High-light-inducible proteins (Hlips) are single-helix transmembrane proteins that are essential for the survival of cyanobacteria under stress conditions. The model cyanobacterium Synechocystis sp. PCC 6803 contains four Hlip isoforms (HliA-D) that associate with Photosystem II (PSII) during its assembly. HliC and HliD are known to form pigmented (hetero)dimers that associate with the newly synthesized PSII reaction center protein D1 in a configuration that allows thermal dissipation of excitation energy. Thus, it is expected that they photoprotect the early steps of PSII biogenesis. HliA and HliB, on the other hand, bind the PSII inner antenna protein CP47, but the mode of interaction and pigment binding have not been resolved. Here, we isolated His-tagged HliA and HliB from Synechocystis and show that these two very similar Hlips do not interact with each other as anticipated, rather they form HliAC and HliBC heterodimers. Both dimers bind Chl and β-carotene in a quenching conformation and associate with the CP47 assembly module as well as later PSII assembly intermediates containing CP47. In the absence of HliC, the cellular levels of HliA and HliB were reduced, and both bound atypically to HliD. We postulate a model in which HliAC-, HliBC-, and HliDC-dimers are the functional Hlip units in Synechocystis. The smallest Hlip, HliC, acts as a 'generalist' that prevents unspecific dimerization of PSII assembly intermediates, while the N-termini of 'specialists' (HliA, B or D) dictate interactions with proteins other than Hlips.

• Keywords
• CP47, Chlorophyll, High-light-inducible proteins, Photosystem II, Synechocystis,
• MeSH
• Bacterial Proteins metabolism   MeSH
• Photosystem II Protein Complex metabolism   MeSH
• Tumor Necrosis Factor Ligand Superfamily Member 14 metabolism   MeSH
• Light-Harvesting Protein Complexes * metabolism   MeSH
• Synechocystis * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Photosystem II Protein Complex MeSH
• Tumor Necrosis Factor Ligand Superfamily Member 14 MeSH
• Light-Harvesting Protein Complexes * MeSH

Carotenoids are conjugated linear molecules built from the repetition of terpene units, which display a large structural diversity in nature. They may, in particular, contain several types of side or end groups, which tune their functional properties, such as absorption position and photochemistry. We report here a detailed experimental study of the absorption and vibrational properties of allene-containing carotenoids, together with an extensive modeling of these experimental data. Our calculations can satisfactorily explain the electronic properties of vaucheriaxanthin, where the allene group introduces the equivalent of one C═C double bond into the conjugated C═C chain. The position of the electronic absorption of fucoxanthin and butanoyloxyfucoxanthin requires long-range corrections to be found correctly on the red side of that of vaucheriaxanthin; however, these corrections tend to overestimate the effect of the conjugated and nonconjugated C═O groups in these molecules. We show that the resonance Raman spectra of these carotenoids are largely perturbed by the presence of the allene group, with the two major Raman contributions split into two components. These perturbations are satisfactorily explained by modeling, through a gain in the Raman intensity of the C═C antisymmetric stretching mode, induced by the presence of the allene group in the carotenoid C═C chain.

• MeSH
• Alkadienes * MeSH
• Electronics MeSH
• Carotenoids * chemistry   MeSH
• Spectrum Analysis, Raman MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Alkadienes * MeSH
• Carotenoids * MeSH
• propadiene MeSH Browser

Life on Earth depends on photosynthesis, the conversion of light energy into chemical energy. Plants collect photons by light harvesting complexes (LHC)-abundant membrane proteins containing chlorophyll and xanthophyll molecules. LHC-like proteins are similar in their amino acid sequence to true LHC antennae, however, they rather serve a photoprotective function. Whether the LHC-like proteins bind pigments has remained unclear. Here, we characterize plant LHC-like proteins (LIL3 and ELIP2) produced in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Both proteins were associated with chlorophyll a (Chl) and zeaxanthin and LIL3 was shown to be capable of quenching Chl fluorescence via direct energy transfer from the Chl Qy state to zeaxanthin S1 state. Interestingly, the ability of the ELIP2 protein to quench can be acquired by modifying its N-terminal sequence. By employing Synechocystis carotenoid mutants and site-directed mutagenesis we demonstrate that, although LIL3 does not need pigments for folding, pigments stabilize the LIL3 dimer.

• MeSH
• Chlorophyll metabolism   MeSH
• Carotenoids metabolism   MeSH
• Protein Multimerization MeSH
• Mutation MeSH
• Energy Transfer MeSH
• Chloroplast Proteins chemistry   genetics   metabolism   MeSH
• Arabidopsis Proteins chemistry   genetics   metabolism   MeSH
• Protein Folding MeSH
• Synechocystis genetics   metabolism   MeSH
• Protein Binding MeSH
• Xanthophylls metabolism   MeSH
• Zeaxanthins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chlorophyll MeSH
• ELIP2 protein, Arabidopsis MeSH Browser
• Carotenoids MeSH
• light-harvesting-like protein 3, Arabidopsis MeSH Browser
• Chloroplast Proteins MeSH
• Arabidopsis Proteins MeSH
• violaxanthin MeSH Browser
• Xanthophylls MeSH
• Zeaxanthins MeSH

Calculating the spectroscopic properties of complex conjugated organic molecules in their relaxed state is far from simple. An additional complexity arises for flexible molecules in solution, where the rotational energy barriers are low enough so that nonminimum conformations may become dynamically populated. These metastable conformations quickly relax during the minimization procedures preliminary to density functional theory calculations, and so accounting for their contribution to the experimentally observed properties is problematic. We describe a strategy for stabilizing these nonminimum conformations in silico, allowing their properties to be calculated. Diadinoxanthin and alloxanthin present atypical vibrational properties in solution, indicating the presence of several conformations. Performing energy calculations in vacuo and polarizable continuum model calculations in different solvents, we found three different conformations with values for the δ dihedral angle of the end ring ca. 0, 180, and 90° with respect to the plane of the conjugated chain. The latter conformation, a nonglobal minimum, is not stable during the minimization necessary for modeling its spectroscopic properties. To circumvent this classical problem, we used a Car-Parinello MD supermolecular approach, in which diadinoxanthin was solvated by water molecules so that metastable conformations were stabilized by hydrogen-bonding interactions. We progressively removed the number of solvating waters to find the minimum required for this stabilization. This strategy represents the first modeling of a carotenoid in a distorted conformation and provides an accurate interpretation of the experimental data.

• Publication type
• Journal Article MeSH

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.

• Keywords
• OCP1, OCP2, Photoactivation, Ultrafast spectroscopy, X-ray footprinting,
• MeSH
• Bacterial Proteins chemistry   MeSH
• Spectrometry, Fluorescence MeSH
• Phycobilisomes chemistry   MeSH
• Cyanobacteria chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Phycobilisomes MeSH

Ferrochelatase (FeCh) is an essential enzyme catalyzing the synthesis of heme. Interestingly, in cyanobacteria, algae, and plants, FeCh possesses a conserved transmembrane chlorophyll a/b binding (CAB) domain that resembles the first and the third helix of light-harvesting complexes, including a chlorophyll-binding motif. Whether the FeCh CAB domain also binds chlorophyll is unknown. Here, using biochemical and radiolabeled precursor experiments, we found that partially inhibited activity of FeCh in the cyanobacterium Synechocystis PCC 6803 leads to overproduction of chlorophyll molecules that accumulate in the thylakoid membrane and, together with carotenoids, bind to FeCh. We observed that pigments bound to purified FeCh are organized in an energy-dissipative conformation and further show that FeCh can exist in vivo as a monomer or a dimer depending on its own activity. However, pigmented FeCh was purified exclusively as a dimer. Separately expressed and purified FeCH CAB domain contained a pigment composition similar to that of full-length FeCh and retained its quenching properties. Phylogenetic analysis suggested that the CAB domain was acquired by a fusion between FeCh and a single-helix, high light-inducible protein early in the evolution of cyanobacteria. Following this fusion, the FeCh CAB domain with a functional chlorophyll-binding motif was retained in all currently known cyanobacterial genomes except for a single lineage of endosymbiotic cyanobacteria. Our findings indicate that FeCh from Synechocystis exists mostly as a pigment-free monomer in cells but can dimerize, in which case its CAB domain creates a functional pigment-binding segment organized in an energy-dissipating configuration.

• Keywords
• Synechocystis, carotenoid, chlorophyll, chloroplast, ferrochelatase, heme, light harvesting complex (LHC)-like proteins, membrane protein, photosynthesis, photosynthetic pigment, pigment binding, plant biochemistry,
• MeSH
• Chlorophyll A metabolism   MeSH
• Chlorophyll metabolism   MeSH
• Dimerization MeSH
• Ferrochelatase chemistry   metabolism   MeSH
• Phylogeny MeSH
• Carotenoids metabolism   MeSH
• Protein Conformation MeSH
• Light-Harvesting Protein Complexes metabolism   MeSH
• Synechocystis enzymology   MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chlorophyll A MeSH
• Chlorophyll MeSH
• chlorophyll b MeSH Browser
• Ferrochelatase MeSH
• Carotenoids MeSH
• Light-Harvesting Protein Complexes MeSH

The soil chromophyte alga Xanthonema (X.) debile contains only non-carbonyl carotenoids and Chl-a. X. debile has an antenna system denoted Xanthophyte light-harvesting complex (XLH) that contains the carotenoids diadinoxanthin, heteroxanthin, and vaucheriaxanthin. The XLH pigment stoichiometry was calculated by chromatographic techniques and the pigment-binding structure studied by resonance Raman spectroscopy. The pigment ratio obtained by HPLC was found to be close to 8:1:2:1 Chl-a:heteroxanthin:diadinoxanthin:vaucheriaxanthin. The resonance Raman spectra suggest the presence of 8-10 Chl-a, all of which are 5-coordinated to the central Mg, with 1-3 Chl-a possessing a macrocycle distorted from the relaxed conformation. The three populations of carotenoids are in the all-trans configuration. Vaucheriaxanthin absorbs around 500-530 nm, diadinoxanthin at 494 nm and heteroxanthin at 487 nm at 4.5 K. The effective conjugation length of heteroxanthin and diadinoxanthin has been determined as 9.4 in both cases; the environment polarizability of the heteroxanthin and diadinoxanthin binding pockets is 0.270 and 0.305, respectively.

• Keywords
• Algae, Carotenoids, Chl-a, Diadinoxanthin, Heteroxanthin, Light-harvesting complex, Resonance Raman,
• MeSH
• Stramenopiles chemistry   MeSH
• Carotenoids chemistry   MeSH
• Protein Conformation MeSH
• Spectrum Analysis, Raman MeSH
• Light-Harvesting Protein Complexes chemistry   MeSH
• Chromatography, High Pressure Liquid MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Carotenoids MeSH
• Light-Harvesting Protein Complexes MeSH

Cyanobacteria possess a family of one-helix high-light-inducible proteins (HLIPs) that are widely viewed as ancestors of the light-harvesting antenna of plants and algae. HLIPs are essential for viability under various stress conditions, although their exact role is not fully understood. The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains four HLIPs named HliA-D, and HliD has recently been isolated in a small protein complex and shown to bind chlorophyll and β-carotene. However, no HLIP has been isolated and characterized in a pure form up to now. We have developed a protocol to purify large quantities of His-tagged HliC from an engineered Synechocystis strain. Purified His-HliC is a pigmented homo-oligomer and is associated with chlorophyll and β-carotene with a 2:1 ratio. This differs from the 3:1 ratio reported for HliD. Comparison of these two HLIPs by resonance Raman spectroscopy revealed a similar conformation for their bound β-carotenes, but clear differences in their chlorophylls. We present and discuss a structural model of HliC, in which a dimeric protein binds four chlorophyll molecules and two β-carotenes.

• Keywords
• Chlorophyll, HLIPs, HliC, Raman spectroscopy, Synechocystis, β-Carotene,
• MeSH
• Bacterial Proteins chemistry   genetics   isolation & purification   metabolism   MeSH
• beta Carotene metabolism   MeSH
• Chlorophyll metabolism   MeSH
• Protein Multimerization MeSH
• Spectrum Analysis, Raman MeSH
• Recombinant Proteins genetics   isolation & purification   metabolism   MeSH
• Light-Harvesting Protein Complexes genetics   metabolism   MeSH
• Synechocystis genetics   metabolism   physiology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• beta Carotene MeSH
• Chlorophyll MeSH
• high light-inducible protein, cyanobacteria MeSH Browser
• Recombinant Proteins MeSH
• Light-Harvesting Protein Complexes MeSH

Photoprotection is fundamental in photosynthesis to avoid oxidative photodamage upon excess light exposure. Excited chlorophylls (Chl) are quenched by carotenoids, but the precise molecular origin remains controversial. The cyanobacterial HliC protein belongs to the Hlip family ancestral to plant light-harvesting complexes, and binds Chl a and β-carotene in 2:1 ratio. We analyzed HliC by watermarked femtosecond stimulated Raman spectroscopy to follow the time evolution of its vibrational modes. We observed a 2 ps rise of the C═C stretch band of the 2Ag- (S1) state of β-carotene upon Chl a excitation, demonstrating energy transfer quenching and fast excess-energy dissipation. We detected two distinct β-carotene conformers by the C═C stretch frequency of the 2Ag- (S1) state, but only the β-carotene whose 2Ag- energy level is significantly lowered and has a lower C═C stretch frequency is involved in quenching. It implies that the low carotenoid S1 energy that results from specific pigment-protein or pigment-pigment interactions is the key property for creating a dissipative energy channel. We conclude that watermarked femtosecond stimulated Raman spectroscopy constitutes a promising experimental method to assess energy transfer and quenching mechanisms in oxygenic photosynthesis.

• Publication type
• Journal Article MeSH

Resonance Raman spectroscopy was used to evaluate pigment-binding site properties in the violaxanthin-chlorophyll-a-binding protein (VCP) from Nannochloropsis oceanica. The pigments bound to this antenna protein are chlorophyll-a, violaxanthin, and vaucheriaxanthin. The molecular structures of bound Chl-a molecules are discussed with respect to those of the plant antenna proteins LHCII and CP29, the crystal structures of which are known. We show that three populations of carotenoid molecules are bound by VCP, each of which is in an all-trans configuration. We assign the lower-energy absorption transition of each of these as follows. One violaxanthin population absorbs at 485 nm, while the second population is red-shifted and absorbs at 503 nm. The vaucheriaxanthin population absorbs at 525 nm, a position red-shifted by 2138 cm-1 as compared to isolated vaucheriaxanthin in n-hexane. The red-shifted violaxanthin is slightly less planar than the blue-absorbing one, as observed for the two central luteins in LHCII, and we suggest that these violaxanthins occupy the two equivalent binding sites in VCP at the centre of the cross-brace. The presence of a highly red-shifted vaucheriaxanthin in VCP is reminiscent of the situation of FCP, in which (even more) highly red-shifted populations of fucoxanthin are present. Tuning carotenoids to absorb in the green-yellow region of the visible spectrum appears to be a common evolutionary response to competition with other photosynthetic species in the aquatic environment.

• Keywords
• Carotenoids, Light-harvesting complex, Nannochloropsis oceanica, Resonance Raman, VCP,
• MeSH
• Chlorophyll chemistry   MeSH
• Carotenoids chemistry   MeSH
• Spectrum Analysis, Raman MeSH
• Light-Harvesting Protein Complexes chemistry   MeSH
• Carrier Proteins chemistry   MeSH
• Xanthophylls chemistry   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chlorophyll MeSH
• Carotenoids MeSH
• Light-Harvesting Protein Complexes MeSH
• Carrier Proteins MeSH
• violaxanthin MeSH Browser
• Xanthophylls MeSH