Plant Photosystem Research Articles

Energetics of primary and secondary electron transfer in Photosystem II membrane particles of spinach revisited on basis of recombination-fluorescence measurements

Photon absorption by one of the roughly 200 chlorophylls of the plant Photosystem II (PSII) results in formation of an equilibrated excited state (Chl200*) and is followed by chlorophyll oxidation (formation of P680+) coupled to reduction of a specific pheophytin (Phe), then electron transfer from Phe− to a firmly bound quinone (QA), and subsequently reduction of P680+ by a redox-active tyrosine residue denoted as Z. The involved free-energy differences (Δ G) and redox potentials are of prime interest. Oxygen-evolving PSII membrane particles of spinach were studied at 5 °C. By analyzing the delayed and prompt Chl fluorescence, we determined the equilibrium constant and thus free-energy difference between Chl200* and the [Z+,QA−] radical pair to be −0.43 ± 0.025 eV, at 10 μs after the photon absorption event for PSII in its S 3-state. On basis of this value and previously published results, the free-energy difference between P680* and [P680+,QA−] is calculated to be −0.50 ± 0.04 eV; the free-energy loss associated with electron transfer from Phe to QA is found to be 0.34 ± 0.04 eV. The given uncertainty ranges do not represent a standard deviation or likely error, but an estimate of the maximal error. Assuming a QA−/QA redox potential of −0.08 V [Krieger et al., 1995, Biochim. Biophys. Acta 1229, 193], the following redox-potential estimates are obtained: +1.25 V for P680/P680+; +1.21 V for Z/Z+ (at 10 μs); −0.42 V for Phe−/Phe; −0.58 V for P680*/P680+.

The low energy emitting states of the Lhca4 subunit of higher plant photosystem I

The selectively red excited emission spectrum, at room temperature, of the in vitro reconstituted Lhca4, has a pronounced non-equilibrium distribution, leading to enhanced emission from the directly excited low-energy pigments. Two different emitting forms (or states), with maximal emission at 713 and 735 nm (F713 and F735) and unusual spectral properties, have been identified. Both high-energy states are populated when selective excitation is into the F735 state and the fluorescence anisotropy spectrum attains the value of 0.3 in the wavelength region where both emission states are present. This indicates that the two states are on the same Lhca4 complex and have transition dipoles with similar orientation.

Size Determination of Cyanobacterial and Higher Plant Photosystem II by Gel Permeation Chromatography, Light Scattering, and Ultracentrifugation

The oxygen-evolving photosystem II core complexes (PSIIcc) from the thermophilic cyanobacterium Thermosynechococcus elongatus (PSIIccTe) and the higher plant Spinacia oleracea (PSIIccSo) have been isolated from the thylakoid membrane by solubilization with n-dodecyl-beta-d-maltoside, purified and characterized by gel permeation chromatography (GPC), dynamic light scattering (DLS), and analytical ultracentrifugation (AUC). DLS suggests that PSIIcc from both organisms exists as a monomer in dilute solution and aggregates with increasing protein concentration. In contrast to DLS, GPC and AUC showed that PSIIcc of both organisms occur as monomers and dimers, and it became clear from our studies that calibration of GPC columns with soluble proteins leads to wrong estimates of the molecular masses of membrane proteins. At a PSIIcc protein concentration of 0.2 mg/mL, molar masses, M, of 756 +/- 18 kDa and 710 +/- 15 kDa for dimeric PSIIccTe and PSIIccSo, respectively, were determined by analytical ultracentrifugation. At very low protein concentrations, at or below 0.05 mg/mL, the dimeric form of PSIIccTe partially dissociates (20-30%) to form monomers. On the basis of these studies 3-dimensional crystals of PSIIccTe were obtained that contain dimers in the asymmetric unit [Zouni, A. et al. (2001) Nature 409, 739-743]. Using synchrotron radiation the crystals diffract to a resolution of 3.8 A, which has been improved recently to 3.2 A [Biesiadka, J., et al. (2004) Phys. Chem. Chem. Phys. 6, 4733-4736].

Photochemical activities of plant photosystem I particles reconstituted into phosphatidylglycerol liposomes

Phosphatidylglycerol (PG) is the only anionic phospholipid in photosynthetic membrane and the important component of photosystem I (PSI). In this study, the interaction of PG with PSI particle from spinach was investigated by using reconstitution method. The results from the properties of electron transport, fluorescence emission, turbidity, and protein secondary structures in PSI complex incorporated into PG liposomes revealed the existence of PSI–PG interactions. A stimulation and an inhibition of oxygen uptake in PSI particle at a low and higher PG/chlorophyll mass ratio, respectively, were observed. Moreover, an additional enhancement and depression of electron flow in the PSI–PG complexes were occurred in the reaction medium containing CaCl 2 at concentrations below and above 5 mM, the aggregation threshold of the reconstituted membranes, respectively. The results demonstrated that the maintenance of the structural optimization was needed for a stimulation of electron transport at a low PG/PSI mass ratio, while a decay of this PSI activity at high PG/PSI ratio was the result of inhibition of the energy transfer from LHCI to PSI reaction center induced by the dissociation of LHCI-680.

Stoichiometry of LHCI antenna polypeptides and characterization of gap and linker pigments in higher plants Photosystem I

We report on the results obtained by measuring the stoichiometry of antenna polypeptides in Photosystem I (PSI) from Arabidopsis thaliana. This analysis was performed by quantification of Coomassie blue binding to individual LHCI polypeptides, fractionation by SDS/PAGE, and by the use of recombinant light harvesting complex of Photosystem I (Lhca) holoproteins as a standard reference. Our results show that a single copy of each Lhca1-4 polypeptide is present in Photosystem I. This is in agreement with the recent structural data on PSI-LHCI complex [Ben Shem, A., Frolow, F. and Nelson, N. (2003) Nature, 426, 630-635]. The discrepancy from earlier estimations based on pigment binding and yielding two copies of each LHCI polypeptide per PSI, is explained by the presence of 'gap' and 'linker' chlorophylls bound at the interface between PSI core and LHCI. We showed that these chlorophylls are lost when LHCI is detached from the PSI core moiety by detergent treatment and that gap and linker chlorophylls are both Chl a and Chl b. Carotenoid molecules are also found at this interface between LHCI and PSI core. Similar experiments, performed on PSII supercomplexes, showed that dissociation into individual pigment-proteins did not produce a significant loss of pigments, suggesting that gap and linker chlorophylls are a peculiar feature of Photosystem I.

Structural Stability of the PsbQ Protein of Higher Plant Photosystem II

We have characterized the stability and folding behavior of the isolated extrinsic PsbQ protein of photosystem II (PSII) from a higher plant, Spinacia oleracea, using intrinsic protein fluorescence emission and near- and far-UV circular dichroism (CD) spectroscopy in combination with differential scanning calorimetry (DSC). Experimental results reveal that both chemical denaturation using guanidine hydrochloride (GdnHCl) and thermal unfolding of PsbQ proceed as a two-state reversible process. The denaturation free-energy changes (DeltaG(D)) at 20 degrees C extrapolated from GdnHCl (4.0 +/- 0.6 kcal mol(-1)) or thermal unfolding (4.4 +/- 0.8 kcal mol(-1)) are very close. Moreover, the far-UV CD spectra of the denatured PsbQ registered at 90 degrees C in the absence and presence of 6.0 M GdnHCl superimpose, leading us to conclude that both denatured states of PsbQ are structurally and energetically similar. The thermal unfolding of PsbQ has been also characterized by CD and DSC over a wide pH range. The stability of PsbQ is at its maximum at pH comprised between 5 and 8, being wider than the optimal pH for oxygen evolution in the lumen of thylakoid membranes. In addition, no significant structural changes were detected in PsbQ between 50 and 55 degrees C in the pH range of 3-8, suggesting that PsbQ behaves as a soluble and stable particle in the lumen when it detaches from PSII under physiological stress conditions such as high temperature (45-50 degrees C) or low pH (<5.0). Sedimentation experiments showed that, in solution at 20 degrees C, the PsbQ protein is a monomer with an elongated shape.

The Long-Wavelength Chlorophyll States of Plant LHCI at Room Temperature: A Comparison with PSI-LHCI

The red antenna states of the external antenna complexes of higher plant photosystem I, known as LHCI, have been analyzed by measurement of their preequilibrium fluorescence upon direct excitation at 280 K. In addition to the previously detected F735 state, a hitherto undetected low-energy state with emission maximum around 713 nm was observed. The 280 K bandwidths (FWHM) are 55 nm for the F735 state and ∼27 nm for the F713-nm state, much greater than for non-red-shifted antenna chlorophylls. The origin absorption band for the F735-nm state was directly detected by determination of its excitation (action) spectrum and lies at 709–710 nm. The absorption spectrum for F735, calculated using the Stepanov expression, closely overlaps the excitation spectrum, indicating that the very large Stokes shift (25 nm) is due to vibrational relaxation within the excited-state manifold and solvent effects can be excluded. Fluorescence anisotropy measurements, with direct excitation of F735, indicate that the transition dipoles of the two red states are parallel. Similar experiments performed in the long-wavelength absorbing tail of PSI-LHCI indicate the presence of emission state(s) that are red-shifted with respect to F735 of isolated LHCI. It is suggested that these are brought about by interactions between the complexes in PSI-LHCI, which occur in some yet undefined way, and which are broken upon solubilization of the component parts.

Proteomic approach to characterize the supramolecular organization of photosystems in higher plants

A project to investigate the supramolecular structure of photosystems was initiated, which is based on protein solubilizations by digitonin, protein separations by Blue native (BN)–polyacrylamide gel electrophoresis (PAGE) and protein identifications by mass spectrometry (MS). Under the conditions applied, nine photosystem supercomplexes could be described for chloroplasts of Arabidopsis, which have apparent molecular masses between 600 and 3200 kDa on BN gels. Identities of the supercomplexes were determined on the basis of their subunit compositions as documented by 2D BN/SDS–PAGE and BN/BN–PAGE. Two supercomplexes of 1060 and ∼1600 kDa represent dimeric and trimeric forms of photosystem I (PSI), which include tightly bound LHCI proteins. Compared to monomeric PSI, these protein complexes are of low abundance. In contrast, photosystem II mainly forms part of dominant supercomplexes of 850, 1000, 1050 and 1300 kDa. According to our interpretation, these supercomplexes contain dimeric PSII, 1–4 LHCII trimers and additionally monomeric LHCII proteins. The 1300-kDa PSII supercomplex (containing four LHCII trimers) is partially converted into the 1000-kDa PSII supercomplex (containing two LHCII trimers) in the presence of dodecylmaltoside on 2D BN/BN gels. Analyses of peptides of the trypsinated 1300-kDa PSII supercomplex by mass spectrometry allowed to identify known subunits of the PSII core complex and additionally LHCII proteins encoded by eight different genes in Arabidopsis. Further application of this experimental approach will allow new insights into the supermolecular organization of photosystems in plants.

Proteomic approach to characterize the supramolecular organization of photosystems in higher plants

Using Blue-native polyacrylamide gel electrophoresis of digitonin solubilized chloroplast fractions from Arabidopsis, nine protein supercomplexes containing either photosystem I or II were resolved in a molecular mass range between 600 and 3200 kDa. A strategy for systematic subunit identifications of supercomplexes is suggested, which employs direct trypsination of complexes and afterwards peptide identifications by mass spectrometry. Based on this approach, eight different proteins of the light harvesting complex II family are shown to form part of a 1300 kDa photosystem II supercomplex. A project to investigate the supramolecular structure of photosystems was initiated, which is based on protein solubilizations by digitonin, protein separations by Blue native (BN)–polyacrylamide gel electrophoresis (PAGE) and protein identifications by mass spectrometry (MS). Under the conditions applied, nine photosystem supercomplexes could be described for chloroplasts of Arabidopsis , which have apparent molecular masses between 600 and 3200 kDa on BN gels. Identities of the supercomplexes were determined on the basis of their subunit compositions as documented by 2D BN/SDS–PAGE and BN/BN–PAGE. Two supercomplexes of 1060 and ∼1600 kDa represent dimeric and trimeric forms of photosystem I (PSI), which include tightly bound LHCI proteins. Compared to monomeric PSI, these protein complexes are of low abundance. In contrast, photosystem II mainly forms part of dominant supercomplexes of 850, 1000, 1050 and 1300 kDa. According to our interpretation, these supercomplexes contain dimeric PSII, 1–4 LHCII trimers and additionally monomeric LHCII proteins. The 1300-kDa PSII supercomplex (containing four LHCII trimers) is partially converted into the 1000-kDa PSII supercomplex (containing two LHCII trimers) in the presence of dodecylmaltoside on 2D BN/BN gels. Analyses of peptides of the trypsinated 1300-kDa PSII supercomplex by mass spectrometry allowed to identify known subunits of the PSII core complex and additionally LHCII proteins encoded by eight different genes in Arabidopsis . Further application of this experimental approach will allow new insights into the supermolecular organization of photosystems in plants.

Magnetic-Field-Induced Orientation of Photosynthetic Reaction Centers, As Revealed by Time-Resolved D-Band Electron Paramagnetic Resonance of Spin-Correlated Radical Pairs. II. Field Dependence of the Alignment

The magnetic-field-induced orientation of photosynthetic reaction centers has been studied by time-resolved D-band (130 GHz) electron paramagnetic resonance (EPR) of the secondary radical pair, , in plant photosystem I (PSI). Experiments have been performed for fresh and lyophilized whole cells of the deuterated cyanobacterium S. lividus. A computer fit of the angle-dependent D-band spectra, measured for two different sample orientations, provides the order parameter SZ‘Z‘ of the symmetry axis, Z‘, of the susceptibility tensor, relative to the magnetic field. The positive sign of this order parameter indicates that membrane proteins are the major source for the anisotropy of the diamagnetic susceptibility, ΔχV. A value for ΔχV has been extracted from the magnetic-field dependence of SZ‘Z‘. The value of ΔχV = 5.7 × 10-27 m3 is in good agreement with an estimate for the susceptibility anisotropy of a cyanobacterial cell. This demonstrates that whole cells are aligned in the magneto-orientation process. The combination of high-field EPR of a magnetically oriented sample with the analysis of quantum beat oscillations allows determination of the three-dimensional structure of in the photosynthetic membrane.

The PSI-O subunit of plant photosystem I is involved in balancing the excitation pressure between the two photosystems.

PSI-O is a subunit of photosystem I in eukaryotes. The function of PSI-O was characterized in Arabidopsis plants using RNA interference. Several transformants with the psaO-RNAi construct were generated, and a high proportion of the plants contained only very little or virtually no residual PSI-O. Plants lacking PSI-O have a 50% reduction in state transitions indicating a role for PSI-O in the balancing of excitation energy between the two photosystems. PSI-H and -L have been shown previously to be involved in state transitions, and immunoblot analysis revealed that plants devoid of PSI-L or -H also have 80-90% reduction in the abundance of PSI-O. In contrast, down-regulation of PSI-O has no negative effect on the content of PSI-H and -L. The interaction between PSI-O and the PSI-L was confirmed by chemical cross-linking. A model of PSI is proposed in which PSI-L as the most ancient subunit is closest to the reaction center, and PSI-O is positioned close to PSI-L on the PSI-H/-L/-I side of the PSI complex. PSI-H, -L, -O, and possibly -I are all involved in forming a domain in PSI that is involved in the interaction with light-harvesting complex II.

Lhca5--an LHC-type protein associated with photosystem I.

The light-harvesting antenna of higher plant photosystem (PS) I is known to be composed of four different types of light-harvesting complex (LHC) proteins (Lhca1-4). However, the genomic sequence of Arabidopsis thaliana contains open reading frames coding for two additional LHC type proteins (Lhca5-6) that are presumably associated with PSI. While Lhca6 might not be expressed at all, ESTs have been detected for the Lhca5 gene in Arabidopsis and a number of other plant species. Here we demonstrate the presence of the Lhca5 gene product in the thylakoid membrane of Arabidopsis as an additional type of Lhca-protein associated with PSI. Lhca5 seems to be regulated differently from the other LHC proteins since Lhca5 mRNA levels increase under high light conditions. Analyses reported here of Lhca5 in plants lacking individual Lhca1-4 proteins show that it is more abundant in plants lacking Lhca1/4, and suggest that it interacts in a direct physical fashion with Lhca2 or Lhca3. We propose that Lhca5 binds chlorophylls in a similar fashion to the other Lhca proteins and is associated with PSI only in sub-stoichiometric amounts.

Light-Harvesting Features Revealed by the Structure of Plant Photosystem I

Oxygenic photosynthesis is driven by two multi-subunit membrane protein complexes, Photosystem I and Photosystem II. In plants and green algae, both complexes are composed of two moieties: a reaction center (RC), where light-induced charge translocation occurs, and a peripheral antenna that absorbs light and funnels its energy to the reaction center. The peripheral antenna of PS I (LHC I) is composed of four gene products (Lhca 1-4) that are unique among the chlorophyll a/b binding proteins in their pronounced long-wavelength absorbance and in their assembly into dimers. The recently determined structure of plant Photosystem I provides the first relatively high-resolution structural model of a super-complex containing a reaction center and its peripheral antenna. We describe some of the structural features responsible for the unique properties of LHC I and discuss the advantages of the particular LHC I dimerization mode over monomeric or trimeric forms. In addition, we delineate some of the interactions between the peripheral antenna and the reaction center and discuss how they serve the purpose of dynamically altering the composition of LHC I in response to environmental pressure. Combining structural insight with spectroscopic data, we propose how altering LHC I composition may protect PS I from excessive light.

Crystal structure of plant photosystem I.

Oxygenic photosynthesis is the principal producer of both oxygen and organic matter on Earth. The conversion of sunlight into chemical energy is driven by two multisubunit membrane protein complexes named photosystem I and II. We determined the crystal structure of the complete photosystem I (PSI) from a higher plant (Pisum sativum var. alaska) to 4.4 A resolution. Its intricate structure shows 12 core subunits, 4 different light-harvesting membrane proteins (LHCI) assembled in a half-moon shape on one side of the core, 45 transmembrane helices, 167 chlorophylls, 3 Fe-S clusters and 2 phylloquinones. About 20 chlorophylls are positioned in strategic locations in the cleft between LHCI and the core. This structure provides a framework for exploration not only of energy and electron transfer but also of the evolutionary forces that shaped the photosynthetic apparatus of terrestrial plants after the divergence of chloroplasts from marine cyanobacteria one billion years ago.

Excited State Trapping and the Stepanov Relation with Reference to Photosystem I

It has been previously demonstrated that the Stepanov equation provides a rather good description of the absorption/fluorescence spectra in Photosystem I, even though excited state equilibration is not rapid with respect to the excited state decay. In the present article this apparent contradiction is examined analytically for two-state systems and numerically for many-state systems. It is demonstrated that, in the special case of the trapping process being associated with the initially populated state, neither very rapid excited state equilibration nor a transfer equilibrium, which approximates a true Boltzmann distribution, are prerequisites to obtaining a very close approximation to a correct Stepanov result. This interesting conclusion is discussed in terms of plant Photosystem I (PSI-200). It is concluded that whereas, in compartmental modeling, photochemical trapping may be formally associated with the bulk antenna pigments due to the strong energy coupling between them and the trap pigments, this is not the case for the red spectral forms.

Crystallization and initial X-ray diffraction studies of higher plant photosystem I.

Complete photosystem I from a higher plant (pea; Pisum sativum) was isolated, purified and crystallized. The crystals diffract to 4 A resolution using synchrotron radiation and belong to the monoclinic crystal system, space group P2(1), with unit-cell parameters a = 181.90, b = 190.24, c = 219.66 A, beta = 90.484 degrees. Data sets from three crystals have been collected and were merged into a 4.4 A resolution data set, taking advantage of the high isomorphicity observed for these crystals. Analysis of the self-rotation Patterson function suggests the possible presence of two PSI complexes in the asymmetric unit.

The single tryptophan of the PsbQ protein of photosystem II is at the end of a 4-alpha-helical bundle domain.

We examined the microenvironment of the single tryptophan and the tyrosine residues of PsbQ, one of the three main extrinsic proteins of green algal and higher plant photosystem II. On the basis of this information and the previous data on secondary structure [Balsera, M., Arellano, J.B., Gutiérrez, J.R., Heredia, P., Revuelta, J.L. & De Las Rivas, J. (2003) Biochemistry42, 1000-1007], we screened structural models derived by combining various threading approaches. Experimental results showed that the tryptophan residue is partially buried in the core of the protein but still in a polar environment, according to the intrinsic fluorescence emission of PsbQ and the fact that fluorescence quenching by iodide was weaker than that by acrylamide. Furthermore, quenching by cesium suggested that a positively charged barrier shields the tryptophan microenvironment. Comparison of the absorption spectra in native and denaturing conditions indicated that one or two out of six tyrosines of PsbQ are buried in the core of the structure. Using threading methods, a 3D structural model was built for the C-terminal domain of the PsbQ protein family (residues 46-149), while the N-terminal domain is predicted to have a flexible structure. The model for the C-terminal domain is based on the 3D structure of cytochrome b562, a mainly alpha-protein with a helical up/down bundle folding. Despite the large sequence differences between the template and PsbQ, the structural and energetic parameters for the explicit model are acceptable, as judged by the corresponding tools. This 3D model is compatible with the experimentally determined environment of the tryptophan residue and with published structural information. The future experimental determination of the 3D structure of the protein will offer a good validation point for our model and the technology used. Until then, the model can provide a starting point for further studies on the function of PsbQ.

EPR at 24 T of the primary donor radical cation from Blastochloris viridis

The g-matrix of photosynthetic pigments has been studied in the last decade due to the advent of high-field EPR techniques. It can be observed when the spectral splitting of the principal g-factor components is larger than the linewidth due to unresolved hyperfine splitting and if there is no g-strain obscuring it. For large organic molecules such as the primary electron donor in photosynthetic reaction centers (RC) this usually requires fields above 11 T, or, for fields between 3 and 11 T, full deuteration and/or single crystal work. Here we present for the first time the fully resolved rhombic EPR spectrum of the primary donor of Blastochloris viridis (formerly called Rhodopseudomonas viridis), a purple photosynthetic bacterium containing bacteriochlorophyll b. As was the case for Rhodobacter sphaeroides, g-strain is negligible for this radical up to a field of 24 T. The temperature dependence of the g-anisotropy is presented and compared with that of the bacteriochlorophyll a-containing Rb. sphaeroides and plant photosystem I. A slight shift in the principal components of the g-matrix is observed at temperatures below 70 K, where it becomes more axial. The experimental work is complemented with theoretical calculations for g using the semi-empirical INDO/S method as implemented in the program ZINDO. The theoretical results generally agree well with the experiment. This indicates that a satisfactory description of the anisotropic g-tensor for radical cations of large planar molecules like the chlorophylls as well as their aggregates, e.g., reaction center primary donor special pairs, is possible with this relatively cheap semi-empirical approach.

Hyperfine structure of the photoexcited triplet state 3P680 in plant PS II reaction centres as determined by pulse ENDOR spectroscopy

The triplet states in plant photosystem II (PS II), 3P680, and from chlorophyll a, 3Chl a, in organic solution have been investigated using pulse ENDOR combined with repetitive laser excitation at cryogenic temperature with the aim to obtain their hyperfine (hf) structure. The large zero field splitting (ZFS) tensor of 3P680 enabled orientation selection via the electron spin resonance (EPR) field setting along the ZFS tensor axes. ENDOR spectra have been obtained for the first time also for the in-plane X- and Y-orientations of the ZFS tensor. This allowed a full determination of the hf-tensors of the three methine protons and one methyl group of 3P680. Based on the orientations of the axes of these hf-tensors, a unique orientation of the axes of the ZFS tensor of 3P680 in the Chl a molecular frame was obtained. These data serve as a structural basis for determining the orientation of 3P680 in the PS II protein complex by EPR on single crystals (see M. Kammel et al. in this issue). The data obtained represent the first complete set of the larger hf-tensors of the triplet state 3P680. They reflect the spin density distribution both in the highest occupied (HOMO) and lowest unoccupied (LUMO) orbitals. The data clearly confirm that 3P680 is a monomeric Chl a species at low temperature ( T=10 K) used, as has been proposed earlier based on D- and E-values obtained from EPR and optically detected magnetic resonance (ODMR) studies. Comparison with the hf data for the cation and anion radicals of Chl a indicates a redistribution of spin densities in particular for the LUMO orbital of the triplet states. The electron spin distribution in the LUMO orbital is of special interest since it harbours the excited electron in the excited P680 singlet state, from which light-induced electron transfer proceeds. Observed shifts of hf couplings from individual nuclei of 3P680 as compared with 3Chl a in organic solution are of special interest, since they indicate specific protein interactions, e.g. hydrogen bonding, which might be used in future studies for assigning 3P680 to a particular chlorophyll molecule in PS II.

Xanthophyll Binding Sites of the CP29 (Lhcb4) Subunit of Higher Plant Photosystem II Investigated by Domain Swapping and Mutation Analysis

The binding sites for xanthophylls in the CP29 antenna protein of higher plant Photosystem II have been investigated using recombinant proteins refolded in vitro. Despite the presence of three xanthophyll species CP29 binds two carotenoids per polypeptide. The localization of neoxanthin was studied producing a chimeric protein constructed by swapping the C-helix domain from CP29 to LHCII. The resulting holoprotein did not bind neoxanthin, confirming that the N1 site is not present in CP29. Neoxanthin in CP29 was, instead, bound to the L2 site, which is thus shown to have a wider specificity with respect to the homologous site L2 in LHCII. Lutein was found in the L1 site of CP29. For each site the selectivity for individual xanthophyll species was studied as well as its role in protein stabilization, energy transfer, and photoprotection. Putative xanthophyll binding sequences, identified by primary structure analysis as a stretch of hydrophobic residues including an acidic term, were analyzed by site-directed mutagenesis or, in one case, by deleting the entire sequence. The mutant proteins were unaffected in their xanthophyll composition, thus suggesting that the target motifs had little influence in determining xanthophyll binding, whereas hydrophobic sequences in the membrane-spanning helices are important.