In situ structure of the red algal phycobilisome-PSII-PSI-LHC megacomplex.

In oxygenic photosynthetic organisms, light energy is captured by antenna systems and transferred to photosystem II (PSII) and photosystem I (PSI) to drive photosynthesis1,2. The antenna systems of red algae consist of soluble phycobilisomes (PBSs) and transmembrane light-harvesting complexes (LHCs)3. Excitation energy transfer pathways from PBS to photosystems remain unclear owing to the lack of structural information. Here we present in situ structures of PBS-PSII-PSI-LHC megacomplexes from the red alga Porphyridium purpureum at near-atomic resolution using cryogenic electron tomography and in situ single-particle analysis4, providing interaction details between PBS, PSII and PSI. The structures reveal several unidentified and incomplete proteins and their roles in the assembly of the megacomplex, as well as a huge and sophisticated pigment network. This work provides a solid structural basis for unravelling the mechanisms of PBS-PSII-PSI-LHC megacomplex assembly, efficient energy transfer from PBS to the two photosystems, and regulation of energy distribution between PSII and PSI.

Structural insight into the mechanism of energy transfer in cyanobacterial phycobilisomes.

Phycobilisomes (PBS) are the major light-harvesting machineries for photosynthesis in cyanobacteria and red algae and they have a hierarchical structure of a core and peripheral rods, with both consisting of phycobiliproteins and linker proteins. Here we report the cryo-EM structures of PBS from two cyanobacterial species, Anabaena 7120 and Synechococcus 7002. Both PBS are hemidiscoidal in shape and share a common triangular core structure. While the Anabaena PBS has two additional hexamers in the core linked by the 4th linker domain of ApcE (LCM). The PBS structures predict that, compared with the PBS from red algae, the cyanobacterial PBS could have more direct routes for energy transfer to ApcD. Structure-based systematic mutagenesis analysis of the chromophore environment of ApcD and ApcF subunits reveals that aromatic residues are critical to excitation energy transfer (EET). The structures also suggest that the linker protein could actively participate in the process of EET in both rods and the cores. These results provide insights into the organization of chromophores and the mechanisms of EET within cyanobacterial PBS.

Marine cyanobacteria tune energy transfer efficiency in their light-harvesting antennae by modifying pigment coupling.

Photosynthetic light harvesting is the first step in harnessing sunlight toward biological productivity. To operate efficiently under a broad and dynamic range of environmental conditions, organisms must tune the harvesting process according to the available irradiance. The marine cyanobacteria Synechococcus WH8102 species is well-adapted to vertical mixing of the water column. By studying its responses to different light regimes, we identify a new photo-acclimation strategy. Under low light, the phycobilisome (PBS) is bigger, with extended rods, increasing the absorption cross-section. In contrast to what was reported in vascular plants and predicted by Forster resonance energy transfer (FRET) calculations, these longer rods transfer energy faster than in the phycobilisomes of cells acclimated to a higher light intensity. Comparison of cultures grown under different blue light intensities, using fluorescence lifetime and emission spectra dependence on temperature at the range of 4-200 K in vivo, indicates that the improved transfer arises from enhanced energetic coupling between the antenna rods' pigments. We suggest two physical models according to which the enhanced coupling strength results either from additional coupled pathways formed by rearranging rod packing or from the coupling becoming non-classical. In both cases, the energy transfer would be more efficient than standard one-dimensional FRET process. These findings suggest that coupling control can be a major factor in photosynthetic antenna acclimation to different light conditions.

Structural basis of energy transfer in Porphyridium purpureum phycobilisome.

Photosynthetic organisms have developed various light-harvesting systems to adapt to their environments1. Phycobilisomes are large light-harvesting protein complexes found in cyanobacteria and red algae2-4, although how the energies of the chromophores within these complexes are modulated by their environment is unclear. Here we report the cryo-electron microscopy structure of a 14.7-megadalton phycobilisome with a hemiellipsoidal shape from the red alga Porphyridium purpureum. Within this complex we determine the structures of 706 protein subunits, including 528 phycoerythrin, 72 phycocyanin, 46 allophycocyanin and 60 linker proteins. In addition, 1,598 chromophores are resolved comprising 1,430 phycoerythrobilin, 48 phycourobilin and 120 phycocyanobilin molecules. The markedly improved resolution of our structure compared with that of the phycobilisome of Griffithsia pacifica5 enabled us to build an accurate atomic model of the P. purpureum phycobilisome system. The model reveals how the linker proteins affect the microenvironment of the chromophores, and suggests that interactions of the aromatic amino acids of the linker proteins with the chromophores may be a key factor in fine-tuning the energy states of the chromophores to ensure the efficient unidirectional transfer of energy.

Biogenic regions of cyanobacterial thylakoids form contact sites with the plasma membrane.

Little is known about how the photosynthetic machinery is arranged in time and space during the biogenesis of thylakoid membranes. Using in situ cryo-electron tomography to image the three-dimensional architecture of the cyanobacterium Synechocystis, we observed that the tips of multiple thylakoids merge to form a substructure called the 'convergence membrane'. This high-curvature membrane comes into close contact with the plasma membrane at discrete sites. We generated subtomogram averages of 70S ribosomes and array-forming phycobilisomes, then mapped these structures onto the native membrane architecture as markers for protein synthesis and photosynthesis, respectively. This molecular localization identified two distinct biogenic regions in the thylakoid network: thylakoids facing the cytosolic interior of the cell that were associated with both marker complexes, and convergence membranes that were decorated by ribosomes but not phycobilisomes. We propose that the convergence membranes perform a specialized biogenic function, coupling the synthesis of thylakoid proteins with the integration of cofactors from the plasma membrane and the periplasmic space.

Phycobilisomes from the mutant cyanobacterium Synechocystis sp. PCC 6803 missing chromophore domain of ApcE.

Phycobilisome (PBS) is a giant photosynthetic antenna associated with the thylakoid membranes of cyanobacteria and red algae. PBS consists of two domains: central core and peripheral rods assembled of disc-shaped phycobiliprotein aggregates and linker polypeptides. The study of the PBS architecture is hindered due to the lack of the data on the structure of the large ApcE-linker also called LCM. ApcE participates in the PBS core stabilization, PBS anchoring to the photosynthetic membrane, transfer of the light energy to chlorophyll, and, very probably, the interaction with the orange carotenoid protein (OCP) during the non-photochemical PBS quenching. We have constructed the cyanobacterium Synechocystis sp. PCC 6803 mutant lacking 235 N-terminal amino acids of the chromophorylated PBLCM domain of ApcE. The altered fluorescence characteristics of the mutant PBSs indicate that the energy transfer to the terminal emitters within the mutant PBS is largely disturbed. The PBSs of the mutant become unable to attach to the thylakoid membrane, which correlates with the identified absence of the energy transfer from the PBSs to the photosystem II. At the same time, the energy transfer from the PBS to the photosystem I was registered in the mutant cells and seems to occur due to the small cylindrical CpcG2-PBSs formation in addition to the conventional PBSs. In contrast to the wild type Synechocystis, the OCP-mediated non-photochemical PBS quenching was not registered in the mutant cells. Thus, the PBLCM domain takes part in formation of the OCP binding site in the PBS.

Structure of phycobilisome from the red alga Griffithsia pacifica.

Life on Earth depends on photosynthesis for its conversion of solar energy to chemical energy. Photosynthetic organisms have developed a variety of light-harvesting systems to capture sunlight. The largest light-harvesting complex is the phycobilisome (PBS), the main light-harvesting antenna in cyanobacteria and red algae. It is composed of phycobiliproteins and linker proteins but the assembly mechanisms and energy transfer pathways of the PBS are not well understood. Here we report the structure of a 16.8-megadalton PBS from a red alga at 3.5 Å resolution obtained by single-particle cryo-electron microscopy. We modelled 862 protein subunits, including 4 linkers in the core, 16 rod-core linkers and 52 rod linkers, and located a total of 2,048 chromophores. This structure reveals the mechanisms underlying specific interactions between linkers and phycobiliproteins, and the formation of linker skeletons. These results provide a firm structural basis for our understanding of complex assembly and the mechanisms of energy transfer within the PBS.

Comparative study of thylakoid membranes in terminal heterocysts and vegetative cells from two cyanobacteria, Rivularia M-261 and Anabaena variabilis, by fluorescence and absorption spectral microscopy.

Heterocyst is a nitrogen-fixing cell differentiated from a cell for oxygen-evolving photosynthesis (vegetative cell) in some filamentous cyanobacteria when fixed nitrogen (e.g., ammonia and nitrate) is limited. Heterocysts appear at multiple separated positions in a single filament with an interval of 10-20 cells in some genera (including Anabaena variabilis). In other genera, a single heterocyst appears only at the basal terminal in a filament (including Rivularia M-261). Such morphological diversity may necessitate different properties of heterocysts. However, possible differences in heterocysts have largely remained unexplored due to the minority of heterocysts among major vegetative cells. Here, we have applied spectroscopic microscopy to Rivularia and A. variabilis to analyze their thylakoid membranes in individual cells. Absorption and fluorescence spectral imaging enabled us to estimate concentrations and interconnections of key photosynthetic components like photosystem I (PSI), photosystem II (PSII) and subunits of light-harvesting phycobilisome including phycocyanin (PC). The concentration of PC in heterocysts of Rivularia is far higher than that of A. variabilis. Fluorescence quantum yield of PC in Rivularia heterocysts was found to be virtually the same as those in its vegetative cells, while fluorescence quantum yield of PC in A. variabilis heterocysts was enhanced in comparison with its vegetative cells. PSI concentration in the thylakoid membranes of heterocysts seems to remain nearly the same as those of the vegetative cells in both the species. The average stoichiometric ratio between PSI monomer and PC hexamer in Rivularia heterocysts is estimated to be about 1:1.

[Cyanobacterial Phycobilisomes and Phycobiliproteins].

In cyanobacteria, phycobilisomes (PBS) act as antennae of the photosynthetic pigment apparatus. They contain brightly colored phycobiliproteins (PBP) and form giant supramolecular complexes (up to 3000-7000 kDa) containing 200 to 500 phycobilin chromophores covalently bound to the proteins. Over ten various PBP are known, which fall into three groups: phycoerythrins, phycocyanins, and allophycocyanins. Hollow disks of PBP trimers and hexamers are arranged into cylinders by colorless linker proteins; the cylinders are then assembled into PBS. Typical semidiscoid PBS consist of a central nucleus formed by three allophycocyanin cylinders and of six lateral cylinders consisting of other PBP and attached as fans to the nucleus. The PBS number, size, and pigment composition in cyanobacteria depend on illumination and other ambient factors. While PBS have certain advantages compared to other antennae, these pigment-protein complexes require more energy than the chlorophyll a/b- and chlorophyll a/c-proteins of oxygenic photosynthetic organisms.

Structural studies show energy transfer within stabilized phycobilisomes independent of the mode of rod-core assembly.

The major light harvesting complex in cyanobacteria and red algae is the phycobilisome (PBS), comprised of hundreds of seemingly similar chromophores, which are protein bound and assembled in a fashion that enables highly efficient uni-directional energy transfer to reaction centers. The PBS is comprised of a core containing 2-5 cylinders surrounded by 6-8 rods, and a number of models have been proposed describing the PBS structure. One of the most critical steps in the functionality of the PBS is energy transfer from the rod substructures to the core substructure. In this study we compare the structural and functional characteristics of high-phosphate stabilized PBS (the standard fashion of stabilization of isolated complexes) with cross-linked PBS in low ionic strength buffer from two cyanobacterial species, Thermosynechococcus vulcanus and Acaryochloris marina. We show that chemical cross-linking preserves efficient energy transfer from the phycocyanin containing rods to the allophycocyanin containing cores with fluorescent emission from the terminal emitters. However, this energy transfer is shown to exist in PBS complexes of different structures as characterized by determination of a 2.4Å structure by X-ray crystallography, single crystal confocal microscopy, mass spectrometry and transmission electron microscopy of negatively stained and cryogenically preserved complexes. We conclude that the PBS has intrinsic structural properties that enable efficient energy transfer from rod substructures to the core substructures without requiring a single unique structure. We discuss the significance of our observations on the functionality of the PBS in vivo.

Organization and flexibility of cyanobacterial thylakoid membranes examined by neutron scattering.

Cyanobacteria are prokaryotes that can use photosynthesis to convert sunlight into cellular fuel. Knowledge of the organization of the membrane systems in cyanobacteria is critical to understanding the metabolic processes in these organisms. We examined the wild-type strain of Synechocystis sp. PCC 6803 and a series of mutants with altered light-harvesting phycobilisome antenna systems for changes in thylakoid membrane architecture under different conditions. Using small-angle neutron scattering, it was possible to resolve correlation distances of subcellular structures in live cells on the nanometer scale and capture dynamic light-induced changes to these distances. Measurements made from samples with varied scattering contrasts confirmed that these distances could be attributed to the thylakoid lamellar system. We found that the changes to the thylakoid system were reversible between light- and dark-adapted states, demonstrating a robust structural flexibility in the architecture of cyanobacterial cells. Chemical disruption of photosynthetic electron transfer diminished these changes, confirming the involvement of the photosynthetic apparatus. We have correlated these findings with electron microscopy data to understand the origin of the changes in the membranes and found that light induces an expansion in the center-to-center distances between the thylakoid membrane layers. These combined data lend a dynamic dimension to the intracellular organization in cyanobacterial cells.

Structural organisation of phycobilisomes from Synechocystis sp. strain PCC6803 and their interaction with the membrane.

In cyanobacteria, the harvesting of light energy for photosynthesis is mainly carried out by the phycobilisome - a giant, multi-subunit pigment-protein complex. This complex is composed of heterodimeric phycobiliproteins that are assembled with the aid of linker polypeptides such that light absorption and energy transfer to photosystem II are optimised. In this work we have studied, using single particle electron microscopy, the phycobilisome structure in mutants lacking either two or all three of the phycocyanin hexamers. The images presented give much greater detail than those previously published, and in the best two-dimensional projection maps a resolution of 13 A was achieved. As well as giving a better overall picture of the assembly of phycobilisomes, these results reveal new details of the association of allophycocyanin trimers within the core. Insights are gained into the attachment of this core to the membrane surface, essential for efficient energy transfer to photosystem II. Comparison of projection maps of phycobilisomes with and without reconstituted ferredoxin:NADP oxidoreductase suggests a location for this enzyme within the complex at the rod-core interface.

Structure and organization of phycobilisomes on membranes of the red alga Porphyridium cruentum.

In the present work, electron microscopy and single particle averaging was performed to investigate the supramolecular architecture of hemiellipsoidal phycobilisomes from the unicellular red alga Porphyridium cruentum. The dimensions were measured as 60 x 41 x 34 nm (length x width x height) for randomly ordered phycobilisomes, seen under high-light conditions. The hemiellipsoidal phycobilisomes were found to have a relatively flexible conformation. In closely packed semi-crystalline arrays, observed under low-light conditions, the width is reduced to 31 or 35 nm, about twice the width of the phycobilisome of the cyanobacterium Synechocystis sp. PCC 6803. Since the latter size matches the width of dimeric PSII, we suggest that one PBS lines up with one PSII dimer in cyanobacteria. In red algae, a similar 1:1 ratio under low-light conditions may indicate that the red algal phycobilisome is enlarged by a membrane-bound peripheral antenna which is absent in cyanobacteria.

Energy transfer processes in Gloeobacter violaceus PCC 7421 that possesses phycobilisomes with a unique morphology.

We examined energy transfer dynamics in phycobilisomes (PBSs) of cyanobacteria in relation to the morphology and pigment compositions of PBSs. We used Gloeobacter violaceus PCC 7421 and measured time-resolved fluorescence spectra in three types of samples, i.e., intact cells, PBSs, and rod assemblies separated from cores. Fremyella diplosiphon, a cyanobacterial species well known for its complementary chromatic adaptation, was used for comparison after growing under red or green light. Spectral data were analyzed by the fluorescence decay-associated spectra with components common in lifetimes with a time resolution of 3 ps/channel and a spectral resolution of 2 nm/channel. This ensured a higher resolution of the energy transfer kinetics than those obtained by global analysis with fewer sampling intervals. We resolved four spectral components in phycoerythrin (PE), three in phycocyanin (PC), two in allophycocyanin, and two in photosystem II. The bundle-like PBSs of G. violaceus showed multiple energy transfer pathways; fast ( approximately 10 ps) and slow ( approximately 100 ps and approximately 500 ps) pathways were found in rods consisting of PE and PC. Energy transfer time from PE to PC was two times slower in G. violaceus than in F. diplosiphon grown under green light.

Three-dimensional architecture of phycobilisomes from Nostoc flagelliforme revealed by single particle electron microscopy.

Phycobilisomes are protein complexes that harvest light and transfer energy to the photo system. Here, the three dimensional structure of intact phycobilisomes from Nostoc flagelliforme is studied by a combination of negative stain electron microscopy and cryo-electron microscopy. Results show that the intact phycobilisomes are composed of a tricylindrical core and six rods. Each allophycocyanin cylinder presents a double-layered structure when viewed from the side and a triangular shape when viewed from the top. These characteristics indicate that allophycocyanin trimers in the intact phycobilisomes are arranged into hexameric oligomers in a parallel manner.

Involvement of phycobilisome diffusion in energy quenching in cyanobacteria.

Nonphotochemical quenching (NPQ) of excitation energy is a well-established phenomenon in green plants, where it serves to protect the photosynthetic apparatus from photodamage under excess illumination. The induction of NPQ involves a change in the function of the light-harvesting apparatus, with the formation of quenching centers that convert excitation energy into heat. Recently, a comparable phenomenon was demonstrated in cyanobacteria grown under iron-starvation. Under these conditions, an additional integral membrane chlorophyll-protein, IsiA, is synthesized, and it is therefore likely that IsiA is required for NPQ in cyanobacteria. We have previously used fluorescence recovery after photobleaching to show that phycobilisomes diffuse rapidly on the membrane surface, but are immobilized when cells are immersed in high-osmotic strength buffers, apparently because the interaction between phycobilisomes and reaction centers is stabilized. Here, we show that when cells of the cyanobacterium Synechocystis sp. PCC 6803 subjected to prolonged iron-deprivation are immersed in 1 m phosphate buffer, NPQ can still be induced as normal by high light. However, the formation of the quenched state is irreversible under these conditions, suggesting that it involves the coupling of free phycobilisomes to an integral-membrane complex, an interaction that is stabilized by 1 m phosphate. Fluorescence spectra are consistent with this idea. Fluorescence recovery after photobleaching measurements confirm that the induction of NPQ in the presence of 1 m phosphate is accompanied by immobilization of the phycobilisomes. We propose as a working hypothesis that a major component of the fluorescence quenching observed in iron-starved cyanobacteria arises from the coupling of free phycobilisomes to IsiA.

Phycobilisome rod mutants in Synechocystis sp. strain PCC6803.

The phycobilisome is a large pigment-protein assembly that harvests light energy for photosynthesis. This supramolecular complex is composed of two main structures: a core substructure and peripheral rods. Linker polypeptides assemble phycobiliproteins within these structures and optimize light absorption and energy transfer. Mutations have been constructed in three rod-linker-coding genes located in the cpc operon of Synechocystis sp. strain PCC6803. The cpcC1 gene encoding the 33 kDa linker is found to be epistatic to cpcC2 encoding the 30 kDa linker, indicating a specific role for each of these two linkers in rod growth. This corroborates studies on the sequential degradation of phycobilisomes upon nitrogen starvation. Three allelic mutants affecting cpcC2 revealed a polar effect of commonly used cassettes (aphI, aadA) on the operon steady-state transcripts and an effect of rod linker availability on the amount of phycocyanin incorporated in the phycobilisome. This led to the proposal that regulation of rod length could occur through processing of transcripts upstream of the cpcC2 gene.

Langmuir-Blodgett film of phycobilisomes from blue-green alga Spirulina platensis.

The phycobilisomes were isolated from blue-green alga Spirulina platensis, and could form monolayer film at air/water interface. The monolayer film of phycobilisomes was transferred to newly cleaved mica, and coated with gold. Scanning tunneling microscope was used to investigate the structure of the Langmuir-Blodgett film of phycobilisomes. It was shown that phycobilisomes in the monolayer arrayed in rows with core attaching on the substrate surface and rods radiating towards the air phase, this phenomenon was similar to the arrangement of phycobilisomes on cytoplasmic surface of thylakoid membrane in vivo. The possible applications of the Langmuir-Blodgett film of phycobilisomes were also discussed.