Intense sunlight is dangerous for photosynthetic organisms. Cyanobacteria, like plants, protect themselves from light-induced stress by dissipating excess absorbed energy as heat. Recently, it was discovered that a soluble orange carotenoid protein, the OCP, is essential for this photoprotective mechanism. Here we show that the OCP is also a member of the family of photoactive proteins; it is a unique example of a photoactive protein containing a carotenoid as the photoresponsive chromophore. Upon illumination with blue-green light, the OCP undergoes a reversible transformation from its dark stable orange form to a red ''active'' form. The red form is essential for the induction of the photoprotective mechanism. The illumination induces structural changes affecting both the carotenoid and the protein. Thus, the OCP is a photoactive protein that senses light intensity and triggers photoprotection.cyanobacteria ͉ nonphotochemical quenching ͉ photoprotection ͉ phycobilisome

Photosynthetic organisms have developed multiple protective mechanisms to survive under high-light conditions. In plants, one of these mechanisms is the thermal dissipation of excitation energy in the membrane-bound chlorophyll antenna of photosystem II. The question of whether or not cyanobacteria, the progenitor of the chloroplast, have an equivalent photoprotective mechanism has long been unanswered. Recently, however, evidence was presented for the possible existence of a mechanism dissipating excess absorbed energy in the phycobilisome, the extramembrane antenna of cyanobacteria. Here, we demonstrate that this photoprotective mechanism, characterized by blue light-induced fluorescence quenching, is indeed phycobilisomerelated and that a soluble carotenoid binding protein, ORANGE CAROTENOID PROTEIN (OCP), encoded by the slr1963 gene in Synechocystis PCC 6803, plays an essential role in this process. Blue light is unable to quench fluorescence in the absence of phycobilisomes or OCP. The fluorescence quenching is not DpH-dependent, and it can be induced in the absence of the reaction center II or the chlorophyll antenna, CP43 and CP47. Our data suggest that OCP, which strongly interacts with the thylakoids, acts as both the photoreceptor and the mediator of the reduction of the amount of energy transferred from the phycobilisomes to the photosystems. These are novel roles for a soluble carotenoid protein.

Pigment-protein and pigment-pigment interactions are of fundamental importance to the light-harvesting and photoprotective functions essential to oxygenic photosynthesis. The orange carotenoid protein (OCP) functions as both a sensor of light and effector of photoprotective energy dissipation in cyanobacteria. We report the atomic-resolution structure of an active form of the OCP consisting of the N-terminal domain and a single noncovalently bound carotenoid pigment. The crystal structure, combined with additional solution-state structural data, reveals that OCP photoactivation is accompanied by a 12 angstrom translocation of the pigment within the protein and a reconfiguration of carotenoid-protein interactions. Our results identify the origin of the photochromic changes in the OCP triggered by light and reveal the structural determinants required for interaction with the light-harvesting antenna during photoprotection.

The orange carotenoid protein (OCP) is a two-domain photoactive protein that noncovalently binds an echinenone (ECN) carotenoid and mediates photoprotection in cyanobacteria. In the dark, OCP assumes an orange, inactive state known as OCPO; blue light illumination results in the red active state, known as OCPR. The OCPR state is characterized by large-scale structural changes that involve dissociation and separation of C-terminal and N-terminal domains accompanied by carotenoid translocation into the N-terminal domain. The mechanistic and dynamic-structural relations between photon absorption and formation of the OCPR state have remained largely unknown. Here, we employ a combination of time-resolved UV–visible and (polarized) mid-infrared spectroscopy to assess the electronic and structural dynamics of the carotenoid and the protein secondary structure, from femtoseconds to 0.5 ms. We identify a hereto unidentified carotenoid excited state in OCP, the so-called S* state, which we propose to play a key role in breaking conserved hydrogen-bond interactions between carotenoid and aromatic amino acids in the binding pocket. We arrive at a comprehensive reaction model where the hydrogen-bond rupture with conserved aromatic side chains at the carotenoid β1-ring in picoseconds occurs at a low yield of <1%, whereby the β1-ring retains a trans configuration with respect to the conjugated π-electron chain. This event initiates structural changes at the N-terminal domain in 1 μs, which allow the carotenoid to translocate into the N-terminal domain in 10 μs. We identified infrared signatures of helical elements that dock on the C-terminal domain β-sheet in the dark and unfold in the light to allow domain separation. These helical elements do not move within the experimental range of 0.5 ms, indicating that domain separation occurs on longer time scales, lagging carotenoid translocation by at least 2 decades of time.

In conditions of fluctuating light, cyanobacteria thermally dissipate excess absorbed energy at the level of the phycobilisome, the light-collecting antenna. The photoactive Orange Carotenoid Protein (OCP) and Fluorescence Recovery Protein (FRP) have essential roles in this mechanism. Absorption of blue-green light converts the stable orange (inactive) OCP form found in darkness into a metastable red (active) form. Using an in vitro reconstituted system, we studied the interactions between OCP, FRP, and phycobilisomes and demonstrated that they are the only elements required for the photoprotective mechanism. In the process, we developed protocols to overcome the effect of high phosphate concentrations, which are needed to maintain the integrity of phycobilisomes, on the photoactivation of the OCP, and on protein interactions. Our experiments demonstrated that, whereas the dark-orange OCP does not bind to phycobilisomes, the binding of only one red photoactivated OCP to the core of the phycobilisome is sufficient to quench all its fluorescence. This binding, which is light independent, stabilizes the red form of OCP. Addition of FRP accelerated fluorescence recovery in darkness by interacting with the red OCP and destabilizing its binding to the phycobilisome. The presence of phycobilisome rods renders the OCP binding stronger and allows the isolation of quenched OCP-phycobilisome complexes. Using the in vitro system we developed, it will now be possible to elucidate the quenching process and the chemical nature of the quencher.

The photoprotective processes of photosynthetic organisms involve the dissipation of excess absorbed light energy as heat. Photoprotection in cyanobacteria is mechanistically distinct from that in plants; it involves the orange carotenoid protein (OCP), a water-soluble protein containing a single carotenoid. The OCP is a new member of the family of blue light-photoactive proteins; blue-green light triggers the OCP-mediated photoprotective response. Here we report structural and functional characterization of the wild type and two mutant forms of the OCP, from the model organism Synechocystis PCC6803. The structural analysis provides high resolution detail of the carotenoid-protein interactions that underlie the optical properties of the OCP, unique among carotenoid-proteins in binding a single pigment per polypeptide chain. Collectively, these data implicate several key amino acids in the function of the OCP and reveal that the photoconversion and photoprotective responses of the OCP to blue-green light can be decoupled.The capture of light energy for oxygenic photosynthesis is arguably one of the most important metabolic processes on earth. It is also inherently risky; the absorbance of excess light energy beyond what can be used in photosynthesis can result in photooxidative damage to the organism. Consequently, photosynthetic organisms have evolved protective mechanisms to dissipate excess captured energy. In plants, one of these mechanisms involves the membrane-embedded chlorophyll-protein antenna of Photosystem II, the light-harvesting complex (for reviews, see Refs. 1-4). Under saturating light conditions, the decrease of the lumen pH activates the xanthophyll cycle (5, 6) and the protonation of PsbS, a Photosystem II subunit (7). Conformational changes in the light-harvesting complex, modifying the interaction between chlorophyll molecules and carotenoids and allowing thermal dissipation, are also involved in this mechanism (8 -10). Energy dissipation is accompanied by a diminution of Photosystem II-related fluorescence emission, also known as non-photochemical quenching (or NPQ; more specifically qE), which usually serves as a measure of the dissipation process.Although the photoprotective mechanism of plants is well studied, only recently have mechanisms for photoprotection in the cyanobacteria been discovered (11-17). One of these occurs at the water-soluble light-harvesting antenna (the phycobilisome) and involves a novel photosensory protein, the orange carotenoid protein (13, 18 -20). Most cyanobacterial species contain the OCP 5 (21,22), and in these organisms, it is constitutively expressed and is also up-regulated under extreme conditions, such as high light, iron starvation, and salt stress (18,(23)(24)(25). The OCP-mediated photoprotective mechanism is completely distinct from any known in plants and algae; it involves the absorption of blue-green light, which induces a shift in the absorbance properties of the OCP; visibly, the protein changes from orange to red (photoconversion). The structur...

We studied the effect of UV-B radiation (280-320 nm) on the donor- and acceptor-side components of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803 by measuring the relaxation of flash-induced variable chlorophyll fluorescence. UV-B irradiation increases the t(1/2) of the decay components assigned to reoxidation of Q(A)(-) by Q(B) from 220 to 330 micros in centers which have the Q(B) site occupied, and from 3 to 6 ms in centers with the Q(B) site empty. In contrast, the t(1/2) of the slow component arising from recombination of the Q(A)Q(B)(-) state with the S(2) state of the water-oxidizing complex decreases from 13 to 1-2 s. In the presence of DCMU, fluorescence relaxation in nonirradiated cells is dominated by a 0.5-0.6 s component, which reflects Q(A)(-) recombination with the S(2) state. After UV-B irradiation, this is partially replaced by much faster components (t(1/2) approximately 800-900 micros and 8-10 ms) arising from recombination of Q(A)(-) with stabilized intermediate photosystem II donors, P680(+) and Tyr-Z(+). Measurement of fluorescence relaxation in the presence of different concentrations of DCMU revealed a 4-6-fold increase in the half-inhibitory concentration for electron transfer from Q(A) to Q(B). UV-B irradiation in the presence of DCMU reduces Q(A) in the majority (60%) of centers, but does not enhance the extent of UV-B damage beyond the level seen in the absence of DCMU, when Q(A) is mostly oxidized. Illumination with white light during UV-B treatment retards the inactivation of PSII. However, this ameliorating effect is not observed if de novo protein synthesis is blocked by lincomycin. We conclude that in intact cyanobacterium cells UV-B light impairs electron transfer from the Mn cluster of water oxidation to Tyr-Z(+) and P680(+) in the same way that has been observed in isolated systems. The donor-side damage of PSII is accompanied by a modification of the Q(B) site, which affects the binding of plastoquinone and electron transport inhibitors, but is not related to the presence of Q(A)(-). White light, at the intensity applied for culturing the cells, provides protection against UV-B-induced damage by enhancing protein synthesis-dependent repair of PSII.

The thermophilic cyanobacterium, Thermosynechococcus elongatus, has been grown in the presence of Sr 2؉ instead of Ca 2؉ with the aim of biosynthetically replacing the Ca 2؉ of the oxygen-evolving enzyme with Sr 2؉. Not only were the cells able to grow normally with Sr 2؉ , they actively accumulated the ion to levels higher than those of Ca 2؉ in the normal cultures. A protocol was developed to purify a fully active Sr The evolution of oxygen as a result of light-driven water oxidation is catalyzed by photosystem II (PSII) 1 in which a cluster of 4 manganese ions acts both as a device for accumulating oxidizing equivalents and as the active site. The reaction center of PSII is made up of two membrane-spanning polypeptides (D1 and D2) that bear the redox cofactors involved in the main electron transfer route. Absorption of a photon results in a charge separation between a chlorophyll molecule (P 680 ), and a pheophytin molecule. The pheophytin anion transfers the electron to a quinone, Q A , and P 680 ϩ is reduced by a tyrosine residue, Tyr Z , that in turn is reduced by the Mn 4 cluster. During the enzyme cycle, the oxidizing side of PSII goes through five different redox states that are denoted S n , n varying from 0 to 4. Oxygen is released during the S 3 to S 0 transition in which S 4 is a transient state (reviewed in Refs.