Can giant chlorosomes be part of the light-harvesting antennae of green bacteria?

Some data on the structure and composition of chlorosomes are in conflict with their energy and kinetic characteristics. Among the latter is the very short excitation lifetime of the dominant pigment C740 in the 3D giant chlorosome (about 1000 pigment molecules per reaction center). Therewith the excitation transfer from C740 to baseplate bacteriochlorophyll B795 and further to the main membrane B860 can hardly be efficient. This result was obtained by modeling the energy migration between these pigment fractions in maximally optimized conditions. The possible reasons and mechanisms responsible for such strong nonphotochemical quenching of electronic excitations in the pigments of giant chlorosomes are substantiated and discussed.     

Identification of a novel light-harvesting complex II protein (LHC IIc′)

The caroteno-chlorophyll-protein, LHC IIc, is a relatively minor component of the PS II antenna. Isolated LHC IIc contains a major protein of 28 kDa along with a 26 kDa subunit in lower abundance. Previously, it was not known if the 26 kDa protein was closely related to the 28 kDa LHC IIc protein or if it was a comigrating LHC IIb contaminating subunit. A sequence of 20 amino acid residues was obtained by direct protein micro-sequencing of an internal cyanogen bromide-derived peptide fragment of the 26 kDa protein isolated from barley. The sequence shows, and antibody reactions confirm, that the 26 kDa protein is similar but distinct from both the 28 kDa LHC IIc and LHC IIb protein sequences, indicating that there remains at least one more cab gene to be identified in higher plants. Furthermore, it is difficult to interpret the data in any way other than that there is a novel LHC II pigment-protein (LHC IIc) that co-migrates with LHC IIc.Abbreviations CC core complex - LHC light-harvesting complex - PVDF polyvinylidene fluoride     

Sensitivity of the photosynthetic apparatus to UV-A radiation: role of light-harvesting complex II–photosystem II supercomplex organization

In this work we study the effect of UV-A radiation on the function of the photosynthetic apparatus in thylakoid membranes with different organization of the light-harvesting complex II–photosystem II (LHCII–PSII) supercomplex. Leaves and isolated thylakoid membranes from a number of previously characterized pea species with different LHCII size and organization were subjected to UV-A treatment. A relationship was found between the molecular organization of the LHCII (ratio of the oligomeric to monomeric forms of LHCII) and UV-A-induced changes both in the energy transfer from PSII to PSI and between the chlorophyll–protein complexes within the LHCII–PSII supercomplex. Dependence on the organization of the LHCII was also found with regard to the degree of inhibition of the photosynthetic oxygen evolution. The susceptibility of energy transfer and oxygen evolution to UV-A radiation decreased with increasing LHCII oligomerization when the UV-A treatment was performed on isolated thylakoid membranes, in contrast to the effect observed in thylakoid membranes isolated from pre-irradiated pea leaves. The data suggest that UV-A radiation leads mainly to damage of the PSIIα centers. Comparison of membranes with different organization of their LHCII–PSII supercomplex shows that the oligomeric forms of LHCII play a key role for sensitivity to UV-A radiation of the photosynthetic apparatus. S. G. Taneva is Associated member of the Institute of Biophysics, Bulgarian Academy of Sciences.     

Importance of trans-Δ3-hexadecenoic acid containing phosphatidylglycerol in the formation of the trimeric light-harvesting complex in Chlamydomonas

Possible roles of trans-Δ³-hexadecenoic acid containing phosphatidylglycerol (PG) in the organisation of photosynthetic complexes were studied using two mutants of Chlamydomonas reinhardtii, mf1 and mf2, that totally lack this lipid and in which the level of the others remaining PG was consequently reduced to about 30% of the wild-type. Both the mutants have lost the capacity to stabilise the light-harvesting chlorophyll a/b–protein complex LHC II in a trimeric state and display an increased instability of the PS I light-harvesting-core complex after detergent mediated solubilisation. In this paper, we show that a very reduced growth rate of the mutant cells largely reduces the extent of these defects, allowing a significant formation of trimeric LHC II and a stabilisation of the PS I complex, in the absence of synthesis of trans-Δ³-hexadecenoic acid or of increased level of PG. These results seem to be at variance with the generally accepted role of trans-Δ³-hexadecenoic fatty acid (16:1(3t)) in the formation of the PS II light-harvesting antenna. On the other hand, they appear to be consistent with the observation that trimeric LHC II can be formed in the presence of 16:1(3t)-lacking PG in a mutant of Arabidopsis thaliana and in chloroplasts from cotyledons of some Orchideae. We conclude that 16:1(3t)-PG is indeed required for the stabilisation of the trimeric LHC II and of the PS I complex under conditions of high biosynthesis rate, and that it is not essential when these components of the photosynthetic membrane are synthesised at low rates.     

Redox-controlled,in vivo and in vitro phosphorylation of the α subunit of the light-harvesting complex I in Rhodobacter capsulatus

Labelling of Rhodobacter capsulatus cells with (³²P)Pi in a phototrophic culture results in phosphorylation of a membrane-bound polypeptide identified as the subunit of the LHI antenna complex of the photosynthetic apparatus. Phosphorylation of the same polypeptide was also observed by incubation of chromatophores with (³²P)ATP or under conditions of photophosphorylation with ADP and (³²P)Pi. The identity of the phosphorylated LHI- subunit was demonstrated by N-terminal protein sequencing of the phosphorylated polypeptide and by failure of labelling in LHI-defective mutants. Pre-aeration of the samples or addition of the oxidant potassium ferrcyanide stimulated the kinase activity whereas the presence of soluble cytoplasmic proteins impaired phosphorylation in an in vitro assay. No effect resulted from addition of reductants to the assay medium. The results indicate the presence of a membrane-bound protein kinase in R. capsulatus that phosphorylates the subunit of the LHI antenna complex under redox control.Abbreviations Pi inorganic phosphate - SDS-PAGE sodium dodecyl-sulfate polyacrylamide gel electrophoresis     

What is the role of the transit peptide in thylakoid integration of the light-harvesting chlorophyll a/b protein?

Whereas it is widely accepted that the transit peptide of the precursor for the light-harvesting chlorophyll a/b protein (preLHCP) is responsible for targeting this polypeptide to chloroplasts, the signals which govern its intraorganellar targeting appears to be transit peptide-mediated for plastocyanin (Smeekins, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365-375) and several other nuclear-encoded, thylakoid luminal proteins. To determine whether a similar mechanism operates for LHCP (an integral thylakoid protein), we have used oligonucleotide-directed mutagenesis to delete the proposed transit sequence from a petunia precursor of this polypeptide. Intact preLHCP and the deletion mutant product have been expressed in vitro, and their abilities to integrate into purified thylakoids have been compared. We have found that both polypeptides insert into thylakoids correctly, provided the latter are supplemented with a membrane-free stromal extract and Mg.ATP. Our results clearly demonstrate that whereas the transit peptide is required for transport into chloroplasts, thylakoid integration of preLHCP is determined by mature portions of the polypeptide. In addition, we note that transit peptide removal has little effect on the apparent solubility of the in vitro translation products.     

Is each light-harvesting complex protein important for plant fitness?

Many of the photosynthetic genes are conserved among all higher plants, indicating that there is strong selective pressure to maintain the genes of each protein. However, mutants of these genes often lack visible growth phenotypes, suggesting that they are important only under certain conditions or have overlapping functions. To assess the importance of specific genes encoding the light-harvesting complex (LHC) proteins for the survival of the plant in the natural environment, we have combined two different scientific traditions by using an ecological fitness assay on a set of genetically modified Arabidopsis plants with differing LHC protein contents. The fitness of all of the LHC-deficient plants was reduced in some of the growth environments, supporting the hypothesis that each of the genes has been conserved because they provide ecological flexibility, which is of great adaptive value given the highly variable conditions encountered in nature.     

Elucidation of structure–function relationships in plant major light-harvesting complex (LHC II) by nonlinear spectroscopy

Conventional linear and time-resolved spectroscopic techniques are often not appropriate to elucidate specific pigment-pigment interactions in light-harvesting pigment-protein complexes (LHCs). Nonlinear (laser-) spectroscopic techniques, including nonlinear polarization spectroscopy in the frequency domain (NLPF) as well as step-wise (resonant) and simultaneous (non-resonant) two-photon excitation spectroscopies may be advantageous in this regard. Nonlinear spectroscopies have been used to elucidate substructure(s) of very complex spectra, including analyses of strong excitonic couplings between chlorophylls and of interactions between (bacterio)chlorophylls and "optically dark" states of carotenoids in LHCs, including the major antenna complex of higher plants, LHC II. This article shortly reviews our previous study and outlines perspectives regarding the application of selected nonlinear laser-spectroscopic techniques to disentangle structure-function relationships in LHCs and other pigment-protein complexes.     

First solid-state NMR analysis of uniformly ¹³C-enriched major light-harvesting complexes from Chlamydomonas reinhardtii and identification of protein and cofactor spin clusters

The light-harvesting complex II (LHCII) is the main component of the antenna system of plants and green algae and plays a major role in the capture of sun light for photosynthesis. The LHCII complexes have also been proposed to play a key role in the optimization of photosynthetic efficiency through the process of state 1-state 2 transitions and are involved in down-regulation of photosynthesis under excess light by energy dissipation through non-photochemical quenching (NPQ). We present here the first solid-state magic-angle spinning (MAS) NMR data of the major light-harvesting complex (LHCII) of Chlamydomonas reinhardtii, a eukaryotic green alga. We are able to identify nuclear spin clusters of the protein and of its associated chlorophyll pigments in 13C-13C dipolar homonuclear correlation spectra on a uniformly 13C-labeled sample. In particular, we were able to resolve several chlorophyll 131 carbon resonances that are sensitive to hydrogen bonding to the 131-keto carbonyl group. The data show that 13C NMR signals of the pigments and protein sites are well resolved, thus paving the way to study possible structural reorganization processes involved in light-harvesting regulation through MAS solid-state NMR.     

Identification of intramembrane hydrogen bonding between 13 keto group of bacteriochlorophyll and serine residue α27 in the LH2 light-harvesting complex

Intramembrane hydrogen bonding and its effect on the structural integrity of purple bacterial light-harvesting complex 2, LH2, have been assessed in the native membrane environment. A novel hydrogen bond has been identified by Raman resonance spectroscopy between a serine residue of the membrane-spanning region of LH2 α-subunit, and the C-13¹ keto carbonyl of bacteriochlorophyll (BChl) B850 bound to the β-subunit. Replacement of the serine by alanine disrupts this strong hydrogen bond, but this neither alters the strongly red-shifted absorption nor the structural arrangement of the BChls, as judged from circular dichroism. It also decreases only slightly the thermal stability of the mutated LH2 in the native membrane environment. The possibility is discussed that weak H-bonding between the C-13¹ keto carbonyl and a methyl hydrogen of the alanine replacing serine(−4) or the imidazole group of the nearby histidine maintains structural integrity in this very stable bacterial light-harvesting complex. A more widespread occurrence of H-bonding to C-13¹ not only in BChl, but also in chlorophyll proteins, is indicated by a theoretical analysis of chlorophyll/polypeptide contacts at <3.5 Å in the high-resolution structure of Photosystem I. Nearly half of the 96 chlorophylls have aa residues suitable as hydrogen bond donors to their keto groups.     

Does a light-harvesting protochlorophyllide a/b-binding protein complex exist?

Recent in vitro studies have led to speculation that a novel light-harvesting protochlorophyllide a/b-binding protein complex (LHPP) might exist in dark-grown angiosperms. Structurally, it has been suggested that LHPP consists of a 5:1 ratio of dark-stable ternary complexes of the light-dependent NADPH: protochlorophyllide oxidoreductases A and B containing nonphotoactive protochlorophyllide b and photoactive protochlorophyllide a, respectively. Functionally, LHPP has been hypothesized to play major roles in establishing the photosynthetic apparatus, in protecting against photo-oxidative damage during greening, and in determining etioplast inner membrane architecture. However, the LHPP model is not compatible with other studies of the pigments and the pigment-protein complexes of dark-grown angiosperms. Protochlorophyllide b, which is postulated to be the major light-harvesting pigment of LHPP, has, for example, never been detected in etiolated seedlings. This raises the question: does LHPP exist?     

Transmembrane orientation of α-helices in the thylakoid membrane and in the light-harvesting complex. A polarized infrared spectroscopy study

The structure and orientation of the major protein constituent of photosynthetic membranes in green plants, the chlorophyll $\text{a}\text{b}$ light-harvesting complex (LHC) have been investigated by ultraviolet circular dichroism (CD) and polarized infrared spectroscopies. The isolated purified LHC has been reconstituted into phosphatidylcholine vesicles and has been compared to the pea thylakoid membrane. The native orientation of the pigments in the LHC reconstituted in vesicles was characterized by monitoring the low-temperature polarized absorption and fluorescence spectra of reconstituted membranes. Conformational analysis of thylakoid and LHC indicate that a large proportion of the thylakoid protein is in the α-helical structure (56 ± 4%), while the LHC is for 44 ± 7% α-helical. By measuring the infrared dichroism of the amide absorption bands of air-dried oriented multilayers of thylakoids and LHC reconstituted in vesicles, we have estimated the degree of orientation of the α-helical chains with respect to the membrane normal. Infrared dichroism data demonstrate that transmembrane α-helices are present in both thylakoid and LHC with the α-helix axes tilted at less than 30° in LHC and 40° in thylakoid with respect to the membrane normal. In thylakoids, an orientation of the polar C=O ester groups of the lipids parallel to the membrane plane is detected. Our results are consistent with the existence of 3–5 transmembrane α-helical segments in the LHC molecules.     

Pigment-protein architecture in the light-harvesting antenna complexes of purple bacteria: does the crystal structure reflect the native pigment-protein arrangement?

Structural analysis of crystallized peripheral (LH2) and core antenna complexes (LH1) of purple bacteria has revealed circular aggregates of high rotational symmetry (C₈, C₉ and C₁₆, respectively). Quantum-chemical calculations indicate that in particular the waterwheel-like arrangements of pigments should show characteristic structure-sensitive spectroscopic behavior in the near infrared absorption region. Laser-spectroscopic data obtained with non-crystallized, isolated LH2 of Rhodospirillum molischianum are in line with a highly symmetric (C₈) circular aggregate, but deviations have been found for LH2 of Rhodobacter sphaeroides and Rhodopseudomonas acidophila. For both the latter, C-shaped incomplete circular aggregates (as seen only recently in electron micrographs of crystallized LH1–reaction center complexes) may be a suitable preliminary model.     

Synthetic analogues of the histidine–chlorophyll complex: a NMR study to mimic structural features of the photosynthetic reaction center and the light-harvesting complex

Mg(II)–porphyrin–ligand and (bacterio)chlorophyl–ligand coordination interactions have been studied by solution and solid-state MAS NMR spectroscopy. ¹H, ¹³C and ¹⁵N coordination shifts due to ring currents, electronic perturbations and structural effects are resolved for imidazole (Im) and 1-methylimidazole (1-MeIm) coordinated axially to Mg(II)-OEP and (B)Chl a. As a consequence of a single axial coordination of Im or 1-MeIm to the Mg(II) ion, 0.9–5.2 ppm ¹H, 0.2–5.5 ppm ¹³C and 2.1–27.2 ppm ¹⁵N coordination shifts were measured for selectively labeled [1,3-¹⁵N]-Im, [1,3-¹⁵N,2-¹³C]-Im and [1,3-¹⁵N,1,2-¹³C]-1-MeIm. The coordination shifts depend on the distance of the nuclei to the porphyrin plane and the perturbation of the electronic structure. The signal intensities in the ¹H NMR spectrum reveal a five-coordinated complex, and the isotropic chemical shift analysis shows a close analogy with the electronic structure of the BChl a–histidine in natural light harvesting 2 complexes. The line broadening of the ligand responses support the complementary IR data and provide evidence for a dynamic coordination bond in the complex.Abbreviations (B)Chl a (bacterio)chlorophyll a - HMBC heteronuclear multiple bond correlation - Im imidazole - LH light-harvesting - 1-MeIm 1-methylimidazole - Mg(II)-Por Mg(II)-porphyrin macrocycle - OEP 2,3,7,8,12,13,17,18-octaethylporphyrin     

Absorption and linear dichroism spectroscopy at room and low temperatures of single crystals of the B800–850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050

The absorbance, polarized absorbance and linear dichroism spectra of single crystals of the B800–850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050 taken at room (298 K) and low (85 K) temperatures are presented. The spectra are compared and contrasted with random phase solution spectra from the same complex. The single crystal spectra display a spectral narrowing at low temperatures in the BChl Q_x (550–650 nm) and carotenoid (450–550 nm) regions similar to that observed from the random phase solution. The single crystal absorption spectra in the BChl Q_y (750–900 nm) region are broader than the solution spectra and remain broad as the temperature is lowered. It is suggested that this broadening is the result of specific exciton interactions between the BChl chromophore Q_y transition dipoles and is a molecular feature which occurs only in the crystalline complex.     

Are syn-ligated (bacterio)chlorophyll dimers energetic traps in light-harvesting systems?

A recent study of the stereochemical details of chlorophyll ligation in photosystem I [Balaban et al., Biochim. Biophys. Acta 1556 (2002) 197-207] has revealed that only 14 chlorophylls out of the total 96 are ligated from the same side (syn) as the 17-propionic acid residue which is esterified with phytol. The syn chlorophylls are carefully surrounding the reaction center forming the inner core antenna system and their ligands have been strongly conserved in several species during evolution. We hypothesize here that the two dimers of closely spaced syn chlorophylls which are encountered within roughly 2 nm of P700 are the ultimate energetic traps of this light-harvesting system. Structurally very similar bacteriochlorophyll a dimers are encountered within the Fenna-Matthews-Olson protein complex and within the B850 ring of the LH2 complex of purple bacteria. The non-random disposal of these dimers lends support to our hypothesis that the syn ligation coupled with a strong excitonic interaction leads to the most red-shifted pigments in light-harvesting systems. We would like to encourage both theoretical and experimental studies to either prove or disprove this intriguing structure-function conjecture in view of designing efficient artificial light-harvesting systems.