Chromophore organization in the higher-plant photosystem II antenna protein CP26

The chlorophyll a/b-xanthophyll-protein CP26 complex belongs to the Lhc protein family. It binds nine chlorophylls and two xanthophylls per 26.6 kDa polypeptide. Determination of the characteristics of each binding site is needed for the understanding of functional organization of individual proteins belonging to the photosystem II supramolecular complex. The biochemical and spectroscopic features of native CP26 are presented here together with identification of pigment binding and energy transitions in different sites. The analysis has been performed via a new approach using recombinant CP26 complexes in which the chromophore content has been experimentally modified. Data were interpreted on the basis of homology with CP29 and LHCII complexes, for which detailed knowledge is available from mutation analysis. We propose that one additional Chl b is present in CP26 as compared to CP29 and that it is located in site B2. We also found that in CP26 three chlorophyll binding sites are selective for Chl a, one of them being essential for the folding of the pigment-protein complex. Two xanthophyll binding sites were identified, one of which (L1) is essential for protein folding and specifically binds lutein. The second site (L2) has lower selectivity and can bind any of the xanthophyll species present in thylakoids.     

The structure of photosystem II in Arabidopsis: localization of the CP26 and CP29 antenna complexes

A genetic approach has been adopted to investigate the organization of the light-harvesting proteins in the photosystem II (PSII) complex in plants. PSII membrane fragments were prepared from wild-type Arabidopis thaliana and plants expressing antisense constructs to Lhcb4 and Lhcb5 genes, lacking CP29 and CP26, respectively (Andersson et al. (2001) Plant Cell 13, 1193-1204). Ordered PS II arrays and PS II supercomplexes were isolated from the membranes of plants lacking CP26 but could not be prepared from those lacking CP29. Membranes and supercomplexes lacking CP26 were less stable than those prepared from the wild type. Transmission electron microscopy aided by single-particle image analysis was applied to the ordered arrays and the isolated PSII complexes. The difference between the images obtained from wild type and antisense plants showed the location of CP26 to be near CP43 and one of the light-harvesting complex trimers. Therefore, the location of the CP26 within PSII was directly established for the first time, and the location of the CP29 complex was determined by elimination. Alterations in the packing of the PSII complexes in the thylakoid membrane also resulted from the absence of CP26. The minor light-harvesting complexes each have a unique location and important roles in the stabilization of the oligomeric PSII structure.     

Time-resolved fluorescence analysis of the recombinant photosystem II antenna complex CP29. Effects of zeaxanthin, pH and phosphorylation.

Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants. The formation of zeaxanthin in the antenna of photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo. We have used recombinant CP29 protein, over-expressed in Escherichia coli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein. These recombinant proteins and the native CP29 were used to measure steady-state chlorophyll fluorescence emission and fluorescence decay kinetics. We found that the presence of zeaxanthin bound to CP29 induces a approximately 35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthin-free reconstituted proteins). Fluorescence decay kinetics showed that four components are always present but lifetimes (tau) as well as relative fluorescence quantum yields (rfqy) of the two long-lived components (tau3 and tau4) are modified by the presence of zeaxanthin. The most relevant changes are observed in the rfqy of tau3 and in the average lifetime ( approximately 2.4 ns with zeaxanthin and 3.2-3.4 ns in the control proteins). When studied in vitro, no significant effect of acidic pH (5.2-5.3) is observed on chlorophyll A fluorescence yield or kinetics. The data presented show that recombinant CP29 is able to bind zeaxanthin and this protein-bound zeaxanthin induces a significant quenching effect.     

Minor complexes at work: light-harvesting by carotenoids in the photosystem II antenna complexes CP24 and CP26

Plant photosynthesis relies on the capacity of chlorophylls and carotenoids to absorb light. One of the roles of carotenoids is to harvest green-blue light and transfer the excitation energy to the chlorophylls. The corresponding dynamics were investigated here for the first time, to our knowledge, in the CP26 and CP24 minor antenna complexes. The results for the two complexes differ substantially. In CP26 fast transfer (80 fs) occurs from the carotenoid S₂ state to chlorophylls a absorbing at 675 and 678 nm, whereas transfer from the hot S₁ state to the lowest energy chlorophylls is observed in <1 ps. In CP24, energy transfer from the S₂ state leads in 80 fs to the population of chlorophylls b and high-energy chlorophylls a absorbing at 670 nm, whereas the low-energy chlorophylls a are populated only in several picoseconds. The results suggest that CP26 has a structural and functional organization similar to that of LHCII, whereas CP24 differs substantially from the other Lhc complexes, especially regarding the lutein L1 binding domain. No energy transfer from the carotenoid S₁ state to chlorophylls was observed in either complex, suggesting that this state is energetically below the chlorophyll Qy state and therefore may play a role in the quenching of chlorophyll excitations.     

Energy transfer pathways in the CP24 and CP26 antenna complexes of higher plant photosystem II: a comparative study

Antenna complexes are key components of plant photosynthesis, the process that converts sunlight, CO₂, and water into oxygen and sugars. We report the first (to our knowledge) femtosecond transient absorption study on the light-harvesting pigment-protein complexes CP26 (Lhcb5) and CP24 (Lhcb6) of Photosystem II. The complexes are excited at three different wavelengths in the chlorophyll (Chl) Qy region. Both complexes show a single subpicosecond Chl b to Chl a transfer process. In addition, a reduction in the population of the intermediate states (in the 660-670 nm range) as compared to light-harvesting complex II is correlated in CP26 to the absence of both Chls a604 and b605. However, Chl forms around 670 nm are still present in the Chl a Qy range, which undergoes relaxation with slow rates (10-15 ps). This reduction in intermediate-state amplitude CP24 shows a distinctive narrow band at 670 nm connected with Chls b and decaying to the low-energy Chl a states in 3-5 ps. This 670 nm band, which is fully populated in 0.6 ps together with the Chl a low-energy states, is proposed to originate from Chl 602 or 603. In this study, we monitored the energy flow within two minor complexes, and our results may help elucidate these structures in the future.     

Evidence that the PsbK polypeptide is associated with the photosystem II core antenna complex CP43

PsbK is encoded by the chloroplast psbK gene and is one of the small polypeptides of photosystem II (PSII). This polypeptide is required for accumulation of the PSII complex. In the present study, we generated an antibody against recombinant mature PsbK of Chlamydomonas and used it in Western blots to localize PsbK in the PSII core complex. PsbK was found in the thylakoid membranes, and purification of the PSII core complex from detergent-solubilized thylakoid membranes showed that PsbK is tightly associated with the PSII core complex. We used potassium thiocyanate to separate PSII into subcore complexes, including the D1/D2/cytochrome b559 reaction center complex, CP47, and CP43, and we found that PsbK co-purifies with one of the core antenna complexes, CP43, during ion exchange chromatography. Subsequent gel filtration chromatography of the purified CP43 confirmed that PsbK is tightly associated with CP43. Steady-state levels of PsbK were also determined in Chlamydomonas mutants expressing various levels of PSII. Quantitative Western blotting revealed that the levels of PsbK in these mutants are approximately equal to those of CP43, suggesting that PsbK is stable only when associated with CP43 in the chloroplast. Together, our results indicate that PsbK is an integral part of the PSII complex and may participate in the assembly and stability of the PSII complex.     

Spectroscopic properties of the CP43 core antenna protein of photosystem II

CP43 is a chlorophyll-protein complex that funnels excitation energy from the main light-harvesting system of photosystem II to the photochemical reaction center. We purified CP43 from spinach photosystem II membranes in the presence of the nonionic detergent n-dodecyl-beta,D-maltoside and recorded its spectroscopic properties at various temperatures between 4 and 293 K by a number of polarized absorption and fluorescence techniques, fluorescence line narrowing, and Stark spectroscopy. The results indicate two "red" states in the Q(y) absorption region of the chlorophylls. The first peaks at 682.5 nm at 4 K, has an extremely narrow bandwidth with a full width at half-maximum of approximately 2.7 nm (58 cm(-1)) at 4 K, and has the oscillator strength of a single chlorophyll. The second peaks at approximately 679 nm, has a much broader bandshape, is caused by several excitonically interacting chlorophylls, and is responsible for all 4 K absorption at wavelengths longer than 685 nm. The Stark spectrum of CP43 resembles the first derivative of the absorption spectrum and has an exceptionally small overall size, which we attribute to opposing orientations of the monomer dipole moments of the excitonically coupled pigments.     

Photoprotection in the antenna complexes of photosystem II: role of individual xanthophylls in chlorophyll triplet quenching

In this work the photoprotective role of all xanthophylls in LHCII, Lhcb4, and Lhcb5 is investigated by laser-induced Triplet-minus-Singlet (TmS) spectroscopy. The comparison of native LHCII trimeric complexes with different carotenoid composition shows that the xanthophylls in sites V1 and N1 do not directly contribute to the chlorophyll triplet quenching. The largest part of the triplets is quenched by the lutein bound in site L1, which is located in close proximity to the chlorophylls responsible for the low energy state of the complex. The lutein in the L2 site is also active in triplet quenching, and it shows a longer triplet lifetime than the lutein in the L1 site. This lifetime difference depends on the occupancy of the N1 binding site, where neoxanthin acts as an oxygen barrier, limiting the access of O(2) to the inner domain of the Lhc complex, thereby strongly contributing to the photostability. The carotenoid triplet decay of monomeric Lhcb1, Lhcb4, and Lhcb5 is mono-exponential, with shorter lifetimes than observed for trimeric LHCII, suggesting that their inner domains are more accessible for O(2). As for trimeric LHCII, only the xanthophylls in sites L1 and L2 are active in triplet quenching. Although the chlorophyll to carotenoid triplet transfer is efficient (95%) in all complexes, it is not perfect, leaving 5% of the chlorophyll triplets unquenched. This effect appears to be intrinsically related to the molecular organization of the Lhcb proteins.     

Minor antenna proteins CP24 and CP26 affect the interactions between photosystem II subunits and the electron transport rate in grana membranes of Arabidopsis

We investigated the function of chlorophyll a/b binding antenna proteins Chlorophyll Protein 26 (CP26) and CP24 in light harvesting and regulation of photosynthesis by isolating Arabidopsis thaliana knockout lines that completely lacked one or both of these proteins. All three mutant lines had a decreased efficiency of energy transfer from trimeric light-harvesting complex II (LHCII) to the reaction center of photosystem II (PSII) due to the physical disconnection of LHCII from PSII and formation of PSII reaction center depleted domains in grana partitions. Photosynthesis was affected in plants lacking CP24 but not in plants lacking CP26: the former mutant had decreased electron transport rates, a lower DeltapH gradient across the grana membranes, reduced capacity for nonphotochemical quenching, and limited growth. Furthermore, the PSII particles of these plants were organized in unusual two-dimensional arrays in the grana membranes. Surprisingly, overall electron transport, nonphotochemical quenching, and growth of the double mutant were restored to wild type. Fluorescence induction kinetics and electron transport measurements at selected steps of the photosynthetic chain suggested that limitation in electron transport was due to restricted electron transport between Q(A) and Q(B), which retards plastoquinone diffusion. We conclude that CP24 absence alters PSII organization and consequently limits plastoquinone diffusion.     

Energy transfer processes in the isolated core antenna complexes CP43 and CP47 of photosystem II

The energy equilibration and transfer processes in the isolated core antenna complexes CP43 and CP47 of photosystem II have been studied by steady-state and ultrafast (femto- to nanosecond) time-resolved spectroscopy at room temperature. The annihilation-free femtosecond absorption data can be described by surprisingly simple sequential kinetic models, in which the excitation energy transfer between blue and red states in both antenna complexes is dominated by sub-picosecond processes and is completed in less than 2 ps. The slowest energy transfer steps with lifetimes in the range of 1-2 ps are assigned to transfer steps between the chlorophyll layers located on the stromal and lumenal sides. We conclude that these ultrafast intra-antenna energy transfer steps do not represent a bottleneck in the rate of the primary processes in intact photosystem II. Since the experimental energy equilibration rates are up to a factor of 3-5 higher than concluded previously, our results challenge the conclusions drawn from theoretical modeling.     

Photophysical behavior and assignment of the low-energy chlorophyll states in the CP43 proximal antenna protein of higher plant photosystem II

We have employed absorption, circular dichroism (CD), and persistent spectral hole-burning measurements at 1.7 K to study the photoconversion properties and exciton coupling of low-energy chlorophylls (Chls) in the CP43 proximal antenna light-harvesting subunit of photosystem II (PSII) isolated from spinach. These approximately 683 nm states act as traps for excitation energy in isolated CP43. They "bleach" at 683 nm upon illumination and photoconvert to a form absorbing in the range approximately 660-680 nm. We present new data that show the changes in the CD spectrum due to the photoconversion process. These changes occur in parallel with those in absorption, providing evidence that the feature undergoing the apparent bleach is a component of a weakly exciton-coupled system. From our photoconversion difference spectra, we assign four states in the Chl long-wavelength region of CP43, two of which are the known trap states and are both highly localized on single Chls. The other two states are associated with weak exciton coupling (maximally approximately 50 cm(-)(1)) to one of these traps. We propose a mechanism for photoconversion that involves Chl-protein hydrogen bonding. New hole-burning data are presented that indicate this mechanism is distinct to that for narrow-band spectral hole burning in CP43. We discuss the photophysical behavior of the Chl trap states in isolated CP43 compared to their behavior in intact PSII preparations. The latter represent a more intact, physiological complex, and we find no clear evidence that they exhibit the photoconversion process reported here.     

Triplet and fluorescing states of the CP47 antenna complex of photosystem II studied as a function of temperature.

Fluorescence emission and triplet-minus-singlet (T-S) absorption difference spectra of the CP47 core antenna complex of photosystem II were measured as a function of temperature and compared to those of chlorophyll a in Triton X-100. Two spectral species were found in the chlorophyll T-S spectra of CP47, which may arise from a difference in ligation of the pigments or from an additional hydrogen bond, similar to what has been found for Chl molecules in a variety of solvents. The T-S spectra show that the lowest lying state in CP47 is at approximately 685 nm and gives rise to fluorescence at 690 nm at 4 K. The fluorescence quantum yield is 0.11 +/- 0.03 at 4 K, the chlorophyll triplet yield is 0.16 +/- 0.03. Carotenoid triplets are formed efficiently at 4 K through triplet transfer from chlorophyll with a yield of 0.15 +/- 0.02. The major decay channel of the lowest excited state in CP47 is internal conversion, with a quantum yield of about 0.58. Increase of the temperature results in a broadening and blue shift of the spectra due to the equilibration of the excitation over the antenna pigments. Upon increasing the temperature, a decrease of the fluorescence and triplet yields is observed to, at 270 K, a value of about 55% of the low temperature value. This decrease is significantly larger than of chlorophyll a in Triton X-100. Although the coupling to low-frequency phonon or vibration modes of the pigments is probably intermediate in CP47, the temperature dependence of the triplet and fluorescence quantum yield can be modeled using the energy gap law in the strong coupling limit of Englman and Jortner (1970. J. Mol. Phys. 18:145-164) for non-radiative decays. This yields for CP47 an average frequency of the promoting/accepting modes of 350 cm-1 with an activation energy of 650 cm-1 for internal conversion and activationless intersystem crossing to the triplet state through a promoting mode with a frequency of 180 cm-1. For chlorophyll a in Triton X-100 the average frequency of the promoting modes for non-radiative decay is very similar, but the activation energy (300 cm-1) is significantly smaller.     

Energy dissipation efficiency in the CP43 assembly intermediate complex of photosystem II

Photosystem II in oxygenic organisms is a large membrane bound rapidly turning over pigment protein complex. During its biogenesis, multiple assembly intermediates are formed, including the CP43-preassembly complex (pCP43). To understand the energy transfer dynamics in pCP43, we first engineered a His-tagged version of the CP43 in a CP47-less strain of the cyanobacterium Synechocystis 6803. Isolated pCP43 from this engineered strain was subjected to advanced spectroscopic analysis to evaluate its excitation energy dissipation characteristics. These included measurements of steady-state absorption and fluorescence emission spectra for which correlation was tested with Stepanov relation. Comparison of fluorescence excitation and absorptance spectra determined that efficiency of energy transfer from β-carotene to chlorophyll a is 39 %. Time-resolved fluorescence images of pCP43-bound Chl a were recorded on streak camera, and fluorescence decay dynamics were evaluated with global fitting. These demonstrated that the decay kinetics strongly depends on temperature and buffer used to disperse the protein sample and fluorescence decay lifetime was estimated in 3.2–5.7 ns time range, depending on conditions. The pCP43 complex was also investigated with femtosecond and nanosecond time-resolved absorption spectroscopy upon excitation of Chl a and β-carotene to reveal pathways of singlet excitation relaxation/decay, Chl a triplet dynamics and Chl a → β-carotene triplet state sensitization process. The latter demonstrated that Chl a triplet in the pCP43 complex is not efficiently quenched by carotenoids. Finally, detailed kinetic analysis of the rise of the population of β-carotene triplets determined that the time constant of the carotenoid triplet sensitization is 40 ns.     

Structure-based simulation of linear optical spectra of the CP43 core antenna of photosystem II

The linear optical spectra (absorbance, linear dichroism, circular dichroism, fluorescence) of the CP43 (PsbC) antenna of the photosystem II core complex (PSIIcc) pertaining to the S(0)?→?S(1) (Q(Y)) transitions of the chlorophyll (Chl) a pigments are simulated by applying a combined quantum chemical/electrostatic method to obtain excitonic couplings and local transition energies (site energies) on the basis of the 2.9?? resolution crystal structure (Guskov et al., Nat Struct Mol Biol 16:334-342, 2009). The electrostatic calculations identify three Chls with low site energies (Chls 35, 37, and 45 in the nomenclature of Loll et al. (Nature 438:1040-1044, 2005). A refined simulation of experimental spectra of isolated CP43 suggests a modified set of site energies within 143?cm(-1) of the directly calculated values (root mean square deviation: 80?cm(-1)). In the refined set, energy sinks are at Chls 37, 43, and 45 in agreement with earlier fitting results (Raszewski and Renger, J Am Chem Soc 130:4431-4446, 2008). The present structure-based simulations reveal that a large part of the redshift of Chl 37 is due to a digalactosyldiacylglycerol lipid. This finding suggests a new role for lipids in PSIIcc, namely the tuning of optical spectra and the creation of an excitation energy funnel towards the reaction center. The analysis of electrostatic pigment-protein interactions is used to identify amino acid residues that are of potential interest for an experimental approach to an assignment of site energies and energy sinks by site-directed mutagenesis.     

Photochemical competence of assembled photosystem II core complex in cyanobacterial plasma membrane

Cyanobacterial cells have two autonomous internal membrane systems, plasma membrane and thylakoid membrane. In these oxygenic photosynthetic organisms the assembly of the large membrane protein complex photosystem II (PSII) is an intricate process that requires the recruitment of numerous protein subunits and cofactors involved in excitation and electron transfer processes. Precise control of this assembly process is necessary because electron transfer reactions in partially assembled PSII can lead to oxidative damage and degradation of the protein complex. In this communication we demonstrate that the activation of PSII electron transfer reactions in the cyanobacterium Synechocystis sp. PCC 6803 takes place sequentially. In this organism partially assembled PSII complexes can be detected in the plasma membrane. We have determined that such PSII complexes can undergo light-induced charge separation and contain a functional electron acceptor side but not an assembled donor side. In contrast, PSII complexes in thylakoid membrane are fully assembled and capable of multiple turnovers. We conclude that PSII reaction center cores assembled in the plasma membrane are photochemically competent and can catalyze single turnovers. We propose that upon transfer of such PSII core complexes to the thylakoid membrane, additional proteins are incorporated followed by binding and activation of various donor side cofactors. Such a stepwise process protects cyanobacterial cells from potentially harmful consequences of performing water oxidation in a partially assembled PSII complex before it reaches its final destination in the thylakoid membrane.     

Photosystem II antenna complexes CP26 and CP29 are essential for nonphotochemical quenching in Chlamydomonas reinhardtii

Photosystems must balance between light harvesting to fuel the photosynthetic process for CO₂ fixation and mitigating the risk of photodamage due to absorption of light energy in excess. Eukaryotic photosynthetic organisms evolved an array of pigment-binding proteins called light harvesting complexes constituting the external antenna system in the photosystems, where both light harvesting and activation of photoprotective mechanisms occur. In this work, the balancing role of CP29 and CP26 photosystem II antenna subunits was investigated in Chlamydomonas reinhardtii using CRISPR-Cas9 technology to obtain single and double mutants depleted of monomeric antennas. Absence of CP26 and CP29 impaired both photosynthetic efficiency and photoprotection: Excitation energy transfer from external antenna to reaction centre was reduced, and state transitions were completely impaired. Moreover, differently from higher plants, photosystem II monomeric antenna proteins resulted to be essential for photoprotective thermal dissipation of excitation energy by nonphotochemical quenching.     

A specific binding site for neoxanthin in the monomeric antenna proteins CP26 and CP29 of Photosystem II

The location of the neoxanthin binding site in CP26 and CP29 was investigated by site-directed mutagenesis. The crystallographic structure of LHCII shows that the binding of neoxanthin to the N1 site is stabilised by an H bond with a tyrosine in the lumenal loop. This residue is conserved in CP26 and CP29. Mutation of this tyrosine into phenylalanine induced specific loss of neoxanthin without affecting violaxanthin binding. In contrast to previous proposals, it is thus concluded that also in these minor antenna complexes neoxanthin is accommodated in the N1 site. The characteristics of this binding site in the different antenna complexes are discussed.     

Identifying the lowest electronic states of the chlorophylls in the CP47 core antenna protein of photosystem II

CP47 is a pigment-protein complex in the core of photosystem II that tranfers excitation energy to the reaction center. Here we report on a spectroscopic investigation of the isolated CP47 complex. By deconvoluting the 77 K absorption and linear dichroism, red-most states at 683 and 690 nm have been identified with oscillator strengths corresponding to approximately 3 and approximately 1 chlorophyll, respectively. Both states contribute to the 4 K emission, and the Stark spectrum shows that they have a large value for the difference polarizability between their ground and excited states. From site-selective polarized triplet-minus-singlet spectra, an excitonic origin for the 683 nm state was found. The red shift of the 690 nm state is most probably due to strong hydrogen bonding to a protein ligand, as follows from the position of the stretch frequency of the chlorophyll 13(1) keto group (1633 cm(-)(1)) in the fluorescence line narrowing spectrum at 4 K upon red-most excitation. We discuss how the 683 and 690 nm states may be linked to specific chlorophylls in the crystal structure [Zouni, A., Witt, H.-T., Kern, J., Fromme, P., Krauss, N., Saenger, W., and Orth, P. (2001) Nature 409, 739-743].     

Spectral substructure and excitonic interactions in the minor photosystem II antenna complex CP29 as revealed by nonlinear polarization spectroscopy in the frequency domain

CP29 (the lhcb4 gene product), a minor photosystem II antenna complex, binds six chlorophyll (Chl) a, two Chl b, and two to three xanthophyll molecules. The Chl a/b Q(y) absorption band substructure of CP29 (purified from spinach) was investigated by nonlinear polarization spectroscopy in the frequency domain (NLPF) at room temperature. A set of NLPF spectra was obtained at 11 probe wavelengths. Seven probe wavelengths were located in the Q(y) spectral region (between 630 and 690 nm) and four in the Soret band (between 450 and 485 nm). Evaluation of the experimental data within the framework of global analysis leads to the following conclusions: (i) The dominant Chl a absorption (with a maximum at 674 nm) splits into (at least) three subbands (centered at 660, 670, and 681.5 nm). (ii) In the Chl b region two subbands can be identified with maxima located at 640 and 646 nm. (iii) The lowest energy Q(y) transition (peaking at 681.5 nm) is assigned to a Chl a which only weakly interacts with other Chl aor b molecules by incoherent F?rster-type excitation energy transfer. (iv) Pronounced excitonic interaction exists between certain Chl a and Chl b molecules, which most likely form a Chl a/b heterodimer. The subbands centered at 640 and 670 nm constitute a strongly coupled Chl a/b pair. The findings of the study indicate that the currently favored view of spectral heterogeneity in CP29 being due essentially to pigment-protein interactions has to be revised.