Characterization and responses to environmental cues of a photosynthetic antenna-deficient mutant of the filamentous cyanobacterium Anabaena sp. PCC 7120

The cyanobacterial phycobilisome (PBS) is a giant pigment-protein complex which harvests light energy for photosynthesis and comprises two structures: a core and peripheral rods. Most studies on PBS structure and function are based on mutants of unicellular strains. In this report, we describe the phenotypic and genetic characterization of a transposon mutant of the filamentous Anabaena sp. strain PCC 7120, denoted LC1, which cannot synthesize the phycobiliprotein phycocyanin (PC), the main component of the rods; in this mutant, the transposon had inserted into the cpcB gene (orf alr0528) which putatively encodes PC-β chain. Mutant LC1 was able to synthesize phycoerythrocyanin (PEC), a phycobiliprotein (PBP) located at the terminal region of the rods; but in the absence of PC, PEC did not attach to the PBSs that only retained the allophycocyanin (APC) core; ferredoxin: NADP⁺-oxidoreductase (FNR) that is associated with the PBS in the wild type, was not found in isolated PBSs from LC1. The performance of the mutant exposed to different environmental conditions was evaluated. The mutant phenotype was successfully complemented by cloning and transfer of the wild type complete cpc operon to mutant LC1. Interestingly, LC1 compensated its mutation by significantly increasing the number of its core-PBS and the effective quantum yield of photosystem II (PSII) photochemistry; this feature suggests a more efficient energy conversion in the mutant which may be useful for biotechnological applications.     

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 2.4 Å 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.     

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 L_CM. 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 PBL_CM 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 PBL_CM domain takes part in formation of the OCP binding site in the PBS.     

Structural organization of an intact phycobilisome and its association with photosystem II

Phycobilisomes (PBSs) are light-harvesting antennae that transfer energy to photosynthetic reaction centers in cyanobacteria and red algae. PBSs are supermolecular complexes composed of phycobiliproteins (PBPs) that bear chromophores for energy absorption and linker proteins. Although the structures of some individual components have been determined using crystallography, the three-dimensional structure of an entire PBS complex, which is critical for understanding the energy transfer mechanism, remains unknown. Here, we report the structures of an intact PBS and a PBS in complex with photosystem II (PSII) from Anabaena sp. strain PCC 7120 using single-particle electron microscopy in combination with biochemical and molecular analyses. In the PBS structure, all PBP trimers and the conserved linker protein domains were unambiguously located, and the global distribution of all chromophores was determined. We provide evidence that ApcE and ApcF are critical for the formation of a protrusion at the bottom of PBS, which plays an important role in mediating PBS interaction with PSII. Our results provide insights into the molecular architecture of an intact PBS at different assembly levels and provide the basis for understanding how the light energy absorbed by PBS is transferred to PSII.     

Structural integrity of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 phycobilisomes evaluated by means of differential scanning calorimetry

Phycobilisomes (PBSs) are supramolecular pigment–protein complexes that serve as light-harvesting antennae in cyanobacteria. They are built up by phycobiliproteins assembled into allophycocyanin core cylinders (ensuring the physical interaction with the photosystems) and phycocyanin rods (protruding from the cores and having light-harvesting function), the whole PBSs structure being maintained by linker proteins. PBSs play major role in light-harvesting optimization in cyanobacteria; therefore, the characterization of their structural integrity in intact cells is of great importance. The present study utilizes differential scanning calorimetry and spectroscopy techniques to explore for the first time, the thermodynamic stability of PBSs in intact Synechocystis sp. PCC 6803 cells and to probe its alteration as a result of mutations or under different growth conditions. As a first step, we characterize the thermodynamic behavior of intact and dismantled PBSs isolated from wild-type cells (having fully assembled PBSs) and from CK mutant cells (that lack phycocyanin rods and contain only allophycocyanin cores), and identified the thermal transitions of phycocyanin and allophycocyanin units in vitro. Next, we demonstrate that in intact cells PBSs exhibit sharp, high amplitude thermal transition at about 63 °C that strongly depends on the structural integrity of the PBSs supercomplex. Our findings implicate that calorimetry could offer a valuable approach for the assessment of the influence of variety of factors affecting the stability and structural organization of phycobilisomes in intact cyanobacterial cells.     

Allophycocyanin trimer stability and functionality are primarily due to polar enhanced hydrophobicity of the phycocyanobilin binding pocket

Allophycocyanin (APC) is the primary pigment-protein component of the cores of the phycobilisome antenna complex. In addition to an extremely high degree of amino acid sequence conservation, the overall structures of APC from both mesophilic and thermophilic species are almost identical at all levels of assembly, yet APC from thermophilic organisms should have structural attributes that prevent thermally induced denaturation. We determined the structure of APC from the thermophilic cyanobacterium Thermosynechococcus vulcanus to 2.9 Å, reaffirming the conservation of structural similarity with APC from mesophiles. We provide spectroscopic evidence that T. vulcanus APC is indeed more stable at elevated temperatures in vitro, when compared with the APC from mesophilic species. APC thermal and chemical stability levels are further enhanced when monitored in the presence of high concentrations of buffered phosphate, which increases the strength of hydrophobic interactions, and may mimic the effect of cytosolic crowding. Absorption spectroscopy, size-exclusion HPLC, and native gel electrophoresis also show that the thermally or chemically induced changes in the APC absorption spectra that result in the loss of the prominent 652-nm band in trimeric APC are not a result of physical monomerization. We propose that the bathochromic shift that occurs in APC upon trimerization is due to the coupling of the hydrophobicity of the α84 phycocyanobilin cofactor environment created by a deep cleft formed by the β subunit with highly charged flanking regions. This arrangement also provides the additional stability required by thermophiles at elevated temperatures. The chemical environment that induces the bathochromic shift in APC trimers is different from the source of shifts in the absorption of monomers of the terminal energy acceptors APC^B and L_CM, as visualized by the building of molecular models.     

Spectroscopic properties of the C-phycocyanin-allophycocyanin conjugate and the isolated phycobilisomes from Spirulina platensis

C-phycocyanin (CPC) and allophycocyanin (APC) were purified from Spirulina platensis, then the CPC was attached covalently to the APC by reacting their ∈-amino groups. The excitation energy could be transferred from the CPC to the APC in the CPC-APC conjugate. Intact phycobilisomes (PBS), consisting of CPC, APC, colourless linker polypeptides, and APC B or Lcm, were isolated from S. platensis. Spectroscopic properties of the isolated PBSs kept at 20 °C for various times showed that the connection between the APC and the APC B or Lcm was looser than that between the CPC and the APC in the isolated PBSs. The CPC-APC conjugate was more stable than the isolated PBSs, and the linker polypeptides had a minor influence on the excitation energy transfer characteristic between different phycobiliproteins in the PBS.     

Tight association of CpcL with photosystem I in Anabaena sp. PCC 7120 grown under iron-deficient conditions

Phycobilisomes (PBSs), which are huge pigment-protein complexes displaying distinctive color variations, bind to photosystem cores for excitation-energy transfer. It is known that isolation of supercomplexes consisting of PBSs and photosystem I (PSI) or PBSs and photosystem II is challenging due to weak interactions between PBSs and the photosystem cores. In this study, we succeeded in purifying PSI-monomer-PBS and PSI-dimer-PBS supercomplexes from the cyanobacterium Anabaena sp. PCC 7120 grown under iron-deficient conditions by anion-exchange chromatography, followed by trehalose density gradient centrifugation. The absorption spectra of the two types of supercomplexes showed apparent bands originating from PBSs, and their fluorescence-emission spectra exhibited characteristic peaks of PBSs. Two-dimensional blue-native (BN)/SDS-PAGE of the two samples showed a band of CpcL, which is a linker protein of PBS, in addition to PsaA/B. Since interactions of PBSs with PSI are easily dissociated during BN-PAGE using thylakoids from this cyanobacterium grown under iron-replete conditions, it is suggested that iron deficiency for Anabaena induces tight association of CpcL with PSI, resulting in the formation of PSI-monomer-PBS and PSI-dimer-PBS supercomplexes. Based on these findings, we discuss interactions of PBSs with PSI in Anabaena.     

Spectroscopic properties of the C-phycocyanin-allophycocyanin conjugate and the isolated phycobilisomes from Spirulina platensis

C-phycocyanin (CPC) and allophycocyanin (APC) were purified from Spirulina platensis, then the CPC was attached covalently to the APC by reacting their ∈-amino groups. The excitation energy could be transferred from the CPC to the APC in the CPC-APC conjugate. Intact phycobilisomes (PBS), consisting of CPC, APC, colourless linker polypeptides, and APC B or Lcm, were isolated from S. platensis. Spectroscopic properties of the isolated PBSs kept at 20 °C for various times showed that the connection between the APC and the APC B or Lcm was looser than that between the CPC and the APC in the isolated PBSs. The CPC-APC conjugate was more stable than the isolated PBSs, and the linker polypeptides had a minor influence on the excitation energy transfer characteristic between different phycobiliproteins in the PBS. This revised version was published online in June 2006 with corrections to the Cover Date.     

Light-induced excitation energy redistribution in Spirulina platensis cells: "spillover" or "mobile PBSs"?

State transitions induced by light and redox were investigated by observing the 77 K fluorescence spectra for the intact cells of Spirulina platensis. To clarify if phycobilisomes (PBSs) take part in the state transition, the contributions of PBSs to light-induced state transition were studied in untreated cells and the cells treated by betaine which fixed PBSs firmly on the thylakoid membranes. It was observed that the betaine-treated cells did not show any light-induced state transition. This result definitely confirmed that the light-induced excitation energy regulation between the two photosystems is mainly dependent on a spatial movement of PBSs on the thylakoid membranes, which makes PBS cores partially decoupled from photosystem II (PSII) while PBS rods more strongly coupled with photosystem I (PSI) during the transition from state 1 to state 2. On the other hand, an energy exchange between the two photosystems was observed in both untreated and betaine-treated cells during redox-induced state transition. These observations suggested that two different mechanisms were involved in the light-induced state transition and the redox-induced one. The former involves only a physical movement of PBSs, while the latter involves not only the movement of PBS but also energy spillover from PSII to PSI. A model for light-induced state transition was proposed based on the current results as well as well known knowledge.     

Concentration-based self-assembly of phycocyanin

Cyanobacteria light-harvesting complexes can change their structure to cope with fluctuating environmental conditions. Studying in vivo structural changes is difficult owing to complexities imposed by the cellular environment. Mimicking this system in vitro is challenging, as well. The in vivo system is highly concentrated, and handling similar in vitro concentrated samples optically is difficult because of high absorption. In this research, we mapped the cyanobacteria antennas self-assembly pathways using highly concentrated solutions of phycocyanin (PC) that mimic the in vivo condition. PC was isolated from the thermophilic cyanobacterium Thermosynechococcus vulcanus and measured by several methods. PC has three oligomeric states: hexamer, trimer, and monomer. We showed that the oligomeric state was changed upon increase of PC solution concentration. This oligomerization mechanism may enable photosynthetic organisms to adapt their light-harvesting system to a wide range of environmental conditions.     

Investigation of Phycobilisome Subunit Interaction Interfaces by Coupled Cross-linking and Mass Spectrometry

The phycobilisome (PBS) is an extremely large light-harvesting complex, common in cyanobacteria and red algae, composed of rods and core substructures. These substructures are assembled from chromophore-bearing phycocyanin and allophycocyanin subunits, nonpigmented linker proteins and in some cases additional subunits. To date, despite the determination of crystal structures of isolated PBS components, critical questions regarding the interaction and energy flow between rods and core are still unresolved. Additionally, the arrangement of minor PBS components located inside the core cylinders is unknown. Different models of the general architecture of the PBS have been proposed, based on low resolution images from electron microscopy or high resolution crystal structures of isolated components. This work presents a model of the assembly of the rods onto the core arrangement and for the positions of inner core components, based on cross-linking and mass spectrometry analysis of isolated, functional intact Thermosynechococcus vulcanus PBS, as well as functional cross-linked adducts. The experimental results were utilized to predict potential docking interactions of different protein pairs. Combining modeling and cross-linking results, we identify specific interactions within the PBS subcomponents that enable us to suggest possible functional interactions between the chromophores of the rods and the core and improve our understanding of the assembly, structure, and function of PBS.     

The phycobilisome core-membrane linkers from Synechocystis sp. PCC 6803 and red-algae assemble in the same topology

The phycobilisomes (PBSs) of cyanobacteria and red-algae are unique megadaltons light-harvesting protein-pigment complexes that utilize bilin derivatives for light absorption and energy transfer. Recently, the high-resolution molecular structures of red-algal PBSs revealed how the multi-domain core-membrane linker (L_CM) specifically organizes the allophycocyanin subunits in the PBS’s core. But, the topology of L_CM in these structures was different than that suggested for cyanobacterial PBSs based on lower-resolution structures. Particularly, the model for cyanobacteria assumed that the Arm2 domain of L_CM connects the two basal allophycocyanin cylinders, whereas the red-algal PBS structures revealed that Arm2 is partly buried in the core of one basal cylinder and connects it to the top cylinder. Here, we show by biochemical analysis of mutations in the apcE gene that encodes L_CM, that the cyanobacterial and red-algal L_CM topologies are actually the same. We found that removing the top cylinder linker domain in L_CM splits the PBS core longitudinally into two separate basal cylinders. Deleting either all or part of the helix-loop-helix domain at the N-terminal end of Arm2, disassembled the basal cylinders and resulted in degradation of the part containing the terminal emitter, ApcD. Deleting the following 30 amino-acids loop severely affected the assembly of the basal cylinders, but further deletion of the amino-acids at the C-terminal half of Arm2 had only minor effects on this assembly. Altogether, the biochemical data are consistent with the red-algal L_CM topology, suggesting that the PBS cores in cyanobacteria and red-algae assemble in the same way.     

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 (≈ 10 ps) and slow (≈ 100 ps and ≈ 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.     

Cyanobacterial Acclimation to Photosystem I or Photosystem II Light

The organization and function of the photochemical apparatus of Synechococcus 6301 was investigated in cells grown under yellow and red light regimes. Broadband yellow illumination is absorbed preferentially by the phycobilisome (PBS) whereas red light is absorbed primarily by the chlorophyll (Chl) pigment beds. Since PBSs are associated exclusively with photosystem II (PSII) and most of the Chl with photosystem I (PSI), it follows that yellow and red light regimes will create an imbalance of light absorption by the two photosystems. The cause and effect relationship between light quality and photosystem stoichiometry in Synechococcus was investigated. Cells grown under red light compensated for the excitation imbalance by synthesis/assembly of more PBS-PSII complexes resulting in high PSII/PSI = 0.71 and high bilin/Chl = 1.30. The adjustment of the photosystem stoichiometry in red light-grown cells was necessary and sufficient to establish an overall balanced absorption of red light by PSII and PSI. Cells grown under yellow light compensated for this excitation imbalance by assembly of more PSI complexes, resulting in low PSII/PSI = 0.27 and low bilin/Chl = 0.42. This adjustment of the photosystem stoichiometry in yellow light-grown cells was necessary but not quite sufficient to balance the absorption of yellow light by the PBS and the Chl pigment beds. A novel excitation quenching process was identified in yellow light-grown cells which dissipated approximately 40% of the PBS excitation, thus preventing over-excitation of PSII under yellow light conditions. It is hypothesized that State transitions in O₂ evolving photosynthetic organisms may serve as the signal for change in the stoichiometry of photochemical complexes in response to light quality conditions.     

Photoinhibition induced alterations in energy transfer process in phycobilisomes of PS II in the cyanobacterium, Spirulina platensis

Exposure of algae or plants to irradiance from above the light saturation point of photosynthesis is known as high light stress. This high light stress induces various responses including photoinhibition of the photosynthetic apparatus. The degree of photoinhibition could be clearly determined by measuring the parameters such as absorption and fluorescence of chromoproteins. In cyanobacteria and red algae, most of the photosystem (PS) II associated light harvesting is performed by a membrane attached complex called the phycobilisome (PBS). The effects of high intensity light (1000-4000 micromol photons m(-2) s(-1)) on excitation energy transfer from PBSs to PS II in a cyanobacterium Spirulina platensis were studied by measuring room temperature PC fluorescence emission spectra. High light (3000 micromol photons m(-2) s(-1)) stress had a significant effect on PC fluorescence emission spectra. On the other hand, light stress induced an increase in the ratio of PC fluorescence intensity of PBS indicating that light stress inhibits excitation energy transfer from PBS to PS II. The high light treatment to 3000 micromol photons m(-2) s(-1) caused disappearance of 31.5 kDa linker polypeptide which is known to link PC discs together. In addition we observed the similar decrease in the other polypeptide contents. Our data concludes that the Spirulina cells upon light treatment causes alterations in the phycobiliproteins (PBPs) and affects the energy transfer process within the PBSs.     

Regulation Mechanism of Excitation Energy Transfer in Phycobilisome-Thylakoid Membrane Complexes

Regulation mechanism of excitation energy transfer between phycobilisomes (PBS) and the photosynthetic reaction centres was studied by the state transition techniques in PBS-thylakoid membrane complexes. DCMU, betaine, and N-ethylmaleimide were applied to search for the details of energy transfer properties based on the steady fluorescence measurement and individual deconvolution spectra at state 2 or state 1. The closure of photosystem (PS) 2 did not influence on fluorescence yields of PS1, i.e., energy could not spill to PS1 from PS2. When the energy transfer pathway from PBS to PS1 was disturbed, the relative fluorescence yield of PS2 was almost the same as that of PS2 in complexes without treatment. If PBSs were fixed by betaine, the state transition process was restrained. Hence PBS may detach from PS2 and become associated to PS1 at state 2. Our results contradict the proposed "spill-over" or "PBS detachment" models and support the mobile "PBS model".     

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.     

Kinetics and dynamics for light state transition in cyanobacterium Spirulina platensis cells

Light state transition in oxygenic organisms was defined as the ability to equalize the excitation of the two photosystems for maximal photosynthetic efficiency. In cyanobacteria, extensive researches on state transition have continuously provided new knowledge in the past decades but the molecular mechanism and physiological significance are still ambiguous. In this work, kinetics and dynamics of the transition from state 1 to state 2 in cyanobacterium Spirulina platensis cells were studied at different intensity of orange light from 10 to 120 μmol m(-2) s(-1). It was revealed that the state transition worked constantly independent of light intensity while the rates varied. The synchronous fluorescence kinetics for phycobilisome (PBS) and photosystem components indicated that the state transition was entirely regulated by "mobile PBS", and continuously changed fluorescence amplitudes suggested a series of intermediate states were involved between state 1 and state 2. The dynamic property of PBS movement during the state transition was revealed by (1,0) distribution of photo-linkable PBSs, indicating a collective movement of all PBSs. The results suggest that state transition in cyanobacteria possesses not only physiological but also photochemical significance.