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.

• Klíčová slova
• Algae, Carotenoids, Chl-a, Diadinoxanthin, Heteroxanthin, Light-harvesting complex, Resonance Raman,
• MeSH
• Heterokontophyta chemie   MeSH
• karotenoidy chemie   MeSH
• konformace proteinů MeSH
• Ramanova spektroskopie MeSH
• světlosběrné proteinové komplexy chemie   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• karotenoidy MeSH
• světlosběrné proteinové komplexy 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.

• Klíčová slova
• Chlorophyll, HLIPs, HliC, Raman spectroscopy, Synechocystis, β-Carotene,
• MeSH
• bakteriální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• beta-karoten metabolismus   MeSH
• chlorofyl metabolismus   MeSH
• multimerizace proteinu MeSH
• Ramanova spektroskopie MeSH
• rekombinantní proteiny genetika   izolace a purifikace   metabolismus   MeSH
• světlosběrné proteinové komplexy genetika   metabolismus   MeSH
• Synechocystis genetika   metabolismus   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• beta-karoten MeSH
• chlorofyl MeSH
• high light-inducible protein, cyanobacteria MeSH Prohlížeč
• rekombinantní proteiny MeSH
• světlosběrné proteinové komplexy 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.

• Klíčová slova
• Carotenoids, Light-harvesting complex, Nannochloropsis oceanica, Resonance Raman, VCP,
• MeSH
• chlorofyl chemie   MeSH
• karotenoidy chemie   MeSH
• Ramanova spektroskopie MeSH
• světlosběrné proteinové komplexy chemie   MeSH
• transportní proteiny chemie   MeSH
• xanthofyly chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chlorofyl MeSH
• karotenoidy MeSH
• světlosběrné proteinové komplexy MeSH
• transportní proteiny MeSH
• violaxanthin MeSH Prohlížeč
• xanthofyly MeSH

Cyanobacteria possess a family of one-helix high light-inducible proteins (Hlips) that are homologous to light-harvesting antenna of plants and algae. An Hlip protein, high light-inducible protein D (HliD) purified as a small complex with the Ycf39 protein is evaluated using resonance Raman spectroscopy. We show that the HliD binds two different β-carotenes, each present in two non-equivalent binding pockets with different conformations, having their (0,0) absorption maxima at 489 and 522 nm, respectively. Both populations of β-carotene molecules were in all-trans configuration and the absorption position of the farthest blue-shifted β-carotene was attributed entirely to the polarizability of the environment in its binding pocket. In contrast, the absorption maximum of the red-shifted β-carotene was attributed to two different factors: the polarizability of the environment in its binding pocket and, more importantly, to the conformation of its β-rings. This second β-carotene has highly twisted β-rings adopting a flat conformation, which implies that the effective conjugation length N is extended up to 10.5 modifying the energetic levels. This increase in N will also result in a lower S1 energy state, which may provide a permanent energy dissipation channel. Analysis of the carbonyl stretching region for chlorophyll a excitations indicates that the HliD binds six chlorophyll a molecules in five non-equivalent binding sites, with at least one chlorophyll a presenting a slight distortion to its macrocycle. The binding modes and conformations of HliD-bound pigments are discussed with respect to the known structures of LHCII and CP29.

• Klíčová slova
• carotenoid, chlorophyll, cyanobacteria, light-harvesting complex (antenna complex), photosynthesis,
• MeSH
• bakteriální proteiny chemie   genetika   MeSH
• beta-karoten chemie   genetika   MeSH
• kvarterní struktura proteinů MeSH
• proteinové domény MeSH
• sekundární struktura proteinů MeSH
• světlosběrné proteinové komplexy chemie   genetika   MeSH
• Synechocystis chemie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• beta-karoten MeSH
• světlosběrné proteinové komplexy MeSH

Raman spectroscopy is a rapid nondestructive technique providing spectroscopic and structural information on both organic and inorganic molecular compounds. Extensive applications for the method in the characterization of pigments have been found. Due to the high sensitivity of Raman spectroscopy for the detection of chlorophylls, carotenoids, scytonemin, and a range of other pigments found in the microbial world, it is an excellent technique to monitor the presence of such pigments, both in pure cultures and in environmental samples. Miniaturized portable handheld instruments are available; these instruments can be used to detect pigments in microbiological samples of different types and origins under field conditions.

• MeSH
• Archaea chemie   MeSH
• Bacteria chemie   MeSH
• biologické pigmenty analýza   MeSH
• houby chemie   MeSH
• Ramanova spektroskopie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• biologické pigmenty MeSH