Excitation-energy transfer dynamics of higher plant photosystem I light-harvesting complexes

Photosystem I (PSI) plays a major role in the light reactions of photosynthesis. In higher plants, PSI is composed of a core complex and four outer antennas that are assembled as two dimers, Lhca1/4 and Lhca2/3. Time-resolved fluorescence measurements on the isolated dimers show very similar kinetics. The intermonomer transfer processes are resolved using target analysis. They occur at rates similar to those observed in transfer to the PSI core, suggesting competition between the two transfer pathways. It appears that each dimer is adopting various conformations that correspond to different lifetimes and emission spectra. A special feature of the Lhca complexes is the presence of an absorption band at low energy, originating from an excitonic state of a chlorophyll dimer, mixed with a charge-transfer state. These low-energy bands have high oscillator strengths and they are superradiant in both Lhca1/4 and Lhca2/3. This challenges the view that the low-energy charge-transfer state always functions as a quencher in plant Lhc's and it also challenges previous interpretations of PSI kinetics. The very similar properties of the low-energy states of both dimers indicate that the organization of the involved chlorophylls should also be similar, in disagreement with the available structural data.     

The PSI-K subunit of photosystem I is involved in the interaction between light-harvesting complex I and the photosystem I reaction center core

PSI-K is a subunit of photosystem I. The function of PSI-K was characterized in Arabidopsis plants transformed with a psaK cDNA in antisense orientation, and several lines without detectable PSI-K protein were identified. Plants without PSI-K have a 19% higher chlorophyll a/b ratio and 19% more P700 than wild-type plants. Thus, plants without PSI-K compensate by making more photosystem I. The photosystem I electron transport in vitro is unaffected in the absence of PSI-K. Light response curves for oxygen evolution indicated that the photosynthetic machinery of PSI-K-deficient plants have less capacity to utilize light energy. Plants without PSI-K have less state 1-state 2 transition. Thus, the redistribution of absorbed excitation energy between the two photosystems is reduced. Low temperature fluorescence emission spectra revealed a 2-nm blue shift in the long wavelength emission in plants lacking PSI-K. Furthermore, thylakoids and isolated PSI without PSI-K had 20-30% less Lhca2 and 30-40% less Lhca3, whereas Lhca1 and Lhca4 were unaffected. During electrophoresis under mildly denaturing conditions, all four Lhca subunits were partially dissociated from photosystem I lacking PSI-K. The observed effects demonstrate that PSI-K has a role in organizing the peripheral light-harvesting complexes on the core antenna of photosystem I.     

Pigment binding of photosystem I light-harvesting proteins

Light-harvesting complexes (LHC) of higher plants are composed of at least 10 different proteins. Despite their pronounced amino acid sequence homology, the LHC of photosystem II show differences in pigment binding that are interpreted in terms of partly different functions. By contrast, there is only scarce knowledge about the pigment composition of LHC of photosystem I, and consequently no concept of potentially different functions of the various LHCI exists. For better insight into this issue, we isolated native LHCI-730 and LHCI-680. Pigment analyses revealed that LHCI-730 binds more chlorophyll and violaxanthin than LHCI-680. For the first time all LHCI complexes are now available in their recombinant form; their analysis allowed further dissection of pigment binding by individual LHCI proteins and analysis of pigment requirements for LHCI formation. By these different approaches a correlation between the requirement of a single chlorophyll species for LHC formation and the chlorophyll a/b ratio of LHCs could be detected, and indications regarding occupation of carotenoid-binding sites were obtained. Additionally the reconstitution approach allowed assignment of spectral features observed in native LHCI-680 to its components Lhca2 and Lhca3. It is suggested that excitation energy migrates from chlorophyll(s) fluorescing at 680 (Lhca3) via those fluorescing at 686/702 nm (Lhca2) or 720 nm (Lhca3) to the photosystem I core chlorophylls.     

Immunogold localization of light-harvesting and photosystem I complexes in the thylakoids ofFucus serratus (Phaeophyceae)

Summary The repartition of light-harvesting complex (LHC) and photosystem I (PS I) complex has been examined in isolated plastids ofFucus serratus by immunocytochemical labelling. LHC is distributed equally all along the length of thylakoid membranes, without any special repartition in the appressed membranes of the three associated thylakoids ofFucus. PS I is present on all the thylakoid membranes, but the external membranes of the three associated thylakoids are largely enriched relatively to the inner ones. This specific repartition of PSI on non-appressed membranes can be compared to the localization of PSI on stroma thylakoid membranes of higher plants and green algae. Consequently, although they share some common features with those of higher plants and green algae, the appressions of thylakoids in brown algae has neither the same structure nor the same functional role as typical grana stacked membranes in the repartition of the harvested energy.Abbreviations BSA bovine serum albumin - GAR goat anti-rabbit immunoglobulin G - LHC light-harvesting complex - PBS phosphatebuffered saline - PS I photosystem I - PS II photosystem II     

Supramolecular organization of photosystem I and light-harvesting complex I in Chlamydomonas reinhardtii

We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of photosystem I and light-harvesting complex I from the unicellular green alga Chlamydomonas reinhardtii. The complex is a monomer, has longest dimensions of 21.3 and 18.2 nm in projection, and is significantly larger than the corresponding complex in spinach. Comparison with photosystem I complexes from other organisms suggests that the complex contains about 14 light-harvesting proteins, two or three of which bind at the side of the PSI-H subunit. We suggest that special light-harvesting I proteins play a role in the binding of phosphorylated light-harvesting complex II in state 2.     

Phosphorylation of the light-harvesting chlorophyll-protein regulates excitation energy distribution between photosystem II and photosystem I

The kinetics of thylakoid membrane protein phosphorylation in the presence of light and adenosine triphosphate is correlated to an incease in the 77 °K fluorescence emission at 735 nm (F735) relative to that at 685 nm (F685). Analysis of detergent-derived submembrane fractions indicate phosphorylation only of the polypeptides of Photosystem II, and the light-harvesting chlorophyll-protein complex serving Photosystem II (LHC-II). Although several polypeptides are phosphorylated, only the dephosphorylation kinetics of LHC-II follow the kinetics of the decrease of the $\text{F735}\text{F685}$ fluorescence emission ratios. The relative quantum yield of Photosystem II was significantly lower in phosphorylated membranes compared to dephosphorylated membranes. Reversible LHC-II phosphorylation thus provides the physiological mechanism for the control of the distribution of absorbed excitation energy between the two photosystems.     

Comparison of the light-harvesting networks of plant and cyanobacterial photosystem I

With the availability of structural models for photosystem I (PSI) in cyanobacteria and plants it is possible to compare the excitation transfer networks in this ubiquitous photosystem from two domains of life separated by over one billion years of divergent evolution, thus providing an insight into the physical constraints that shape the networks' evolution. Structure-based modeling methods are used to examine the excitation transfer kinetics of the plant PSI-LHCI supercomplex. For this purpose an effective Hamiltonian is constructed that combines an existing cyanobacterial model for structurally conserved chlorophylls with spectral information for chlorophylls in the Lhca subunits. The plant PSI excitation migration network thus characterized is compared to its cyanobacterial counterpart investigated earlier. In agreement with observations, an average excitation transfer lifetime of approximately 49 ps is computed for the plant PSI-LHCI supercomplex with a corresponding quantum yield of 95%. The sensitivity of the results to chlorophyll site energy assignments is discussed. Lhca subunits are efficiently coupled to the PSI core via gap chlorophylls. In contrast to the chlorophylls in the vicinity of the reaction center, previously shown to optimize the quantum yield of the excitation transfer process, the orientational ordering of peripheral chlorophylls does not show such optimality. The finding suggests that after close packing of chlorophylls was achieved, constraints other than efficiency of the overall excitation transfer process precluded further evolution of pigment ordering.     

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

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

Chlorophyll-protein complexes of barley photosystem I

Photosystem I (PSI) preparations with a chlorophyll a/b ratio of 6.0 were isolated from barley thylakoids using two different methods. The high-molecular-mass complex (CP1a) which is resolved by non-denaturing gel electrophoresis had the same properties as a PSI preparation (PSI-200) isolated by Triton X-100 solubilisation of thylakoids followed by sucrose gradient ultracentrifugation. This material had a chlorophyll:P700 ratio of 208:1 and was composed of three different chlorophyll-protein complexes which could be separated from each other by solubilising the PSI preparation in dodecyl maltoside followed by sucrose gradient ultracentrifugation. Approximately half of the chlorophyll, including all the chlorophyll b, was located in two antenna complexes designated LHCI-680 and LHCI-730, which were identified by their characteristic low-temperature fluorescence emission spectra. The rest of the chlorophyll a was associated with the PSI reaction centre, P700 Chla-P1, which fluoresced at 720 nm. Each chlorophyll-protein complex had a unique polypeptide composition and characteristic circular dichroic and absorption spectra. The use of dodecyl maltoside instead of dodecyl sulphate resulted in a less denatured form of LHCI-680, which fluoresced at 690 nm at 77 K. One of the sucrose gradient fractions contained a complex consisting of only LHCI-730 and P700 Chla-P1 which fluoresced at 731 nm, indicating that LHCI-730 is structurally associated with P700 Chla-P1 and quenches its fluorescence. Approximately three-quarters of the light-harvesting antenna chlorophyll was in LHCI-730, but only about one-quarter of the normal complement of LHCI-730 was required to quench the reaction centre. By reducing the amount of Triton relative to the chlorophyll concentration, a PSI preparation (chlorophyll a/b ratio of 3.5) with a chlorophyll:P700 ratio of 300:1 was isolated. It contained no photosystem II, but a significant amount of LHCII which was functionally connected to the PSI reaction centre. Reconstitution studies demonstrated that excitation energy transfer from LHCII to PSI requires the presence of LHCI-680, and we propose that, in PSI, the following linear excitation energy transfer sequence occurs: LHCII----LHCI-680----LHCI-730----P700 Chla-P1.     

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

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

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

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

The Lhca antenna complexes of higher plants photosystem I

The Lhca antenna complexes of photosystem I (PSI) have been characterized by comparison of native and recombinant preparations. Eight Lhca polypeptides have been found to be all organized as dimers in the PSI-LHCI complex. The red emission fluorescence is associated not only with Lhca1-4 heterodimer, but also with dimers containing Lhca2 and/or Lhca3 complexes. Reconstitution of Lhca1 and Lhca4 monomers as well as of the Lhca1-4 dimer in vitro was obtained. The biochemical and spectroscopic features of these three complexes are reported. The monomers Lhca1 and Lhca4 bind 10 Chls each, while the Chl a/b ratio is lower in Lhca4 as compared to Lhca1. Three carotenoid binding sites have been found in Lhca1, while only two are present in Lhca4. Both complexes contain lutein and violaxanthin while beta-carotene is selectively bound to the Lhca1-4 dimer in substoichiometric amounts upon dimerization. Spectral analysis revealed the presence of low energy absorption forms in Lhca1 previously thought to be exclusively associated with Lhca4. It is shown that the process of dimerization changes the spectroscopic properties of some chromophores and increases the amplitude of the red absorption tail of the complexes. The origin of these spectroscopic features is discussed.     

The Lhca antenna complexes of higher plants photosystem I

The Lhca antenna complexes of photosystem I (PSI) have been characterized by comparison of native and recombinant preparations. Eight Lhca polypeptides have been found to be all organized as dimers in the PSI-LHCI complex. The red emission fluorescence is associated not only with Lhca1-4 heterodimer, but also with dimers containing Lhca2 and/or Lhca3 complexes. Reconstitution of Lhca1 and Lhca4 monomers as well as of the Lhca1-4 dimer in vitro was obtained. The biochemical and spectroscopic features of these three complexes are reported. The monomers Lhca1 and Lhca4 bind 10 Chls each, while the Chl a/b ratio is lower in Lhca4 as compared to Lhca1. Three carotenoid binding sites have been found in Lhca1, while only two are present in Lhca4. Both complexes contain lutein and violaxanthin while β-carotene is selectively bound to the Lhca1-4 dimer in substoichiometric amounts upon dimerization. Spectral analysis revealed the presence of low energy absorption forms in Lhca1 previously thought to be exclusively associated with Lhca4. It is shown that the process of dimerization changes the spectroscopic properties of some chromophores and increases the amplitude of the red absorption tail of the complexes. The origin of these spectroscopic features is discussed.     

Single-molecule spectroscopic characterization of light-harvesting 2 complexes reconstituted into model membranes

The spectroscopic properties of the light-harvesting 2 complexes (LH2) from the purple bacterium Rhodopseudomonas acidophila (strain 10050) in detergent micelles and reconstituted into lipid membranes have been studied by single-molecule spectroscopy. When LH2 complexes are solubilized from their host biological membranes by nondenaturing detergents, such as LDAO, there is a small 2-nm spectral shift of the B850 absorption band in the ensemble spectrum. This is reversed when the LH2 complexes are put back into phospholipid vesicles, i.e., into a more native-like environment. The spectroscopic properties on the single-molecule level of the detergent-solubilized LH2 complexes were compared with those reconstituted into the lipid membranes to see if their detailed spectroscopic behavior was influenced by these small changes in the position of the B850 absorption band. A detailed analysis of the low-temperature single-molecule fluorescence-excitation spectra of the LH2 complexes in these two different conditions showed no significant differences. In particular, the distribution of the spectral splitting between the circular k = +/-1 exciton states of the B850 absorption band and the distribution of the mutual angle between the k = +/-1 exciton states are identical in both cases. It can be concluded, therefore, that the LH2 complexes from Rps. acidophila are equally stable when solubilized in detergent micelles as they are when membrane reconstituted. Moreover, when they are solubilized in a suitable detergent and spin coated onto a surface for the single-molecule experiments they do not display any more structural disorder than when in a phospholipid membrane.     

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.     

The importance of PS I chlorophyll red forms in light-harvesting by leaves

We have investigated the importance of the long wavelength absorbing spectral forms (red forms) of Photosystem I in photosynthetic light harvesting by leaves. To this end leaf spectra were simulated by using a linear combination of absorption (OD) spectra of purified Photosystem I, Photosystem II and LHC II, multiplied by an empirical multiple scattering chloroplast/leaf conversion function. In this way it is demonstrated that while the PS I red forms account for only about 4–5% of light absorption in a normal daylight environment, in different shadelight environments these long wavelength pigments may be responsible for up to 40% of total photon capture. In the context of maximising the photosynthetic quantum efficiency under the low light conditions of shadelight, this relative increase in the absorption cross section of PS I can be understood by considering the increased synthesis of the major PS II antenna complex, LHC II, known to occur in plants growing under these light conditions. It is demonstrated that for plants in a moderate to deep shadelight regime the PS II cross section needs to increase by 50% to 100% via LHC II synthesis to balance the increased PS I absorption by the red forms. The possibility that under shade light conditions the increased PS I cross section may serve in cyclic phosphorylation is also discussed.     

The composition and spectral properties of three different forms of light-harvesting complex II

Different aggregates of LHC II play a very important role in regulating the light absorption and excitation energy transfer of plant. Trimeric LHC II was purified from spinach thylakoid membrane. In order to obtain the dimeric and monomeric LHC II, the trimer was treated with the mixture of 2% OGP and 10 μg/mL PLA₂, then loaded onto the sucrose density gradient in the presence of 0.06% triton X-100. The LHC II trimer, dimer and monomer isolated by sucrose density gradient all contained three polypeptides with molecular weight of 29, 28 and 26 kd respectively. The pigment composition showed much difference in the content of Chl b and xanthophyll among three forms of LHC II. To study the light capture and excitation energy transfer in different forms of LHC II, the absorption and fluorescence spectra were analyzed. The results clearly showed that the efficiency of energy absorption and transfer was different in the three kinds of LHC II, the highest for trimeric LHC II, intermediate for dimeric LHC II, and the lowest for monomeric LHC II. It was suggested that there might be a physiological homeostasis of different aggregates of LHC II in plants, which is significant for the plant self-regulating upon exposure to variable light environment.     

Structural analysis and comparison of light-harvesting complexes I and II

Photosynthesis is a fundamental biological process involving the conversion of solar energy into chemical energy. The initial photochemical and photophysical events of photosynthesis are mediated by photosystem II (PSII) and photosystem I (PSI). Both PSII and PSI are multi-subunit supramolecular machineries composed of a core complex and a peripheral antenna system. The antenna system serves to capture light energy and transfer it to the core efficiently. Both PSII and PSI in the green lineage (plants and green algae) and PSI in red algae have an antenna system comprising a series of chlorophyll- and carotenoid-binding membrane proteins belonging to the light-harvesting complex (LHC) superfamily, including LHCII and LHCI. However, the antenna size and subunit composition vary considerably in the two photosystems from diverse organisms. On the basis of the plant and algal LHCII and LHCI structures that have been solved by X-ray crystallography and single-particle cryo-electron microscopy we review the detailed structural features and characteristic pigment properties of these LHCs in PSII and PSI. This article is part of a Special Issue entitled Light harvesting, edited by Dr. Roberta Croce.     

Green plant photosystem I binds light-harvesting complex I on one side of the complex

We report a structural characterization by electron microscopy of green plant photosystem I solubilized by the mild detergent n-dodecyl-alpha-D-maltoside. It is shown by immunoblotting that the isolated complexes contain all photosystem I core proteins and all peripheral light-harvesting proteins. The electron microscopic analysis is based on a large data set of 14 000 negatively stained single-particle projections and reveals that most of the complexes are oval-shaped monomers. The monomers have a tendency to associate into artificial dimers, trimers, and tetramers in which the monomers are oppositely oriented. Classification of the dimeric complexes suggests that some of the monomers lack a part of the peripheral antenna. On the basis of a comparison with projections from trimeric photosystem I complexes from cyanobacteria, we conclude that light-harvesting complex I only binds to the core complex at the side of the photosystem I F/J subunits and does not cause structural hindrances for the type of trimerization observed in cyanobacterial photosystem I.