The apoprotein precursor of the major light-harvesting complex of photosystem II (LHCIIb) is inserted primarily into stromal lamellae and subsequently migrates to the grana

The formation of the lateral distribution of the major antenna complex of photosystem II (LHCIIb) between the granal and stromal lamellae was studied. Specifically, the localization of the insertion and the assembly of the precursor of the apoprotein of LHCIIb (pLHCP) were studied with isolated thylakoids. After insertion of pLHCP into isolated thylakoids, fractionation of the latter into granal and stromal lamellar was performed. At 25 °C most of the precursor was located in the granal lamellae, although both highly purified granal and stromal lamellar fractions demonstrated a similar capability to insert pLHCP. When the insertion reaction to the thylakoids was performed at 10 °C, followed by their separation into stromal and granal lamellae, the labelled pLHCP was localized in the stromal ones. To examine whether pLHCP inserts into both granal and stromal lamellae, or preferentially into stromal lamellae and subsequently migrating to granal lamellae, a chase experiment was performed. Insertion of pLHCP at 10 °C was followed by chase of the radioactive precursor with excess of non-radioactive pLHCP at 25 °C. From the results presented it is evident that the level of pLHCP in stromal lamellae was gradually reduced, while it gradually accumulated in the granal lamellae. Furthermore, the pLHCP in the stromal lamellae was found to be in a free form, while after migrating to the granal lamellae it assembled into the pigmented LHCIIb.     

Novel insights into plant light-harvesting complex II phosphorylation and 'state transitions'

Plants need a highly responsive regulatory system to keep photosynthetic light reactions in balance with the needs and restrictions of the downstream metabolism. This mechanism optimises plant growth under naturally fluctuating light conditions. In this opinion article, we present a model addressing the biological role of the light intensity-controlled phosphorylation of light-harvesting complex II (LHCII) proteins and its relation with the non-photochemical quenching of excitation energy (NPQ). We overturn a long held view of the possible role of 'state transitions'. Instead, we discuss the interplay between LHCII protein phosphorylation and NPQ, a mechanism that is crucial for regulating excitation energy distribution to the two photosystems (PSII and PSI) and balancing the intersystem electron flow despite constant fluctuations in light intensity.     

Assembly of the precursor and processed light-harvesting chlorophyll a/b protein of Lemna into the light-harvesting complex II of barley etiochloroplasts

When the in vitro synthesized precursor of a light-harvesting chlorophyll a/b binding protein (LHCP) from Lemna gibba is imported into barley etiochloroplasts, it is processed to a single form. Both the processed form and the precursor are found in the thylakoid membranes, assembled into the light-harvesting complex of photosystem II. Neither form can be detected in the stromal fraction. The relative amounts of precursor and processed forms observed in the thylakoids are dependent on the developmental stage of the plastids used for uptake. The precursor as well as the processed form can also be detected in thylakoids of greening maize plastids used in similar uptake experiments. This detection of a precursor in the thylakoids, which has not been previously reported, could be a result of using rapidly developing plastids and/or using an heterologous system. Our results demonstrate that the extent of processing of LHCP precursor is not a prerequisite for its inclusion in the complex. They are also consistent with the possibility that the processing step can occur after insertion of the protein into the thylakoid membrane.     

Degradation of the main Photosystem II light-harvesting complex.

Many factors trigger the degradation of proteins, including changes in environmental conditions, genetic mutations, and limitations in the availability of cofactors. Despite the importance for viability, still very little is known about protein degradation and its regulation. The degradation of the most abundant membrane protein on Earth, the light-harvesting complex of Photosystem II (LHC II), is highly regulated under different environmental conditions, e.g. light stress, to prevent photochemical damage of the reaction center. However, despite major effort to identify the protease/proteases involved in the degradation of the apoproteins of LHC II the molecular details of this important process remain obscure. LHC II belongs to the family of chlorophyll a/b binding proteins (CAB proteins) and is located in the thylakoid membrane of the plant chloroplast. The results of biochemical experiments to isolate and characterize the protease degrading LHC II are summarized here and compared to our own recent finding indicating that a metalloprotease of the FtsH family is involved in this process.     

Role of spermidine in the stabilization of the apoprotein of the light-harvesting chlorophyll a/b-protein complex of photosystem II during leaf senescence process

Barley leaf discs maintained in dark accumulated a massive amount of putrescine (Put), lost chlorophyll and senescenced rapidly. At the same time RNase activity increased significantly. Exogenous spermidine (Spd) inhibited RNase activity, the loss of chlorophyll and degradation of the proteins from thylakoid membranes. Using SDS-PAGE and immunoblot analysis it was shown that spermidine was effective in the retardation of the loss of LHCPII observed in water-treated detached leaves. Analysis of PSII particles isolated from leaf fragments floated in water in the dark revealed the presence of Put, Spd and Spm. In spermidine treated leaves the level of this polyamine in photosystem II was above 5-fold higher than in control. The experimental findings obtained in this study provide evidence that applied spermidine interacts directly with thylakoid membranes so that they become more stable to degradation during senescence.     

Diffusion of light-harvesting complex II in the thylakoid membranes

The light-harvesting complex II (LHCII) is the main energy absorber for photosynthesis in green plants, and its translocation between photosystems I and II is the primary means of energy redistribution between them. Using single-particle tracking, we performed the first measurement of the mobility of LHCII in the photosynthetic membranes in both the nonphosphorylated and the phosphorylated (P-LHCII) conformations. These are part of an important, reversible, energy re-equilibration process called the state transition. We found that the population of P-LHCII in unappressed membranes is more mobile than the population of non-P-LHCII from the same regions.     

De-epoxidation of violaxanthin after reconstitution into different carotenoid binding sites of light-harvesting complex II

In higher plants, the de-epoxidation of violaxanthin (Vx) to antheraxanthin and zeaxanthin is required for the pH-dependent dissipation of excess light energy as heat and by that process plays an important role in the protection against photo-oxidative damage. The de-epoxidation reaction was investigated in an in vitro system using reconstituted light-harvesting complex II (LHCII) and a thylakoid raw extract enriched in the enzyme Vx de-epoxidase. Reconstitution of LHCII with varying carotenoids was performed to replace lutein and/or neoxanthin, which are bound to the native complex, by Vx. Recombinant LHCII containing either 2 lutein and 1 Vx or 1.6 Vx and 1.1 neoxanthin or 2.8 Vx per monomer were studied. Vx de-epoxidation was inducible for all complexes after the addition of Vx de-epoxidase but to different extents and with different kinetics in each complex. Analysis of the kinetics indicated that the three possible Vx binding sites have at least two, and perhaps three, specific rate constants for de-epoxidation. In particular, Vx bound to one of the two lutein binding sites of the native complex, most likely L1, was not at all or only at a slow rate convertible to Zx. In reisolated LHCII, newly formed Zx almost stoichiometrically replaced the transformed Vx, indicating that LHCII and Vx de-epoxidase stayed in close contact during the de-epoxidation reactions and that no release of carotenoids occurred.     

Assembly of the major light-harvesting complex II in lipid nanodiscs

Self-aggregation of isolated plant light-harvesting complexes (LHCs) upon detergent extraction is associated with fluorescence quenching and is used as an in vitro model to study the photophysical processes of nonphotochemical quenching (NPQ). In the NPQ state, in vivo induced under excess solar light conditions, harmful excitation energy is safely dissipated as heat. To prevent self-aggregation and probe the conformations of LHCs in a lipid environment devoid from detergent interactions, we assembled LHCII trimer complexes into lipid nanodiscs consisting of a bilayer lipid matrix surrounded by a membrane scaffold protein (MSP). The LHCII nanodiscs were characterized by fluorescence spectroscopy and found to be in an unquenched, fluorescent state. Remarkably, the absorbance spectra of LHCII in lipid nanodiscs show fine structure in the carotenoid and Q_y region that is different from unquenched, detergent-solubilized LHCII but similar to that of self-aggregated, quenched LHCII in low-detergent buffer without magnesium ions. The nanodisc data presented here suggest that 1), LHCII pigment-protein complexes undergo conformational changes upon assembly in nanodiscs that are not correlated with downregulation of its light-harvesting function; and 2), these effects can be separated from quenching and aggregation-related phenomena. This will expand our present view of the conformational flexibility of LHCII in different microenvironments.     

Amino-terminal sequence of the 21 kDa apoprotein of a minor light-harvesting pigment-protein complex of the Photosystem II antenna (LHC IId/CP24)

The 21 kDa apoprotein of LHC IId, a minor light-harvesting antenna component of Photosystem II, has been isolated and subjected to N-terminal protein sequencing. A sequence of 66 residues was obtained which contains regions of considerable homology to both those reported for LHC II and LHC I, but which is obviously distinct from them. The proposed occurrence of an identical 21 kDa LHC subunit in both photosystems I and II is shown to be incorrect.     

The effect of amphiphilic peptide surfactants on the light-harvesting complex II

The peptide surfactants are amphiphilic peptides which have a hydrophobic tail and a hydrophilic head, and have been reported to stabilize and protect some membrane proteins more effectively than conventional surfactants. The effects of a class of peptide surfactants on the structure and thermal stability of the photosynthetic membrane protein lightharvesting complex II (LHCII) in aqueous media have been investigated. After treatment with the cationic peptide surfactants A₆K, V₆K₂, I₅K₂ and I₅R₂, the absorption at 436 nm and 470 nm decreased and the absorption at 500–510 nm and 684–690 nm increased. Moreover, the circular dichroism (CD) signal intensity in the Soret region also decreased significantly, indicating the conformation of some chlorophyll (Chl) a, Chl b, and the xanthophyll molecules distorted upon cationic peptide surfactants treatment. The anionic peptide surfactants A₆D and V₆D₂ had no obvious effect on the absorption and CD spectra. Except for A₆D, these peptides all decreased the thermal stability of LHCII, indicating that these peptides may reconstitute protein into a less stable conformation. In addition, the cationic peptide surfactants resulted in LHCII aggregation, as shown by sucrose gradient ultracentrifugation and fluorescence spectra.     

Stark spectroscopy of the light-harvesting complex II in different oligomerisation states

The electric field-induced absorption changes (Stark effect) of light-harvesting complex II (LHCII) in different oligomerisation states-monomeric, trimeric and aggregated-have been probed at 77 K. All the chlorophyll (Chl) a molecules exhibit electro-optic properties in the Q(y) absorption region characterized by a change in dipole moment /Deltamu-->/ =0.6+/-0.06D/f and polarizability, Tr(Deltaalpha;) approximately 55+/-5 A(3)/f(2) upon electronic excitation, which are similar to those of unbound monomeric Chl a, indicating the absence of strong delocalization of the excitations which would be expected in the presence of strong excitonic interactions. The Stark effect in the Chl b absorption region is significantly bigger with /Deltamu-->/ values of the order of 2.0+/-0.2 D/f and it is attributed to strong interactions with neoxanthin molecules. Clear oligomerisation-dependent differences are observed in the carotenoid region, mainly due to the appearance of a new xanthophyll absorption band at 509 in the spectra of trimers and oligomers. It is ascribed to some lutein molecules, in agreement with previous experimental observations. The electro-optic properties of these lutein molecules are significantly different from those of the other xanthophylls in LHCII, which do not exhibit such a big change in dipole moment upon electronic excitation (/Deltamu-->/ =14.6+/-2.0 D/f). Upon aggregation of LHCII some extra absorption appears on the red side of the main Chl a Q(y) absorption band. In contrast to an earlier suggestion [J. Phys. Chem., A 103 (1999) 2422], no indications are found for the charge-transfer character of the corresponding band. The assignments of the S(2) electronic transitions of neoxanthin and lutein in LHCII and possible origins of the Stark effect are discussed.     

Proteolytic activity against the light-harvesting complex and the D1/D2 core proteins of Photosystem II in close association to the light-harvesting complex II trimer

Light-harvesting complex II (LHCII) prepared from isolated thylakoids of either broken or intact chloroplasts by three independent methods, exhibits proteolytic activity against LHCII. This activity is readily detectable upon incubation of these preparations at 37 °C (without addition of any chemicals or prior pre-treatment), and can be monitored either by the LHCII immunostain reduction on Western blots or by the Coomassie blue stain reduction in substrate-containing “activity gels”. Upon SDS-sucrose density gradient ultracentrifugation of SDS-solubilized thylakoids, a method which succeeds in the separation of the pigment-protein complexes in their trimeric and monomeric forms, the protease activity copurifies with the LHCII trimer, its monomer exhibiting no activity. This LHCII trimer, apart from being “self-digested”, also degrades the Photosystem II (PSII) core proteins (D1, D2) when added to an isolated PSII core protein preparation containing the D1/D2 heterodimer. Under our experimental conditions, 50% of LHCII or the D1, D2 proteins are degraded by the LHCII-protease complex within 30 min at 37 °C and specific degradation products are observed. The protease is light-inducible during chloroplast biogenesis, stable in low concentrations of SDS, activated by Mg²⁺, and inhibited by Zn²⁺, Cd²⁺, EDTA and p-hydroxy-mercury benzoate (pOHMB), suggesting that it may belong to the cysteine family of proteases. Upon electrophoresis of the LHCII trimer on substrate-containing “activity gels” or normal Laemmli gels, the protease is released from the complex and runs in the upper part of the gel, above the LHCII trimer. A polypeptide of 140 kDa that exhibits proteolytic activity against LHCII, D1 and D2 has been identified as the protease. We believe that this membrane-bound protease is closely associated to the LHCII complex in vivo, as an LHCII-protease complex, its function being the regulation of the PSII unit assembly and/or adaptation.     

Proteolytic activity against the light-harvesting complex and the D1/D2 core proteins of Photosystem II in close association to the light-harvesting complex II trimer

Light-harvesting complex II (LHCII) prepared from isolated thylakoids of either broken or intact chloroplasts by three independent methods, exhibits proteolytic activity against LHCII. This activity is readily detectable upon incubation of these preparations at 37 degrees C (without addition of any chemicals or prior pre-treatment), and can be monitored either by the LHCII immunostain reduction on Western blots or by the Coomassie blue stain reduction in substrate-containing "activity gels". Upon SDS-sucrose density gradient ultracentrifugation of SDS-solubilized thylakoids, a method which succeeds in the separation of the pigment-protein complexes in their trimeric and monomeric forms, the protease activity copurifies with the LHCII trimer, its monomer exhibiting no activity. This LHCII trimer, apart from being "self-digested", also degrades the Photosystem II (PSII) core proteins (D1, D2) when added to an isolated PSII core protein preparation containing the D1/D2 heterodimer. Under our experimental conditions, 50% of LHCII or the D1, D2 proteins are degraded by the LHCII-protease complex within 30 min at 37 degrees C and specific degradation products are observed. The protease is light-inducible during chloroplast biogenesis, stable in low concentrations of SDS, activated by Mg(2+), and inhibited by Zn(2+), Cd(2+), EDTA and p-hydroxy-mercury benzoate (pOHMB), suggesting that it may belong to the cysteine family of proteases. Upon electrophoresis of the LHCII trimer on substrate-containing "activity gels" or normal Laemmli gels, the protease is released from the complex and runs in the upper part of the gel, above the LHCII trimer. A polypeptide of 140 kDa that exhibits proteolytic activity against LHCII, D1 and D2 has been identified as the protease. We believe that this membrane-bound protease is closely associated to the LHCII complex in vivo, as an LHCII-protease complex, its function being the regulation of the PSII unit assembly and/or adaptation.     

Predicting the structure of the light-harvesting complex II of Rhodospirillum molischianum.

We attempted to predict through computer modeling the structure of the light-harvesting complex II (LH-II) of Rhodospirillum molischianum, before the impending publication of the structure of a homologous protein solved by means of X-ray diffraction. The protein studied is an integral membrane protein of 16 independent polypeptides, 8 alpha-apoproteins and 8 beta-apoproteins, which aggregate and bind to 24 bacteriochlorophyll-a's and 12 lycopenes. Available diffraction data of a crystal of the protein, which could not be phased due to a lack of heavy metal derivatives, served to test the predicted structure, guiding the search. In order to determine the secondary structure, hydropathy analysis was performed to identify the putative transmembrane segments and multiple sequence alignment propensity analyses were used to pinpoint the exact sites of the 20-residue-long transmembrane segment and the 4-residue-long terminal sequence at both ends, which were independently verified and improved by homology modeling. A consensus assignment for the secondary structure was derived from a combination of all the prediction methods used. Three-dimensional structures for the alpha- and the beta-apoprotein were built by comparative modeling. The resulting tertiary structures are combined, using X-PLOR, into an alpha beta dimer pair with bacteriochlorophyll-a's attached under constraints provided by site-directed mutagenesis and spectral data. The alpha beta dimer pairs were then aggregated into a quaternary structure through further molecular dynamics simulations and energy minimization. The structure of LH-II so determined is an octamer of alpha beta heterodimers forming a ring with a diameter of 70 A.     

Structure,function and assembly of Photosystem II and its light-harvesting proteins

Photosystem II (PSII) is a multisubunit chlorophyll–protein complex that drives electron transfer from water to plastoquinone using energy derived from light. In green plants, the native form of PSII is surrounded by the light-harvesting complex (LHCII complex) and thus it is called the PSII–LHCII supercomplex. Over the past several years, understanding of the structure, function, and assembly of PSII and LHCII complexes has increased considerably. The unicellular green alga Chlamydomonas reinhardtii has been an excellent model organism to study PSII and LHCII complexes, because this organism grows heterotrophically and photoautotrophically and it is amenable to biochemical, genetic, molecular biological and recombinant DNA methodology. Here, the genes encoding and regulating components of the C. reinhardtii PSII–LHCII supercomplex have been thoroughly catalogued: they include 15 chloroplast and 20 nuclear structural genes as well as 13 nuclear genes coding for regulatory factors. This review discusses these molecular genetic data and presents an overview of the structure, function and assembly of PSII and LHCII complexes.     

Localization of light-harvesting complex II to the occluded surfaces of photosynthetic membranes

The photosynthetic membranes of green plants are organized into stacked regions interconnected by nonstacked regions that have been shown to be biochemically and structurally distinct. Because the stacking process occludes the surfaces of appressed membranes, it has been impossible to conduct structural or biochemical studies of the outer surfaces of the photosynthetic membrane in regions of membrane stacking. Although stacking is mediated at this surface, it has not been possible to determine whether membrane components implicated in the stacking process, including a major light-harvesting complex (LHC-II), are in fact exposed at the membrane surface. We have been able to expose this surface for study in the electron microscope and directly label it with antibodies to determine protein exposure. The appearance of the newly exposed outer stacked surface highlights the extreme lateral heterogeneity of the photosynthetic membrane. The surface is smooth in contrast to the neighboring nonstacked surface that is covered with distinct particles. Although some investigators have suggested the existence of a cytochrome b6/f-rich boundary region between stacked and nonstacked membranes, our results provide no structural support for this concept. To explore the biochemical nature of the occluded membrane surface, we have used an mAb against the amino terminal region of the LHC-II. This mAb clearly labels the newly exposed outer stacked surface but does not label the inner surface or the outer nonstacked surface. These experimental results confirm the presence of the amino terminal region of this complex at the outer surface of the membrane in stacked regions, and also show that this complex is largely absent from nonstacked membranes.     

The light-harvesting chlorophyll a/b complex can be reconstituted in vitro from its completely unfolded apoprotein

The major light-harvesting chlorophyll a/b protein (LHCIIb) of higher plants is one of the few membrane proteins that can be refolded in vitro. During folding, the apoprotein is assembled with pigments to form a structurally authentic and functional pigment--protein complex. All reconstitution procedures used so far include solubilization of the apoprotein in sodium dodecyl sulfate (SDS) where the protein adopts approximately half of its alpha-helical folding present in the native structure. This paper shows that this preformed alpha-helix is not a prerequisite for LHCIIb folding in vitro. The apoprotein can also be reconstituted starting from a solution in guanidinium hydrochloride (Gnd) where the protein contains no detectable helical structure. Reconstitution yields are somewhat lower in the Gnd than in the SDS procedure, but the reconstitution products exhibit very similar biochemical and spectroscopic properties. The kinetics of LHCIIb assembly, as assessed by time-resolved fluorescence measurements, are virtually the same in both reconstitution procedures. This demonstrates that the initiation of alpha-helix formation is not a rate-limiting step in LHCIIb apoprotein folding.     

Carotenoid-binding sites of the major light-harvesting complex II of higher plants.

Recombinant light-harvesting complex II (LHCII) proteins with modified carotenoid composition have been obtained by in vitro reconstitution of the Lhcb1 protein overexpressed in bacteria. The monomeric protein possesses three xanthophyll-binding sites. The L1 and L2 sites, localized by electron crystallography in the helix A/helix B cross, have the highest affinity for lutein, but also bind violaxanthin and zeaxanthin with lower affinity. The latter xanthophyll causes disruption of excitation energy transfer. The occupancy of at least one of these sites, probably L1, is essential for protein folding. Neoxanthin is bound to a distinct site (N1) that is highly selective for this species and whose occupancy is not essential for protein folding. Whereas xanthophylls in the L1 and L2 sites interact mainly with chlorophyll a, neoxanthin shows strong interaction with chlorophyll b, inducing the hyperchromic effect of the 652 nm absorption band. This observation explains the recent results of energy transfer from carotenoids to chlorophyll b obtained by femtosecond absorption spectroscopy. Whereas xanthophylls in the L1 and L2 sites are active in photoprotection through chlorophyll-triplet quenching, neoxanthin seems to act mainly in (1)O(2)(*) scavenging.     

Single molecule spectroscopy on the light-harvesting complex II of higher plants.

Spectroscopic and polarization properties of single light-harvesting complexes of higher plants (LHC-II) were studied at both room temperature and T < 5 K. Monomeric complexes emit roughly linearly polarized fluorescence light thus indicating the existence of only one emitting state. Most probably this observation is explained by efficient triplet quenching restricted to one chlorophyll a (Chl a) molecule or by rather irreversible energy transfer within the pool of Chl a molecules. LHC-II complexes in the trimeric (native) arrangement bleach in a number of steps, suggesting localization of excitations within the monomeric subunits. Interpretation of the fluorescence polarization properties of trimers requires the assumption of transition dipole moments tilted out of the symmetry plane of the complex. Low-temperature fluorescence emission of trimers is characterized by several narrow spectral lines. Even at lowest excitation intensities, we observed considerable spectral diffusion most probably due to low temperature protein dynamics. These results also indicate weak interaction between Chls belonging to different monomeric subunits within the trimer thus leading to a localization of excitations within the monomer. The experimental results demonstrate the feasibility of polarization sensitive studies on single LHC-II complexes and suggest an application for determination of the Chl transition-dipole moment orientations, a key issue in understanding the structure-function relationships.     

A study on the heterogeneity of the light-harvesting complex II from Ectothiorhodospira sp. after acid/chaotropic treatment

The light-harvesting complex II of the purple bacteria has two strong near infrared electronic absorption bands, around 800 (B800) and 850 (B850) nm, arising from the Qy transitions of bacteriochlorophyll a. It was previously reported that under some specific acid/chaotropic conditions the B850 bacteriochlorophylls of the light-harvesting complex II of Ectothiorhodospira sp. are strongly reorganised. Part of these pigments absorbs at 843 nm while another set absorbs around 858 nm. The current work should investigate whether a mix of two different complexes could generate the 843- and 858-nm bands. Acid/chaotropic conditions inducing the reorganisation of B850 were reproduced on a sample bound to an ionic-exchange column. The chromatographic pattern was found strongly homogeneous. The findings indicate that the heterogeneity of the reorganised B850 results from two forms of differently structured bacteriochlorophylls bound to the same polypeptide backbone.