Remodeling of excitation energy transfer in extremophilic red algal PSI-LHCI complex during light adaptation

Photosynthetic PSI-LHCI complexes from an extremophilic red alga C. merolae grown under varying light regimes are characterized by decreasing size of LHCI antenna with increasing illumination intensity [1]. In this study we applied time-resolved fluorescence spectroscopy to characterize the kinetics of energy transfer processes in three types of PSI-LHCI supercomplexes isolated from the low (LL), medium (ML) and extreme high light (EHL) conditions. We show that the average rate of fluorescence decay is not correlated with the size of LHCI antenna and is twice faster in complexes isolated from ML-grown cells (~25–30 ps) than from both LL- and EHL-exposed cells (~50–55 ps). The difference is mainly due to a contribution of a long ~100-ps decay component detected only for the latter two PSI samples. We propose that the lack of this phase in ML complexes is caused by perfect coupling of this antenna to PSI core and lack of low-energy chlorophylls in LHCI. On the other hand, the presence of the slow, ~100-ps, fluorescence decay component in LL and EHL complexes may be due to the weak coupling between PSI core and LHCI antenna complex, and due to the presence of particularly low-energy or red chlorophylls in LHCI. Our study has revealed the remarkable functional flexibility of light harvesting strategies that have evolved in the extremophilic red algae in response to harsh or limiting light conditions involving accumulation of low energy chlorophylls that exert two distinct functions: as energy traps or as far-red absorbing light harvesting antenna, respectively.     

The photochemical trapping rate from red spectral states in PSI-LHCI is determined by thermal activation of energy transfer to bulk chlorophylls

The average fluorescence decay lifetimes, due to reaction centre photochemical trapping, were calculated for wavelengths in the 690- to 770-nm interval from the published fluorescence decay-associated emission spectra for Photosystem I (PSI)-light-harvesting complex of Photosystem I (LHCI) [Biochemistry 39 (2000) 6341] at 280 and 170 K. For 280 K, the overall trapping time at 690 nm is 81 ps and increases with wavelength to reach 103 ps at 770 nm. For 170 K, the 690-nm value is 115 ps, increasing to 458 ps at 770 nm. This underlines the presence of kinetically limiting processes in the PSI antenna (diffusion limited). The explanation of these nonconstant values for the overall trapping time band is sought in terms of thermally activated transfer from the red absorbing states to the "bulk" acceptor chlorophyll (chl) states in the framework of the Arrhenius-Eyring theory. It is shown that the wavelength-dependent "activation energies" come out in the range between 1.35 and 2.7 kcal mol(-1), increasing with the emission wavelength within the interval 710-770 nm. These values are in good agreement with the Arrhenius activation energy determined for the steady-state fluorescence yield over the range 130-280 K for PSI-LHCI. We conclude that the variable trapping time in PSI-LHCI can be accounted for entirely by thermally activated transfer from the low-energy chl states to the bulk acceptor states and therefore that the position of the various red states in the PSI antenna seems not to be of significant importance. The analysis shows that the bulk antenna acceptor states are on the low-energy side of the bulk antenna absorption band.     

Lhca5 interaction with plant photosystem I

In the outer antenna (LHCI) of higher plant photosystem I (PSI) four abundantly expressed light-harvesting protein of photosystem I (Lhca)-type proteins are organized in two heterodimeric domains (Lhca1/Lhca4 and Lhca2/Lhca3). Our cross-linking studies on PSI-LHCI preparations from wildtype Arabidopsis and pea plants indicate an exclusive interaction of the rarely expressed Lhca5 light-harvesting protein with LHCI in the Lhca2/Lhca3-site. In PSI particles with an altered LHCI composition Lhca5 assembles in the Lhca1/Lhca4 site, partly as a homodimer. This flexibility indicates a binding-competitive model for the LHCI assembly in plants regulated by molecular interactions of the Lhca proteins with the PSI core.     

Fluorescence decay and spectral evolution in intact photosystem I of higher plants

A photosystem I preparation from maize, containing its full antenna complement (PSI-200) and in which detergent effects on chlorophyll coupling are almost completely absent, has been studied by time-resolved fluorescence techniques with approximately 5 ps resolution at 280 and 170 K in the wavelength interval of 690-780 nm. The data have been analyzed in terms of both the decay-associated spectra (DAS) and the time-resolved emission spectra (TRES). As in a previous room temperature study [Turconi, S., Weber, N., Schweitzer, D., Strotmann, H., and Holzwarth, A. R. (1994) Biochim. Biophys. Acta 1187, 324-334], the 280 K decay is well described by three DAS components in the 11-130 ps time range, the fastest of which displays both positive and negative amplitudes characteristic of excitation transfer from the bulk to the red antenna forms. Both the 57 and 130 ps components have all positive amplitudes and describe complex decay and equilibration processes involving the red forms. At 170 K, four major components in the 10-715 ps time range are required to describe the decay. The fastest represents bulk to red form transfer processes, while the 55, 216, and 715 ps decays, with all positive amplitudes, have maxima near 720, 730, and 740 nm, respectively, in accord with previous steady-state fluorescence measurements. The width and asymmetry of these DAS indicate that they are spectrally complex and represent decay and equilibration processes involving the red forms. Spectral evolution during the fluorescence decay process was analyzed in terms of the TRES. The red shifting of the TRES was analyzed in terms of the first central spectral moment (mean spectral energy) which is biexponential at both temperatures. The slower component, which describes equilibration between the red forms, leads to spectral red shifting during the entire fluorescence decay process, and the mean lifetimes of the spectral moments at 280 and 170 K (86 and 291 ps, respectively) are similar to the mean lifetimes of the fluorescence decays (119 and 384 ps, respectively). Thus, both spectral evolution and the trapping-associated fluorescence decay occur on a similar time scale, and both processes display a very similar temperature sensitivity. On the basis of these data, it is concluded that trapping in PSI-200 is to a large extent rate-limited by excitation diffusion in the antenna and in particular by the slow "uphill" transfer from the low-energy forms to the bulk and/or inner core chlorophyll molecules.     

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 approximately 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.     

Comparison of the subunit compositions of the PSI-LHCI supercomplex and the LHCI in the green alga Chlamydomonas reinhardtii

Although the light-harvesting chlorophyll protein complex I (LHCI) of photosystem I (PSI) is intimately associated with the PSI core complex and forms the PSI-LHCI supercomplex, the LHCI is normally synthesized in PSI-deficient mutants. In this paper, we compared the subunit compositions of the PSI-LHCI supercomplex and the LHCI by immunoblot analysis and two-dimensional gel electrophoresis combined with mass spectrometry. The PSI-LHCI supercomplex and the LHCI were purified by sucrose density gradient centrifugation and (diethylamino)ethyl column chromatography from n-dodecyl-beta-D-maltoside-solubilized thylakoids of the wild-type and DeltapsaB mutant of the green alga Chlamydomonas reinhardtii. The PSI-LHCI supercomplex contained all of the nine Lhca polypeptides (Lhca1-9) that are detected in wild-type thylakoids. In contrast, the LHCI retained only six Lhca polypeptides, whereas Lhca3 and two minor polypeptides, Lhca2 and Lhca9, were lost during the purification procedure. Sucrose density gradient centrifugation showed that the purified LHCI retains an oligomeric structure with an apparent molecular mass of 300-400 kDa. We therefore concluded that Lhca2, Lhca3, and Lhca9 are not required for the stable oligomeric structure of the LHCI and that the association of these polypeptides in the LHCI is stabilized by the presence of the PSI core complex. Finally, we discuss the possible localization and function of Lhca polypeptides in the LHCI.     

Structure of the higher plant light harvesting complex I: in vivo characterization and structural interdependence of the Lhca proteins

We have investigated the structure of the higher plant light harvesting complex of photosystem I (LHCI) by analyzing PSI-LHCI particles isolated from a set of Arabidopsis plant lines, each lacking a specific Lhca (Lhca1-4) polypeptide. Functional antenna size measurements support the recent finding that there are four Lhca proteins per PSI in the crystal structure [Ben-Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630-635]. According to HPLC analyses the number of pigment molecules bound within the LHCI is higher than expected from reconstitution studies or analyses of isolated native LHCI. Comparison of the spectra of the particles from the different lines reveals chlorophyll absorption bands peaking at 696, 688, 665, and 655 nm that are not present in isolated PSI or LHCI. These bands presumably originate from "gap" or "linker" pigments that are cooperatively coordinated by the Lhca and/or PSI proteins, which we have tentatively localized in the PSI-LHCI complex.     

The photochemical trapping rate from red spectral states in PSI–LHCI is determined by thermal activation of energy transfer to bulk chlorophylls

The average fluorescence decay lifetimes, due to reaction centre photochemical trapping, were calculated for wavelengths in the 690- to 770-nm interval from the published fluorescence decay-associated emission spectra for Photosystem I (PSI)–light-harvesting complex of Photosystem I (LHCI) [Biochemistry 39 (2000) 6341] at 280 and 170 K. For 280 K, the overall trapping time at 690 nm is 81 ps and increases with wavelength to reach 103 ps at 770 nm. For 170 K, the 690-nm value is 115 ps, increasing to 458 ps at 770 nm. This underlines the presence of kinetically limiting processes in the PSI antenna (diffusion limited). The explanation of these nonconstant values for the overall trapping time band is sought in terms of thermally activated transfer from the red absorbing states to the “bulk” acceptor chlorophyll (chl) states in the framework of the Arrhenius–Eyring theory. It is shown that the wavelength-dependent “activation energies” come out in the range between 1.35 and 2.7 kcal mol⁻¹, increasing with the emission wavelength within the interval 710–770 nm. These values are in good agreement with the Arrhenius activation energy determined for the steady-state fluorescence yield over the range 130–280 K for PSI–LHCI. We conclude that the variable trapping time in PSI–LHCI can be accounted for entirely by thermally activated transfer from the low-energy chl states to the bulk acceptor states and therefore that the position of the various red states in the PSI antenna seems not to be of significant importance. The analysis shows that the bulk antenna acceptor states are on the low-energy side of the bulk antenna absorption band.     

Light harvesting in photosystem I supercomplexes

In photosynthetic membranes of cyanobacteria, algae, and higher plants, photosystem I (PSI) mediates light-driven transmembrane electron transfer from plastocyanin or cytochrome c6 to the ferredoxin-NADP complex. The oxidoreductase function of PSI is sensitized by a reversible photooxidation of primary electron donor P700, which launches a multistep electron transfer via a series of redox cofactors of the reaction center (RC). The excitation energy for the functioning of the primary electron donor in the RC is delivered via the chlorophyll core antenna in the complex with peripheral light-harvesting antennas. Supermolecular complexes of the PSI acquire remarkably different structural forms of the peripheral light-harvesting antenna complexes, including distinct pigment types and organizational principles. The PSI core antenna, being the main functional unit of the supercomplexes, provides an increased functional connectivity in the chlorophyll antenna network due to dense pigment packing resulting in a fast spread of the excitation among the neighbors. Functional connectivity within the network as well as the spectral overlap of antenna pigments allows equilibration of the excitation energy in the depth of the whole membrane within picoseconds and loss-free delivery of the excitation to primary donor P700 within 20-40 ps. Low-light-adapted cyanobacteria under iron-deficiency conditions extend this capacity via assembly of efficiently energy coupled rings of CP43-like complexes around the PSI trimers. In green algae and higher plants, less efficient energy coupling in the eukaryotic PSI-LHCI supercomplexes is probably a result of the structural adaptation of the Chl a/b binding LHCI peripheral antenna that not only extends the absorption cross section of the PSI core but participates in regulation of excitation flows between the two photosystems as well as in photoprotection.     

The light-harvesting complex of photosystem I in Chlamydomonas reinhardtii: protein composition, gene structures and phylogenic implications

Light-harvesting chlorophyll a/b-binding proteins (LHCI) associated with photosystem I (PSI) and the genes encoding these proteins have been characterized in the unicellular green alga Chlamydomonas reinhardtii, extending previous studies of the PSII-LHCII [Teramoto et al. (2001) Plant Cell Physiol. 42: 849]. In order to assign LHCI proteins in the thylakoid membranes, the PSI-LHCI supercomplex that retains all of the major LHCI proteins was purified. Seven distinct LHCI proteins were resolved from the purified supercomplex by a high-resolution SDS polyacrylamide gel electrophoresis, and their N-terminal amino acid sequences were determined. One LHCI protein (band e) was newly found, although the other six LHCI proteins corresponded to those previously reported. Genomic clones encoding these seven LHCI proteins were newly isolated and the nucleotide sequences were determined. A comprehensive characterization of all members of Lhc gene family in this alga revealed that LHCI proteins are more highly diverged than LHCII, suggesting functional differentiation of the protein components in LHCI. Neighbor joining trees were constructed for LHC proteins from C. reinhardtii and those of Arabidopsis thaliana or Galdieria sulphuraria to assess evolutionary relationships. Phylogenetic analysis revealed that (1). green algal LHCI and LHCII proteins are more closely related to one another than to LHCI proteins in red algae, (2). green algae and higher plants possess seven common lineages of LHC proteins, and (3). Type I and III LHCI proteins are conserved between green algae and higher plants, while Type II and IV are not. These findings are discussed in the context of evolution of multiple diverse antenna complexes.     

Three structures of PSI-LHCI from Chlamydomonas reinhardtii suggest a resting state re-activated by ferredoxin

Photosystem I (PSI) from the green alga Chlamydomonas reinhardtii, with various numbers of membrane bound antenna complexes (LHCI), has been described in great detail. In contrast, structural characterization of soluble binding partners is less advanced. Here, we used X-ray crystallography and single particle cryo-EM to investigate three structures of the PSI-LHCI supercomplex from Chlamydomonas reinhardtii. An X-ray structure demonstrates the absence of six chlorophylls from the luminal side of the LHCI belts, suggesting these pigments were either physically absent or less stably associated with the complex, potentially influencing excitation transfer significantly. CryoEM revealed extra densities on luminal and stromal sides of the supercomplex, situated in the vicinity of the electron transfer sites. These densities disappeared after the binding of oxidized ferredoxin to PSI-LHCI. Based on these structures, we propose the existence of a PSI-LHCI resting state with a reduced active chlorophyll content, electron donors docked in waiting positions and regulatory binding partners positioned at the electron acceptor site. The resting state PSI-LHCI supercomplex would be recruited to its active form by the availability of oxidized ferredoxin.     

Uphill energy transfer in photosystem I from <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>. Time-resolved fluorescence measurements at 77 K

Energetic properties of chlorophylls in photosynthetic complexes are strongly modulated by their interaction with the protein matrix and by inter-pigment coupling. This spectral tuning is especially striking in photosystem I (PSI) complexes that contain low-energy chlorophylls emitting above 700 nm. Such low-energy chlorophylls have been observed in cyanobacterial PSI, algal and plant PSI–LHCI complexes, and individual light-harvesting complex I (LHCI) proteins. However, there has been no direct evidence of their presence in algal PSI core complexes lacking LHCI. In order to determine the lowest-energy states of chlorophylls and their dynamics in algal PSI antenna systems, we performed time-resolved fluorescence measurements at 77 K for PSI core and PSI–LHCI complexes isolated from the green alga Chlamydomonas reinhardtii. The pool of low-energy chlorophylls observed in PSI cores is generally smaller and less red-shifted than that observed in PSI–LHCI complexes. Excitation energy equilibration between bulk and low-energy chlorophylls in the PSI–LHCI complexes at 77 K leads to population of excited states that are less red-shifted (by ~?12 nm) than at room temperature. On the other hand, analysis of the detection wavelength dependence of the effective trapping time of bulk excitations in the PSI core at 77 K provided evidence for an energy threshold at ~?675 nm, above which trapping slows down. Based on these observations, we postulate that excitation energy transfer from bulk to low-energy chlorophylls and from bulk to reaction center chlorophylls are thermally activated uphill processes that likely occur via higher excitonic states of energy accepting chlorophylls.     

Ultrafast primary processes in PS I from Synechocystis sp. PCC 6803: roles of P700 and A(0)

The excitation transport and trapping kinetics of core antenna-reaction center complexes from photosystem I of wild-type Synechocystis sp. PCC 6803 were investigated under annihilation-free conditions in complexes with open and closed reaction centers. For closed reaction centers, the long-component decay-associated spectrum (DAS) from global analysis of absorption difference spectra excited at 660 nm is essentially flat (maximum amplitude <10(-5) absorbance units). For open reaction centers, the long-time spectrum (which exhibits photobleaching maxima at approximately 680 and 700 nm, and an absorbance feature near 690 nm) resembles one previously attributed to (P700(+) - P700). For photosystem I complexes excited at 660 nm with open reaction centers, the equilibration between the bulk antenna and far-red chlorophylls absorbing at wavelengths >700 nm is well described by a single DAS component with lifetime 2.3 ps. For closed reaction centers, two DAS components (2.0 and 6.5 ps) are required to fit the kinetics. The overall trapping time at P700 ( approximately 24 ps) is very nearly the same in either case. Our results support a scenario in which the time constant for the P700 --> A(0) electron transfer is 9-10 ps, whereas the kinetics of the subsequent A(0) --> A(1) electron transfer are still unknown.     

Binding of ferredoxin NADP+ oxidoreductase (FNR) to plant photosystem I

The binding of FNR to PSI has been postulated long ago, however, a clear evidence is still missing. In this work, using isothermal titration calorimetry (ITC), we found that FNR binds to photosystem I with its light harvesting complex I (PSI-LHCI) from C. reinhardtii with a 1:1 stoichiometry, a Kd of ~0.8 μM and ?H of ?20.7 kcal/mol. Titrations at different temperatures were used to determine the heat capacity change, ?CP, of the binding, through which the size of the interface area between the proteins was assessed as ~3000 Ų. In a different set of ITC experiments, introduction of various sucrose concentrations was used to estimate that ~95 water molecules are released to the solvent. These observations support the notion of a binding site shared by few of the photosystem I - light harvesting complex I (PSI-LHCI) subunits in addition to PsaE. Based on these results, a hypothetical model was built for the binding site of FNR at PSI, using known crystallographic structures of: cyanobacterial PSI in complex with ferredoxin (Fd), plant PSI-LHCI and Fd:FNR complex from cyanobacteria. FNR binding site location is proposed to be at the foot of the stromal ridge and above the inner LHCI belt. It is expected to form contacts with PsaE, PsaB, PsaF and at least one of the LHCI. In addition, a ~4.5-fold increased affinity between FNR and PSI-LHCI under crowded 1 M sucrose environment led us to conclude that in C. reinhardtii FNR also functions as a subunit of PSI-LHCI.     

Solving the structure of plant photosystem I--biochemistry is vital.

The recently determined structure of plant photosystem I (PSI) provides the first relatively high-resolution structural model of a supercomplex containing a reaction center and its peripheral antenna. Large amounts of highly purified PSI were required to get enough crystals amenable for structural determination by X-ray crystallography. In addition, a deep biochemical understanding of the large supercomplex was vital for achieving the goal. The stability of PSI was analyzed by sucrose gradient centrifugation and gel electrophoresis. Small amounts of LHCI were detached from PSI following a 12 day incubation under crystallization conditions. The interaction between the reaction center and the peripheral antenna of PSI (LHCI) as well as the interactions among the LHCI monomers are flexible. Nevertheless, the pure and homogeneous preparation of PSI allows for relatively tight crystal packing, which holds promise for obtaining atomic resolution in the future.     

Structural and functional changes of PSI-LHCI supercomplexes of Chlamydomonas reinhardtii cells grown under high salt conditions

The effect of high salt concentration (100 mM NaCl) on the organization of photosystem I-light harvesting complex I supercomplexes (PSI-LHCI) of Chlamydomonas reinhardtii was studied. The electron transfer activity was reduced by 39% in isolated PSI-LHCI supercomplexes. The visible circular dichroism (CD) spectra associated with strongly coupled chlorophyll (Chl) dimers were reduced in intensity, indicating that pigment–pigment interactions were disrupted. This data is consistent with results from fluorescence streak camera spectroscopy, which suggest that red-shifted pigments in the PSI-LHCI antenna had been lost. Denaturing gel electrophoresis and immunoblot analysis reveals that levels of the PSI reaction center proteins PsaD, PsaE and PsaF were reduced due to salt stress. PsaE is almost completely absent under high salt conditions. It is known that the membrane-extrinsic subunits PsaD and E form the ferredoxin-docking site. Our results indicate that the PSI-LHCI supercomplex is damaged by reactive oxygen species at high salt concentration, with particular impact on the ferredoxin-docking site and the PSI-LHCI interface.     

Antenna structure and excitation dynamics in photosystem I. II. Studies with mutants of Chlamydomonas reinhardtii lacking photosystem II.

Using time-resolved single photon counting, fluorescence decay in photosystem I (PS I) was analyzed in mutant strains of Chlamydomonas reinhardtii that lack photosystem II. Two strains are compared: one with a wild-type PS I core antenna (120 chlorophyll a/P700) and a second showing an apparent reduction in core antenna size (60 chlorophyll a/P700). These data were calculated from the lifetimes of core antenna excited states (75 and 45 ps, respectively) and from pigment stoichiometries. Fluorescence decay in wild type PS I is composed of two components: a fast 75-ps decay that represents the photochemically limited lifetime of excited states in the core antenna, and a minor (less than 10%) 300-800 ps component that has spectral characteristics of both peripheral and core antenna pigments. Temporal and spectral properties of the fast PS I decay indicate that (a) excitations are nearly equilibrated among the range of spectral forms present in the PS I core antenna, (b) an average excitation visits a representative distribution of core antenna spectral forms on all pigment-binding subunits regardless of the origin of the excitation, (c) reduction in core antenna size does not alter the range of antenna spectral forms present, and (d) transfer from peripheral antennae to the PS I core complex is rapid (less than 5 ps).     

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.     

Identification and Characterization of an Assembly Intermediate Subcomplex of Photosystem I in the Green Alga Chlamydomonas reinhardtii

Photosystem I (PSI) is a multiprotein complex consisting of the PSI core and peripheral light-harvesting complex I (LHCI) that together form the PSI-LHCI supercomplex in algae and higher plants. The supercomplex is synthesized in steps during which 12–15 core and 4–9 LHCI subunits are assembled. Here we report the isolation of a PSI subcomplex that separated on a sucrose density gradient from the thylakoid membranes isolated from logarithmic growth phase cells of the green alga Chlamydomonas reinhardtii. Pulse-chase labeling of total cellular proteins revealed that the subcomplex was synthesized de novo within 1 min and was converted to the mature PSI-LHCI during the 2-h chase period, indicating that the subcomplex was an assembly intermediate. The subcomplex was functional; it photo-oxidized P700 and demonstrated electron transfer activity. The subcomplex lacked PsaK and PsaG, however, and it bound PsaF and PsaJ weakly and was not associated with LHCI. It seemed likely that LHCI had been integrated into the subcomplex unstably and was dissociated during solubilization and/or fractionation. We, thus, infer that PsaK and PsaG stabilize the association between PSI core and LHCI complexes and that PsaK and PsaG bind to the PSI core complex after the integration of LHCI in one of the last steps of PSI complex assembly.     

Protein-protein interactions by molecular modeling and biochemical characterization of PSI-LHCI supercomplexes from Chlamydomonas reinhardtii

The physiological function of Photosystem I (PSI) is a sunlight energy converter, catalyzing one of the initial steps in driving oxygenic photosynthesis in cyanobacteria, algae and higher plants. The Chlamydomonas reinhardtii PSI structure was not known since it contains a unique structure having additional light harvesting complex I (LHCI) subunits, which play a major role in the transfer of sunlight energy to the reaction center. Here, individual subunits of LHC and core subunits are built based on the PDB taken from RCSB Protein Data Bank. The model gives information about the geometrical existence of subunits following a flanking order of Lhca5, Lhca1, Lhca6, Lhca4, Lhca2, Lhca8, Lhca9, Lhca7, and Lhca3. The new subunit PsaO is located close to the PsaH, PsaI and PsaL subunits, thus it may be involved in the state transition mechanism and stabilization of PSI-LHCI supercomplexes. The modeled PSI-LHCI structure of C. reinhardtii shows a unique arrangement of PsaN, PsaO of PSI core subunits and Lhca5 to Lhca9 of LHCI subunits. There are many non-covalent interactions among the PSI and LHCI subunits, which suggest that C. reinhardtii PSI-LHCI supercomplexes are more complex than higher plants. These results strongly support the experimental data that, even with harsh treatment of the PSI-LHCI supercomplexes with detergent, the complexes do not dissociate due to strong interactions between the PSI core and LHCI. Thus, our 3D model may give valid structural information of the PSI-LHCI arrangement and its physiological role in C. reinhardtii.