Kaku Uehara

Raman spectra of chlorophyll forms

Abstract The Raman spectra of chlorophyll a (Chl) forms in aqueous poly(vinyl alcohol) (PVA) and in dimethyl sulfoxide (DMSO)—water mixtures were recorded at 457.9 nm excitation and their structures were characterized by comparison with the spectra of the following well-known chlorophyll forms: (1) monomers (Chl) 1 in the polar solvents of group (A), i.e., diethyl ether, tetrahydrofuran, acetone, N , N -dimethylformamide and DMSO, of which the oxygen atom is expected to coordinate to the central magnesium atom; (2) monomers (Chl) 1 in the polar solvents of group (B), i.e., methanol, ethanol, 1-propanol, 2-propanol and 1-butanol, which are supposed to form a hydrogen-bond to the C 9 =O group in addition to the coordination-bond to the Mg atom; (3) dehydrated aggregates (Chl) n in dry non-polar solvents, i.e., carbon tetrachloride, n-hexane and n-octane; and (4) hydrated aggregates (Chl·2H 2 O) n in wet non-polar solvents, i.e., n-hexane and n-octane. The frequency of the C 9 = O stretching Raman line of each of the above chlorophyll forms was: (1) 1702—1680 cm −1 ; (2) 1673—1668 cm −1 ; (3) around 1655 cm −1 ; (4) around 1645 cm −1 . The frequency proved to be a marker of intermolecular interaction of the Chl molecules. The spectral patterns in the 1650—700 cm −1 region of (1), (2) and (3) were similar. However, the relative intensities of Raman lines of (4), which was ascribed to a one-dimensional, regular stacking of the Chl macrocycles, were quite different from those of (1)—(3). The chlorophyll form in PVA aqueous solution was identified as (Chl·2H 2 O) n by spectral comparison. The chlorophyll forms present in the DMSO—water mixtures were highly dependent on the DMSO content. It is suggested that (Chl) 1 having hydrogen-bonded H 2 O should be present in 10% DMSO solution, and that a new chlorophyll form (Chl·DMSO) n having (a) a stoichiometric intermolecular interaction with DMSO and (b) a regular stacking of the chlorophyll macrocycles, should be predominant in 50% DMSO aqueous solution.

The formation and characterization of the in vitro polymeric aggregates of bacteriochlorophyllc homologs fromChlorobium limicola in aqueous suspension in the presence of monogalactosyl diglyceride.

Artificial aggregates of bacteriochlorophyllc (BChlc) were formed in an aqueous medium in the presence of a lipid, monogalactosyl diglyceride (MGDG), and the optical properties of those aggregates were studied by absorption and circular dichroism (CD) mainly. Four BChlc homologs, ([E,E]BChlcF, [P,E]BChlcF, [E,M]BChlcF and [I,E]BChlcF), were isolated from the green photosynthetic bacteriumChlorobium limicola strain 6230. Above 0.0004%, MGDG induced a red-shift of the absorption maxima of BChlc aggregates. At 0.003% MGDG BChlc aggregates showed absorption maxima in the range of 724 to 745 (±3) nm with a shift of 12 to 24 (±3) nm depending on the homolog species. Four kinds of BChlc-MGDG aggregates showed characteristic CD spectra. [E,M]BChlcF gave rise to a CD spectrum similar to that of chlorosomes, while the other three gave spectra of opposite sign. These aggregates are sensitive to 1-hexanol treatment; in a saturating amount (0.85%) of 1-hexanol, all the homologs gave a monomer-like absorption spectrum peaking at 670nm. At an intermediate concentration (0.5%), [E,M]BChlcF showed an enhanced CD intensity, as observed in native chlorosomes. Resonance Raman spectra of the monomer-like BChlc samples indicated that the keto vibrational band at ca. 1640 cm−1 was considerably weakened by the 0.85% 1-hexanol treatment, however the 1680 cm−1 band characteristic of a free keto group did not appear. These results indicate that the artificial aggregates formed by purified BChlc homologs and MGDG are good models for studying chlorosomes structure.

Aggregation of bacteriochlorophyll c homologs to dimers, tetramers, and polymers in water-saturated carbon tetrachloride

Three homologs of BChl c, 2-(R)-(1-hydroxyethyl)-4-n-propyl-5-ethyl-farnesyl BChl c (PEF-BChl c), 2-(R)-(1-hydroxyethyl)-4-ethyl-5-ethyl-farnesyl BChl c (EEF-BChl c), and 2-(S)-(1-hydroxyethyl)-4-isobutyl-5-methyl/ethyl-farnesyl BChl c (iBM/EF-BChl c), formed aggregates in water-saturated carbon tetrachloride (H2O-satd CCl4). The water content was about 100 times higher than that of the dried CCl4 previously used. Absorption spectra were recorded for 8 concentrations for the three homologs of BChl c and were deconvoluted in terms of standard spectra of monomer, dimer, tetramer and polymer (747-nm aggregate, Olson and Pedersen (1990) Photosynthe Res 25: 25). PEF- and EEF-BChl c formed dimers (680 nm maximum) and tetramers (705–710 nm maximum), but iBM/EF-BChl c formed polymers. Inhibition of dimer formation by water faciliated the study of the initial stages of the polymerization of BChl c. When the logarithm of polymer concentration was plotted versus the logarithm of the monomer concentration for iBM/EF-BChl c, the initial slope was 30±10 and indicated the cooperation of 20–40 BChl c molecules to form a polymer from a seed. Circular dichroism spectra of the polymers with positive and negative bands at 743 and 760 nm, respectively, were similar to those for chlorosomes (Brune et al. (1990) Photosynth Res 24: 253).

The Effects of Epimerization at the 31‐position of Bacteriochlorophylls c on their Aggregation in Chlorosomes of Green Sulfur Bacteria. Control of the Ratio of 31 Epimers by Light Intensity

R‐ and S‐epimerization at the 31 position of bacteriochlorophyll (BChl) c and the formation of rod‐like aggregates in chlorosomes of green sulfur bacteria were markedly affected in Chlorobium (Cb.) tepidum and Cb.limicola by cultivation under various light intensities (photon fluence rate). The stronger the light, the higher the ratio of the S‐epimer to the R‐epimer for each homolog of BChl c in the bacteria. S[P,E] BChl cF and S[I,E] BChl cF were found to be the major S‐epimers in Cb. tepidum and Cb. limicola, respectively. R[P,E] BChl cF decreased markedly compared to R[E,E] BChl cF in Cb. tepidum, whereas no observable change in the ratio of R[P,E]/R[E,E] was detected for Cb. limicola. With increase in light intensity the Qy absorption maximum of the bacteria shifted to shorter wavelengths. In vitro spectroscopic studies of the aggregates showed a marked difference in the formation of aggregates from R‐ and S‐epimers of BChl c; the S‐epimers formed aggregates much more slowly than did the R‐epimers. These results suggest that the ratio of the epimers of BChl c might significantly affect the aggregation of BChl in the chlorosome. We propose different roles for the R‐ and S‐epimers in chlorosomes of Cb. limicola and Cb. tepidum.

SPECTRAL ANALYSIS OF CHLOROPHYLL a AGGREGATES IN THE PRESENCE OF WATER-SOLUBLE MACROMOLECULES

Abstract— The absorption and fluorescence spectra of chlorophyll a (Chi a) aggregates formed in aqueous solutions of polyvinyl alcohol) (PVA), polyvinyl pyrrolidone) (PVP), and bovine serum albumin (BSA) were analyzed by curve‐fitting methods in the wavelength region from 650 to 800 nm. The results indicated that the aggregation of Chi a to polymeric forms such as (Chia–2H20), was suppressed in the presence of the macromolecules. The suppression was due to a coordination of macromolecule bound ligands to Chi a and was strongest in BSA and weaker in PVA. There were differences in the spectra even though the same types of polymeric Chi a forms were observed due to characteristically different composition of these forms. Fluorescence patterns indicated that energy was transferred from the shorter to the longer wavelength forms.

The Role of Carotenoids in the Photoadaptation of the Brown-colored Sulfur Bacterium Chlorobium phaeobacteroides

The brown‐colored sulfur bacterium Chlorobium (Cb.) phaeobacteroides 1549 (new name, Chlorobaculum limnaeum 1549) contains many kinds of carotenoids as well as bacteriochlorophyll (BChl) e. These carotenoids were identified with C18‐high‐performance liquid chromatography, absorption, mass and proton nuclear magnetic resonance spectroscopies and were divided into two groups: the first is carotenoid with one or two φ‐end groups such as isorenieratene and β‐isorenieratene and the second is carotenoid with one or two β‐end groups such as β‐zeacarotene, β‐carotene and 7,8‐dihydro‐β‐carotene. The latter 7,8‐dihydro‐β‐carotene was found to be a novel carotenoid in nature. OH‐γ‐Carotene glucoside laurate and OH‐chlorobactene glucoside laurate were also found as minor components. The distribution of BChl e homologs in Cb. phaeobacteroides cultivated under various light intensities did not change, but the carotenoid to BChl e ratio changed markedly: carotenoid with the φ‐end group maintained the same ratio to BChl e, whereas that with the β‐end group increased with increasing light intensity. The cells cultured under low‐light intensity contained more φ‐end carotenoids than β‐end. In Cb. phaeobacteroides the wavelength of the Qy band of BChl e aggregates did not change. We suggested that Cb. phaeobacteroides photoadapts to light intensity by changing the carotenoid composition.

SPECTROSCOPIC STUDIES OF CHLOROPHYLL a AGGREGATES FORMED BY AQUEOUS DIMETHYL SULFOXIDE

Characteristic chlorophyll (Chl) a aggregates formed in aqueous dimethyl sulfoxide (DMSO) were investigated spectroscopically. Four chlorophyll forms were found with increasing DMSO concentration; they are called A‐672, A‐683, A‐695 and A‐665 according to the wavelengths of their absorption maxima. Transformation occurred only in this order. Reverse transformation could not be realized. A‐683 and A‐695 were apparently formed by the interaction of Chl a with DMSO in the linear dimer and linear polymer arrangements, respectively. Coordination of the Mg atom with a DMSO O atom and interaction between the S atom of one DMSO molecule and the O atom of an other DMSO molecule should lead to formation of a sandwich‐type complex of partially overlapping chlorophyll macrocycles (Chl a‐DMSO)n. A‐672 and A‐665 were assigned to Chl a micelles and to dissolved monomeric Chl a in DMSO, respectively. Fluorescence spectra showed that the A‐683 was highly fluorescent, while the A‐695 was less fluorescent. Energy migration within the A‐695 form to a trap with a low fluorescence yield might be responsible for this difference in the emission intensity.

How the formation process influences the structure of BChl c aggregates

The change of absorption spectra has been measured during the drying process of (31 R)bacteriochlorophyll (BChl) c from diethyl ether, dichloromethane (CH2Cl2) and carbon tetrachloride (CCl4) solutions. Absorption maxima of the Qy(0–0) transition of BChl c appear at 659 nm in diethyl ether, 680 nm in CH2Cl2 and 710 nm in CCl4. All these peaks are red-shifted to about 740 nm with formation of solid high aggregates when the solutions are completely dried. Fourier transform infrared spectra of the three solid aggregates are almost identical. However, magnetic circular dichroism and circular dichroism spectra are different and can be explained in terms of variations in stacking size of the aggregates and molecular arrangement of BChl c. Small-angle X-ray diffraction has been observed only for the aggregates treated with CH2Cl2, and the same sample gave rise to highly resolved cross polarization/magic angle spinning 13C nuclear magnetic resonance spectrum. The results suggest that molecular ordering of the solid-state BChl c aggregates is highly dependent on the formation process which is largely determined by the solvent used.

Redox reaction at the two-layer interface between aluminum and electropolymerized poly(3-methylthiophene) thin solid films

The corrosion of an aluminum (Al) layer vacuum-deposited onto the electropolymerized poly(3-methylthiophene) (PMeT) film grown on the gold electrode was studied by visible absorption, ESR spectroscopy and XPS and by SEM analysis from the viewpoint of the redox reaction in the dark at the two-layer interface between Al and PMeT for the Al/doped PMeT/Au sandwich cell. The electrochemical undoping of the perchlorate anions from a PMeT induced a remarkable suppression of the redox reaction at the two-layer interface leading to avoid a corrosion of Al electrode vacuum-deposited onto the PMeT film. It was suggested that the rectifying and photovoltaic effects for the Au/undoped PMeT/Al sandwich cell might be explained under the light of the redox reaction between Al and PMeT under biased voltage and under photo-illumination, respectively.

The Effects of pH and Ionic Strength on the Aggregation of Bacteriochlorophyll c in Aqueous Organic Media: The Possibility of Two Kinds of Aggregates

The aggregation behavior of two homologs of bacteriochlorophyll c (BChl c) in various media was investigated for the effects of pH and salt, and the corresponding structures were analyzed by Fourier transform (FT)‐IR spectroscopy. R‐[P, E] BChl cF (31‐R‐form of BChl c with a propyl group at the C‐8 position and an ethyl group at the C‐12 position) and R‐[E, E] BChl cF (31‐R‐form of BChl c with two ethyl groups at positions C‐8 and C‐12) were isolated from the green sulfur bacterium Chloro‐bium limicola. Aggregates of each homolog showed a pH‐dependent shift of the absorption maximum; at low pH, the peak moved to the red. This tendency was also revealed by circular dichroic spectra. A similar red shift of the peak was also induced by a high concentration of salt (NaCl) or buffer for both homologs. The FT‐IR spectrum indicates that at low pH, both homologs formed a rather amorphous aggregate. On the other hand, a regular structure of R‐[P, E] BChl cF was indicated in an acetone‐water mixture. This structure was stabilized by a triangular interaction among three pigment molecules through the Mg‐OH (3>) O = C (131) linkage. This structure was not found for R‐[E, E] BChl cF. These results indicate that the replacement of the side chain at the C‐8 position on the macrocycle induces a change in aggregation behavior. A possible heterogeneity of the in vivo rod structure of chlorosomes in green sulfur bacteria is discussed based on the above results.