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.     

Mass spectrometry footprinting reveals the structural rearrangements of cyanobacterial orange carotenoid protein upon light activation

The orange carotenoid protein (OCP), a member of the family of blue light photoactive proteins, is required for efficient photoprotection in many cyanobacteria. Photoexcitation of the carotenoid in the OCP results in structural changes within the chromophore and the protein to give an active red form of OCP that is required for phycobilisome binding and consequent fluorescence quenching. We characterized the light-dependent structural changes by mass spectrometry-based carboxyl footprinting and found that an α helix in the N-terminal extension of OCP plays a key role in this photoactivation process. Although this helix is located on and associates with the outside of the β-sheet core in the C-terminal domain of OCP in the dark, photoinduced changes in the domain structure disrupt this interaction. We propose that this mechanism couples light-dependent carotenoid conformational changes to global protein conformational dynamics in favor of functional phycobilisome binding, and is an essential part of the OCP photocycle.     

Site of non-photochemical quenching of the phycobilisome by orange carotenoid protein in the cyanobacterium Synechocystis sp. PCC 6803

In cyanobacteria, the thermal dissipation of excess absorbed energy at the level of the phycobilisome (PBS)-antenna is triggered by absorption of strong blue-green light by the photoactive orange carotenoid protein (OCP). This process known as non-photochemical quenching, whose molecular mechanism remains in many respects unclear, is revealed in vivo as a decrease in phycobilisome fluorescence. In vitro reconstituted system on the interaction of the OCP and the PBS isolated from the cyanobacterium Synechocystis sp. PCC 6803 presents evidence that the OCP is not only a photosensor, but also an effecter that makes direct contacts with the PBS and causes dissipation of absorbed energy. To localize the site(s) of quenching, we have analyzed the role of chromophorylated polypeptides of the PBS using PBS-deficient mutants in conjunction with in vitro systems of assembled PBS and of isolated components of the PBS core. The results demonstrated that L(CM), the core-membrane linker protein and terminal emitter of the PBS, could act as the docking site for OCP in vitro. The ApcD and ApcF terminal emitters of the PBS core are not directly subjected to quenching. The data suggests that there could be close contact between the phycocyanobilin chromophore of L(CM) and the 3'-hydroxyechinenone chromophore present in OCP and that L(CM) could be involved in OCP-induced quenching. According to the reduced average life-time of the PBS-fluorescence and linear dependence of fluorescence intensity of the PBS on OCP concentration, the quenching has mostly dynamic character. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Chemical activation of the cyanobacterial orange carotenoid protein

The effects of the Hofmeister series of ions on the activation of the orange carotenoid protein (OCP) from the inactive orange form to the active red form were tested. Kosmotropes led to lower OCP activation, whereas chaotropes led to greater OCP activation. Concentrations of thiocyanate exceeding 1.5 M dark activate the orange carotenoid protein to its red form. This chemically activated OCP was studied by UV–vis and circular dichroism spectroscopies. The chemically-activated OCP quenches the fluorescence of phycobilisomes in vitro, to a level comparable to that of the light-activated OCP.     

Elucidation of the essential amino acids involved in the binding of the cyanobacterial Orange Carotenoid Protein to the phycobilisome

The Orange Carotenoid Protein (OCP) is a soluble photoactive protein involved in cyanobacterial photoprotection. It is formed by the N-terminal domain (NTD) and C-terminal (CTD) domain, which establish interactions in the orange inactive form and share a ketocarotenoid molecule. Upon exposure to intense blue light, the carotenoid molecule migrates into the NTD and the domains undergo separation. The free NTD can then interact with the phycobilisome (PBS), the extramembrane cyanobacterial antenna, and induces thermal dissipation of excess absorbed excitation energy. The OCP and PBS amino acids involved in their interactions remain undetermined. To identify the OCP amino acids essential for this interaction, we constructed several OCP mutants (23) with modified amino acids located on different NTD surfaces. We demonstrated that only the NTD surface that establishes interactions with the CTD in orange OCP is involved in the binding of OCP to PBS. All amino acids surrounding the carotenoid β1 ring in the OCP^R-NTD (L51, P56, G57, N104, I151, R155, N156) are important for binding OCP to PBS. Additionally, modification of the amino acids influences OCP photoactivation and/or recovery rates, indicating that they are also involved in the translocation of the carotenoid.     

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.     

Role of the PB-loop in ApcE and phycobilisome core function in cyanobacterium Synechocystis sp. PCC 6803

The phycobilisome (PBS) is a giant highly-structured pigment-protein antenna of cyanobacteria and red algae. PBS is composed of the phycobiliproteins and several linker polypeptides. The large core-membrane linker protein (L_CM or ApcE) influences many features and functions of PBS and consists of several domains including the chromophorylated PB-domain. Being homologous to the phycobiliprotein α-subunits this domain includes a so-called PB-loop insertion whose functions are still unknown. We have created the photoautotrophic mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 with lacking PB-loop. Using various spectral techniques we have demonstrated that this mutation does not destroy the PBS integrity and the internal PBS excitation energy transfer pathways. At the same time, the deletion of the PB-loop leads to the decrease of connectivity between the PBS and thylakoid membrane and to the compensatory increase of the relative photosystem II content. Mutation provokes the violation of the thylakoid membranes arrangement, the inability to perform state transitions, and diminishing of the OCP-dependent non-photochemical PBS quenching. In essence, even such a minute mutation of the PBS polypeptide component, like the PB-loop deletion, becomes important for the concerted function of the photosynthetic apparatus.     

Synthetic OCP heterodimers are photoactive and recapitulate the fusion of two primitive carotenoproteins in the evolution of cyanobacterial photoprotection

The orange carotenoid protein (OCP) governs photoprotection in the majority of cyanobacteria. It is structurally and functionally modular, comprised of a C‐terminal regulatory domain (CTD), an N‐terminal effector domain (NTD) and a ketocarotenoid; the chromophore spans the two domains in the ground state and translocates fully into the NTD upon illumination. Using both the canonical OCP1 from Fremyella diplosiphon and the presumably more primitive OCP2 paralog from the same organism, we show that an NTD‐CTD heterodimer forms when the domains are expressed as separate polypeptides. The carotenoid is required for the heterodimeric association, assembling an orange complex which is stable in the dark. Both OCP1 and OCP2 heterodimers are photoactive, undergoing light‐driven heterodimer dissociation, but differ in their ability to reassociate in darkness, setting the stage for bioengineering photoprotection in cyanobacteria as well as for developing new photoswitches for biotechnology. Additionally, we reveal that homodimeric CTD can bind carotenoid in the absence of NTD, and name this truncated variant the C‐terminal domain‐like carotenoid protein (CCP). This finding supports the hypothesis that the OCP evolved from an ancient fusion event between genes for two different carotenoid‐binding proteins ancestral to the NTD and CTD. We suggest that the CCP and its homologs constitute a new family of carotenoproteins within the NTF2‐like superfamily found across all kingdoms of life.     

Protective dissipation of excess absorbed energy by photosynthetic apparatus of cyanobacteria: role of antenna terminal emitters

Two mechanisms of photoprotective dissipation of the excessively absorbed energy by photosynthetic apparatus of cyanobacteria are described that divert energy from reaction centers. Energy dissipation, monitored as nonphotochemical fluorescence quenching, occurs at different steps of energy transfer within the phycobilisomes or core antenna of photosystem I. Although these mechanisms differ significantly, in both cases, energy dissipates mainly from terminal emitters: allophycocyanin B or core membrane linker protein (L_CM) in phycobilisomes, or the longest-wavelength chlorophylls in photosystem I antenna. It is supposed that carotenoid-induced energy dissipation in phycobilisomes is triggered by light-induced transformation of the nonquenched state of antenna into quenched state due to conformation changes caused by orange carotinoid-binding protein (OCP)–phycobilisome interaction. Fluorescence of the longest-wavelength chlorophylls of photosystem I antenna is strongly quenched by P700 cation radical or by P700 triplet state, dependent on redox state of the acceptor side cofactors of photosystem I.     

The crystal structure of a cyanobacterial water-soluble carotenoid binding protein

Carotenoids undergo a wide range of photochemical reactions in animal, plant, and microbial systems. In photosynthetic organisms, in addition to light harvesting, they perform an essential role in protecting against light-induced damage by quenching singlet oxygen, superoxide anion radicals, or triplet-state chlorophyll. We have determined the crystal structure of a water-soluble orange carotenoid protein (OCP) isolated from the cyanobacterium Arthrospira maxima at a resolution of 2.1 A. OCP forms a homodimer with one carotenoid molecule per monomer. The carotenoid binding site is lined by a striking number of methionine residues. The structure reveals several possible ways in which the protein environment influences the spectral properties of the pigment and provides insight into how the OCP carries out its putative functions in photoprotection.     

Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein

Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCP^O) into the red-shifted, active state (OCP^R) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCP^R is converted to OCP^O and dissociates from PBs; however, the mode and site of OCP^R/FRP interactions remain elusive. Recently, we have introduced the purple OCP^W288A mutant as a competent model for the signaling state OCP^R (Sluchanko et al., Biochim Biophys Acta 1858:1–11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCP^W288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCP^O, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCP^O, a substantial compaction of the OCP^W288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCP^W288A or the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.     

Assembly of photoactive orange carotenoid protein from its domains unravels a carotenoid shuttle mechanism

The photoswitchable orange carotenoid protein (OCP) is indispensable for cyanobacterial photoprotection by quenching phycobilisome fluorescence upon photoconversion from the orange OCP^O to the red OCP^R form. Cyanobacterial genomes frequently harbor, besides genes for orange carotenoid proteins (OCPs), several genes encoding homologs of OCP’s N- or C-terminal domains (NTD, CTD). Unlike the well-studied NTD homologs, called Red Carotenoid Proteins (RCPs), the role of CTD homologs remains elusive. We show how OCP can be reassembled from its functional domains. Expression of Synechocystis OCP-CTD in carotenoid-producing Escherichia coli yielded violet-colored proteins, which, upon mixing with the RCP-apoprotein, produced an orange-like photoswitchable form that further photoconverted into a species that quenches phycobilisome fluorescence and is spectroscopically indistinguishable from RCP, thus demonstrating a unique carotenoid shuttle mechanism. Spontaneous carotenoid transfer also occurs between canthaxanthin-coordinating OCP-CTD and the OCP apoprotein resulting in formation of photoactive OCP. The OCP-CTD itself is a novel, dimeric carotenoid-binding protein, which can coordinate canthaxanthin and zeaxanthin, effectively quenches singlet oxygen and interacts with the Fluorescence Recovery Protein. These findings assign physiological roles to the multitude of CTD homologs in cyanobacteria and explain the evolutionary process of OCP formation.

     

Functional interaction of low-homology FRPs from different cyanobacteria with Synechocystis OCP

Photosynthesis requires a balance between efficient light harvesting and protection against photodamage. The cyanobacterial photoprotection system uniquely relies on the functioning of the photoactive orange carotenoid protein (OCP) that under intense illumination provides fluorescence quenching of the light-harvesting antenna complexes, phycobilisomes. The recently identified fluorescence recovery protein (FRP) binds to the photoactivated OCP and accelerates its relaxation into the basal form, completing the regulatory circle. The molecular mechanism of FRP functioning is largely controversial. Moreover, since the available knowledge has mainly been gained from studying Synechocystis proteins, the cross-species conservation of the FRP mechanism remains unexplored. Besides phylogenetic analysis, we performed a detailed structural-functional analysis of two selected low-homology FRPs by comparing them with Synechocystis FRP (SynFRP). While adopting similar dimeric conformations in solution and preserving binding preferences of SynFRP towards various OCP variants, the low-homology FRPs demonstrated distinct binding stoichiometries and differentially accentuated features of this functional interaction. By providing clues to understand the FRP mechanism universally, our results also establish foundations for upcoming structural investigations necessary to elucidate the FRP-dependent regulatory mechanism.     

Oligomerization processes limit photoactivation and recovery of the orange carotenoid protein

The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection by quenching of the excess of light-harvested energy. The photoactivation mechanism remains elusive, in part due to absence of data pertaining to the timescales over which protein structural changes take place. It also remains unclear whether or not oligomerization of the dark-adapted and light-adapted OCP could play a role in the regulation of its energy-quenching activity. Here, we probed photoinduced structural changes in OCP by a combination of static and time-resolved X-ray scattering and steady-state and transient optical spectroscopy in the visible range. Our results suggest that oligomerization partakes in regulation of the OCP photocycle, with different oligomers slowing down the overall thermal recovery of the dark-adapted state of OCP. They furthermore reveal that upon non-photoproductive excitation a numbed state forms, which remains in a non-photoexcitable structural state for at least ≈0.5 μs after absorption of a first photon.     

Protein-protein interactions in carotenoid triggered quenching of phycobilisome fluorescence in Synechocystis sp. PCC 6803

An inquiry into the effect of temperature on carotenoid triggered quenching of phycobilisome (PBS) fluorescence in a photosystem II-deficient mutant of Synechocystis sp. results in identification of two temperature-dependent processes: one is responsible for the quenching rate, and one determines the yield of PBS fluorescence. Non-Arrhenius behavior of the light-on quenching rate suggests that carotenoid-absorbed light triggers a process that bears a strong resemblance to soluble protein folding, showing temperature-dependent enthalpy of activated complex formation. The response of PBS fluorescence yield to hydration changing additives and to passing of the membrane lipid phase transition point indicates that the pool size of PBSs subject to quenching depends on the state of some membrane component.     

Ultrafast spectroscopy tracks carotenoid configurations in the orange and red carotenoid proteins from cyanobacteria

A quenching mechanism mediated by the orange carotenoid protein (OCP) is one of the ways cyanobacteria protect themselves against photooxidative stress. Here, we present a femtosecond spectroscopic study comparing OCP and RCP (red carotenoid protein) samples binding different carotenoids. We confirmed significant changes in carotenoid configuration upon OCP activation reported by Leverenz et al. (Science 348:1463–1466. doi:  10.1126/science.aaa7234, 2015) by comparing the transient spectra of OCP and RCP. The most important marker of these changes was the magnitude of the transient signal associated with the carotenoid intramolecular charge-transfer (ICT) state. While OCP with canthaxanthin exhibited a weak ICT signal, it increased significantly for canthaxanthin bound to RCP. On the contrary, a strong ICT signal was recorded in OCP binding echinenone excited at the red edge of the absorption spectrum. Because the carbonyl oxygen responsible for the appearance of the ICT signal is located at the end rings of both carotenoids, the magnitude of the ICT signal can be used to estimate the torsion angles of the end rings. Application of two different excitation wavelengths to study OCP demonstrated that the OCP sample contains two spectroscopically distinct populations, none of which is corresponding to the photoactivated product of OCP.     

Structural and spectroscopic characterization of HCP2

The Helical Carotenoid Proteins (HCPs) are a large group of newly identified carotenoid-binding proteins found in ecophysiologically diverse cyanobacteria. They likely evolved before becoming the effector (quenching) domain of the modular Orange Carotenoid Protein (OCP). The number of discrete HCP families—at least nine—suggests they are involved in multiple distinct functions. Here we report the 1.7?Å crystal structure of HCP2, one of the most widespread HCPs found in nature, from the chromatically acclimating cyanobacterium Tolypothrix sp. PCC 7601. By purifying HCP2 from the native source we are able to identify its natively-bound carotenoid, which is exclusively canthaxanthin. In solution, HCP2 is a monomer with an absorbance maximum of 530?nm. However, the HCP2 crystals have a maximum absorbance at 548?nm, which is accounted by the stacking of the β1 rings of the carotenoid in the two molecules in the asymmetric unit. Our results demonstrate how HCPs provide a valuable system to study carotenoid-protein interactions and their spectroscopic implications, and contribute to efforts to understand the functional roles of this large, newly discovered family of pigment proteins, which to-date remain enigmatic.     

Structure and Function of the Water-Soluble Carotenoid-Binding Proteins of Cyanobacteria

The orange carotenoid protein (OCP) and its derivative, the red carotenoid protein (RCP), appear to play important photoprotective roles in cyanobacteria. Structural and functional characterization is gradually elucidating the specific details of how carotenoid-protein interactions, including the role of six methionine residues oriented toward the pigment, contribute to the spectral and functional properties of these proteins.     

Biophysical modeling of in vitro and in vivo processes underlying regulated photoprotective mechanism in cyanobacteria

Non-photochemical quenching (NPQ) is a mechanism responsible for high light tolerance in photosynthetic organisms. In cyanobacteria, NPQ is realized by the interplay between light-harvesting complexes, phycobilisomes (PBs), a light sensor and effector of NPQ, the photoactive orange carotenoid protein (OCP), and the fluorescence recovery protein (FRP). Here, we introduced a biophysical model, which takes into account the whole spectrum of interactions between PBs, OCP, and FRP and describes the experimental PBs fluorescence kinetics, unraveling interaction rate constants between the components involved and their relative concentrations in the cell. We took benefit from the possibility to reconstruct the photoprotection mechanism and its parts in vitro, where most of the parameters could be varied, to develop the model and then applied it to describe the NPQ kinetics in the Synechocystis sp. PCC 6803 mutant lacking photosystems. Our analyses revealed  that while an excess of the OCP over PBs is required to obtain substantial PBs fluorescence quenching in vitro, in vivo the OCP/PBs ratio is less than unity, due to higher local concentration of PBs, which was estimated as ~10^?5 M, compared to in vitro experiments. The analysis of PBs fluorescence recovery on the basis of the generalized model of enzymatic catalysis resulted in determination of the FRP concentration in vivo close to 10% of the OCP concentration. Finally, the possible role of the FRP oligomeric state alteration in the kinetics of PBs fluorescence was shown. This paper provides the most comprehensive model of the OCP-induced PBs fluorescence quenching to date and the results are important for better understanding of the regulatory molecular mechanisms underlying NPQ in cyanobacteria.

     

The time course of non-photochemical quenching in phycobilisomes of Synechocystis sp. PCC6803 as revealed by picosecond time-resolved fluorimetry

As high-intensity solar radiation can lead to extensive damage of the photosynthetic apparatus, cyanobacteria have developed various protection mechanisms to reduce the effective excitation energy transfer (EET) from the antenna complexes to the reaction center. One of them is non-photochemical quenching (NPQ) of the phycobilisome (PB) fluorescence. In Synechocystis sp. PCC6803 this role is carried by the orange carotenoid protein (OCP), which reacts to high-intensity light by a series of conformational changes, enabling the binding of OCP to the PBs reducing the flow of energy into the photosystems. In this paper the mechanisms of energy migration in two mutant PB complexes of Synechocystis sp. were investigated and compared. The mutant CK is lacking phycocyanin in the PBs while the mutant ΔPSI/PSII does not contain both photosystems. Fluorescence decay spectra with picosecond time resolution were registered using a single photon counting technique. The studies were performed in a wide range of temperatures — from 4 to 300 K. The time course of NPQ and fluorescence recovery in darkness was studied at room temperature using both steady-state and time-resolved fluorescence measurements. The OCP induced NPQ has been shown to be due to EET from PB cores to the red form of OCP under photon flux densities up to 1000 μmol photons m^− 2 s^− 1. The gradual changes of the energy transfer rate from allophycocyanin to OCP were observed during the irradiation of the sample with blue light and consequent adaptation to darkness. This fact was interpreted as the revelation of intermolecular interaction between OCP and PB binding site. At low temperatures a significantly enhanced EET from allophycocyanin to terminal emitters has been shown, due to the decreased back transfer from terminal emitter to APC. The activation of OCP not only leads to fluorescence quenching, but also affects the rate constants of energy transfer as shown by model based analysis of the decay associated spectra. The results indicate that the ability of OCP to quench the fluorescence is strongly temperature dependent. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.