Structures of a phycobilisome in light-harvesting and photoprotected states.

Phycobilisome (PBS) structures are elaborate antennae in cyanobacteria and red algae1,2. These large protein complexes capture incident sunlight and transfer the energy through a network of embedded pigment molecules called bilins to the photosynthetic reaction centres. However, light harvesting must also be balanced against the risks of photodamage. A known mode of photoprotection is mediated by orange carotenoid protein (OCP), which binds to PBS when light intensities are high to mediate photoprotective, non-photochemical quenching3-6. Here we use cryogenic electron microscopy to solve four structures of the 6.2 MDa PBS, with and without OCP bound, from the model cyanobacterium Synechocystis sp. PCC 6803. The structures contain a previously undescribed linker protein that binds to the membrane-facing side of PBS. For the unquenched PBS, the structures also reveal three different conformational states of the antenna, two previously unknown. The conformational states result from positional switching of two of the rods and may constitute a new mode of regulation of light harvesting. Only one of the three PBS conformations can bind to OCP, which suggests that not every PBS is equally susceptible to non-photochemical quenching. In the OCP-PBS complex, quenching is achieved through the binding of four 34 kDa OCPs organized as two dimers. The complex reveals the structure of the active form of OCP, in which an approximately 60 Å displacement of its regulatory carboxy terminal domain occurs. Finally, by combining our structure with spectroscopic properties7, we elucidate energy transfer pathways within PBS in both the quenched and light-harvesting states. Collectively, our results provide detailed insights into the biophysical underpinnings of the control of cyanobacterial light harvesting. The data also have implications for bioengineering PBS regulation in natural and artificial light-harvesting systems.

Antenna Modification Leads to Enhanced Nitrogenase Activity in a High Light-Tolerant Cyanobacterium.

Biological nitrogen fixation is an energy-intensive process that contributes significantly toward supporting life on this planet. Among nitrogen-fixing organisms, cyanobacteria remain unrivaled in their ability to fuel the energetically expensive nitrogenase reaction with photosynthetically harnessed solar energy. In heterocystous cyanobacteria, light-driven, photosystem I (PSI)-mediated ATP synthesis plays a key role in propelling the nitrogenase reaction. Efficient light transfer to the photosystems relies on phycobilisomes (PBS), the major antenna protein complexes. PBS undergo degradation as a natural response to nitrogen starvation. Upon nitrogen availability, these proteins are resynthesized back to normal levels in vegetative cells, but their occurrence and function in heterocysts remain inconclusive. Anabaena 33047 is a heterocystous cyanobacterium that thrives under high light, harbors larger amounts of PBS in its heterocysts, and fixes nitrogen at higher rates compared to other heterocystous cyanobacteria. To assess the relationship between PBS in heterocysts and nitrogenase function, we engineered a strain that retains large amounts of the antenna proteins in its heterocysts. Intriguingly, under high light intensities, the engineered strain exhibited unusually high rates of nitrogenase activity compared to the wild type. Spectroscopic analysis revealed altered PSI kinetics in the mutant with increased cyclic electron flow around PSI, a route that contributes to ATP generation and nitrogenase activity in heterocysts. Retaining higher levels of PBS in heterocysts appears to be an effective strategy to enhance nitrogenase function in cyanobacteria that are equipped with the machinery to operate under high light intensities. IMPORTANCE The function of phycobilisomes, the large antenna protein complexes in heterocysts has long been debated. This study provides direct evidence of the involvement of these proteins in supporting nitrogenase activity in Anabaena 33047, a heterocystous cyanobacterium that has an affinity for high light intensities. This strain was previously known to be recalcitrant to genetic manipulation and, hence, despite its many appealing traits, remained largely unexplored. We developed a genetic modification system for this strain and generated a ΔnblA mutant that exhibited resistance to phycobilisome degradation upon nitrogen starvation. Physiological characterization of the strain indicated that PBS degradation is not essential for acclimation to nitrogen deficiency and retention of PBS is advantageous for nitrogenase function.

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.

Binding of red form of Orange Carotenoid Protein (OCP) to phycobilisome is not sufficient for quenching.

The Orange Carotenoid Protein (OCP) is responsible for photoprotection in many cyanobacteria. Absorption of blue light drives the conversion of the orange, inactive form (OCPO) to the red, active form (OCPR). Concomitantly, the N-terminal domain (NTD) and the C-terminal domain (CTD) of OCP separate, which ultimately leads to the formation of a quenched OCPR-PBS complex. The details of the photoactivation of OCP have been intensely researched. Binding site(s) of OCPR on the PBS core have also been proposed. However, the post-binding events of the OCPR-PBS complex remain unclear. Here, we demonstrate that PBS-bound OCPR is not sufficient as a PBS excitation energy quencher. Using site-directed mutagenesis, we generated a suite of single point mutations at OCP Leucine 51 (L51) of Synechocystis 6803. Steady-state and time-resolved fluorescence analyses demonstrated that all mutant proteins are unable to quench the PBS fluorescence, owing to either failed OCP binding to PBS, or, if bound, an OCP-PBS quenching state failed to form. The SDS-PAGE and Western blot analysis support that the L51A (Alanine) mutant binds to the PBS and therefore belongs to the second category. We hypothesize that upon binding to PBS, OCPR likely reorganizes and adopts a new conformational state (OCP3rd) different than either OCPO or OCPR to allow energy quenching, depending on the cross-talk between OCPR and its PBS core-binding counterpart.

Trimeric organization of photosystem I is required to maintain the balanced photosynthetic electron flow in cyanobacterium Synechocystis sp. PCC 6803.

In Synechocystis sp. PCC 6803 and some other cyanobacteria photosystem I reaction centres exist predominantly as trimers, with minor contribution of monomeric form, when cultivated at standard optimized conditions. In contrast, in plant chloroplasts photosystem I complex is exclusively monomeric. The functional significance of trimeric organization of cyanobacterial photosystem I remains not fully understood. In this study, we compared the photosynthetic characteristics of PSI in wild type and psaL knockout mutant. The results show that relative to photosystem I trimer in wild-type cells, photosystem I monomer in psaL- mutant has a smaller P700+ pool size under low and moderate light, slower P700 oxidation upon dark-to-light transition, and slower P700+ reduction upon light-to-dark transition. The mutant also shows strongly diminished photosystem I donor side limitations [quantum yield Y(ND)] at low, moderate and high light, but enhanced photosystem I acceptor side limitations [quantum yield Y(NA)], especially at low light (22 µmol photons m-2 s-1). In line with these functional characteristics are the determined differences in the relative expression genes encoding of selected electron transporters. The psaL- mutant showed significant (ca fivefold) upregulation of the photosystem I donor cytochrome c6, and downregulation of photosystem I acceptors (ferredoxin, flavodoxin) and proteins of alternative electron flows originating in photosystem I acceptor side. Taken together, our results suggest that photosystem I trimerization in wild-type Synechocystis cells plays a role in the protection of photosystem I from photoinhibition via maintaining enhanced donor side electron transport limitations and minimal acceptor side electron transport limitations at various light intensities.

Exploring the low photosynthetic efficiency of cyanobacteria in blue light using a mutant lacking phycobilisomes.

The ubiquitous chlorophyll a (Chl a) pigment absorbs both blue and red light. Yet, in contrast to green algae and higher plants, most cyanobacteria have much lower photosynthetic rates in blue than in red light. A plausible but not yet well-supported hypothesis is that blue light results in limited energy transfer to photosystem II (PSII), because cyanobacteria invest most Chl a in photosystem I (PSI), whereas their phycobilisomes (PBS) are mostly associated with PSII but do not absorb blue photons. In this paper, we compare the photosynthetic performance in blue and orange-red light of wildtype Synechocystis sp. PCC 6803 and a PBS-deficient mutant. Our results show that the wildtype had much lower biomass, Chl a content, PSI:PSII ratio and O2 production rate per PSII in blue light than in orange-red light, whereas the PBS-deficient mutant had a low biomass, Chl a content, PSI:PSII ratio, and O2 production rate per PSII in both light colors. More specifically, the wildtype displayed a similar low photosynthetic efficiency in blue light as the PBS-deficient mutant in both light colors. Our results demonstrate that the absorption of light energy by PBS and subsequent transfer to PSII are crucial for efficient photosynthesis in cyanobacteria, which may explain both the low photosynthetic efficiency of PBS-containing cyanobacteria and the evolutionary success of chlorophyll-based light-harvesting antennae in environments dominated by blue light.

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.

The photocycle of orange carotenoid protein conceals distinct intermediates and asynchronous changes in the carotenoid and protein components.

The 35-kDa Orange Carotenoid Protein (OCP) is responsible for photoprotection in cyanobacteria. It acts as a light intensity sensor and efficient quencher of phycobilisome excitation. Photoactivation triggers large-scale conformational rearrangements to convert OCP from the orange OCPO state to the red active signaling state, OCPR, as demonstrated by various structural methods. Such rearrangements imply a complete, yet reversible separation of structural domains and translocation of the carotenoid. Recently, dynamic crystallography of OCPO suggested the existence of photocycle intermediates with small-scale rearrangements that may trigger further transitions. In this study, we took advantage of single 7 ns laser pulses to study carotenoid absorption transients in OCP on the time-scale from 100 ns to 10 s, which allowed us to detect a red intermediate state preceding the red signaling state, OCPR. In addition, time-resolved fluorescence spectroscopy and the assignment of carotenoid-induced quenching of different tryptophan residues derived thereof revealed a novel orange intermediate state, which appears during the relaxation of photoactivated OCPR to OCPO. Our results show asynchronous changes between the carotenoid- and protein-associated kinetic components in a refined mechanistic model of the OCP photocycle, but also introduce new kinetic signatures for future studies of OCP photoactivity and photoprotection.

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.

Acclimation to High CO2 Requires the ω Subunit of the RNA Polymerase in Synechocystis.

Inactivation of the nonessential ω-subunit of the RNA polymerase core in the ΔrpoZ strain of the model cyanobacterium Synechocystis sp. PCC 6803 leads to a unique high-CO2-sensitive phenotype. Supplementing air in the growth chamber with 30 mL L-1 (3%) CO2 accelerated the growth rate of the control strain (CS) 4-fold, whereas ΔrpoZ did not grow faster than under ambient air. The slow growth of ΔrpoZ during the first days in high CO2 was due to the inability of the mutant cells to adjust photosynthesis to high CO2 The light-saturated photosynthetic activity of ΔrpoZ in high CO2 was only half of that measured in CS, Rubisco content was one-third lower, and cells of ΔrpoZ were not able to increase light-harvesting phycobilisome antenna like CS upon high-CO2 treatment. In addition, altered structural and functional organization of photosystem I and photosystem II were detected in the ΔrpoZ strain compared with CS when cells were grown in high CO2 but not in ambient air. Moreover, respiration of ΔrpoZ did not acclimate to high CO2 Unlike the photosynthetic complexes, the RNA polymerase complex and ribosomes were produced in high CO2 similarly as in CS Our results indicate that the deletion of the ω-subunit specifically affects photosynthesis and respiration, but transcription and translation remain active. Thus, the specific effect of the ω-subunit on photosynthesis but not on all household processes suggests that the ω-subunit might have a regulatory function in cyanobacteria.

Orange carotenoid protein burrows into the phycobilisome to provide photoprotection.

In cyanobacteria, photoprotection from overexcitation of photochemical centers can be obtained by excitation energy dissipation at the level of the phycobilisome (PBS), the cyanobacterial antenna, induced by the orange carotenoid protein (OCP). A single photoactivated OCP bound to the core of the PBS affords almost total energy dissipation. The precise mechanism of OCP energy dissipation is yet to be fully determined, and one question is how the carotenoid can approach any core phycocyanobilin chromophore at a distance that can promote efficient energy quenching. We have performed intersubunit cross-linking using glutaraldehyde of the OCP and PBS followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS-MS) to identify cross-linked residues. The only residues of the OCP that cross-link with the PBS are situated in the linker region, between the N- and C-terminal domains and a single C-terminal residue. These links have enabled us to construct a model of the site of OCP binding that differs from previous models. We suggest that the N-terminal domain of the OCP burrows tightly into the PBS while leaving the OCP C-terminal domain on the exterior of the complex. Further analysis shows that the position of the small core linker protein ApcC is shifted within the cylinder cavity, serving to stabilize the interaction between the OCP and the PBS. This is confirmed by a ΔApcC mutant. Penetration of the N-terminal domain can bring the OCP carotenoid to within 5-10 Å of core chromophores; however, alteration of the core structure may be the actual source of energy dissipation.

[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.

Regulation of Orange Carotenoid Protein Activity in Cyanobacterial Photoprotection.

Plants, algae, and cyanobacteria have developed mechanisms to decrease the energy arriving at reaction centers to protect themselves from high irradiance. In cyanobacteria, the photoactive Orange Carotenoid Protein (OCP) and the Fluorescence Recovery Protein are essential elements in this mechanism. Absorption of strong blue-green light by the OCP induces carotenoid and protein conformational changes converting the orange (inactive) OCP into a red (active) OCP. Only the red orange carotenoid protein (OCP(r)) is able to bind to phycobilisomes, the cyanobacterial antenna, and to quench excess energy. In this work, we have constructed and characterized several OCP mutants and focused on the role of the OCP N-terminal arm in photoactivation and excitation energy dissipation. The N-terminal arm largely stabilizes the closed orange OCP structure by interacting with its C-terminal domain. This avoids photoactivation at low irradiance. In addition, it slows the OCP detachment from phycobilisomes by hindering fluorescence recovery protein interaction with bound OCP(r). This maintains thermal dissipation of excess energy for a longer time. Pro-22, at the beginning of the N-terminal arm, has a key role in the correct positioning of the arm in OCP(r), enabling strong OCP binding to phycobilisomes, but is not essential for photoactivation. Our results also show that the opening of the OCP during photoactivation is caused by the movement of the C-terminal domain with respect to the N-terminal domain and the N-terminal arm.

Impacts of varying light regimes on phycobiliproteins of Nostoc sp. HKAR-2 and Nostoc sp. HKAR-11 isolated from diverse habitats.

The adaptability of cyanobacteria in diverse habitats is an important factor to withstand harsh conditions. In the present investigation, the impacts of photosynthetically active radiation (PAR; 400-700 nm), ultraviolet-B (UV-B; 280-315 nm), and PAR + UV-B radiations on two cyanobacteria viz., Nostoc sp. HKAR-2 and Nostoc sp. HKAR-11 inhabiting diverse habitats such as hot springs and rice fields, respectively, were studied. Cell viability was about 14 % in Nostoc sp. HKAR-2 and <10 % in Nostoc sp. HKAR-11 after 48 h of UV-B exposure. PAR had negligible negative impact on the survival of both cyanobacteria. The continuous exposure of UV-B and PAR + UV-B showed rapid uncoupling, bleaching, fragmentation, and degradation in both phycocyanin (C-PC) and phycoerythrin (C-PE) subunits of phycobiliproteins (PBPs). Remarkable bleaching effect of C-PE and C-PC was not only observed with UV-B or PAR + UV-B radiation, but longer period (24-48 h) of exposure with PAR alone also showed noticeable negative impact. The C-PE and C-PC subunits of the rice field isolate Nostoc sp. HKAR-11 were severely damaged in comparison to the hot spring isolate Nostoc sp. HKAR-2 with rapid wavelength shifting toward shorter wavelengths denoting the bleaching of both the accessory light harvesting pigments. The results indicate that PBPs of the hot spring isolate Nostoc sp. HKAR-2 were more stable under various light regimes in comparison to the rice field isolate Nostoc sp. HKAR-11 that could serve as a good source of valuable pigments to be used in various biomedical and biotechnological applications.

Features of temporal behavior of fluorescence recovery in Synechocystis sp. PCC6803.

Under high photon flux density of solar radiation, the photosynthetic apparatus can be damaged. To prevent this photodestruction, cyanobacteria developed special mechanisms of non-photochemical quenching (NPQ) of excitation energy in phycobilisomes. In Synechocystis, NPQ is triggered by the orange carotenoid protein (OCP), which is sensitive to blue-green illumination allowing it to bind to the phycobilisome reducing the flow of energy to the photosystems. Consequent decoupling of OCP and recovery of phycobilisome fluorescence in vivo is controlled by the so called fluorescence recovery protein (FRP). In this work, the role of the phycobilisome core components, apcD and apcF, in non-photochemical quenching and subsequent fluorescence recovery in the phycobilisomes of the cyanobacterium Synechocystis sp. PCC6803 has been investigated. Using a single photon counting technique, we have registered fluorescence decay spectra with picosecond time resolution during fluorescence recovery. In order to estimate the activation energy for the photocycle, spectroscopic studies in dependency on the temperature from 5 to 45 °C have been performed. It was found that fluorescence quenching and recovery were strongly temperature dependent for all strains exhibiting characteristic non-linear time courses. The rise of the fluorescence intensity during fluorescence recovery after NPQ can be completely described by the increase of the phycobilisome core fluorescence lifetime. It was shown that fluorescence recovery of apcD- and apcF-deficient mutants is characterized by a significantly lower activation energy barrier compared to wild type. This phenomenon indicates that apcD and apcF gene products may be required for proper interaction of FRP and OCP coupled to the phycobilisome core. In addition, we found that the rate of fluorescence recovery decreases with an increase of the non-photochemical quenching amplitude, probably due to depletion of substrate for the enzymatic reaction catalyzed by FRP.

Light adaptation of the unicellular red alga, Cyanidioschyzon merolae, probed by time-resolved fluorescence spectroscopy.

Photosynthetic organisms change the quantity and/or quality of their pigment-protein complexes and the interactions among these complexes in response to light conditions. In the present study, we analyzed light adaptation of the unicellular red alga Cyanidioschyzon merolae, whose pigment composition is similar to that of cyanobacteria because its phycobilisomes (PBS) lack phycoerythrin. C. merolae were grown under different light qualities, and their responses were measured by steady-state absorption, steady-state fluorescence, and picosecond time-resolved fluorescence spectroscopies. Cells were cultivated under four monochromatic light-emitting diodes (blue, green, yellow, and red), and changes in pigment composition and energy transfer were observed. Cells grown under blue and green light increased their relative phycocyanin levels compared with cells cultured under white light. Energy-transfer processes to photosystem I (PSI) were sensitive to yellow and red light. The contribution of direct energy transfer from PBS to PSI increased only under yellow light, while red light induced a reduction in energy transfer from photosystem II to PSI and an increase in energy transfer from light-harvesting chlorophyll protein complex I to PSI. Differences in pigment composition, growth, and energy transfer under different light qualities are discussed.

Energy transfer pathways among phycobilin chromophores and fluorescence emission spectra of the phycobilisome core at 293 and 77 K.

Energy transfer pathways between phycobiliproteins chromophores in the phycobilisome (PBS) core of the cyanobacterium Synechocystis sp. PCC 6803 were investigated. The computer 3D model of the PBS core with determination of chromophore to chromophore distance was created. Our kinetic equations based on this model allowed us to describe the relative intensities of the fluorescence emission of the short(peaked at 665 nm) and long-wavelength (peaked at 680 nm) chromophores in the PBS core at low and room temperatures. The difference of emissions of the PBS core at 77 and 293 K are due to the back energy transfer, which is observed at room temperature and is negligible at 77 K.

Specificity of the cyanobacterial orange carotenoid protein: influences of orange carotenoid protein and phycobilisome structures.

Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the reaction centers by increasing thermal energy dissipation at the level of the phycobilisome (PB), the extramembranous light-harvesting antenna. This mechanism is triggered by the photoactive Orange Carotenoid Protein (OCP), which acts both as the photosensor and the energy quencher. The OCP binds the core of the PB. The structure of this core differs in diverse cyanobacterial strains. Here, using two isolated OCPs and four classes of PBs, we demonstrated that differences exist between OCPs related to PB binding, photoactivity, and carotenoid binding. Synechocystis PCC 6803 (hereafter Synechocystis) OCP, but not Arthrospira platensis PCC 7345 (hereafter Arthrospira) OCP, can attach echinenone in addition to hydroxyechinenone. Arthrospira OCP binds more strongly than Synechocystis OCP to all types of PBs. Synechocystis OCP can strongly bind only its own PB in 0.8 m potassium phosphate. However, if the Synechocystis OCP binds to the PB at very high phosphate concentrations (approximately 1.4 m), it is able to quench the fluorescence of any type of PB, even those isolated from strains that lack the OCP-mediated photoprotective mechanism. Thus, the determining step for the induction of photoprotection is the binding of the OCP to PBs. Our results also indicated that the structure of PBs, at least in vitro, significantly influences OCP binding and the stabilization of OCP-PB complexes. Finally, the fact that the OCP induced large fluorescence quenching even in the two-cylinder core of Synechococcus elongatus PBs strongly suggested that OCP binds to one of the basal allophycocyanin cylinders.

Effect of temperature on photosynthesis and growth in marine Synechococcus spp.

In this study, we develop a mechanistic understanding of how temperature affects growth and photosynthesis in 10 geographically and physiologically diverse strains of Synechococcus spp. We found that Synechococcus spp. are able to regulate photochemistry over a range of temperatures by using state transitions and altering the abundance of photosynthetic proteins. These strategies minimize photosystem II (PSII) photodamage by keeping the photosynthetic electron transport chain (ETC), and hence PSII reaction centers, more oxidized. At temperatures that approach the optimal growth temperature of each strain when cellular demand for reduced nicotinamide adenine dinucleotide phosphate (NADPH) is greatest, the phycobilisome (PBS) antenna associates with PSII, increasing the flux of electrons into the ETC. By contrast, under low temperature, when slow growth lowers the demand for NADPH and linear ETC declines, the PBS associates with photosystem I. This favors oxidation of PSII and potential increase in cyclic electron flow. For Synechococcus sp. WH8102, growth at higher temperatures led to an increase in the abundance of PBS pigment proteins, as well as higher abundance of subunits of the PSII, photosystem I, and cytochrome b6f complexes. This would allow cells to increase photosynthetic electron flux to meet the metabolic requirement for NADPH during rapid growth. These PBS-based temperature acclimation strategies may underlie the larger geographic range of this group relative to Prochlorococcus spp., which lack a PBS.

Crystal structure of the FRP and identification of the active site for modulation of OCP-mediated photoprotection in cyanobacteria.

Photosynthetic reaction centers are sensitive to high light conditions, which can cause damage because of the formation of reactive oxygen species. To prevent high-light induced damage, cyanobacteria have developed photoprotective mechanisms. One involves a photoactive carotenoid protein that decreases the transfer of excess energy to the reaction centers. This protein, the orange carotenoid protein (OCP), is present in most cyanobacterial strains; it is activated by high light conditions and able to dissipate excess energy at the site of the light-harvesting antennae, the phycobilisomes. Restoration of normal antenna capacity involves the fluorescence recovery protein (FRP). The FRP acts to dissociate the OCP from the phycobilisomes by accelerating the conversion of the active red OCP to the inactive orange form. We have determined the 3D crystal structure of the FRP at 2.5 Å resolution. Remarkably, the FRP is found in two very different conformational and oligomeric states in the same crystal. Based on amino acid conservation analysis, activity assays of FRP mutants, FRP:OCP docking simulations, and coimmunoprecipitation experiments, we conclude that the dimer is the active form. The second form, a tetramer, may be an inactive form of FRP. In addition, we have identified a surface patch of highly conserved residues and shown that those residues are essential to FRP activity.