Insights into energy quenching mechanisms and carotenoid uptake by orange carotenoid protein homologs: HCP4 and CTDH.

Photodamage to the photosynthetic apparatus by excessive light radiation has led to the evolution of a variety of energy dissipation mechanisms. A mechanism that exists in some cyanobacterial species, enables non-photochemical quenching of excitation energy within the phycobilisome (PBS) antenna complex by the Orange Carotenoid Protein (OCP). The OCP contains an active N-terminal domain (NTD) and a regulatory C-terminal domain (CTD). Some cyanobacteria also have genes encoding for homologs to both the CTD (CTDH) and the NTD (referred to as helical carotenoid proteins, HCP). The CTDH facilitates uptake of carotenoids from the thylakoid membranes to be transferred to the HCPs. Holo-HCPs exhibit diverse functionalities such as carotenoid carriers, singlet oxygen quenchers, and in the case of HCP4, constitutive OCP-like energy quenching. Here, we present the first crystal structure of the holo-HCP4 binding canthaxanthin molecule and an improved structure of the apo-CTDH from Anabaena sp. PCC 7120. We propose here models of the binding of the HCP4 to the PBS and the associated energy quenching mechanism. Our results show that the presence of the carotenoid is essential for fluorescence quenching. We also examined interactions within OCP-like species, including HCP4 and CTDH, providing the basis for mechanisms of carotenoid transfer from CTDH to HCPs.

Orange Carotenoid Protein in Mesoporous Silica: A New System towards the Development of Colorimetric and Fluorescent Sensors for pH and Temperature.

Orange carotenoid protein (OCP) is a photochromic carotenoprotein involved in the photoprotection of cyanobacteria. It is activated by blue-green light to a red form OCPR capable of dissipating the excess of energy of the cyanobacterial photosynthetic light-harvesting systems. Activation to OCPR can also be achieved in the dark. In the present work, activation by pH changes of two different OCPs-containing echinenone or canthaxanthin as carotenoids-is investigated in different conditions. A particular emphasis is put on OCP encapsulated in SBA-15 mesoporous silica nanoparticles. It is known that in these hybrid systems, under appropriate conditions, OCP remains photoactive. Here, we show that when immobilised in SBA-15, the OCP visible spectrum is sensitive to pH changes, but such a colorimetric response is very different from the one observed for OCP in solution. In both cases (SBA-15 matrices and solutions), pH-induced colour changes are related either by orange-to-red OCP activation, or by carotenoid loss from the denatured protein. Of particular interest is the response of OCP in SBA-15 matrices, where a sudden change in the Vis absorption spectrum and in colour is observed for pH changing from 2 to 3 (in the case of canthaxanthin-binding OCP in SBA-15: λMAX shifts from 454 to 508 nm) and for pH changing from 3 to 4 (in the case of echinenone-binding OCP in SBA-15: λMAX shifts from 445 to 505 nm). The effect of temperature on OCP absorption spectrum and colour (in SBA-15 matrices) has also been investigated and found to be highly dependent on the properties of the used mesoporous silica matrix. Finally, we also show that simultaneous encapsulation in selected surface-functionalised SBA-15 nanoparticles of appropriate fluorophores makes it possible to develop OCP-based pH-sensitive fluorescent systems. This work therefore represents a proof of principle that OCP immobilised in mesoporous silica is a promising system in the development of colorimetric and fluorometric pH and temperature sensors.

Light-induced infrared difference spectroscopy on three different forms of orange carotenoid protein: focus on carotenoid vibrations.

Orange carotenoid protein (OCP) is a photoactive carotenoprotein involved in photoprotection of cyanobacteria, which uses a keto-catorenoid as a chromophore. When it absorbs blue-green light, it converts from an inactive OCPO orange form to an activated OCPR red form, the latter being able to bind the light-harvesting complexes facilitating thermal dissipation of the excess of absorbed light energy. Several research groups have focused their attention on the photoactivation mechanism, characterized by several steps, involving both carotenoid photophysics and protein conformational changes. Among the used techniques, time-resolved IR spectroscopy have the advantage of providing simultaneously information on both the chromophore and the protein, giving thereby the possibility to explore links between carotenoid dynamics and protein dynamics, leading to a better understanding of the mechanism. However, an appropriate interpretation of data requires previous assignment of marker IR bands, for both the carotenoid and the protein. To date, some assignments have concerned specific α-helices of the OCP backbone, but no specific marker band for the carotenoid was identified on solid ground. This paper provides evidence for the assignment of putative marker bands for three carotenoids bound in three different OCPs: 3'-hydroxyechineone (3'-hECN), echinenone (ECN), canthaxanthin (CAN). Light-induced FTIR difference spectra were recorded in H2O and D2O and compared with spectra of isolated carotenoids. The use of DFT calculations allowed to propose a description for the vibrations responsible of several IR bands. Interestingly, most bands are located at the same wavenumber for the three kinds of OCPs suggesting that the conformation of the three carotenoids is the same in the red and in the orange form. These results are discussed in the framework of recent time-resolved IR studies on OCP.

Is orange carotenoid protein photoactivation a single-photon process?

In all published photoactivation mechanisms of orange carotenoid protein (OCP), absorption of a single photon by the orange dark state starts a cascade of red-shifted OCP ground-state intermediates that subsequently decay within hundreds of milliseconds, resulting in the formation of the final red form OCPR, which is the biologically active form that plays a key role in cyanobacteria photoprotection. A major challenge in deducing the photoactivation mechanism is to create a uniform description explaining both single-pulse excitation experiments, involving single-photon absorption, and continuous light irradiation experiments, where the red-shifted OCP intermediate species may undergo re-excitation. We thus investigated photoactivation of Synechocystis OCP using stationary irradiation light with a biologically relevant photon flux density coupled with nanosecond laser pulse excitation. The kinetics of photoactivation upon continuous and nanosecond pulse irradiation light show that the OCPR formation quantum yield increases with photon flux density; thus, a simple single-photon model cannot describe the data recorded for OCP in vitro. The results strongly suggest a consecutive absorption of two photons involving a red intermediate with ≈100 millisecond lifetime. This intermediate is required in the photoactivation mechanism and formation of the red active form OCPR.

The success of the bloom-forming cyanobacteria Planktothrix: Genotypes variability supports variable responses to light and temperature stress.

Cyanobacterial blooms can modify the dynamic of aquatic ecosystems and have harmful consequences for human activities. Moreover, cyanobacteria can produce a variety of cyanotoxins, including microcystins, but little is known about the role of environmental factors on the prevalence of microcystin producers in the cyanobacterial bloom dynamics. This study aimed to better understand the success of Planktothrix in various environments by unveiling the variety of strategies governing cell responses to sudden changes in light intensity and temperature. The cellular responses (photosynthesis, photoprotection, heat shock response and metabolites synthesis) of four Planktothrix strains to high-light or high-temperature were studied, focusing on how distinct ecotypes (red- or green-pigmented) and microcystin production capability affect cyanobacteria's ability to cope with such abiotic stimuli. Our results showed that high-light and high-temperature impact different cellular processes and that Planktothrix responses are heterogeneous, specific to each strain and thus, to genotype. The ability of cyanobacteria to cope with sudden increase in light intensity and temperature was not related to red- or green-pigmented ecotype or microcystin production capability. According to our results, microcystin producers do not cope better to high-light or high-temperature and microcystin content does not increase in response to such stresses.

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.

Structure-function-dynamics relationships in the peculiar Planktothrix PCC7805 OCP1: Impact of his-tagging and carotenoid type.

The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection. Here, we report on the functional, spectral and structural characteristics of the peculiar Planktothrix PCC7805 OCP (Plankto-OCP). We show that this OCP variant is characterized by higher photoactivation and recovery rates, and a stronger energy-quenching activity, compared to other OCP studied thus far. We characterize the effect of the functionalizing carotenoid and of his-tagging on these reactions, and identify the time scales on which these modifications affect photoactivation. The presence of a his-tag at the C-terminus has a large influence on photoactivation, thermal recovery and PBS-fluorescence quenching, and likewise for the nature of the carotenoid that additionally affects the yield and characteristics of excited states and the ns-s dynamics of photoactivated OCP. By solving the structures of Plankto-OCP in the ECN- and CAN-functionalized states, each in two closely-related crystal forms, we further unveil the molecular breathing motions that animate Plankto-OCP at the monomer and dimer levels. We finally discuss the structural changes that could explain the peculiar properties of Plankto-OCP.

Unifying Perspective of the Ultrafast Photodynamics of Orange Carotenoid Proteins from Synechocystis: Peril of High-Power Excitation, Existence of Different S* States, and Influence of Tagging.

A substantial number of Orange Carotenoid Protein (OCP) studies have aimed to describe the evolution of singlet excited states leading to the formation of a photoactivated form, OCPR. The most recent one suggests that 3 ps-lived excited states are formed after the sub-100 fs decay of the initial S2 state. The S* state, which has the longest reported lifetime of a few to tens of picoseconds, is considered to be the precursor of the first red photoproduct P1. Here, we report the ultrafast photodynamics of the OCP from Synechocystis PCC 6803 carried out using visible-near infrared femtosecond time-resolved absorption spectroscopy as a function of the excitation pulse power and wavelength. We found that a carotenoid radical cation can form even at relatively low excitation power, obscuring the determination of photoactivation yields for P1. Moreover, the comparison of green (540 nm) and blue (470 nm) excitations revealed the existence of an hitherto uncharacterized excited state, denoted as S∼, living a few tens of picoseconds and formed only upon 470 nm excitation. Because neither the P1 quantum yield nor the photoactivation speed over hundreds of seconds vary under green and blue continuous irradiation, this S∼ species is unlikely to be involved in the photoactivation mechanism leading to OCPR. We also addressed the effect of His-tagging at the N- or C-termini on the excited-state photophysical properties. Differences in spectral signatures and lifetimes of the different excited states were observed at a variance with the usual assumption that His-tagging hardly influences protein dynamics and function. Altogether our results advocate for the careful consideration of the excitation power and His-tag position when comparing the photoactivation of different OCP variants and beg to revisit the notion that S* is the precursor of photoactivated OCPR.

A kaleidoscope of photosynthetic antenna proteins and their emerging roles.

Photosynthetic light-harvesting antennae are pigment-binding proteins that perform one of the most fundamental tasks on Earth, capturing light and transferring energy that enables life in our biosphere. Adaptation to different light environments led to the evolution of an astonishing diversity of light-harvesting systems. At the same time, several strategies have been developed to optimize the light energy input into photosynthetic membranes in response to fluctuating conditions. The basic feature of these prompt responses is the dynamic nature of antenna complexes, whose function readily adapts to the light available. High-resolution microscopy and spectroscopic studies on membrane dynamics demonstrate the crosstalk between antennae and other thylakoid membrane components. With the increased understanding of light-harvesting mechanisms and their regulation, efforts are focusing on the development of sustainable processes for effective conversion of sunlight into functional bio-products. The major challenge in this approach lies in the application of fundamental discoveries in light-harvesting systems for the improvement of plant or algal photosynthesis. Here, we underline some of the latest fundamental discoveries on the molecular mechanisms and regulation of light harvesting that can potentially be exploited for the optimization of photosynthesis.

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 ß1 ring in the OCPR-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.

Inverse regulation of light harvesting and photoprotection is mediated by a 3'-end-derived sRNA in cyanobacteria.

Phycobilisomes (PBSs), the principal cyanobacterial antenna, are among the most efficient macromolecular structures in nature, and are used for both light harvesting and directed energy transfer to the photosynthetic reaction center. However, under unfavorable conditions, excess excitation energy needs to be rapidly dissipated to avoid photodamage. The orange carotenoid protein (OCP) senses light intensity and induces thermal energy dissipation under stress conditions. Hence, its expression must be tightly controlled; however, the molecular mechanism of this regulation remains to be elucidated. Here, we describe the discovery of a posttranscriptional regulatory mechanism in Synechocystis sp. PCC 6803 in which the expression of the operon encoding the allophycocyanin subunits of the PBS is directly and in an inverse fashion linked to the expression of OCP. This regulation is mediated by ApcZ, a small regulatory RNA that is derived from the 3'-end of the tetracistronic apcABC-apcZ operon. ApcZ inhibits ocp translation under stress-free conditions. Under most stress conditions, apc operon transcription decreases and ocp translation increases. Thus, a key operon involved in the collection of light energy is functionally connected to the expression of a protein involved in energy dissipation. Our findings support the view that regulatory RNA networks in bacteria evolve through the functionalization of mRNA 3'-UTRs.

State transitions and photosystems spatially resolved in individual cells of the cyanobacterium Synechococcus elongatus.

State transitions are a low-light acclimation response through which the excitation of Photosystem I (PSI) and Photosystem II (PSII) is balanced; however, our understanding of this process in cyanobacteria remains poor. Here, picosecond fluorescence kinetics was recorded for the cyanobacterium Synechococcus elongatus using fluorescence lifetime imaging microscopy (FLIM), both upon chlorophyll a and phycobilisome (PBS) excitation. Fluorescence kinetics of single cells obtained using FLIM were compared with those of ensembles of cells obtained with time-resolved fluorescence spectroscopy. The global distribution of PSI and PSII and PBSs was mapped making use of their fluorescence kinetics. Both radial and lateral heterogeneity were found in the distribution of the photosystems. State transitions were studied at the level of single cells. FLIM results show that PSII quenching occurs in all cells, irrespective of their state (I or II). In S. elongatus cells, this quenching is enhanced in State II. Furthermore, the decrease of PSII fluorescence in State II was homogeneous throughout the cells, despite the inhomogeneous PSI/PSII ratio. Finally, some disconnected PBSs were resolved in most State II cells. Taken together our data show that PSI is enriched in the inner thylakoid, while state transitions occur homogeneously throughout the cell.

Effects of Modification of Light Parameters on the Production of Cryptophycin, Cyanotoxin with Potent Anticancer Activity, in Nostoc sp.

Cryptophycin-1 is a cyanotoxin produced by filamentous cyanobacteria. It has been evaluated as an anticancer agent with great potential. However, its synthesis provides insufficient yield for industrial use. An alternative solution for metabolite efficient production is to stress cyanobacteria by modifying the environmental conditions of the culture (Nostoc sp. ATCC 53789). Here, we examined the effects of light photoperiod, wavelength, and intensity. In light photoperiod, photoperiods 24:0 and 16:8 (light:dark) were tested while in wavelength, orange-red light was compared with blue. Medium, high, and very high light intensity experiments were performed to test the effect of light stress. For a 10-day period, growth was measured, metabolite concentration was calculated through HPLC, and the related curves were drawn. The differentiation of light wavelength had a major effect on the culture, as orange-red filter contributed to noticeable increase in both growth and doubled the cyanotoxin concentration in comparison to blue light. Remarkably, constant light provides higher cryptophycin yield, but slightly lower growth rate. Lastly, the microorganism prefers medium light intensities for both growth and metabolite expression. The combination of these optimal conditions would contribute to the further exploitation of cryptophycin.

State transitions in cyanobacteria studied with picosecond fluorescence at room temperature.

Cyanobacteria can rapidly regulate the relative activity of their photosynthetic complexes photosystem I and II (PSI and PSII) in response to changes in the illumination conditions. This process is known as state transitions. If PSI is preferentially excited, they go to state I whereas state II is induced either after preferential excitation of PSII or after dark adaptation. Different underlying mechanisms have been proposed in literature, in particular i) reversible shuttling of the external antenna complexes, the phycobilisomes, between PSI and PSII, ii) reversible spillover of excitation energy from PSII to PSI, iii) a combination of both and, iv) increased excited-state quenching of the PSII core in state II. Here we investigated wild-type and mutant strains of Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803 using time-resolved fluorescence spectroscopy at room temperature. Our observations support model iv, meaning that increased excited-state quenching of the PSII core occurs in state II thereby balancing the photochemistry of photosystems I and II.

Structural dynamics in the C terminal domain homolog of orange carotenoid Protein reveals residues critical for carotenoid uptake.

The structural features enabling carotenoid translocation between molecular entities in nature is poorly understood. Here, we present the three-dimensional X-ray structure of an expanded oligomeric state of the C-terminal domain homolog (CTDH) of the orange carotenoid protein, a key water-soluble protein in cyanobacterial photosynthetic photo-protection, at 2.9 Å resolution. This protein binds a canthaxanthin carotenoid ligand and undergoes structural reorganization at the dimeric level, which facilitates cargo uptake and delivery. The structure displays heterogeneity revealing the dynamic nature of its C-terminal tail (CTT). Molecular dynamics (MD) simulations based on the CTDH structures identified specific residues that govern the dimeric transition mechanism. Mutagenesis based on the crystal structure and these MD simulations then confirmed that these specific residues within the CTT are critical for carotenoid uptake, encapsulation and delivery processes. We present a mechanism that can be applied to other systems that require cargo uptake.

Revisiting cyanobacterial state transitions.

Photosynthetic organisms are exposed to a fluctuating environment in which light intensity and quality change continuously. Specific illumination of either photosystem (PSI or PSII) creates an energy imbalance, leading to the reduction or oxidation of the intersystem electron transport chain. This redox imbalance could trigger the formation of dangerous reactive oxygen species. Cyanobacteria, like plants and algae, have developed a mechanism to re-balance this preferential excitation of either reaction center, called state transitions. State transitions are triggered by changes in the redox state of the membrane-soluble plastoquinone (PQ) pool. In plants and green algae, these changes in redox potential are sensed by Cytochrome b6f, which interacts with a specific kinase that triggers the movement of the main PSII antenna (the light-harvesting complex II). By contrast, although cyanobacterial state transitions have been studied extensively, there is still no agreement about the molecular mechanism, the PQ redox state sensor and the signaling pathways involved. In this review, we aimed to critically evaluate the results published on cyanobacterial state transitions, and discuss the "new" and "old" models in the subject. The phycobilisome and membrane contributions to this physiological process were addressed and the current hypotheses regarding its signaling transduction pathway were discussed.

Light stress in green and red Planktothrix strains: The orange carotenoid protein and its related photoprotective mechanism.

Photosynthetic organisms need to sense and respond to fluctuating environmental conditions, to perform efficient photosynthesis and avoid the formation of harmful reactive oxygen species. 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, the extramembranal light-harvesting antenna. This mechanism is triggered by the photoactive orange carotenoid protein (OCP). In this study, we characterized OCP and the related photoprotective mechanism in non-stressed and light-stressed cells of three different strains of Planktothrix that can form impressive blooms. In addition to changing lake ecosystemic functions and biodiversity, Planktothrix blooms can have adverse effects on human and animal health as they produce toxins (e.g., microcystins). Three Planktothrix strains were selected: two green strains, PCC 10110 (microcystin producer) and PCC 7805 (non-microcystin producer), and one red strain, PCC 7821. The green strains colonize shallow lakes with higher light intensities while red strains proliferate in deep lakes. Our study allowed us to conclude that there is a correlation between the ecological niche in which these strains proliferate and the rates of induction and recovery of OCP-related photoprotection. However, differences in the resistance to prolonged high-light stress were correlated to a better replacement of damaged D1 protein and not to differences in OCP photoprotection. Finally, microcystins do not seem to be involved in photoprotection as was previously suggested.

Changing Color for Photoprotection: The Orange Carotenoid Protein.

Under high irradiance, light becomes dangerous for photosynthetic organisms and they must protect themselves. Cyanobacteria have developed a simple mechanism, involving a photoactive soluble carotenoid protein, the orange carotenoid protein (OCP), which increases thermal dissipation of excess energy by interacting with the cyanobacterial antenna, the phycobilisome. Here, we summarize our knowledge of the OCP-related photoprotective mechanism, including the remarkable progress that has been achieved in recent years on OCP photoactivation and interaction with phycobilisomes, as well as with the fluorescence recovery protein, which is necessary to end photoprotection. A recently discovered unique mechanism of carotenoid transfer between soluble proteins related to OCP is also described.

Interdomain interactions reveal the molecular evolution of the orange carotenoid protein.

The photoactive orange carotenoid protein (OCP) is a blue-light intensity sensor involved in cyanobacterial photoprotection. Three OCP families co-exist (OCPX, OCP1 and OCP2), having originated from the fusion of ancestral domain genes. Here, we report the characterization of an OCPX and the evolutionary characterization of OCP paralogues focusing on the role of the linker connecting the domains. The addition of the linker with specific amino acids enabled the photocycle of the OCP ancestor. OCPX is the paralogue closest to this ancestor. A second diversification gave rise to OCP1 and OCP2. OCPX and OCP2 present fast deactivation and weak antenna interaction. In OCP1, OCP deactivation became slower and interaction with the antenna became stronger, requiring a further protein to detach OCP from the antenna and accelerate its deactivation. OCP2 lost the tendency to dimerize, unlike OCPX and OCP1, and the role of its linker is slightly different, giving less controlled photoactivation.