Structure and distinct supramolecular organization of a PSII-ACPII dimer from a cryptophyte alga Chroomonas placoidea.

Cryptophyte algae are an evolutionarily distinct and ecologically important group of photosynthetic unicellular eukaryotes. Photosystem II (PSII) of cryptophyte algae associates with alloxanthin chlorophyll a/c-binding proteins (ACPs) to act as the peripheral light-harvesting system, whose supramolecular organization is unknown. Here, we purify the PSII-ACPII supercomplex from a cryptophyte alga Chroomonas placoidea (C. placoidea), and analyze its structure at a resolution of 2.47 Å using cryo-electron microscopy. This structure reveals a dimeric organization of PSII-ACPII containing two PSII core monomers flanked by six symmetrically arranged ACPII subunits. The PSII core is conserved whereas the organization of ACPII subunits exhibits a distinct pattern, different from those observed so far in PSII of other algae and higher plants. Furthermore, we find a Chl a-binding antenna subunit, CCPII-S, which mediates interaction of ACPII with the PSII core. These results provide a structural basis for the assembly of antennas within the supercomplex and possible excitation energy transfer pathways in cryptophyte algal PSII, shedding light on the diversity of supramolecular organization of photosynthetic machinery.

Structures and organizations of PSI-AcpPCI supercomplexes from red tidal and coral symbiotic photosynthetic dinoflagellates.

Marine photosynthetic dinoflagellates are a group of successful phytoplankton that can form red tides in the ocean and also symbiosis with corals. These features are closely related to the photosynthetic properties of dinoflagellates. We report here three structures of photosystem I (PSI)-chlorophylls (Chls) a/c-peridinin protein complex (PSI-AcpPCI) from two species of dinoflagellates by single-particle cryoelectron microscopy. The crucial PsaA/B subunits of a red tidal dinoflagellate Amphidinium carterae are remarkably smaller and hence losing over 20 pigment-binding sites, whereas its PsaD/F/I/J/L/M/R subunits are larger and coordinate some additional pigment sites compared to other eukaryotic photosynthetic organisms, which may compensate for the smaller PsaA/B subunits. Similar modifications are observed in a coral symbiotic dinoflagellate Symbiodinium species, where two additional core proteins and fewer AcpPCIs are identified in the PSI-AcpPCI supercomplex. The antenna proteins AcpPCIs in dinoflagellates developed some loops and pigment sites as a result to accommodate the changed PSI core, therefore the structures of PSI-AcpPCI supercomplex of dinoflagellates reveal an unusual protein assembly pattern. A huge pigment network comprising Chls a and c and various carotenoids is revealed from the structural analysis, which provides the basis for our deeper understanding of the energy transfer and dissipation within the PSI-AcpPCI supercomplex, as well as the evolution of photosynthetic organisms.

Structure of a unique PSII-Pcb tetrameric megacomplex in a chlorophyll d-containing cyanobacterium.

Acaryochloris marina is a unique cyanobacterium using chlorophyll d (Chl d) as its major pigment and thus can use far-red light for photosynthesis. Photosystem II (PSII) of A. marina associates with a number of prochlorophyte Chl-binding (Pcb) proteins to act as the light-harvesting system. We report here the cryo-electron microscopic structure of a PSII-Pcb megacomplex from A. marina at a 3.6-angstrom overall resolution and a 3.3-angstrom local resolution. The megacomplex is organized as a tetramer consisting of two PSII core dimers flanked by sixteen symmetrically related Pcb proteins, with a total molecular weight of 1.9 megadaltons. The structure reveals the detailed organization of PSII core consisting of 15 known protein subunits and an unknown subunit, the assembly of 4 Pcb antennas within each PSII monomer, and possible pathways of energy transfer within the megacomplex, providing deep insights into energy transfer and dissipation mechanisms within the PSII-Pcb megacomplex involved in far-red light utilization.

Structural insights into photosystem II supercomplex and trimeric FCP antennae of a centric diatom Cyclotella meneghiniana.

Diatoms are dominant marine algae and contribute around a quarter of global primary productivity, the success of which is largely attributed to their photosynthetic capacity aided by specific fucoxanthin chlorophyll-binding proteins (FCPs) to enhance the blue-green light absorption under water. We purified a photosystem II (PSII)-FCPII supercomplex and a trimeric FCP from Cyclotella meneghiniana (Cm) and solved their structures by cryo-electron microscopy (cryo-EM). The structures reveal detailed organizations of monomeric, dimeric and trimeric FCP antennae, as well as distinct assemblies of Lhcx6_1 and dimeric FCPII-H in PSII core. Each Cm-PSII-FCPII monomer contains an Lhcx6_1, an FCP heterodimer and other three FCP monomers, which form an efficient pigment network for harvesting energy. More diadinoxanthins and diatoxanthins are found in FCPs, which may function to quench excess energy. The trimeric FCP contains more chlorophylls c and fucoxanthins. These diversified FCPs and PSII-FCPII provide a structural basis for efficient light energy harvesting, transfer, and dissipation in C. meneghiniana.

Structure of a diatom photosystem II supercomplex containing a member of Lhcx family and dimeric FCPII.

Diatoms rely on fucoxanthin chlorophyll a/c-binding proteins (FCPs) for their great success in oceans, which have a great diversity in their pigment, protein compositions, and subunit organizations. We report a unique structure of photosystem II (PSII)-FCPII supercomplex from Thalassiosira pseudonana at 2.68-Å resolution by cryo-electron microscopy. FCPIIs within this PSII-FCPII supercomplex exist in dimers and monomers, and a homodimer and a heterodimer were found to bind to a PSII core. The FCPII homodimer is formed by Lhcf7 and associates with PSII through an Lhcx family antenna Lhcx6_1, whereas the heterodimer is formed by Lhcf6 and Lhcf11 and connects to the core together with an Lhcf5 monomer through Lhca2 monomer. An extended pigment network consisting of diatoxanthins, diadinoxanthins, fucoxanthins, and chlorophylls a/c is revealed, which functions in efficient light harvesting, energy transfer, and dissipation. These results provide a structural basis for revealing the energy transfer and dissipation mechanisms and also for the structural diversity of FCP antennas in diatoms.

Origin of Energy Dissipation in the Oligomeric Fucoxanthin-Chlorophyll a/c Binding Proteins.

Fucoxanthin-chlorophyll proteins (FCPs) are a family of photosynthetic light-harvesting complex (LHC) proteins found in diatoms. They efficiently capture photons and regulate their functions, ensuring diatom survival in highly fluctuating light. FCPs are present in different oligomeric states in vivo, but functional differences among these FCP oligomers are not yet fully understood. Here we characterized two types of antenna complexes (FCP-B/C dimers and FCP-A tetramers) that coexist in the marine centric diatom Chaetoceros gracilis using both time-resolved fluorescence and transient absorption spectroscopy. We found that the FCP-B/C complex did not show fluorescence quenching, whereas FCP-A was severely quenched, via an ultrafast excitation energy transfer (EET) pathway from Chl a Qy to the fucoxanthin S1/ICT state. These results highlight the functional differences between FCP dimers and tetramers and indicate that the EET pathway from Chl a to carotenoids is an energy dissipation mechanism conserved in a variety of photosynthetic organisms.

Structural and functional properties of different types of siphonous LHCII trimers from an intertidal green alga Bryopsis corticulans.

Light-harvesting complexes of photosystem II (LHCIIs) in green algae and plants are vital antenna apparatus for light harvesting, energy transfer, and photoprotection. Here we determined the structure of a siphonous-type LHCII trimer from the intertidal green alga Bryopsis corticulans by X-ray crystallography and cryo-electron microscopy (cryo-EM), and analyzed its functional properties by spectral analysis. The Bryopsis LHCII (Bry-LHCII) structures in both homotrimeric and heterotrimeric form show that green light-absorbing siphonaxanthin and siphonein occupied the sites of lutein and violaxanthin in plant LHCII, and two extra chlorophylls (Chls) b replaced Chls a. Binding of these pigments expands the blue-green light absorption of B. corticulans in the tidal zone. We observed differences between the Bry-LHCII homotrimer crystal and cryo-EM structures, and also between Bry-LHCII homotrimer and heterotrimer cryo-EM structures. These conformational changes may reflect the flexibility of Bry-LHCII, which may be required to adapt to light fluctuations from tidal rhythms.

Structural insights into a unique PSI-LHCI-LHCII-Lhcb9 supercomplex from moss Physcomitrium patens.

Photosystem I (PSI) possesses a variable supramolecular organization among different photosynthetic organisms to adapt to different light environments. Mosses are evolutionary intermediates that diverged from aquatic green algae and evolved into land plants. The moss Physcomitrium patens (P. patens) has a light-harvesting complex (LHC) superfamily more diverse than those of green algae and higher plants. Here, we solved the structure of a PSI-LHCI-LHCII-Lhcb9 supercomplex from P. patens at 2.68 Å resolution using cryo-electron microscopy. This supercomplex contains one PSI-LHCI, one phosphorylated LHCII trimer, one moss-specific LHC protein, Lhcb9, and one additional LHCI belt with four Lhca subunits. The complete structure of PsaO was observed in the PSI core. One Lhcbm2 in the LHCII trimer interacts with PSI core through its phosphorylated N terminus, and Lhcb9 mediates assembly of the whole supercomplex. The complicated pigment arrangement provided important information for possible energy-transfer pathways from the peripheral antennae to the PSI core.

Photoinduced chlorophyll charge transfer state identified in the light-harvesting complex II from a marine green alga Bryopsis corticulans.

The light-harvesting complex II of Bryopsis corticulans (B-LHCII), a green alga, differs from that of spinach (S-LHCII) in chlorophyll (Chl) and carotenoid (Car) compositions. We investigated ultrafast excitation dynamics of B-LHCII with visible-to-near infrared time-resolved absorption spectroscopy. Absolute fluorescence quantum yield (Φ FL) of LHCII and spectroelectrochemical (SEC) spectra of Chl a and b were measured to assist the spectral analysis. Red-light excitation at Chl Qy-band, but not Car-band, induced transient features resembling the characteristic SEC spectra of Chl a ⋅+ and Chl b ⋅-, indicating ultrafast photogeneration of Chl-Chl charge transfer (CT) species; Φ FL and 3Car∗ declined whereas CT species increased upon prolonging excitation wavelength, showing positive correlation of 1Chl∗ deactivation with Chl-Chl CT formation. Moreover, ultrafast Chl b-to-Chl a and Car-to-Chl singlet excitation transfer were illustrated. The red-light induction of Chl-Chl CT species, as also observed for S-LHCII, is considered a general occurrence for LHCIIs in light-harvesting form.

Cryo-electron microscopy structure of the intact photosynthetic light-harvesting antenna-reaction center complex from a green sulfur bacterium.

The photosynthetic reaction center complex (RCC) of green sulfur bacteria (GSB) consists of the membrane-imbedded RC core and the peripheric energy transmitting proteins called Fenna-Matthews-Olson (FMO). Functionally, FMO transfers the absorbed energy from a huge peripheral light-harvesting antenna named chlorosome to the RC core where charge separation occurs. In vivo, one RC was found to bind two FMOs, however, the intact structure of RCC as well as the energy transfer mechanism within RCC remain to be clarified. Here we report a structure of intact RCC which contains a RC core and two FMO trimers from a thermophilic green sulfur bacterium Chlorobaculum tepidum at 2.9 Å resolution by cryo-electron microscopy. The second FMO trimer is attached at the cytoplasmic side asymmetrically relative to the first FMO trimer reported previously. We also observed two new subunits (PscE and PscF) and the N-terminal transmembrane domain of a cytochrome-containing subunit (PscC) in the structure. These two novel subunits possibly function to facilitate the binding of FMOs to RC core and to stabilize the whole complex. A new bacteriochlorophyll (numbered as 816) was identified at the interspace between PscF and PscA-1, causing an asymmetrical energy transfer from the two FMO trimers to RC core. Based on the structure, we propose an energy transfer network within this photosynthetic apparatus.

A Possible Mechanism for Aggregation-Induced Chlorophyll Fluorescence Quenching in Light-Harvesting Complex II from the Marine Green Alga Bryopsis corticulans.

The light-harvesting complex II of a green alga Bryopsis corticulans (B-LHCII) is peculiar in that it contains siphonein and siphonaxathin as carotenoid (Car). Since the S1 state of siphonein and siphonaxathin lies substantially higher than the Qy state of chlorophyll a (Chl a), the Chl a(Qy)-to-Car(S1) excitation energy transfer is unfeasible. To understand the photoprotective mechanism of algal photosynthesis, we investigated the influence of temperature on the excitation dynamics of B-LHCII in trimeric and aggregated forms. At room temperature, the aggregated form showed a 10-fold decrease in fluorescence intensity and lifetime than the trimeric form. Upon lowering the temperature, the characteristic 680 nm fluorescence (F-680) of B-LHCII in both forms exhibited systematic intensity enhancement and spectral narrowing; however, only the aggregated form showed a red emission extending over 690-780 nm (F-RE) with pronounced blueshift, lifetime prolongation, and intensity boost. The remarkable T-dependence of F-RE is ascribed to the Chl-Chl charge transfer (CT) species involved directly in the aggregation-induced Chl deactivation. The CT-quenching mechanism, which is considered to be crucial for B. corticulans photoprotection, draws strong support from the positive correlation of the Chl deactivation rate with the CT state population, as revealed by comparing the fluorescence dynamics of B-LHCII with that of the plant LHCII.

Effects of mutations of D1-R323, D1-N322, D1-D319, D1-H304 on the functioning of photosystem II in Thermosynechococcus vulcanus.

Photosystem II (PSII) has a number of hydrogen-bonding networks connecting the manganese cluster with the lumenal bulk solution. The structure of PSII from Thermosynechococcus vulcanus (T. vulcanus) showed that D1-R323, D1-N322, D1-D319 and D1-H304 are involved in one of these hydrogen-bonding networks located in the interfaces between the D1, CP43 and PsbV subunits. In order to investigate the functions of these residues in PSII, we generated seven site-directed mutants D1-R323A, D1-R323E, D1-N322R, D1-D319L, D1-D319R, D1-D319Y and D1-H304D of T. vulcanus and examined the effects of these mutations on the growth and functions of the oxygen-evolving complex. The photoautotrophic growth rates of these mutants were similar to that of the wild type, whereas the oxygen-evolving activities of the mutant cells were decreased differently to 63-91% of that of the wild type at pH 6.5. The mutant cells showed a higher relative activity at higher pH region than the wild type cells, suggesting that higher pH facilitated proton egress in the mutants. In addition, oxygen evolution of thylakoid membranes isolated from these mutants showed an apparent decrease compared to that of the cells. This is due to the loss of PsbU during purification of the thylakoid membranes. Moreover, PsbV was also lost in the PSII core complexes purified from the mutants. Taken together, D1-R323, D1-N322, D1-D319 and D1-H304 are vital for the optimal function of oxygen evolution and functional binding of extrinsic proteins to PSII core, and may be involved in the proton egress pathway mediated by YZ.

Structural elucidation of vascular plant photosystem I and its functional implications.

In vascular plants, bryophytes and algae, the photosynthetic light reaction takes place in the thylakoid membrane where two transmembrane supercomplexes PSII and PSI work together with cytochrome b 6 f and ATP synthase to harvest the light energy and produce ATP and NADPH. Vascular plant PSI is a 600-kDa protein-pigment supercomplex, the core complex of which is partly surrounded by peripheral light-harvesting complex I (LHCI) that captures sunlight and transfers the excitation energy to the core to be used for charge separation. PSI is unique mainly in absorption of longer-wavelengths than PSII, fast excitation energy transfer including uphill energy transfer, and an extremely high quantum efficiency. From the early 1980s, a lot of effort has been dedicated to structural and functional studies of PSI-LHCI, leading to the current understanding of how more than 200 cofactors are kept at the correct distance and geometry to facilitate fast energy transfer in this supercomplex at an atomic level. In this review, we review the history of studies on vascular plant PSI-LHCI, summarise the present research progress on its structure, and present some new and further questions to be answered in future studies.

Architecture of the chloroplast PSI-NDH supercomplex in Hordeum vulgare.

The chloroplast NADH dehydrogenase-like (NDH) complex is composed of at least 29 subunits and has an important role in mediating photosystem I (PSI) cyclic electron transport (CET)1-3. The NDH complex associates with PSI to form the PSI-NDH supercomplex and fulfil its function. Here, we report cryo-electron microscopy structures of a PSI-NDH supercomplex from barley (Hordeum vulgare). The structures reveal that PSI-NDH is composed of two copies of the PSI-light-harvesting complex I (LHCI) subcomplex and one NDH complex. Two monomeric LHCI proteins, Lhca5 and Lhca6, mediate the binding of two PSI complexes to NDH. Ten plant chloroplast-specific NDH subunits are presented and their exact positions as well as their interactions with other subunits in NDH are elucidated. In all, this study provides a structural basis for further investigations on the functions and regulation of PSI-NDH-dependent CET.

LHCA4 residues surrounding red chlorophylls allow for fine-tuning of the spectral region for photosynthesis in Arabidopsis thaliana.

Improving far-red light utilization could be an approach to increasing crop production under suboptimal conditions. In land plants, only a small part of far-red light can be used for photosynthesis, which is captured by the antenna proteins LHCAs of photosystem I (PSI) through the chlorophyll (Chl) pair a603 and a609. However, it is unknown how the energy level of Chls a603-a609 is fine-tuned by the local protein environment in vivo. In this study, we investigated how changing the amino acid ligand for Chl a603 in LHCA4, the most red-shifted LHCA in Arabidopsis thaliana, or one amino acid near Chl a609, affected the energy level of the resulting PSI-LHCI complexes in situ and in vitro. Substitutions of the Chl a603 ligand N99 caused a blue shift in fluorescence emission, whereas the E146Q substitution near Chl a609 expanded the emission range to the red. Purified PSI-LHCI complexes with N99 substitutions exhibited the same fluorescence emission maxima as their respective transgenic lines, while the extent of red shift in purified PSI-LHCI with the E146Q substitution was weaker than in the corresponding transgenic lines. We propose that substituting amino acids surrounding red Chls can tune their energy level higher or lower in vivo, while shifting the absorption spectrum more to the red could prove more difficult than shifting to the blue end of the spectrum. Here, we report the first in vivo exploration of changing the local protein environment on the energy level of the red Chls, providing new clues for engineering red/blue-shifted crops.

Structural insights into cyanobacterial photosystem II intermediates associated with Psb28 and Tsl0063.

Photosystem II (PSII) is a multisubunit pigment-protein complex and catalyses light-induced water oxidation, leading to the conversion of light energy into chemical energy and the release of dioxygen. We analysed the structures of two Psb28-bound PSII intermediates, Psb28-RC47 and Psb28-PSII, purified from a psbV-deletion strain of the thermophilic cyanobacterium Thermosynechococcus vulcanus, using cryo-electron microscopy. Both Psb28-RC47 and Psb28-PSII bind one Psb28, one Tsl0063 and an unknown subunit. Psb28 is located at the cytoplasmic surface of PSII and interacts with D1, D2 and CP47, whereas Tsl0063 is a transmembrane subunit and binds at the side of CP47/PsbH. Substantial structural perturbations are observed at the acceptor side, which result in conformational changes of the quinone (QB) and non-haem iron binding sites and thus may protect PSII from photodamage during assembly. These results provide a solid structural basis for understanding the assembly process of native PSII.

A unique photosystem I reaction center from a chlorophyll d-containing cyanobacterium Acaryochloris marina.

Photosystem I (PSI) is a large protein supercomplex that catalyzes the light-dependent oxidation of plastocyanin (or cytochrome c6 ) and the reduction of ferredoxin. This catalytic reaction is realized by a transmembrane electron transfer chain consisting of primary electron donor (a special chlorophyll (Chl) pair) and electron acceptors A0 , A1 , and three Fe4 S4 clusters, FX , FA , and FB . Here we report the PSI structure from a Chl d-dominated cyanobacterium Acaryochloris marina at 3.3 Å resolution obtained by single-particle cryo-electron microscopy. The A. marina PSI exists as a trimer with three identical monomers. Surprisingly, the structure reveals a unique composition of electron transfer chain in which the primary electron acceptor A0 is composed of two pheophytin a rather than Chl a found in any other well-known PSI structures. A novel subunit Psa27 is observed in the A. marina PSI structure. In addition, 77 Chls, 13 α-carotenes, two phylloquinones, three Fe-S clusters, two phosphatidyl glycerols, and one monogalactosyl-diglyceride were identified in each PSI monomer. Our results provide a structural basis for deciphering the mechanism of photosynthesis in a PSI complex with Chl d as the dominating pigments and absorbing far-red light.

Structure of plant photosystem I-light harvesting complex I supercomplex at 2.4 Å resolution.

Photosystem I (PSI) is one of the two photosystems in photosynthesis, and performs a series of electron transfer reactions leading to the reduction of ferredoxin. In higher plants, PSI is surrounded by four light-harvesting complex I (LHCI) subunits, which harvest and transfer energy efficiently to the PSI core. The crystal structure of PSI-LHCI supercomplex has been analyzed up to 2.6 Å resolution, providing much information on the arrangement of proteins and cofactors in this complicated supercomplex. Here we have optimized crystallization conditions, and analyzed the crystal structure of PSI-LHCI at 2.4 Å resolution. Our structure showed some shift of the LHCI, especially the Lhca4 subunit, away from the PSI core, suggesting the indirect connection and inefficiency of energy transfer from this Lhca subunit to the PSI core. We identified five new lipids in the structure, most of them are located in the gap region between the Lhca subunits and the PSI core. These lipid molecules may play important roles in binding of the Lhca subunits to the core, as well as in the assembly of the supercomplex. The present results thus provide novel information for the elucidation of the mechanisms for the light-energy harvesting, transfer and assembly of this supercomplex.

Structure of photosystem I-LHCI-LHCII from the green alga Chlamydomonas reinhardtii in State 2.

Photosystem I (PSI) and II (PSII) balance their light energy distribution absorbed by their light-harvesting complexes (LHCs) through state transition to maintain the maximum photosynthetic performance and to avoid photodamage. In state 2, a part of LHCII moves to PSI, forming a PSI-LHCI-LHCII supercomplex. The green alga Chlamydomonas reinhardtii exhibits state transition to a far larger extent than higher plants. Here we report the cryo-electron microscopy structure of a PSI-LHCI-LHCII supercomplex in state 2 from C. reinhardtii at 3.42 Å resolution. The result reveals that the PSI-LHCI-LHCII of C. reinhardtii binds two LHCII trimers in addition to ten LHCI subunits. The PSI core subunits PsaO and PsaH, which were missed or not well-resolved in previous Cr-PSI-LHCI structures, are observed. The present results reveal the organization and assembly of PSI core subunits, LHCI and LHCII, pigment arrangement, and possible pathways of energy transfer from peripheral antennae to the PSI core.

Antenna arrangement and energy-transfer pathways of PSI-LHCI from the moss Physcomitrella patens.

Plants harvest light energy utilized for photosynthesis by light-harvesting complex I and II (LHCI and LHCII) surrounding photosystem I and II (PSI and PSII), respectively. During the evolution of green plants, moss is at an evolutionarily intermediate position from aquatic photosynthetic organisms to land plants, being the first photosynthetic organisms that landed. Here, we report the structure of the PSI-LHCI supercomplex from the moss Physcomitrella patens (Pp) at 3.23 Å resolution solved by cryo-electron microscopy. Our structure revealed that four Lhca subunits are associated with the PSI core in an order of Lhca1-Lhca5-Lhca2-Lhca3. This number is much decreased from 8 to 10, the number of subunits in most green algal PSI-LHCI, but the same as those of land plants. Although Pp PSI-LHCI has a similar structure as PSI-LHCI of land plants, it has Lhca5, instead of Lhca4, in the second position of Lhca, and several differences were found in the arrangement of chlorophylls among green algal, moss, and land plant PSI-LHCI. One chlorophyll, PsaF-Chl 305, which is found in the moss PSI-LHCI, is located at the gap region between the two middle Lhca subunits and the PSI core, and therefore may make the excitation energy transfer from LHCI to the core more efficient than that of land plants. On the other hand, energy-transfer paths at the two side Lhca subunits are relatively conserved. These results provide a structural basis for unravelling the mechanisms of light-energy harvesting and transfer in the moss PSI-LHCI, as well as important clues on the changes of PSI-LHCI after landing.