Photosystem I, an improved model of the stromal subunits PsaC, PsaD, and PsaE.
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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. (+info)
Structural features and assembly of the soluble overexpressed PsaD subunit of photosystem I.
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PsaD is a peripheral protein on the reducing side of photosystem I (PS I). We expressed the psaD gene from the thermophilic cyanobacterium Mastigocladus laminosus in Escherichia coli and obtained a soluble protein with a polyhistidine tag at the carboxyl terminus. The soluble PsaD protein was purified by Ni-affinity chromatography and had a mass of 16716 Da by MALDI-TOF. The N-terminal amino acid sequence of the overexpressed PsaD matched the N-terminal sequence of the native PsaD from M. laminosus. The soluble PsaD could assemble into the PsaD-less PS I. As determined by isothermal titration calorimetry, PsaD bound to PS I with 1.0 binding site per PS I, the binding constant of 7.7x10(6) M-1, and the enthalpy change of -93.6 kJ mol-1. This is the first time that the binding constant and binding heat have been determined in the assembly of any photosynthetic membrane protein. To identify the surface-exposed domains, purified PS I complexes and overexpressed PsaD were treated with N-hydroxysuccinimidobiotin (NHS-biotin) and biotin-maleimide, and the biotinylated residues were mapped. The Cys66, Lys21, Arg118 and Arg119 residues were exposed on the surface of soluble PsaD whereas the Lys129 and Lys131 residues were not exposed on the surface. Consistent with the X-ray crystallographic studies on PS I, circular dichroism spectroscopy revealed that PsaD contains a small proportion of alpha-helical conformation. (+info)
The cysteine-proximal aspartates in the Fx-binding niche of photosystem I. Effect of alanine and lysine replacements on photoautotrophic growth, electron transfer rates, single-turnover flash efficiency, and EPR spectral properties.
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The FX electron acceptor in Photosystem I (PS I) is a highly electronegative (Em = -705 mV) interpolypeptide [4Fe-4S] cluster ligated by cysteines 556 and 565 on PsaB and cysteines 574 and 583 on PsaA in Synechocystis sp. PCC 6803. An aspartic acid is adjacent to each of these cysteines on PsaB and adjacent to the proline-proximal cysteine on PsaA. We investigated the effect of D566PsaB and D557PsaB on electron transfer through FX by changing each aspartate to the neutral alanine or to the positively charged lysine either singly (D566APsaB, D557APsaB, D566KPsaB, and D557KPsaB) or in pairs (D557APsaB/D566APsaB and D557KPsaB/D566APsaB). All mutants except for D557KPsaB/D566APsaB grew photoautotrophically, but the growth of D557KPsaB and D557APsaB/D566APsaB was impaired under low light. The doubling time was increased, and the chlorophyll content per cell was lower in D557KPsaB and D557APsaB/D566APsaB relative to the wild type and the other mutants. Nevertheless, the rates of NADP+ photoreduction in PS I complexes from all mutants were no less than 75% of that of the wild type. The kinetics of back-reaction of the electron acceptors on a single-turnover flash showed efficient electron transfer to the terminal acceptors FA and FB in PS I complexes from all mutants. The EPR spectrum of FX was identical to that in the wild type in all but the single and double D566APsaB mutants, where the high-field resonance was shifted downfield. We conclude that the impaired growth of some of the mutants is related to a reduced accumulation of PS I rather than to photosynthetic efficiency. The chemical nature and the charge of the amino acids adjacent to the cysteine ligands on PsaB do not appear to be significant factors in the efficiency of electron transfer through FX. (+info)
Photosystem I is indispensable for photoautotrophic growth, CO2 fixation, and H2 photoproduction in Chlamydomonas reinhardtii.
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Certain Chlamydomonas reinhardtii mutants deficient in photosystem I due to defects in psaA mRNA maturation have been reported to be capable of CO2 fixation, H2 photoevolution, and photoautotrophic growth (Greenbaum, E., Lee, J. W., Tevault, C. V., Blankinship, S. L. , and Mets, L. J. (1995) Nature 376, 438-441 and Lee, J. W., Tevault, C. V., Owens, T. G.; Greenbaum, E. (1996) Science 273, 364-367). We have generated deletions of photosystem I core subunits in both wild type and these mutant strains and have analyzed their abilities to grow photoautotrophically, to fix CO2, and to photoevolve O2 or H2 (using mass spectrometry) as well as their photosystem I content (using immunological and spectroscopic analyses). We find no instance of a strain that can perform photosynthesis in the absence of photosystem I. The F8 strain harbored a small amount of photosystem I, and it could fix CO2 and grow slowly, but it lost these abilities after deletion of either psaA or psaC; these activities could be restored to the F8-psaADelta mutant by reintroduction of psaA. We observed limited O2 photoevolution in mutants lacking photosystem I; use of 18O2 indicated that this O2 evolution is coupled to O2 uptake (i.e. respiration) rather than CO2 fixation or H2 evolution. We conclude that the reported instances of CO2 fixation, H2 photoevolution, and photoautotrophic growth of photosystem I-deficient mutants result from the presence of unrecognized photosystem I. (+info)
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.
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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. (+info)
The Qo site of cytochrome b6f complexes controls the activation of the LHCII kinase.
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We created a Qo pocket mutant by site-directed mutagenesis of the chloroplast petD gene in Chlamydomonas reinhardtii. We mutated the conserved PEWY sequence in the EF loop of subunit IV into PWYE. The pwye mutant did not grow in phototrophic conditions although it assembled wild-type levels of cytochrome b6f complexes. We demonstrated a complete block in electron transfer through the cytochrome b6f complex and a loss of plastoquinol binding at Qo. The accumulation of cytochrome b6f complexes lacking affinity for plastoquinol enabled us to investigate the role of plastoquinol binding at Qo in the activation of the light-harvesting complex II (LHCII) kinase during state transitions. We detected no fluorescence quenching at room temperature in state II conditions relative to that in state I. The quantum yield spectrum of photosystem I charge separation in the two state conditions displayed a trough in the absorption region of the major chlorophyll a/b proteins, demonstrating that the cells remained locked in state I. 33Pi labeling of the phosphoproteins in vivo demonstrated that the antenna proteins remained poorly phosphorylated in both state conditions. Thus, the absence of state transitions in the pwye mutant demonstrates directly that plastoquinol binding in the Qo pocket is required for LHCII kinase activation. (+info)
Oxidizing side of the cyanobacterial photosystem I. Evidence for interaction between the electron donor proteins and a luminal surface helix of the PsaB subunit.
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Photosystem I (PSI) interacts with plastocyanin or cytochrome c6 on the luminal side. To identify sites of interaction between plastocyanin/cytochrome c6 and the PSI core, site-directed mutations were generated in the luminal J loop of the PsaB protein from Synechocystis sp. PCC 6803. The eight mutant strains differed in their photoautotrophic growth. Western blotting with subunit-specific antibodies indicated that the mutations affected the PSI level in the thylakoid membranes. PSI proteins could not be detected in the S600R/G601C/N602I, N609K/S610C/T611I, and M614I/G615C/W616A mutant membranes. The other mutant strains contained different levels of PSI proteins. Among the mutant strains that contained PSI proteins, the H595C/L596I, Q627H/L628C/I629S, and N638C/N639S mutants showed similar levels of PSI-mediated electron transfer activity when either cytochrome c6 or an artificial electron donor was used. In contrast, cytochrome c6 could not function as an electron donor to the W622C/A623R mutant, even though the PSI activity mediated by an artificial electron donor was detected in this mutant. Thus, the W622C/A623R mutation affected the interaction of the PSI complex with cytochrome c6. Biotin-maleimide modification of the mutant PSI complexes indicated that His-595, Trp-622, Leu-628, Tyr-632, and Asn-638 in wild-type PsaB may be exposed on the surface of the PSI complex. The results presented here demonstrate the role of an extramembrane loop of a PSI core protein in the interaction with soluble electron donor proteins. (+info)
Spectroscopic and molecular characterization of a long wavelength absorbing antenna of Ostreobium sp.
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One of the strains of the marine green alga Ostreobium sp. possesses an exceptionally large number of long wavelength absorbing chlorophylls (P. Haldall, Biol. Bull. 134, 1968, 411-424) as evident from a distinct shoulder in the absorption spectrum at around 710 nm while in the other strain this shoulder is absent. Therefore, Ostreobium offers a unique possibility to explore the origin of these red-shifted chlorophylls, because strains with and without these spectral forms can be compared. Here, we characterize these red forms spectroscopically by absorption, fluorescence and CD spectroscopy. In the CD spectra at least three spectroscopic red forms are identified which lead to an unusual room temperature fluorescence spectrum that peaks at 715 nm. The gel electrophoretic pattern from thylakoids of Ostreobium sp. shows an intense band at 22 kDa which correlates with the presence or absence of long wavelength absorbing pigments. By protein sequencing of the N-terminus of the 22-kDa polypeptide and sequence alignments, this was identified as an Lhca1-type light-harvesting complex. The abundance of this polypeptide - and a possibly co-migrating one - in Ostreobium sp. indicates an antenna size of approximately 340 chlorophyll molecules (Chl a and Chl b) per PS IIalpha reaction center, which is significantly larger than in higher plants ( approximately 240). The red forms are more abundant in the interior of the thalli where a 'shade-light' light field is expected than in the white-light exposed surface. This demonstrates that algae exist which may be able to up-regulate the synthesis of large amounts of LHCI and associated red forms under appropriate illumination conditions. (+info)