Tadashi Mizoguchi

Structure of the light-harvesting bacteriochlorophyll c assembly in chlorosomes from Chlorobium limicola determined by solid-state NMR

We have determined the atomic structure of the bacteriochlorophyll c (BChl c) assembly in a huge light-harvesting organelle, the chlorosome of green photosynthetic bacteria, by solid-state NMR. Previous electron microscopic and spectroscopic studies indicated that chlorosomes have a cylindrical architecture with a diameter of ≈10 nm consisting of layered BChl molecules. Assembly structures in huge noncrystalline chlorosomes have been proposed based mainly on structure-dependent chemical shifts and a few distances acquired by solid-state NMR, but those studies did not provide a definite structure. Our approach is based on 13C dipolar spin-diffusion solid-state NMR of uniformly 13C-labeled chlorosomes under magic-angle spinning. Approximately 90 intermolecular CC distances were obtained by simultaneous assignment of distance correlations and structure optimization preceded by polarization-transfer matrix analysis. It was determined from the ≈90 intermolecular distances that BChl c molecules form piggyback-dimer-based parallel layers. This finding rules out the well known monomer-based structures. A molecular model of the cylinder in the chlorosome was built by using this structure. It provided insights into the mechanisms of efficient light harvesting and excitation transfer to the reaction centers. This work constitutes an important advance in the structure determination of huge intact systems that cannot be crystallized.

Structural Transformation Among the Aggregate Forms of Bacteriochlorophyll c as Determined by Electronic-Absorption and NMR Spectroscopies: Dependence on the Stereoisomeric Configuration and on the Bulkiness of the 8-C Side Chain ‡

Transformation among the aggregate forms of bacteriochlorophyll (BChl) c characterized by the wavelength of the Qy absorption, i.e. the dimer (B675), B705, B720 and B745, was traced by electronic‐absorption spectroscopy for each of the isomers including R[E,E], R[P,E], R[I,E], S[P,E] and S[I,E] suspended in the mixtures of methylene chloride and n‐hexane. A combination of NMR spectroscopy determining the structural motifs and calculation of the shift of the Qy absorption reflecting the long‐range transition dipole–transition dipole interactions among the macrocycles in the entire aggregate structures proposed the following models: B705d (B705d′), a linear array of straight (inclined) columns consisting of a pair of the piggyback dimers; B720d and B745d, an assembly of two and five shifted‐inclined columns consisting of more than six piggyback dimers; and B720m and B745m, an assembly of one and two parallel stepwise stacking of ∼30 monomers. Calculations of the steric energies rationalized two different pathways of transformations: the dimer → B705d (B705d′) → B720d → B745d for the R isomers; and the monomer → (B720m) → B745m for the S isomers. Addition of S[I,E] seems to trigger the B745d → B745m transformation of the R isomers.

The Structure of the Aggregate Form of Bacteriochlorophyll c Showing the Qy Absorption above 740 nm as Determined by the Ring-current Effects on 1H and 13C Nuclei and by 1H–1H Intermolecular NOE Correlations

13C‐enriched bacteriochlorophyll c (S[I, E] BChl cF) was suspended in a 1:3 mixture of methylene chloride and carbon tetrachloride to form an aggregate showing the Qy absorption above 740 nm; changes in the 13C chemical shifts were traced when methanol was titrated to dissolve the aggregate, and then, the changes were correlated to the ring‐current effects due to the neighboring macrocycles in the aggregate. A pair of aggregate structures has been proposed based on the ring‐current effects on both 1H and 13C nuclei; the monomeric units are stacked together to form an inclined column with different sliding directions, in which the y‐axis of the molecule is parallel to the long axis of the column. In order to confirm this pair of models, the ring‐current effects on the 1H and 13C nuclei were calculated based on both the magnetic‐dipole and the loop‐current approximations. Further, an application of three‐dimensional F1 13C‐edited F3 13C‐filtered heteronuclear single‐quantum correlation‐nuclear Overhauser effect spectroscopy to the above aggregate consisting of a 1:1 mixture of 13C‐labeled and unlabeled BChl c succeeded in detecting selectively the 1H–1H intermolecular nuclear Overhauser effect correlations, which established the coexistence of the above pair of stacked structures in the aggregate.

Composition and localization of bacteriochlorophyll a intermediates in the purple photosynthetic bacterium Rhodopseudomonas sp. Rits

Rhodopseudomonas sp. Rits is a recently isolated new species of photosynthetic bacteria and found to accumulate a significantly high amount of bacteriochlorophyll (BChl) a intermediates possessing non-, di- and tetra-hydrogenated geranylgeranyl groups at the 17-propionate as well as normal phytylated BChl a (Mizoguchi T etxa0al. (2006) FEBS Lett 580:137–143). A phylogenetic analysis showed that this bacterium was closely related to Rhodopseudomonas palustris. The strain Rits synthesizes light-harvesting complexes 2 and 4 (LH2/4), as peripheral antennas, as well as the reaction center and light-harvesting 1 core complex (RC-LH1 core). The amounts of these complexes were dependent upon the incident light intensities, which was also a typical behavior of Rhodopseudomonas palustris. HPLC analyses of extracted pigments indicated that all four BChls a were associated with the purified photosynthetic pigment–protein, as complexes described above. The results suggested that this bacterium could use these pigments as functional molecules within the LH2/4 and RC-LH1 core. Pigment compositional analyses in several purple photosynthetic bacteria showed that such BChl a intermediates were always detected and were more widely distributed than expected. Long chains in the propionate moiety of BChl a would be one of the important factors for assembly of LH systems in purple photosynthetic bacteria.

Time-dependent Changes in the Carotenoid Composition and Preferential Binding of Spirilloxanthin to the Reaction Center and Anhydrorhodovibrin to the LH1 Antenna Complex in Rhodobium marinum ¶

Carotenoids were isolated from the cells of Rhodobium marinum, and their structures were determined by mass spectrometry and 1H nuclear magnetic resonance spectroscopy; the carotenoids include lycopene, rhodopin, anhydrorhodovibrin, rhodovibrin and spirilloxanthin. Time‐dependent changes in the carotenoid composition in the reaction center (RC) and the light‐harvesting complex 1 (LH1) were traced by high‐performance liquid chromatography analysis of the extracts. The carotenoid composition changed according to the spirilloxanthin biosynthetic pathway. However, spirilloxanthin having the longest conjugated chain was always preferentially bound to the RC, and anhydrorhodovibrin and other precursors to the LH1.

The structure of the aggregate form of bacteriochlorophyll c showing the Qy absorption above 740 nm: a 1H-NMR study

Abstract Bacteriochlorophyll c (3 1 S, 8-isobutyl-12-ethyl, farnesyl) was dissolved in a mixture of methylene chloride and carbon tetrachloride (1 : 3), and changes in the 1 H-NMR spectrum caused by the titration of methanol were traced. On the basis of the changes in chemical shift due to the ring-current effect of the neighboring macrocycles and in peak intensity (broadening) due to their stacking, the structure of the aggregate form showing the Q y absorption band above 740 nm is proposed: the macrocycles are stacked to form a one-dimensional inclined column, the y axis of each macrocycle being parallel to the long axis of the column.

1H NMR, electronic-absorption and resonance-Raman spectra of isomeric okenone as compared with those of isomeric β-carotene, canthaxanthin, β-apo-8′-carotenal and spheroidene

Abstract Eleven cis-trans isomers of okenone were isolated by means of HPLC using a silica-gel column from an isomeric mixture which was obtained by iodine-sensitized photo-isomerization of the all-trans isomer. The configurations of eight isomers among them were determined by NMR spectroscopy using the isomerization shifts of the olefinic 1Hs and the 1H–1H NOE correlations to be all-trans, 7-cis, 7-cis,8-s-cis, 9-cis, 9′-cis, 13-cis, 13′-cis and 9,9′-di-cis, and their electronic-absorption and resonance-Raman spectra were recorded. Based on the results: (1) the chemical shifts of the olefinic 1Hs in NMR; (2) the wavelength of the Ag−→Bu+ transition; and (3) the relative intensity of the Ag−→Ag+ versus the Ag−→Bu+ transition in electronic absorption; (4) the Cue605C stretching frequency; and (5) the relative intensity of the C10–C11 (C10′–C11′) versus the C14–C15 (C14′–C15′) stretching vibration in resonance Raman were compared among the all-trans, 7-cis, 9-cis (9′-cis) and 13-cis (13′-cis) isomers of β-carotene, canthaxanthin, β-apo-8′-carotenal, neurosporene, spheroidene and okenone. Relevance of the systematic changes in the above five different parameters originally found in β-carotene was examined in the rest of the carotenoids, and the effects of the peripheral groups on them were explained in terms of the length and asymmetry of the conjugated system consisting of the Cue605C and Cue605O bonds.

Aggregation forms of 8-ethyl-12-ethyl farnesyl bacteriochlorophyll c in methanol-chloroform mixtures as revealed by 1H NMR spectroscopy

Abstract 8-Ethyl-12-ethyl farnesyl bacteriochlorophyll c was dissolved in chloroform and changes in the 1 H NMR spectrum due to the titration of methanol were traced. Based on the changes in chemical shift and in peak intensity, the structures of two aggregate forms have been proposed. Form I showing the Q y electron absorption at 675 nm was ascribed to a dimer which has a “piggy-back” stacking of the macrocycles and a pair of MgŻOH coordination bonds. This structure was confirmed by an intermolecular 1 Hue5f8 1 H nuclear Overhauser effect correlation. However, Form II showing the Q y absorption at 705 nm was ascribed to an oligomer in which the dimer units are stacked to form an inclined column.

Self-aggregation of synthetic zinc chlorophyll derivatives possessing multi-perfluoroalkyl chains in perfluorinated solvents

Zinc 3(1)-hydroxy-13(1)-oxo-chlorins possessing two, three, four and six perfluorooctyl chains were synthesized from naturally occurring chlorophyll-a. Only the synthetic zinc chlorin possessing six perfluorooctyl chains was directly dissolved in perfluorinated solvents due to its high fluorine content in molecular weight (over 50%). In this solution, visible absorption spectra gave a red-shifted Q(y) band at 723 nm (compared to 648 nm in THF) and showed the formation of well-ordered self-aggregates. No monomeric form was observed in the solution from any fluorescence emission spectra from visible absorption spectra. In the aggregate solution, no precipitation occurred during either standing for a long period or heating at 70 degrees C. This showed that the supramolecular structure was stabilized by F-F interactions on its surface among the perfluorooctyl chains on the 17-position and perfluorinated solvents. The core part of the supramolecular structure was constructed by a special intramolecular bond of Zn ... O3(2)-H ... O=C13(1), which was confirmed from resonance Raman spectral analysis.

Central-Cis isomers of lutein found in the major light-harvesting complex of Photosystem II (LHC IIb) of higher plants

Lutein (β,∈-carotene-3,3′-diol) is the major carotenoid of the light-harvesting systems of higher plants. Lutein was isolated at 4°C and in complete darkness from the bulk light-harvesting complex of Photosystem II of spinach (LHC IIb) and from BBY particles. Separation using normal-phase HPLC (with 2D detection) in comparison to the authentic isomers (prepared by iodine-sensitised isomerization) showed the presence of a number of geometrical isomers of this xanthophyll in PS II, namely all-trans (the major component); 13-cis, 13′-cis and 15-cis-lutein. Iodine-sensitised photo-isomerization of all-trans lutein produced six geometrical isomers of lutein as determined by HPLC. The configuration of five of these isomers was determined by 1H-NMR to be all-trans, 9-cis, 9′-cis, 13-cis and 13′-cis. In addition, small amounts of another isomer have been tentatively identified to be 15-cis lutein on the basis of its electronic absorption spectrum. The possible functional significance of the presence of cis-isomers of this carotenoid in LHC IIb is discussed.