusters FA and FB of photosystem I.

Two [4Fe-4S] clusters, FA and FB, function as terminal electron carriers in Photosystem I (PS I), a thylakoid membrane-bound protein-pigment complex. To probe the function of these two clusters in photosynthetic electron transport, site-directed mutants were created in the transformable cyanobacterium Synechocystis sp. PCC 6803. Cysteine ligands in positions 14 or 51 to FB and FA, respectively, were replaced with aspartate, serine, or alanine, and the effect on the genetic, physiological, and biochemical characteristics of PS I complexes from the mutant strains were studied. All mutant strains were unable to grow photoautotrophically, and compared with wild type, mixotrophic growth was inhibited under normal light intensity. The mutant cells supported lower rates of whole-chain photosynthetic electron transport. Thylakoids isolated from the aspartate and serine mutants have lower levels of PS I subunits PsaC, PsaD, and PsaE and lower rates of PS I-mediated substrate photoreduction compared with the wild type. The alanine and double aspartate mutants have no detectable levels PsaC, PsaD, and PsaE. Electron transfer rates, measured by cytochrome c6-mediated NADP+ photoreduction, were lower in purified PS I complexes from the aspartate and serine mutants. By measuring the P700(+) kinetics after a single turnover flash, a large percentage of the backreaction in the aspartate and serine mutants was found to be derived from A1 and FX, indicating an inefficiency at the FX --> FA/FB electron transfer step. The alanine and double aspartate mutants failed to show any backreaction from [FA/FB]-. These results indicate that the various mutations of the cysteine 14 and 51 ligands to FB and FA affect biogenesis and electron transfer differently depending on the type of substitution, and that the effects of mutations on biogenesis and function can be biochemically separated and analyzed.

Cyanobacteria↗
PubMed1999

Photosystem I, an improved model of the stromal subunits PsaC, PsaD, and PsaE.

An improved electron density map of photosystem I (PSI) calculated at 4-A resolution yields a more detailed structural model of the stromal subunits PsaC, PsaD, and PsaE than previously reported. The NMR structure of the subunit PsaE of PSI from Synechococcus sp. PCC7002 (Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062) has been used as a model to interpret the region of the electron density map corresponding to this subunit. The spatial orientation with respect to other subunits is described as well as the possible interactions between the stromal subunits. A first model of PsaD consisting of a four-stranded beta-sheet and an alpha-helix is suggested, indicating that this subunit partly shields PsaC from the stromal side. In addition to the improvements on the stromal subunits, the structural model of the membrane-integral region of PSI is also extended. The current electron density map allows the identification of the N and C termini of the subunits PsaA and PsaB. The 11-transmembrane alpha-helices of these subunits can now be assigned uniquely to the hydrophobic segments identified by hydrophobicity analyses.

Amino Acid Sequence↗
PubMed2005

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.

Aspartic Acid↗
PubMed1990

Reconstitution of electron transport in photosystem I with PsaC and PsaD proteins expressed in Escherichia coli.

A fusion protein, denoted PsaC1, which contains an amino-terminal extension of five amino acids (MEHSM...) and is derived from an in vitro modified form of the psaC gene of Synechococcus sp. PCC 7002, has been over-expressed in Escherichia coli. The product of the psaD gene of Nostoc sp. PCC 8009 has similarly been over-expressed. The PsaC1 and PsaD proteins can be combined with the photosystem I core protein of Synechococcus sp. PCC 6301 to reconstitute electron transport from P700 to the terminal FA/FB acceptors. Reconstitution was found to be absolutely dependent on reinsertion of the iron-sulfur clusters in the PsaC1 apoprotein and on the presence of the PsaD protein. This implies that the PsaC1 holoprotein does not bind solely to the PsaA/PsaB heterodimer but rather that its interaction with these proteins is mediated through the PsaD protein.

Amino Acid Sequence↗