Yoshitaka Saga

Synthesis of homologously pure bacteriochlorophyll-e and f analogues from BChls-c/d via transformation of the 7-methyl to formyl group and self-aggregation of synthetic zinc methyl bacteriopheophorbides-c/d/e/f in non-polar organic solvent

Abstract Homologously pure methyl bacteriopheophorbides-e and f (BPhes-e/fM) were prepared from modification of naturally occurring bacteriochlorophylls-c and d (BChls-c/d), respectively, by transformation of the methyl to formyl group at the 7-position. The absolute configuration of the 1-hydroxyethyl group at the 3-position of (Zn-)BPhes-e/fM was determined from comparison with structurally known BChl-c/d epimers. Visible spectra of synthetic (Zn-)BPhe-c/d/e/fM showed that the 71-oxidation and the 82/121/20-methylation affected Soret, Qx and Qy bands of both the monomeric (in a polar organic solvent) and oligomeric species (in a non-polar solvent).

Spectroscopic Studies on Self-aggregation of Bacteriochlorophyll-e in Nonpolar Organic Solvents: Effects of Stereoisomeric Configuration at the 31-Position and Alkyl Substituents at the 81-Position¶

Abstract Self-aggregation of naturally occurring bacteriochlorophyll (BChl)-e in nonpolar organic solvents was investigated by visible absorption, fluorescence emission and circular dichroism spectra. Cultured brown-colored photosynthetic bacteria have several BChl-e as light-harvesting antenna pigments. Three major BChl-e homologs were separated from the extracts of the culture by reverse-phase high-performance liquid chromatography (HPLC) and characterized by 1H-NMR and fast-atom bombardment mass spectroscopy: 8-ethyl-12-ethyl ([E,E])-, 8-propyl-12-ethyl- and 8-isobutyl-12-ethyl-BChl-e farnesyl esters. All the homologs consisted of a mixture of the 31-epimers, and epimerically pure BChl-e were also given by HPLC separation. All the separated BChl-e epimers, the epimeric mixtures and the homologous mixtures formed self-aggregates in 2% dichloromethane/hexane, giving visible absorption spectra similar to that of the whole cells, which showed two peaks (or shoulders) around 430–450 and 520 nm at the Soret region as well as a red-shifted Qy band relative to the monomeric. The spectral properties of the Soret band were basically unchanged among the epimers or epimeric/homologous mixtures. In contrast, the Qy band of aggregates of epimeric mixtures (except [E,E]) and homologous mixtures red-shifted and broadened compared with the epimerically pure. The red-shift and broadening of the Qy band are advantageous for efficient energy transfer from BChl-e aggregates to BChl-a in a baseplate in chlorosomes because their spectral overlap increases.

Isolation and structure determination of a complete set of bacteriochlorophyll-d homologs and epimers from a green sulfur bacterium Chlorobium vibrioforme and their aggregation properties in hydrophobic solvents

Eight bacteriochlorophyll (BChl)-d homologs and epimers were isolated from a strain of the green sulfur bacterium Chlorobium vibrioforme. By a combination of mass spectrometry and 1H-NMR spectroscopy using the chemical shifts of meso- and 3(1)-protons and 1H-1H NOE correlations, the molecular structures were determined as (3(1)R)-8-ethyl-12-methyl, (3R)-8-ethyl-12-ethyl, (3(1)R)-8-propyl-12-methyl, (3(1)S)-8-propyl-12-methyl, (3(1)R)-8-propyl-12-ethyl, (3(1)S)-8-propyl-12-ethyl, (3(1)S)-8-isobutyl-12-methyl and (3(1)S)-8-isobutyl-12-ethyl. The aggregation behavior of the epimerically pure BChls-d in hydrophobic organic solvents was examined to investigate the absolute configuration of the 3-(1-hydroxyethyl) group as well as the bulkiness of the C8 and C12 side-chains by using electronic-absorption and fluorescence-emission spectroscopies At high concentration of the BChls-d in CH2Cl2, the absolute configuration of the 3-(1-hydroxyethyl) group governed the formation of a subunit as a building block for the subsequent higher assembly. Upon dilution of the resulting subunit with hexane, the bulkiness of the C8 and C12 side-chains were found to affect the association of the subunits differently: the bulkiness of the C8 side-chain acted as a promoter for the association due to a stabilized hydrophobic interaction among the relevant larger side-chain, whereas the bulkiness of the C12 side-chain acted as an inhibitor for that association due to introduction of a particular steric-hindrance around the side-chain in the aggregates.

Spectral heterogeneity in single light-harvesting chlorosomes from green sulfur photosynthetic bacterium chlorobium tepidum.

Abstract The fluorescence emission properties of single chlorosomes from the green sulfur photosynthetic bacterium Chlorobium (Chl.) tepidum are studied for the first time, using a total internal reflection fluorescence microscope. The fluorescence peak positions of bacteriochlorophyll (BChl)-c self-aggregates in a single chlorosome of Chl. tepidum were widely distributed in the wavelength region between 750 and 768 nm, and the standard deviation (s.d. = 4.1 nm, n = 51) was larger than that of single chlorosomes of Chloroflexus (Cfl.) (s.d. = 1.9 nm, n = 50). The spectral heterogeneity among single chlorosomes from Chl. tepidum was in sharp contrast to those from Cfl. aurantiacus. The difference of chlorosomal spectral properties between Chl. tepidum and Cfl. aurantiacus at the single-unit level would be ascribed to the homolog composition of BChl-c—chlorosomes of Chl. tepidum have BChl substituted with various alkyl groups at both the 8- and 12-positions, whereas light-harvesting BChl-c molecules in Cfl. chlorosomes have the same substituents at the 8- (ethyl group) and 12- (methyl group) positions.

In vitro activity of C-20 methyltransferase, BchU, involved in bacteriochlorophyll c biosynthetic pathway in green sulfur bacteria

The activity of a methyltransferase, BchU, which catalyzes methylation at the C‐20 position of chlorin ring in the biosynthetic pathway of bacteriochlorophyll c, was investigated in vitro. The bchU gene derived from the photosynthetic green sulfur bacterium, Chlorobium tepidum, was overexpressed in Escherichia coli as a His‐tagged protein (His6‐BchU), and the enzyme was purified. In the presence of S‐adenosylmethionine, His6‐BchU methylated zinc bacteriopheophorbide d at the C‐20 position to give zinc bacteriopheophorbide c. Metal‐free bacteriopheophorbide d could not be methylated by the BchU, indicating that the central metal in the chlorin should be required for the recognition by the BchU.

Bacteriochlorophyll-c Homolog Composition in Green Sulfur Photosynthetic Bacterium Chlorobium vibrioformeDependent on the Concentration of Sodium Sulfide in Liquid Cultures

Green sulfur photosynthetic bacteria Chlorobium (Chl.) vibrioforme (DSM 263 strain and NCIB 8327 substrain possessing BChl-c) and Chl. tepidum (ATCC 49652) were photoautotrophically grown in liquid cultures containing different concentrations of sodium sulfide (Na2S). BChl-c homologs possessing a methyl group at the 12-position tended to increase in cells of the two strains of Chl. vibrioforme cultured under high Na2S concentrations. In contrast, the Na2S concentration in liquid cultures did not affect the relative composition of BChl-c homologs in Chl. tepidum. 8-Propyl-12-methyl([P,M])-BChl-c homolog, which has been little observed in usual cultivations, could be isolated by reverse-phase high-performance liquid chromatography from the cells of Chl. vibrioforme grown under high Na2S contents. The [P,M]-BChl-c homolog has the R-configuration at the 31-position, which was determined by 1H-NMR analyses.

Fluorescence spectroscopy of single photosynthetic light-harvesting supramolecular systems.

Recent spectroscopic studies of photosynthetic light-harvesting supramolecular complexes at the single supramolecule level are reviewed. This report describes the “single-molecule” investigation on light-harvesting complex 2 (LH2) of purple photosynthetic bacteria, phycobiliproteins of cyanobacteria and red algae, light-harvesting complex 2 (LHC2) of higher plants, and chlorosomes of green photosynthetic bacteria. Unique behaviors and spectral features of single light-harvesting apparatus have been unraveled that were hidden by the ensemble averaging of many of the complexes. The information obtained with be useful for understanding the electronic structures and energy-transfer mechanism of photosynthetic light-harvesting supramolecular systems.

Self-Assembly of synthetic zinc chlorins in a silicate micelle prepared by sol-gel process

Zinc methyl 3-devinyl-3-hydroxymethyl-pyropheophorbide-a (1), a good model compound of light-harvesting pigments of green photosynthetic bacteria, formed self-aggregates in the presence of octadecyltriethoxysilane and tetraethoxysilane in an aqueous solution to exhibit visible absorption spectra similar to the natural antenna. Base-catalyzed cross-linked polymerization of the additive Si-ORs (R=ethyl and H) afforded the formation of a siloxane network (Si-O-Si) on the surface of the self-assemblies of 1. The resulting microcapsules were stable to tolerate the deaggregation to monomeric 1 by addition of surfactant Triton X-100 more largely than the corresponding micelles before polymerization.

Different sensitivities to oxygen between two strains of the photosynthetic green sulfur bacterium Chlorobium vibrioforme NCIB 8327 with bacteriochlorophyll c and d

Two sub-strains of the anoxygenic photosynthetic green sulfur bacterium Chlorobium vibrioforme NCIB 8327 were derived from the same clone and could be discriminated only by their possession of either bacteriochlorophyll (BChl) c or d as the major pigment in the peripheral light-harvesting antenna system, chlorosome (Saga Y etxa0al. (2003) Anal Sci 19: 1575–1579). In the presence of a proper amount of oxygen in the initial culture medium, the BChl d strain showed longer retardation on its growth initiation than the BChl c strain, indicating that the latter was advantageous for survival under aerobic light conditions which produced reactive oxygen species in vivo. The result would be ascribable to the difference of the midpoint potentials between two kinds of chlorosomes formed by self-aggregates of BChl c and d as measured by their fluorescence quenching.

Effect of metal ion exchange on the photocurrent response from bacteriorhodopsin on tin oxide electrodes

The transient photocurrent response from bacteriorhodopsin (bR) on tin oxide electrodes was strongly influenced by metal ions bound to bR molecules. The photocurrent polarity reversal pH, which corresponded to the pH value for the reversal of the proton release/uptake sequence in the bR photocycle, of cation-substituted purple membrane (PM) was shifted to lower pH with the increase in the cation affinities to carboxyl groups and a close correlation was noted between the two values. This suggests that the metal ion present in the extracellular region of a bR molecule modulates the pK(a) of proton release groups of bR by stabilizing the ionized state of the proton-releasing glutamic acids. The behavior of photocurrents at light-off in alkaline media, reflecting the proton uptake by bR, was unchanged by binding monovalent (Na(+) and K(+)) or divalent cations (Mg(2+) and Ca(2+)), but was drastically changed by binding La(3+) ions. This can be explained by invoking a substantial slowing of the proton uptake process in the presence of La(3+).