Influence of zeaxanthin and echinenone binding on the activity of the Orange Carotenoid Protein

In most cyanobacteria high irradiance induces a photoprotective mechanism that downregulates photosynthesis by increasing thermal dissipation of the energy absorbed by the phycobilisome, the water-soluble antenna. The light activation of a soluble carotenoid protein, the Orange-Carotenoid-Protein (OCP), binding hydroxyechinenone, a keto carotenoid, is the key inducer of this mechanism. Light causes structural changes within the carotenoid and the protein, leading to the conversion of a dark orange form into a red active form. Here, we tested whether echinenone or zeaxanthin can replace hydroxyechinenone in a study in which the nature of the carotenoid bound to the OCP was genetically changed. In a mutant lacking hydroxyechinenone and echinenone, the OCP was found to bind zeaxanthin but the stability of the binding appeared to be lower and light was unable to photoconvert the dark form into a red active form. Moreover, in the strains containing zeaxanthin-OCP, blue-green light did not induce the photoprotective mechanism. In contrast, in mutants in which echinenone is bound to the OCP, the protein is photoactivated and photoprotection is induced. Our results strongly suggest that the presence of the carotenoid carbonyl group that distinguishes echinenone and hydroxyechinenone from zeaxanthin is essential for the OCP activity.     

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.

     

Essential role of two tyrosines and two tryptophans on the photoprotection activity of the Orange Carotenoid Protein

Photosynthetic organisms have developed photoprotective mechanisms to protect themselves from lethal high light intensities. One of these mechanisms involves the dissipation of excess absorbed light energy into heat. In cyanobacteria, light activation of a soluble carotenoid protein, the Orange Carotenoid Protein (OCP), binding a keto carotenoid, is the key inducer of this mechanism. Blue-green light absorption triggers structural changes within the carotenoid and the protein, leading to the conversion of a dark orange form into a red active form. Here we report the role in photoconversion and photoprotection of individual conserved tyrosines and tryptophans surrounding the rings of the carotenoid. Our results demonstrate that the interaction between the keto group of the carotenoid and Tyr201 and Trp288 is essential for OCP photoactivity. In addition, these amino acids are responsible for carotenoid affinity and specificity. We have already demonstrated that the aromatic character of Tyr44 and Trp110 interacting with the hydroxyl ring is critical. Here we show that the replacement of Tyr44 by Ser affects the stability of the red form avoiding its accumulation at any temperature, while Trp110Ser is affected in the energy necessary to the orange to red conversion and in the interaction with the antenna. Collectively our data support the idea that the red form is essential for photoprotection but not sufficient. Specific conformational changes occurring in the protein seem to be critical to the events leading to energy dissipation.     

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.     

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.     

Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle

Orange carotenoid protein (OCP), responsible for the photoprotection of the cyanobacterial photosynthetic apparatus under excessive light conditions, undergoes significant rearrangements upon photoconversion and transits from the stable orange to the signaling red state. This is thought to involve a 12-Å translocation of the carotenoid cofactor and separation of the N- and C-terminal protein domains. Despite clear recent progress, the detailed mechanism of the OCP photoconversion and associated photoprotection remains elusive. Here, we labeled the OCP of Synechocystis with tetramethylrhodamine-maleimide (TMR) and obtained a photoactive OCP-TMR complex, the fluorescence of which was highly sensitive to the protein state, showing unprecedented contrast between the orange and red states and reflecting changes in protein conformation and the distances from TMR to the carotenoid throughout the photocycle. The OCP-TMR complex was sensitive to the light intensity, temperature, and viscosity of the solvent. Based on the observed Förster resonance energy transfer, we determined that upon photoconversion, the distance between TMR (donor) bound to a cysteine in the C-terminal domain and the carotenoid (acceptor) increased by 18 Å, with simultaneous translocation of the carotenoid into the N-terminal domain. Time-resolved fluorescence anisotropy revealed a significant decrease of the OCP rotation rate in the red state, indicating that the light-triggered conversion of the protein is accompanied by an increase of its hydrodynamic radius. Thus, our results support the idea of significant structural rearrangements of OCP, providing, to our knowledge, new insights into the structural rearrangements of OCP throughout the photocycle and a completely novel approach to the study of its photocycle and non-photochemical quenching. We suggest that this approach can be generally applied to other photoactive proteins.     

A kinetic model of non-photochemical quenching in cyanobacteria

High light poses a threat to oxygenic photosynthetic organisms. Similar to eukaryotes, cyanobacteria evolved a photoprotective mechanism, non-photochemical quenching (NPQ), which dissipates excess absorbed energy as heat. An orange carotenoid protein (OCP) has been implicated as a blue-green light sensor that induces NPQ in cyanobacteria. Discovered in vitro, this process involves a light-induced transformation of the OCP from its dark, orange form (OCP(o)) to a red, active form, however, the mechanisms of NPQ in vivo remain largely unknown. Here we show that the formation of the quenching state in vivo is a multistep process that involves both photoinduced and dark reactions. Our kinetic analysis of the NPQ process reveals that the light induced conversion of OCP(o) to a quenching state (OCP(q)) proceeds via an intermediate, non-quenching state (OCP(i)), and this reaction sequence can be described by a three-state kinetic model. The conversion of OCP(o) to OCP(i) is a photoinduced process with the effective absorption cross section of 4.5 × 10(-3)?2 at 470 nm. The transition from OCP(i) to OCP(q) is a dark reaction, with the first order rate constant of approximately 0.1s(-1) at 25°C and the activation energy of 21 kcal/mol. These characteristics suggest that the reaction rate may be limited by cis-trans proline isomerization of Gln224-Pro225 or Pro225-Pro226, located at a loop near the carotenoid. NPQ decreases the functional absorption cross-section of Photosystem II, suggesting that formation of the quenched centers reduces the flux of absorbed energy from phycobilisomes to the reaction centers by approximately 50%.     

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.     

Role of inter-domain cavity in the attachment of the orange carotenoid protein to the phycobilisome core and to the fluorescence recovery protein

Using molecular modeling and known spatial structure of proteins, we have derived a universal 3D model of the orange carotenoid protein (OCP) and phycobilisome (PBS) interaction in the process of non-photochemical PBS quenching. The characteristic tip of the phycobilin domain of the core-membrane linker polypeptide (L_CM) forms the attachment site on the PBS core surface for interaction with the central inter-domain cavity of the OCP molecule. This spatial arrangement has to be the most advantageous one because the L_CM, as the major terminal PBS-fluorescence emitter, accumulates energy from the most other phycobiliproteins within the PBS before quenching by OCP. In agreement with the constructed model, in cyanobacteria, the small fluorescence recovery protein is wedged in the OCP’s central cavity, weakening the PBS and OCP interaction. The presence of another one protein, the red carotenoid protein, in some cyanobacterial species, which also can interact with the PBS, also corresponds to this model.     

Historical deposition behaviors of organochlorine pesticides (OCPs) in the sediments of a shallow eutrophic lake in Eastern China: Roles of the sources and sedimentological conditions

Organochlorine pesticides (OCPs) were quantified in sediments from Lake Chaohu, a shallow eutrophic lake in Eastern China, to investigate their historical deposition behaviors and to reconstruct their use history. The OCP concentrations ranged from 2.17 to 18.61 ng g⁻¹ in the surface sediments in the lake region and from .28 to 86.27 ng g⁻¹ in the inflowing river. The highest values of the aforementioned variables were attributed to urban-industrial pollution sources in the west lake region and decreased with distance from the river inlets. Historically, OCP contamination displayed three stages in Lake Chaohu: an initial increase before the 1930s, a sharp increase until the 1980s, and a decrease due the implementation of policies banning their use in the 1980s. During the second stage, the OCP concentrations increased rapidly from .69, .58, 1.95 and .02 ng g⁻¹ in the C4, C5, C6 and C10 samples in the 1930s to 8.68, 61.89, 24.14 and 3.53 ng g⁻¹, respectively, in the early 1980s. This temporal trend of OCP concentrations corresponded with historically intense anthropogenic activities, indicating that contamination by OCPs was accompanied by industrialization and civilization prior to their prohibition. In addition, dichlorodiphenyltrichloroethane (DDT) contamination was derived from the historical use of technical DDTs, whereas hexachlorocyclohexane (BHC) contamination was attributed to the historical use of technical BHCs with Lindane and new inputs of illegal DDTs. Strong positive relationships between the OCP concentrations and the total organic carbon (TOC) concentrations, sediment grain sizes (<4 μm), nutrient contents and heavy metal contents indicated that the sedimentary conditions and human activities affected the depositional characteristics of the OCPs. The DDT residues and their metabolites, particularly those in the inlet rivers, should be of concern because they result in an ecotoxicological risk in the catchment of Lake Chaohu.     

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.     

Toxicity of basil and orange essential oils and their components against two coleopteran stored products insect pests

Two commercialized essential oils and their constituent compounds were investigated for fumigant and contact activities against two grain storage insects, adults of the maize weevil (Sitophilus zeamais) and the red flour beetle (Tribolium castaneum). The two commercialized basil and orange oils showed strong fumigant and contact activities against S. zeamais and T. castaneum. The constituents of the basil oil were linalool (21.83%), estragole (74.29%), and α-humulene (2.17%), and those of the orange oil were α-pinene (0.54%), sabinene (0.38%), β-myrcene (1.98%), limonene (96.5%), and linalool (0.6%). As a toxic fumigant, the basil oil was more effective (24-h LC₅₀ = 0.014 and 0.020 mg cm^− 3) than the orange oil (24-h LC₅₀ = 0.106 and 0.130 mg cm^− 3) against S. zeamais and T. castaneum adults, respectively. Among the constituents of the two essential oils, the toxicity of estragole was the highest (0.004 and 0.013), followed by linalool (0.016 and 0.023), limonene (0.122 and 0.171), α-pinene (0.264 and 0.273), and β-myrcene (0.274 and 0.275) based on 24-h LC₅₀ values (mg cm^− 3). Similar results were obtained in a contact toxicity test. The contact activity of basil oil was more toxic than orange oil, and estragole and linalool showed pronounced contact toxicity against S. zeamais and T. castaneum adults. Alpha-humulene had no activity as a fumigant at the tested doses, but it did have an effect as a contact poison, having 24-h LD₅₀ values of 0.040 and 0.045 mg adult^− 1 to S. zeamais and T. castaneum, respectively. Although basil oil, orange oil, and their components displayed both contact and fumigant toxicities, their effects were mainly exerted by fumigant action via the vapor phase. Thus, basil oil, orange oil, and their components could be potential candidates as new fumigants for the control of S. zeamais and T. castaneum adults.     

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.     

The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher

Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the photosynthetic reaction centers under high-light conditions. The photoactive orange carotenoid protein (OCP) is essential in this mechanism as a light sensor and energy quencher. When OCP is photoactivated by strong blue-green light, it is able to dissipate excess energy as heat by interacting with phycobilisomes. As a consequence, charge separation and recombination leading to the formation of singlet oxygen diminishes. Here, we demonstrate that OCP has another essential role. We observed that OCP also protects Synechocystis cells from strong orange-red light, a condition in which OCP is not photoactivated. We first showed that this photoprotection is related to a decrease of singlet oxygen concentration due to OCP action. Then, we demonstrated that, in vitro, OCP is a very good singlet oxygen quencher. By contrast, another carotenoid protein having a high similarity with the N-terminal domain of OCP is not more efficient as a singlet oxygen quencher than a protein without carotenoid. Although OCP is a soluble protein, it is able to quench the singlet oxygen generated in the thylakoid membranes. Thus, OCP has dual and complementary photoprotective functions as an energy quencher and a singlet oxygen quencher.     

Structural Determinants Underlying Photoprotection in the Photoactive Orange Carotenoid Protein of Cyanobacteria

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.     

Optimization of carotenoid production by Rhodotorula graminis DBVPG 7021 as a function of trace element concentration by means of response surface analysis

As the final step of a study aiming at the optimization of culture conditions for the production of carotenoids by red yeasts, a statistically-based experimental design has been applied to assess the influence of selected trace elements on carotenogenesis in Rhodotorula graminis DBVPG 7021. In particular, a central composite design scheme has been used to evaluate the influence of Fe³⁺, Co²⁺, Mn²⁺, Al²⁺ and Zn²⁺ (within the range 0–50 ppm) on various responses, namely biomass (B), total carotenoid production (TC) and percentage of specific carotenoids (β-CAR, β-carotene; γ-CAR, γ-carotene; TN, torulene; TD, torularhodin) on total carotenoids. Second-order polynomial models were calculated and reduced equations were designed by neglecting non-significant (P < 0.01) regression coefficients. Reduced equations were used to calculate the optimal concentration of trace elements in view of maximising the level of B, TC, β-CAR, γ-CAR, TN and TD. After optimization, average final values total carotenoids (TC = 803.2 μg/g DW) was about 370% of value observed as central point of the central composite design scheme. Under the same condition, average final values of other responses were: B = 5.40 g/L; β-CAR = 50.3%; γ-CAR = 15.4%; TN = 22.7%; TD = 11.6%. All above experimental data are in good agreement with calculated ones, thus confirming the reliability of the proposed empirical model in describing carotenoid production by R. graminis as a function of trace element concentrations.     

Changes in carotenoid and chlorophyll content of black tomatoes (Lycopersicone sculentum L.) during storage at various temperatures

Black tomatoes have a unique color and higher lycopene content than typical red tomatoes. Here, black tomatoes were investigated how maturation stage and storage temperature affected carotenoid and chlorophyll accumulation. Immature fruits were firmer than mature fruits, but failed to develop their distinctive color and contained less lycopene when stored at 8 °C. Hunter a^* values of black tomatoes increased with storage temperature and duration; storage of immature fruits at high temperature favored lycopene accumulation. Chlorophyll levels of black tomatoes declined during storage, but differences between mature and immature tomatoes stored at 12 °C were minimal. β-Carotene levels of black tomatoes increased during early storage, but rapidly declined beginning 13 d post-harvest. The highest lycopene and chlorophyll levels were observed in mature black tomatoes stored at 12 °C for 13 d; these conditions also yielded the best quality fruit. Thus, the unique pigmentation properties of black tomatoes can be precisely controlled by standardizing storage conditions.     

Morphological and physiological response of sour orange (Citrus aurantium L.) seedlings to the inoculation of taxonomically characterized bacterial endophytes

Entophytic bacteria (EBs) are very diverse and found in virtually all plant species studied. These natural EBs live insides the host plant and can be used to maximize crop and fruit yield by exploiting their potential. In this paper, EBs characterization from various citrus genotypes and their influence on the morphological and physiological functioning of sour orange (Citrus aurantium) seedlings are described. To assess the influence of 10 distinct EBs, three different techniques (injection, soil mix, and spray) were applied for single and mixed inoculation on sour orange (C. aurantium) seedlings. The selected strains were identified as firmicutes (Enterococcus faecalis, Bacillus safensis, Bacillus cereus, Bacillus megaterium, Brevibacillus borstelensis & Staphylococcus haemolyticus), and gamma Proteobacteria (Enterobacter hormachaei, Proteus mirabilis, Pseudomonas aeruginosa, & Pseudomonas sp.) by 16S rRNA gene sequencing. To investigate the influence of these EBs on host plant morphology, different parameters (morphometric) were recorded after five WOI (weeks of inoculation), including shoot/root length, shoot/root fresh and dry biomass, and biophysical analyses i.e., relative water content (RLWC). Physiological markers such as chlorophyll & carotenoid content, protein content, proline content, phenolics, and flavonoids were also analyzed to determine the influence of endophytes on sour orange seedlings. Five strains such as SM-34, SM-20, SM-36, SM-68, and SM-56 significantly improved the development and physiology of sour orange seedlings. Bacillus cereus and Pseudomonas aeruginosa produced the best outcomes in terms of plant growth. The relative quantification of bacterial inoculums was determined using real-time PCR. A rise in the number of bacterial cells in inoculated treatment suggests that bacterial strains survived and colonized successfully, and also shown their competitiveness with native bacterial community structure. As per the results of inoculation methods, soil mixing, and injection methods were determined to be effective for bacterial inoculation to plants but a variable trend was found for different parameters with test bacterial strains. After testing their impact on field conditions, these strains can be applied as fertilizers as an alternative to conventional chemical fertilizer, although in the context of mixed inoculation of bacterial strains, 5 M and 6 M performed best and enhanced plant growth-promoting activity.     

Characterization of a naringinase from Aspergillus oryzae 11250 and its application in the debitterization of orange juice

Naringinase plays a rather important role in reducing the bitterness of juice by hydrolyzing naringin. A novel extracellular naringinase was purified from Aspergillus oryzae 11250 cultured in the presence of orange peel. A 26.78-fold purification rate was achieved by salt-induced precipitation, followed by anion-exchange and gel filtration chromatography with 32% recovery and specific activity of 2194.62 units per mg protein (U/mg). The optimum pH and temperature for naringinase activity were 5.0 and 45 °C, respectively. This enzyme was stable at 30 °C for 5 h. The K_m and V_max of naringinase toward naringin determined by Lineweaver-Burk method were 1.60 ± 0.13 mM and 126.21 ± 5.52 μmol/(min mg), respectively. The enzyme activity was inhibited completely by Ag⁺ at 10 mM. Naringinase is capable of hydrolyzing naringin, neohesperidin, and some other glycosides. A supplement of 6 U/mL of this naringinase in citrus juice sufficiently removed naringin to relieve the bitterness of citrus juice. These properties make the enzyme an ideal candidate for commercial application in the debitterization of orange juice.     

Comparative ultrafast spectroscopy and structural analysis of OCP1 and OCP2 from Tolypothrix

The orange carotenoid protein (OCP) is a structurally and functionally modular photoactive protein involved in cyanobacterial photoprotection. Recently, based on bioinformatic analysis and phylogenetic relationships, new families of OCP have been described, OCP2 and OCPx. The first characterization of the OCP2 showed both faster photoconversion and back-conversion, and lower fluorescence quenching of phycobilisomes relative to the well-characterized OCP1. Moreover, OCP2 is not regulated by the fluorescence recovery protein (FRP). In this work, we present a comprehensive study combining ultrafast spectroscopy and structural analysis to compare the photoactivation mechanisms of OCP1 and OCP2 from Tolypothrix PCC 7601. We show that despite significant differences in their functional characteristics, the spectroscopic properties of OCP1 and OCP2 are comparable. This indicates that the OCP functionality is not directly related to the spectroscopic properties of the bound carotenoid. In addition, the structural analysis by X-ray footprinting reveals that, overall, OCP1 and OCP2 have grossly the same photoactivation mechanism. However, the OCP2 is less reactive to radiolytic labeling, suggesting that the protein is less flexible than OCP1. This observation could explain fast photoconversion of OCP2.