Excitation energy trapping in photosystem I complexes depleted in Lhca1 and Lhca4

We report a time-resolved fluorescence spectroscopy characterization of photosystem I (PSI) particles prepared from Arabidopsis lines with knock-out mutations against the peripheral antenna proteins of Lhca1 or Lhca4. The first mutant retains Lhca2 and Lhca3 while the second retains one other light-harvesting protein of photosystem I (Lhca) protein, probably Lhca5. The results indicate that Lhca2/3 and Lhca1/4 each provides about equally effective energy transfer routes to the PSI core complex, and that Lhca5 provides a less effective energy transfer route. We suggest that the specific location of each Lhca protein within the PSI-LHCI supercomplex is more important than the presence of so-called red chlorophylls in the Lhca proteins.     

The low energy emitting states of the Lhca4 subunit of higher plant photosystem I

The selectively red excited emission spectrum, at room temperature, of the in vitro reconstituted Lhca4, has a pronounced non-equilibrium distribution, leading to enhanced emission from the directly excited low-energy pigments. Two different emitting forms (or states), with maximal emission at 713 and 735nm (F713 and F735) and unusual spectral properties, have been identified. Both high-energy states are populated when selective excitation is into the F735 state and the fluorescence anisotropy spectrum attains the value of 0.3 in the wavelength region where both emission states are present. This indicates that the two states are on the same Lhca4 complex and have transition dipoles with similar orientation.     

Molecular reorganization induced by Ca2+ of plant photosystem I reconstituted into phosphatidylglycerol liposomes

The interaction of divalent cations with biomembranes is important for a number of biological processes. In this study, the regulatory effect of Ca2+ on the interaction between plant spinach photosystem I (PSI) particles and negatively charged lipid phosphatidylglycerol (PG) was investigated by circular dichroism (CD) spectroscopy. It was found that in the absence of CaCl2, PG causes an increase in alpha-helix and a decrease in disordered conformations of protein secondary structures of PSI, the beta-sheet and turns being almost unaffected. Meanwhile, the same effect also enhances the excitonic interactions relating to Chl a and Chl b from the PSI core complex and external antenna light-harvesting complex (LHCI). By contrast, in the presence of CaCl2, PG hardly interferes with the structure of the proteins' skeleton of PSI, but it can depress the excitonic interactions for Chl b of LHCI and for PSI core complex Chl a at (-) 433.5 nm of the CD signal which is accompanied by a blue shift of its peak. It is most likely that the neutralization of the phosphate groups in the PSI-PG complex and the negative surface charges of PSI, and partial dehydration in the vicinity of the ester CO region of the PG polar head group by the Ca-ions modify the interaction between PSI and PG, thereby inducing molecular reorganization of protein and pigments within both the external antenna LHCI and PSI core complex in proteoliposomes.     

Chloramphenicol effects on chlorophyll degradation and photosystem I assembly in the chlorina CD3 wheat mutant

We previously reported that applications of chloramphenicol to the chlorina wheat mutant, CD3, decreased the leaf Chl a/b ratio and enhanced accumulations of LHC proteins and LHC complexes during greening (Duysen et al. 1985). We have now examined Chl degradation and the change in Chl a/b ratios in wheat leaves kept in the dark as a measure of LHC destruction. Chl b was stable in chloroplasts of the CD3 wheat kept in darkness up to 5 days. Chloramphenicol significantly increased Chl b accumulations and impaired Chl a degradation in both CD3 mutant and normal wheat relative to untreated plants. Our Chl data suggest that the chloramphenicol induced accumulation of the LHC complex in the mutant wheat results from enhanced processing of LHC into the membrane rather than impairment of LHC degradation. The photosystem I (PSI) fraction of the CD3 wheat mutant was examined relative to that of normal wheat after 3 days greening. PSI was deficient in 25, 26, 26.5 kD LHCI protein in the mutant but both wheats accumulated low quantities of the 27–29 kD LHCII protein as detected by Western blot analysis. Chloramphenicol enhanced accumulations of several LHCI proteins primarily near 25 kD in the mutant and the 27–29 kD LHCII protein in normal wheat. The fluorescence emission and absorbance spectra suggest that chloramphenicol enhances accumulations of dissociated LHC in the PSI preparation of normal and CD3 mutant wheat.A contribution of North Dakota Agricultural Experiment Station. Published with approval of the Director as Journal Paper Number 1563.     

The long-wavelength chlorophyll states of plant LHCI at room temperature: a comparison with PSI-LHCI

The red antenna states of the external antenna complexes of higher plant photosystem I, known as LHCI, have been analyzed by measurement of their preequilibrium fluorescence upon direct excitation at 280 K. In addition to the previously detected F735 state, a hitherto undetected low-energy state with emission maximum around 713 nm was observed. The 280 K bandwidths (FWHM) are 55 nm for the F735 state and approximately 27 nm for the F713-nm state, much greater than for non-red-shifted antenna chlorophylls. The origin absorption band for the F735-nm state was directly detected by determination of its excitation (action) spectrum and lies at 709-710 nm. The absorption spectrum for F735, calculated using the Stepanov expression, closely overlaps the excitation spectrum, indicating that the very large Stokes shift (25 nm) is due to vibrational relaxation within the excited-state manifold and solvent effects can be excluded. Fluorescence anisotropy measurements, with direct excitation of F735, indicate that the transition dipoles of the two red states are parallel. Similar experiments performed in the long-wavelength absorbing tail of PSI-LHCI indicate the presence of emission state(s) that are red-shifted with respect to F735 of isolated LHCI. It is suggested that these are brought about by interactions between the complexes in PSI-LHCI, which occur in some yet undefined way, and which are broken upon solubilization of the component parts.     

Functional analysis of Photosystem I light-harvesting complexes (Lhca) gene products of Chlamydomonas reinhardtii

The outer antenna system of Chlamydomonas reinhardtii Photosystem I is composed of nine gene products, but due to difficulty in purification their individual properties are not known. In this work, the functional properties of the nine Lhca antennas of Chlamydomonas, have been investigated upon expression of the apoproteins in bacteria and refolding in vitro of the pigment-protein complexes. It is shown that all Lhca complexes have a red-shifted fluorescence emission as compared to the antenna complexes of Photosystem II, similar to Lhca from higher plants, but less red-shifted. Three complexes, namely Lhca2, Lhca4 and Lhca9, exhibit emission maxima above 707 nm and all carry an asparagine as ligand for Chl 603. The comparison of the protein sequences and the biochemical/spectroscopic properties of the refolded Chlamydomonas complexes with those of the well-characterized Arabidopsis thaliana Lhcas shows that all the Chlamydomonas complexes have a chromophore organization similar to that of A. thaliana antennas, particularly to Lhca2, despite low sequence identity. All the major biochemical and spectroscopic properties of the Lhca complexes have been conserved through the evolution, including those involved in “red forms” absorption. It has been proposed that in Chlamydomonas PSI antenna size and polypeptide composition can be modulated in vivo depending on growth conditions, at variance as compared to higher plants. Thus, the different properties of the individual Lhca complexes can be functional to adapt the architecture of the PSI-LHCI supercomplex to different environmental conditions.     

Characterization of a full-length petunia cDNA encoding a polypeptide of the light-harvesting complex associated with photosystem I

We have isolated and characterized a full-length cDNA clone (LHCI-15) which specifies a new chlorophyll-binding protein. This protein is associated with the light-harvesting complex of photosystem I (LHCI). The DNA sequence predicts a precursor protein of 270 amino acids, which shares significant homology with the amino acid sequence of another chlorophyll-binding protein; the chlorophyll a/b-binding (Cab) protein of the photosystem II light-harvesting complex (LHCII). There are two extensive regions of homology (at least 45 residues each) which have approximately 50% amino acid sequence identity. These regions coincide with two of the proposed membrane-spanning alpha helices in the Cab proteins of the LHCII and probably include conserved chlorophyll-binding sites. The LHCI-15 cDNA hybridizes to at least 7 genomic EcoRI DNA fragments, which are very closely related at the nucleotide sequence level.     

Protein domains required for formation of stable monomeric Lhca1- and Lhca4-complexes

The peripheral light-harvesting complex of Photosystem I consists of two subpopulations, LHC I-680 and LHC I-730. The latter is composed of the two apoproteins Lhca1 and Lhca4. Recently, reconstitution of monomeric LHC I using bacterially overexpressed Lhca1 or Lhca4 was achieved. In order to obtain insight into the structure requirements for formation of monomeric light-harvesting complexes, we produced a series of N- and C-terminal deletion mutants and used the overexpressed proteins for reconstitution experiments. We found the entire extrinsic N-terminal region dispensable for monomer formation in Lhca1 and Lhca4. Also at the C-terminus, both subunits revealed similarity since all amino acids up to the end of the fourth helix could be removed without abolishing monomer formation. In connection with former corresponding results for Lhcb1, the dispensability of these regions appears to be a general feature in LHC-formation. In LHC I, however, a stabilising effect can be ascribed to these regions since the yield of complexes was decreased. In the majority of the mutant LHC I versions no effect on pigment binding was detected. However, in the LHC with the most extensively N-terminally truncated mutant of Lhca4 a dramatic shift in the 77 K fluorescence emission to shorter wavelengths was observed. This suggests that chlorophylls involved in long wavelength fluorescence emission are located in the chlorophyll array located towards the stromal face of the thylakoid membrane assuming a pigment arrangement corresponding to that in LHC II and CP29. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photosystem I: its biogenesis and function in higher plants

Photosystem I (PSI), the plastocyanin-ferredoxin oxidoreductase of the photosynthetic electron transport chain, is one of the largest bioenergetic complexes known. It is composed of subunits encoded in both the chloroplast genome and the nuclear genome and thus, its assembly requires an intricate coordination of gene expression and intensive communication between the two compartments. In this review, we first briefly describe PSI structure and then focus on recent findings on the role of the two small chloroplast genome-encoded subunits PsaI and PsaJ in the stability and function of PSI in higher plants. We then address the sequence of PSI biogenesis, discuss the role of auxiliary proteins involved in cofactor insertion into the PSI apoproteins and in the establishment of protein-protein interactions during subunit assembly. Finally, we consider potential limiting steps of PSI biogenesis, and how they may contribute to the control of PSI accumulation.     

Excitation dynamics in Photosystem I from Chlamydomonas reinhardtii. Comparative studies of isolated complexes and whole cells

Identical time-resolved fluorescence measurements with ~ 3.5-ps resolution were performed for three types of PSI preparations from the green alga, Chlamydomonas reinhardtii: isolated PSI cores, isolated PSI–LHCI complexes and PSI–LHCI complexes in whole living cells. Fluorescence decay in these types of PSI preparations has been previously investigated but never under the same experimental conditions. As a result we present consistent picture of excitation dynamics in algal PSI. Temporal evolution of fluorescence spectra can be generally described by three decay components with similar lifetimes in all samples (6–8 ps, 25–30 ps, 166–314 ps). In the PSI cores, the fluorescence decay is dominated by the two fastest components (~ 90%), which can be assigned to excitation energy trapping in the reaction center by reversible primary charge separation. Excitation dynamics in the PSI–LHCI preparations is more complex because of the energy transfer between the LHCI antenna system and the core. The average trapping time of excitations created in the well coupled LHCI antenna system is about 12–15 ps longer than excitations formed in the PSI core antenna. Excitation dynamics in PSI–LHCI complexes in whole living cells is very similar to that observed in isolated complexes. Our data support the view that chlorophylls responsible for the long-wavelength emission are located mostly in LHCI. We also compared in detail our results with the literature data obtained for plant PSI.     

Modification of excitation energy distribution to photosystem I by protein phosphorylation and cation depletion during thylakoid biogenesis in wheat

The effects of protein phosphorylation and cation depletion on the electron transport rate and fluorescence emission characteristics of photosystem I at two stages of chloroplast development in light-grown wheat leaves are examined. The light-harvesting chlorophyll a/b protein complex associated with photosystem I (LHC I) was absent from the thylakoids at the early stage of development, but that associated with photosystem II (LHC II) was present. Protein phosphorylation produced an increase in the light-limited rate of photosystem I electron transport at the early stage of development when chlorophyll b was preferentially excited, indicating that LHC I is not required for transfer of excitation energy from phosphorylated LHC II to the core complex of photosystem I. However, no enhancement of photosystem I fluorescence at 77 K was observed at this stage of development, demonstrating that a strict relationship between excitation energy density in photosystem I pigment matrices and the long-wavelength fluorescence emission from photosystem I at 77 K does not exist. Depletion of Mg²⁺ from the thylakoids produced a stimulation of photosystem I electron transport at both stages of development, but a large enhancement of the photosystem I fluorescence emission was observed only in the thylakoids containing LHC I. It is suggested that the enhancement of PS I electron transport by Mg²⁺-depletion and phosphorylation of LHC II is associated with an enhancement of fluorescence at 77 K from LHC I and not from the core complex of PS I.     

Spectroelectrochemistry of P700 in native photosystem I particles and diethyl ether-treated thylakoid membranes from spinach and Thermosynechococcus elongatus

The redox potentials (E(composite function')) of P700 in intact and diethyl ether-treated thylakoid membranes as well as native photosystem (PS) I particles from spinach and Thermosynechococcus elongatus have been measured by a spectroelectrochemistry with an error range of +/-2-3 mV. Stepwise removal of antenna pigments by ether treatment caused distinct shifts of the E( composite function') value with increasing degree of water saturation in ether; negatively from +471 to +428 mV for spinach, but positively from +423 to +436 mV for T. elongatus. Such a contrasting behavior is discussed by invoking the mode of action of ether on the microenvironments around P700.     

A previously found thylakoid membrane protein of 14kDa (TMP14) is a novel subunit of plant photosystem I and is designated PSI-P

We show that the thylakoid membrane phosphoprotein TMP14 is a novel subunit of plant photosystem I (PSI). Blue native/SDS-PAGE and sucrose gradient fractionation demonstrated the association of the protein exclusively with PSI. We designate the protein PSI-P. The presence of PSI-P subunit in Arabidopsis mutants lacking other PSI subunits was analyzed and suggested a location in the proximity of PSI-L, -H and -O subunits. The PSI-P protein was not differentially phosphorylated in state 1 and state 2.     

Isolation and characterization of a large photosystem I–light‐harvesting complex II supercomplex with an additional Lhca1–a4 dimer in Arabidopsis

The biological conversion of light energy into chemical energy is performed by a flexible photosynthetic machinery located in the thylakoid membranes. Photosystems I and II (PSI and PSII) are the two complexes able to harvest light. PSI is the last complex of the electron transport chain and is composed of multiple subunits: the proteins building the catalytic core complex that are well conserved between oxygenic photosynthetic organisms, and, in green organisms, the membrane light‐harvesting complexes (Lhc) necessary to increase light absorption. In plants, four Lhca proteins (Lhca1–4) make up the antenna system of PSI, which can be further extended to optimize photosynthesis by reversible binding of LHCII, the main antenna complex of photosystem II. Here, we used biochemistry and electron microscopy in Arabidopsis to reveal a previously unknown supercomplex of PSI with LHCII that contains an additional Lhca1–a4 dimer bound on the PsaB–PsaI–PsaH side of the complex. This finding contradicts recent structural studies suggesting that the presence of an Lhca dimer at this position is an exclusive feature of algal PSI. We discuss the features of the additional Lhca dimer in the large plant PSI–LHCII supercomplex and the differences with the algal PSI. Our work provides further insights into the intricate structural plasticity of photosystems.     

Structure of a large photosystem II supercomplex from Acaryochloris marina

Acaryochloris marina is a prochlorophyte-like cyanobacterium containing both phycobilins and chlorophyll d as light harvesting pigments. We show that the chlorophyll d light harvesting system, composed of Pcb proteins, functionally associates with the photosystem II (PSII) reaction center (RC) core to form a giant supercomplex. This supercomplex has a molecular mass of about 2300 kDa and dimensions of 385 A x 240 A. It is composed of two PSII-RC core dimers arranged end-to-end, flanked by eight symmetrically related Pcb proteins on each side. Thus each PSII-RC monomer has four Pcb subunits acting as a light harvesting system which increases the absorption cross section of the PSII-RC core by almost 200%.     

LhcaR1 of the red alga Porphyridium cruentum encodes a polypeptide of the LHCI complex with seven potential chlorophyll a-binding residues that are conserved in most LHCs

The accessory light-harvesting polypeptides associated with photosystem I (LHCI) in Porphyridium cruentum bind chlorophyll a, zeaxanthin and -carotene. A cDNA library of P. cruentum was screened with an antiserum specific to the LHCI polypeptides, and an 0.9 kb fragment was identified as coding for an LHCI polypeptide. This cDNA, which we named LhcaR1, has an open reading frame encoding 222 amino acid residues including a putative transit peptide of 28 amino acids. Hydropathy analysis suggests that there are three transmembrane helices in the mature polypeptide. Each of the amino acid residues that bind chlorophyll (six residues) and serve in stabilizing the helices in higher-plant LHCs are conserved in helices 1 and 3 of P. cruentum LhcaR1. The N-terminal flanking regions of these two helices also show high sequence conservation with other LHCs. Helix 2 contains a seventh putative chlorophyll-binding site, but resembles helix 2 of higher-plant LHCs to a lesser degree. A sequence motif of 11 residues found near the N-terminus and in each of the three helices suggests the possibility that the red algal LhcaR1 derives from a gene duplication. Polypeptides of the expected molecular weight in six other red algae (Achrochaetium, Bangia, Callithamnion, Cyanidium, Polysiphonia, Spermothamnion) were recognized by the antiserum to P. cruentum LHCI, indicating a wide distribution of LHCI in rhodophytes.     

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.     

Characterization of the Carotenoidless Strain of Scenedesmus obliquus,Mutant C-6E,a Living Photosystem I Model*

When grown heterotrophically in the dark on enriched culture medium, the pigment-deficient strain of Scenedesmus obliquus, mutant C-6E, is uniquely characterized by a complete deficiency in carotenoids and chlorophyll b while retaining a low level of chlorophyll a which is exclusively utilized in photosystem I-type reactions. The strain lacks photosystem II activity but exhibits all PS-I reactions tested, including P700 redox reactions, photoreduction of CO₂ with hydrogen as electron donor, and O₂ uptake following methyl viologen reduction. The mutant contains 10 times more P700 per chlorophyll than the wild type and develops the pigment-protein complex of PS-I, CP-I. The action spectrum for methyl viologen reduction compares favorable to the low temperature absorption spectrum of whole cells. Both the chlorophyll fluorescence excitation and emission spectra of pigment-protein complexes derived from cells of C-6E show patterns typical of PS-I. The strain lacks the LHCs and CP-II as well as their respective apoproteins. The absence of carotenoids appears to prevent the development of the normal variety of pigment-protein complexes and the accumulation of Chl b. This inability is also expressed by the presence of only single stranded thylakoid membranes in the chloroplast of C-6E. When heterotrophically grown cells of this mutant are exposed to white light of 8 or 22 W m^?2, 50% of its chlorophyll is lost by photooxidation within 4 or 1.5 hours, respectively.