Immunogold localization of photosystem I and photosystem II light-harvesting complexes in cryptomonad thylakoids

Summary— The molecular organization of the thylakoids of Cryptomonas rufescens was studied by immunoelectron microscopy employing antibodies against photosystem (PS)-I and two PS-II antenna proteins. The PS-I complex and the 19-kDa chlorophyll a/c light-harvesting (LH) protein are both localized along the length of the thylakoid membranes. The external membranes of the paired thylakoids are enriched in PS-I whereas the chlorophyll a/c LH protein is more concentrated in the internal or appressed membranes. However, unlike the situation in higher plants and Chlamydomonas, there is not a marked asymmetry in the concentration of PS-I and chorophyll a/c LH protein in the two types of membranes. Double labelling studies of sections and isolated PE-PS-II particles with anti-phycoerythrin and anti-LH confirmed that phycoerythrin is localized in the thylakoid lumen and that this pigment exists in two forms, a fraction closely associated with the thylakoid membranes and another fraction free in the lumen. These results confirm the uniqueness of cryptomonad thylakoids.     

Structural characterization of a complex of photosystem I and light-harvesting complex II of Arabidopsis thaliana

Chloroplasts are central to the provision of energy for green plants. Their photosynthetic membrane consists of two major complexes converting sunlight: photosystem I (PSI) and photosystem II (PSII). The energy flow toward both photosystems is regulated by light-harvesting complex II (LHCII), which after phosphorylation can move from PSII to PSI in the so-called state 1 to state 2 transition and can move back to PSII after dephosphorylation. To investigate the changes of PSI and PSII during state transitions, we studied the structures and frequencies of all major membrane complexes from Arabidopsis thaliana chloroplasts at conditions favoring either state 1 or state 2. We solubilized thylakoid membranes with digitonin and analyzed the complete set of complexes immediately after solubilization by electron microscopy and image analysis. Classification indicated the presence of a PSI-LHCII supercomplex consisting of one PSI-LHCI complex and one LHCII trimer, which was more abundant in state 2 conditions. The presence of LHCII was confirmed by excitation spectra of the PSI emission of membranes in state 1 or state 2. The PSI-LHCII complex could be averaged with a resolution of 16 A, showing that LHCII has a specific binding site at the PSI-A, -H, -L, and -K subunits.     

Structural and functional diversification of the light-harvesting complexes in photosynthetic eukaryotes

Eukaryotes acquired photosynthetic metabolism over a billion years ago, and during that time the light-harvesting antennae have undergone significant structural and functional divergence. The antenna systems are generally used to harvest and transfer excitation energy into the reaction centers to drive photosynthesis, but also have the dual role of energy dissipation. Phycobilisomes formed the first antenna system in oxygenic photoautotrophs, and this soluble protein complex continues to be the dominant antenna in extant cyanobacteria, glaucophytes, and red algae. However, phycobilisomes were lost multiple times during eukaryotic evolution in favor of a thylakoid membrane-integral light-harvesting complex (LHC) antenna system found in the majority of eukaryotic taxa. While photosynthesis spread across different eukaryotic kingdoms via endosymbiosis, the antenna systems underwent extensive modification as photosynthetic groups optimized their light-harvesting capacity and ability to acclimate to changing environmental conditions. This review discusses the different classes of LHCs within photosynthetic eukaryotes and examines LHC diversification in different groups in a structural and functional context.     

Chlorophyll b is involved in long-wavelength spectral properties of light-harvesting complexes LHC I and LHC II.

Chlorophyll (Chl) molecules attached to plant light-harvesting complexes (LHC) differ in their spectral behavior. While most Chl a and Chl b molecules give rise to absorption bands between 645 nm and 670 nm, some special Chls absorb at wavelengths longer than 700 nm. Among the Chl a/b-antennae of higher plants these are found exclusively in LHC I. In order to assign this special spectral property to one chlorophyll species we reconstituted LHC of both photosystem I (Lhca4) and photosystem II (Lhcb1) with carotenoids and only Chl a or Chl b and analyzed the effect on pigment binding, absorption and fluorescence properties. In both LHCs the Chl-binding sites of the omitted Chl species were occupied by the other species resulting in a constant total number of Chls in these complexes. 77-K spectroscopic measurements demonstrated that omission of Chl b in refolded Lhca4 resulted in a loss of long-wavelength absorption and 730-nm fluorescence emission. In Lhcb1 with only Chl b long-wavelength emission was preserved. These results clearly demonstrate the involvement of Chl b in establishing long-wavelength properties.     

Structural stability and properties of three isoforms of the major light-harvesting chlorophyll a/b complexes of photosystem II

Three isoforms of the major light-harvesting chlorophyll (Chl) a/b complexs of photosystem II (LHCIIb) in the pea, namely, Lhcb1, Lhcb2, and Lhcb3, were obtained by overexpression of apoprotein in Escherichia coli and by successfully refolding these isoforms with thylakoid pigments in vitro. The sequences of the protein, pigment stoichiometries, spectroscopic characteristics, thermo- and photostabilities of different isoforms were analysed. Comparison of their spectroscopic properties and structural stabilities revealed that Lhcb3 differed strongly from Lhcb1 and Lhcb2 in both respects. It showed the lowest Qy transition energy, with its reddest absorption about 2 nm red-shifted, and the highest photostability under strong illuminations. Among the three isoforms, Lhcb 2 showed lowest thermal stability regarding energy transfer from Chl b to Chl a in the complexes, which implies that the main function of Lhcb 2 under high temperature stress is not the energy transfer.     

Structural and functional heterogeneity in the major light-harvesting complexes of higher plants

The major light-harvesting complex (LHC IIb) of higher plants plays a crucial role in capturing light energy for photosynthesis and in regulating the flow of energy within the photosynthetic apparatus. Multiple isoforms of the protein bind chlorophyll and xanthophyll chromophores, but it is commonly believed that the pigment-binding properties of different LHC IIb complexes are conserved within and between species. We have investigated the structure and function of different LHC IIb complexes isolated from Arabidopsis thaliana grown under different light conditions. LHC IIb isolated from low light-grown plants shows increased amounts of the Lhcb2 gene product, increased binding of chlorophyll a, and altered energy transfer characteristics. We suggest that Lhcb2 specifically binds at least one additional chlorophyll a compared to the Lhcb1 gene product, and that differences in the functioning of LHC IIb from high and low light-grown plants are a direct consequence of the change in polypeptide composition. We show that changes in LHC IIb composition are accompanied by changes in photosynthetic function in vivo and discuss the possible functional significance of LHC IIb heterogeneity.     

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.     

Structural stability and properties of three isoforms of the major light-harvesting chlorophyll a/b complexes of photosystem II

Three isoforms of the major light-harvesting chlorophyll (Chl) a/b complexs of photosystem II (LHCIIb) in the pea, namely, Lhcb1, Lhcb2, and Lhcb3, were obtained by overexpression of apoprotein in Escherichia coli and by successfully refolding these isoforms with thylakoid pigments in vitro. The sequences of the protein, pigment stoichiometries, spectroscopic characteristics, thermo- and photostabilities of different isoforms were analysed. Comparison of their spectroscopic properties and structural stabilities revealed that Lhcb3 differed strongly from Lhcb1 and Lhcb2 in both respects. It showed the lowest Qy transition energy, with its reddest absorption about 2 nm red-shifted, and the highest photostability under strong illuminations. Among the three isoforms, Lhcb 2 showed lowest thermal stability regarding energy transfer from Chl b to Chl a in the complexes, which implies that the main function of Lhcb 2 under high temperature stress is not the energy transfer.     

Structural and spectroscopic comparison of five-coordinate cobalt(II) and nickel(II) thiolato complexes with the related four-coordinate complexes

Five-coordinate thiolato complexes, [L1M(SMeIm)] (M = Co and Ni) (L1 = hydrotris(3,5-diisopropyl-1-pyrazolyl)borate anion and HSMeIm = 2-mercapto-1-methylimidazole), were synthesized. These complexes were compared with the corresponding Cu(II) and Zn(II) complexes with the same ligands and were also compared with the related four-coordinate complexes [L1M(SC₆F₅)] (HSC₆F₅ = pentafluorobenzenthiol). All the complexes were characterized by X-ray crystallography and UV-Vis absorption, IR, ¹H NMR, and other spectroscopic techniques. All five-coordinate thiolato complexes, [L1M(SMeIm)] (M = Co, Ni, and Cu), form a distorted square pyramidal structure with a high spin state, and only [L1Zn(SMeIm)] takes a four-coordinate structure with a distorted tetrahedral configuration. The N21-M-S bond angles of the obtained five-coordinate complexes were proportional to the corresponding d value, which comes from between the equatorial basal plane with N4S ligand donor sets and metal ion. These observations and M-S bond distances affect on UV-Vis and far-IR spectral behavior.     

Structural and functional organization of the peripheral light-harvesting system in Photosystem I

This review centers on the structural and functional organization of the light-harvesting system in the peripheral antenna of Photosystem I (LHC I) and its energy coupling to the Photosystem I (PS I) core antenna network in view of recently available structural models of the eukaryotic Photosystem I–LHC I complex, eukaryotic LHC II complexes and the cyanobacterial Photosystem I core. A structural model based on the 3D homology of Lhca4 with LHC II is used for analysis of the principles of pigment arrangement in the LHC I peripheral antenna, for prediction of the protein ligands for the pigments that are unique for LHC I and for estimates of the excitonic coupling in strongly interacting pigment dimers. The presence of chlorophyll clusters with strong pigment–pigment interactions is a structural feature of PS I, resulting in the characteristic red-shifted fluorescence. Analysis of the interactions between the PS I core antenna and the peripheral antenna leads to the suggestion that the specific function of the red pigments is likely to be determined by their localization with respect to the reaction center. In the PS I core antenna, the Chl clusters with a different magnitude of low energy shift contribute to better spectral overlap of Chls in the reaction center and the Chls of the antenna network, concentrate the excitation around the reaction center and participate in downhill enhancement of energy transfer from LHC II to the PS I core. Chlorophyll clusters forming terminal emitters in LHC I are likely to be involved in photoprotection against excess energy.     

Reconstitution of light-harvesting complexes and photosystem II cores into galactolipid and phospholipid liposomes

Chlorophyll a/b light-harvesting complexes (chl a/b LHC) and photosystem II (PSII) cores were isolated from an octyl glucoside-containing sucrose gradient after solubilization of barley thylakoid membranes with Triton X-100 and octyl glucoside. No cation precipitation step was necessary to collect the chl a/b LHC. PAGE under mildly denaturing and fully denaturing conditions showed that the chl a/b LHC fraction contained chlorophyll-protein complexes CP27, CP29, and CP64. The PSII core material contained CP43 and CP47, and little contamination by other nonpigmented polypeptides. Freeze-fracture electron microscopy of the chl a/b LHC after reconstitution into digalactosyldiglyceride (DG) or phosphatidylcholine (PC) vesicles showed that the protein particles (approximately 7.5 +/- 1.6 nm) were approximately 99 and 90% randomly dispersed, respectively, in the liposomes. Addition of Mg++ produced particle aggregation and membrane adhesion in chl a/b LHC-DG liposomes in a manner analogous to that described for LHC-PC liposomes. Reconstitution of PSII cores into DG vesicles also produced proteoliposomes with randomly dispersed particles (approximately 7.5 +/- 1.6 nm). In contrast, PSII-PC mixtures formed convoluted networks of tubular membranes that exhibited very few fracture faces. Most of the protein particles (approximately 7.0 +/- 1.5 nm) were seen trapped between, rather than embedded in, the membranes. The interaction between the zwitterionic head group of the phosphatidyl choline and the negatively charged PSII core may be responsible for the unusual membrane structures observed.     

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.     

Comparative analysis of the composition of two chlorophyll-b-containing light-harvesting complexes

The major light-harvesting complexes from Mantoniella squamata (Prasinophyceae) and from Chlorella fusca (Chlorophyceae) were analyzed with respect to polypeptide composition and pigmentation. It was found that the polypeptides of Mantoniella are smaller than those of Chlorella and bind twice the amount of pigment. We assume that the amount of pigment per polypeptide is of ecological as well as of taxonomical importance.Abbreviations Chl chlorophyll - LHC light-harvesting complex - Xan xanthophyll We thank the support by the Deutsche Forschungsgemeinschaft.     

The low-energy forms of photosystem I light-harvesting complexes: spectroscopic properties and pigment-pigment interaction characteristics

In this work the spectroscopic properties of the special low-energy absorption bands of the outer antenna complexes of higher plant Photosystem I have been investigated by means of low-temperature absorption, fluorescence, and fluorescence line-narrowing experiments. It was found that the red-most absorption bands of Lhca3, Lhca4, and Lhca1-4 peak, respectively, at 704, 708, and 709 nm and are responsible for 725-, 733-, and 732-nm fluorescence emission bands. These bands are more red shifted compared to "normal" chlorophyll a (Chl a) bands present in light-harvesting complexes. The low-energy forms are characterized by a very large bandwidth (400-450 cm(-1)), which is the result of both large homogeneous and inhomogeneous broadening. The observed optical reorganization energy is untypical for Chl a and resembles more that of BChl a antenna systems. The large broadening and the changes in optical reorganization energy are explained by a mixing of an Lhca excitonic state with a charge transfer state. Such a charge transfer state can be stabilized by the polar residues around Chl 1025. It is shown that the optical reorganization energy is changing through the inhomogeneous distribution of the red-most absorption band, with the pigments contributing to the red part of the distribution showing higher values. A second red emission form in Lhca4 was detected at 705 nm and originates from a broad absorption band peaking at 690 nm. This fluorescence emission is present also in the Lhca4-N-47H mutant, which lacks the 733-nm emission band.     

Structural characterization of high 800 nm-absorbing light-harvesting complexes from Rhodospirillales from their resonance Raman spectra

Resonance Raman spectroscopy provided evidence that high 800 nm-absorbing antennae from Rhodopseudomonas (Rps.) acidophila and Rps. palustris have similar structures around their dweller bacteriochlorophylls. These host-site structures are different from those of B 850-800 complexes from Chromatiaceae, which also exhibit a high absorbance at 800 nm. As also shown by previous biochemical data, these complexes might be stoichiometrically different from other antenna complexes, having one more BChl per minimal size unit of protein. A new classification of B 850-800 complexes is proposed, on the basis of resonance Raman and biochemical data: this classification distinguishes a class of B 850-800 S (involving the B 850-800 complexes from sulfur purple bacteria), two classes of B 850-800 NS (involving the B 850-800 complexes from non sulfur purple bacteria) and a class of H 800 complexes (involving the B 850-800 complexes from non sulfur purple bacteria exhibiting a high absorbance at 800 nm).     

Structural analysis of the reaction center light-harvesting complex I photosynthetic core complex of Rhodospirillum rubrum using atomic force microscopy

The bacterium Rhodospirillum rubrum contains a simple photosynthetic system, in which the reaction center (RC) receives energy from the light-harvesting (LH1) complex. We have used high-resolution atomic force microscopy (AFM) to image two-dimensional crystals of the RC-LH1 complex of R. rubrum. The AFM topographs show that the RC-LH1 complex is approximately 94 A in height, the RC-H subunit protrudes from the cytoplasmic face of the membrane by 40 A, and it sits 21 A above the highest point of the surrounding LH1 ring. In contrast, the RC on the periplasmic side is at a lower level than LH1, which protrudes from the membrane by 12 A. The RC-LH1 complex can adopt an irregular shape in regions of uneven packing forces in the crystal; this reflects a likely flexibility in the natural membrane, which might be functionally important by allowing the export of quinol formed as a result of RC photochemistry. Nanodissection of the RC by the AFM tip removes the RC-H subunit and reveals the underlying RC-L and -M subunits. LH1 complexes completely lacking the RC were also found, providing ideal conditions for imaging both rings of LH1 polypeptides for the first time by AFM. In addition, we demonstrate the ellipticity of the LH1 ring at the cytoplasmic and periplasmic sides of the membrane, in both the presence and absence of the RC. These AFM measurements have been reconciled with previous electron microscopy and NMR data to produce a model of the RC-LH1 complex.