The Lhca antenna complexes of higher plants photosystem I

The Lhca antenna complexes of photosystem I (PSI) have been characterized by comparison of native and recombinant preparations. Eight Lhca polypeptides have been found to be all organized as dimers in the PSI-LHCI complex. The red emission fluorescence is associated not only with Lhca1-4 heterodimer, but also with dimers containing Lhca2 and/or Lhca3 complexes. Reconstitution of Lhca1 and Lhca4 monomers as well as of the Lhca1-4 dimer in vitro was obtained. The biochemical and spectroscopic features of these three complexes are reported. The monomers Lhca1 and Lhca4 bind 10 Chls each, while the Chl a/b ratio is lower in Lhca4 as compared to Lhca1. Three carotenoid binding sites have been found in Lhca1, while only two are present in Lhca4. Both complexes contain lutein and violaxanthin while beta-carotene is selectively bound to the Lhca1-4 dimer in substoichiometric amounts upon dimerization. Spectral analysis revealed the presence of low energy absorption forms in Lhca1 previously thought to be exclusively associated with Lhca4. It is shown that the process of dimerization changes the spectroscopic properties of some chromophores and increases the amplitude of the red absorption tail of the complexes. The origin of these spectroscopic features is discussed.     

Photosystem I of Chlamydomonas reinhardtii contains nine light-harvesting complexes (Lhca) located on one side of the core

In this work we have purified the Photosystem I (PSI) complex of Chlamydomonas reinhardtii to homogeneity. Biochemical, proteomic, spectroscopic, and structural analyses reveal the main properties of this PSI-LHCI supercomplex. The data show that the largest purified complex is composed of one core complex and nine Lhca antennas and that it contains all Lhca gene products. A projection map at 15 ? resolution obtained by electron microscopy reveals that the Lhcas are organized on one side of the core in a double half-ring arrangement, in contrast with previous suggestions. A series of stable disassembled PSI-LHCI intermediates was purified. The analysis of these complexes suggests the sequence of the assembly/disassembly process. It is shown that PSI-LHCI of C. reinhardtii is larger but far less stable than the complex from higher plants. Lhca2 and Lhca9 (the red-most antenna complexes), although present in the largest complex in 1:1 ratio with the core, are only loosely associated with it. This can explain the large variation in antenna composition of PSI-LHCI from C. reinhardtii found in the literature. The analysis of several subcomplexes with reduced antenna size allows determination of the position of Lhca2 and Lhca9 and leads to a proposal for a model of the organization of the Lhcas within the PSI-LHCI supercomplex.     

The Lhca antenna complexes of higher plants photosystem I

The Lhca antenna complexes of photosystem I (PSI) have been characterized by comparison of native and recombinant preparations. Eight Lhca polypeptides have been found to be all organized as dimers in the PSI-LHCI complex. The red emission fluorescence is associated not only with Lhca1-4 heterodimer, but also with dimers containing Lhca2 and/or Lhca3 complexes. Reconstitution of Lhca1 and Lhca4 monomers as well as of the Lhca1-4 dimer in vitro was obtained. The biochemical and spectroscopic features of these three complexes are reported. The monomers Lhca1 and Lhca4 bind 10 Chls each, while the Chl a/b ratio is lower in Lhca4 as compared to Lhca1. Three carotenoid binding sites have been found in Lhca1, while only two are present in Lhca4. Both complexes contain lutein and violaxanthin while β-carotene is selectively bound to the Lhca1-4 dimer in substoichiometric amounts upon dimerization. Spectral analysis revealed the presence of low energy absorption forms in Lhca1 previously thought to be exclusively associated with Lhca4. It is shown that the process of dimerization changes the spectroscopic properties of some chromophores and increases the amplitude of the red absorption tail of the complexes. The origin of these spectroscopic features is discussed.     

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.     

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.     

Lhca5 interaction with plant photosystem I

In the outer antenna (LHCI) of higher plant photosystem I (PSI) four abundantly expressed light-harvesting protein of photosystem I (Lhca)-type proteins are organized in two heterodimeric domains (Lhca1/Lhca4 and Lhca2/Lhca3). Our cross-linking studies on PSI-LHCI preparations from wildtype Arabidopsis and pea plants indicate an exclusive interaction of the rarely expressed Lhca5 light-harvesting protein with LHCI in the Lhca2/Lhca3-site. In PSI particles with an altered LHCI composition Lhca5 assembles in the Lhca1/Lhca4 site, partly as a homodimer. This flexibility indicates a binding-competitive model for the LHCI assembly in plants regulated by molecular interactions of the Lhca proteins with the PSI core.     

Pigment binding, fluorescence properties, and oligomerization behavior of Lhca5, a novel light-harvesting protein

A new potential light-harvesting protein, named Lhca5, was recently detected in higher plants. Because of the low amount of Lhca5 in thylakoid membranes, the isolation of a native Lhca5 pigment-protein complex has not been achieved to date. Therefore, we used in vitro reconstitution to analyze whether Lhca5 binds pigments and is actually an additional light-harvesting protein. By this approach we could demonstrate that Lhca5 binds pigments in a unique stoichiometry. Analyses of pigment requirements for light-harvesting complex formation by Lhca5 revealed that chlorophyll b is the only indispensable pigment. Fluorescence measurements showed that ligated chlorophylls and carotenoids are arranged in a way that allows directed energy transfer within the light-harvesting complex. Reconstitutions of Lhca5 together with other Lhca proteins resulted in the formation of heterodimers with Lhca1. This result demonstrates that Lhca5 is indeed a protein belonging to the light-harvesting antenna of photosystem I. The properties of Lhca5 are compared with those of previously characterized Lhca proteins, and the consequences of an additional Lhca protein for the composition of the light-harvesting antenna of photosystem I are discussed in view of the recently published photosystem I structure of the pea.     

Evidence for two spectroscopically different dimers of light-harvesting complex I from green plants

A preparation consisting of isolated dimeric peripheral antenna complexes from green plant photosystem I (light-harvesting complex I or LHCI) has been characterized by means of (polarized) steady-state absorption and fluorescence spectroscopy at low temperatures. We show that this preparation can be described reasonably well by a mixture of two types of dimers. In the first dimer about 10% of all Q(y)() absorption of the chlorophylls arises from two chlorophylls with absorption and emission maxima at about 711 and 733 nm, respectively, whereas in the second about 10% of the absorption arises from two chlorophylls with absorption and emission maxima at about 693 and 702 nm, respectively. The remaining chlorophylls show spectroscopic properties comparable to those of the related peripheral antenna complexes of photosystem II. We attribute the first dimer to a heterodimer of the Lhca1 and Lhca4 proteins and the second to a hetero- or homodimer of the Lhca2 and/or Lhca3 proteins. We suggest that the chlorophylls responsible for the 733 nm emission (F-730) and 702 nm emission (F-702) are excitonically coupled dimers and that F-730 originates from one of the strongest coupled pair of chlorophylls observed in nature.     

Excitation-energy transfer dynamics of higher plant photosystem I light-harvesting complexes

Photosystem I (PSI) plays a major role in the light reactions of photosynthesis. In higher plants, PSI is composed of a core complex and four outer antennas that are assembled as two dimers, Lhca1/4 and Lhca2/3. Time-resolved fluorescence measurements on the isolated dimers show very similar kinetics. The intermonomer transfer processes are resolved using target analysis. They occur at rates similar to those observed in transfer to the PSI core, suggesting competition between the two transfer pathways. It appears that each dimer is adopting various conformations that correspond to different lifetimes and emission spectra. A special feature of the Lhca complexes is the presence of an absorption band at low energy, originating from an excitonic state of a chlorophyll dimer, mixed with a charge-transfer state. These low-energy bands have high oscillator strengths and they are superradiant in both Lhca1/4 and Lhca2/3. This challenges the view that the low-energy charge-transfer state always functions as a quencher in plant Lhc's and it also challenges previous interpretations of PSI kinetics. The very similar properties of the low-energy states of both dimers indicate that the organization of the involved chlorophylls should also be similar, in disagreement with the available structural data.     

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.     

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.     

Organization of the Light-Harvesting Complex of Photosystem I and Its Assembly during Plastid Development

Photosystem I (PSI) holocomplexes were fractionated to study the organization of the light-harvesting complex I (LHC I) pigment-proteins in barley (Hordeum vulgare) plastids. LHC Ia and LHC Ib can be isolated as oligomeric, presumably trimeric, pigment-protein complexes. The LHC Ia oligomeric complex contains both the 24- and the 21.5-kD apoproteins encoded by the Lhca3 and Lhca2 genes and is slightly larger than the oligomeric LHC Ib complex containing the Lhca1 and Lhca4 gene products of 21 and 20 kD. The synthesis and assembly of LHC I during light-driven development of intermittent light-grown plants occurs rapidly upon exposure to continuous illumination. Complete PSI complexes are detected by nondenaturing Deriphat (disodium N-dodecyl-[beta]-iminodipropionate-160)-PAGE after 2 h of illumination, and their appearance correlates with that of the 730- to 740-nm emission characteristic of assembled LHC I. However, the majority of the newly synthesized LHC I apoproteins are present as monomeric complexes in the thylakoids during the early hours of greening. We propose that during development of the protochloroplast the LHC I apoproteins are first assembled into monomeric pigmented complexes that then aggregate into trimers before becoming attached to the pre-existing core complex to form a complete PSI holocomplex.     

Structure of the higher plant light harvesting complex I: in vivo characterization and structural interdependence of the Lhca proteins

We have investigated the structure of the higher plant light harvesting complex of photosystem I (LHCI) by analyzing PSI-LHCI particles isolated from a set of Arabidopsis plant lines, each lacking a specific Lhca (Lhca1-4) polypeptide. Functional antenna size measurements support the recent finding that there are four Lhca proteins per PSI in the crystal structure [Ben-Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630-635]. According to HPLC analyses the number of pigment molecules bound within the LHCI is higher than expected from reconstitution studies or analyses of isolated native LHCI. Comparison of the spectra of the particles from the different lines reveals chlorophyll absorption bands peaking at 696, 688, 665, and 655 nm that are not present in isolated PSI or LHCI. These bands presumably originate from "gap" or "linker" pigments that are cooperatively coordinated by the Lhca and/or PSI proteins, which we have tentatively localized in the PSI-LHCI complex.     

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.     

N-terminal processing of Lhca3 Is a key step in remodeling of the photosystem I-light-harvesting complex under iron deficiency in Chlamydomonas reinhardtii

Iron deficiency induces a remodeling of the photosynthetic apparatus in Chlamydomonas reinhardtii. In this study we showed that a key mechanistic event in the remodeling process of photosystem I (PSI) and its associated light-harvesting proteins (LHCI) is the N-terminal processing of Lhca3. N-terminal processing of Lhca3 is documented independently by two-dimensional gel electrophoresis and tandem mass spectrometric (MS/MS) analysis as well as by quantitative comparative MS/MS peptide profiling using isotopic labeling of proteins. Dynamic remodeling of the LHCI complex under iron deficiency is further exemplified by depletion of Lhca5 and up-regulation of Lhca4 and Lhca9 polypeptides in respect to photosystem I. Most importantly, the induction of N-terminal processing of Lhca3 by progression of iron deficiency correlates with the functional drop in excitation energy transfer efficiency between LHCI and PSI as assessed by low temperature fluorescence emission spectroscopy. Using an RNA interference (RNAi) strategy, we showed that the truncated form of Lhca3 is essential for the structural stability of LHCI. Depletion of Lhca3 by RNAi strongly impacted the efficiency of excitation energy transfer between PSI and LHCI, as is the case for iron deficiency. However, in contrast to iron deficiency, comparative MS/MS peptide profiling using isotopic labeling of proteins demonstrated that RNAi depletion of Lhca3 caused strong reduction of almost all Lhca proteins in isolated PSI particles.     

Probing the structure of Lhca3 by mutation analysis

Lhc proteins constitute a family of transmembrane proteins which share homology in sequence and similarity in the general organisation although members can be strongly differentiated such as in the case of PsbS and ELIPs. In this work, we report on the structure of Lhca3, a pigment-protein subunit component of the antenna system of higher plants Photosystem I, through the effect of point mutations in critical sites. Based on the structure of PSI-LHCI (Ben Shem et al., PDB file 1QZV remark 999) it has been suggested that Lhca3 may have different folding as compared to other members of the Lhc family. In particular, it was proposed that the two central helices may be swapped and chlorophylls in sites 1013 and 1023 are not present. This different folding would imply that the chlorophylls coordinated to the two central helices have different ligands in Lhca3 with respect to the other Lhc complexes. The structural model was tested by substituting the putative binding residues with residues unable to coordinate chlorophyll and the spectroscopic properties of the individual pigments were used as structural probes. The results indicate that Lhca3 folds in the same way as the other antenna proteins. Moreover, the low-energy absorption form originates from interaction between chlorophylls in site 1015 and 1025, like for the other PSI antenna subunits. Evidence is also shown for the presence in Lhca3 of chlorophylls in sites 1013 and 1023.     

The properties of the chlorophyll a/b-binding proteins Lhca2 and Lhca3 studied in vivo using antisense inhibition

The specific functions of the light-harvesting proteins Lhca2 and Lhca3 were studied in Arabidopsis ecotype Colombia antisense plants in which the proteins were individually repressed. The antisense effect was specific in each plant, but levels of Lhca proteins other than the targeted products were also affected. The contents of Lhca1 and Lhca4 were unaffected, but Lhca3 (in Lhca2-repressed plants) was almost completely depleted, and Lhca2 decreased to about 30% of wild-type levels in Lhca3-repressed plants. This suggests that the Lhca2 and Lhca3 proteins are in physical contact with each other and that they require each other for stability. Photosystem I fluorescence at 730 nm is thought to emanate from pigments bound to Lhca1 and Lhca4. However, fluorescence emission and excitation spectra suggest that Lhca2 and Lhca3, which fluoresce in vitro at 680 nm, also could contribute to far-red fluorescence in vivo. Spectral forms with absorption maxima at 695 and 715 nm, apparently with emission maxima at 702 and 735 nm, respectively, might be associated with Lhca2 and Lhca3.     

Probing the structure of Lhca3 by mutation analysis

Lhc proteins constitute a family of transmembrane proteins which share homology in sequence and similarity in the general organisation although members can be strongly differentiated such as in the case of PsbS and ELIPs. In this work, we report on the structure of Lhca3, a pigment-protein subunit component of the antenna system of higher plants Photosystem I, through the effect of point mutations in critical sites. Based on the structure of PSI-LHCI (Ben Shem et al., PDB file 1QZV remark 999) it has been suggested that Lhca3 may have different folding as compared to other members of the Lhc family. In particular, it was proposed that the two central helices may be swapped and chlorophylls in sites 1013 and 1023 are not present. This different folding would imply that the chlorophylls coordinated to the two central helices have different ligands in Lhca3 with respect to the other Lhc complexes. The structural model was tested by substituting the putative binding residues with residues unable to coordinate chlorophyll and the spectroscopic properties of the individual pigments were used as structural probes. The results indicate that Lhca3 folds in the same way as the other antenna proteins. Moreover, the low-energy absorption form originates from interaction between chlorophylls in site 1015 and 1025, like for the other PSI antenna subunits. Evidence is also shown for the presence in Lhca3 of chlorophylls in sites 1013 and 1023.     

Proteomics of light-harvesting proteins in different plant species. Analysis and comparison by liquid chromatography-electrospray ionization mass spectrometry. Photosystem I

The light-harvesting proteins (Lhca) of photosystem I (PSI) from four monocot and five dicot species were extracted from plant material, separated by reversed-phase high-performance liquid chromatography (HPLC) and subsequently identified on the basis of their intact molecular masses upon on-line hyphenation with electrospray ionization mass spectrometry. Although their migration behavior in gel electrophoresis was very similar, the elution times among the four antenna types in reversed-phase-HPLC differed significantly, even more than those observed for the light-harvesting proteins of photosystem II. Identification of proteins is based on the good agreement between the measured intact molecular masses and the values calculated on the basis of their nucleotide-derived amino acid sequences, which makes the intact molecular masses applicable as intact mass tags. These values match excellently for Arabidopsis, most probably because of the availability of high-quality DNA sequence data. In all species examined, the four antennae eluted in the same order, namely Lhca1 > Lhca3 > Lhca4 > Lhca2. These characteristic patterns enabled an unequivocal assignment of the proteins in preparations from different species. Interestingly, in all species examined, Lhca1 and Lhca2 were present in two or three isoforms. A fifth antenna protein, corresponding to the Lhca6 gene, was found in tomato (Lycopersicon esculentum). However PSI showed a lower heterogeneity than photosystem II. In most plant species, Lhca2 and Lhca4 proteins are the most abundant PSI antenna proteins. The HPLC method used in this study was found to be highly reproducible, and the chromatograms may serve as a highly confident fingerprint for comparison within a single and among different species for future studies of the PSI antenna.