Megumi Isaji

Coherent Oscillations in Chlorosome Elucidated by Two-Dimensional Electronic Spectroscopy.

Chlorosomes are the most efficient photosynthetic light-harvesting complexes found in nature and consist of many bacteriochlorophyll (BChl) molecules self-assembled into supramolecular aggregates. Here we elucidate the presence and the origin of coherent oscillations in chlorosome at cryogenic temperature using 2D electronic spectroscopy. We observe coherent oscillations of multiple frequencies superimposed on the ultrafast amplitude decay of 2D spectra. Comparison of oscillatory features in the rephasing and nonrephasing 2D spectra suggests that an oscillation of 620 cm(-1) frequency arises from electronic coherence. However, this coherent oscillation can be enhanced by vibronic coupling with intermolecular vibrations of BChl aggregate, and thus it might originate from vibronic coherence rather than pure electronic coherence. Although the 620 cm(-1) oscillation dephases rapidly, the electronic (or vibronic) coherence may still take part in the initial step of energy transfer in chlorosome, which is comparably fast.

Isolation and pigment composition of the reaction centers from purple photosynthetic bacterium Rhodopseudomonas palustris species

The reaction centers (RCs) from several species of a purple photosynthetic bacterium, Rhodopseudomonas palustris, were first isolated by ammonium-sulfate fractionation of the isolated core complexes, and were successfully purified by anion-exchange and gel-filtration chromatography as well as sucrose-density gradient centrifugation. The RCs were characterized by spectroscopic and biochemical analyses, indicating that they were sufficiently pure and had conserved their redox activity. The pigment composition of the purified RCs was carefully analyzed by LCMS. Significant accumulation of both bacteriochlorophyll(BChl)-a and bacteriopheophytin(BPhe)-a esterified with various isoprenoid alcohols in the 17-propionate groups was shown in RCs for the first time. Moreover, a drastic decrease in BPhe-a with the most dehydrogenated and rigid geranylgeranyl(GG) ester was observed, indicating that BPhe-a in RC preferably took partially hydrogenated and flexible ester groups, i.e. dihydro-GG and tetrahydro-GG in addition to phytyl. Based on the reported X-ray crystal structures of purple bacterial RCs, the meaning of flexibility of the ester groups in BChl-a and BPhe-a as the cofactors of RCs is proposed.

Structural determination of the Δ2, 10-phytadienyl substituent in the 17-propionate of bacteriochlorophyll-b from Halorhodospira halochloris

Bacteriochlorophyll(BChl)-b, having a unique phytadienyl group in the propionate-type residue at the 17-position instead of a usual phytyl group, was isolated from the thermophilic purple photosynthetic bacterium Halorhodospira (Hlr.) halochloris. The structure of the propionate, especially for the positions of two C=C double bonds, was unambiguously determined to be C2=C3 and C10=C11 (Δ2, 10-phytadienyl) by means of NMR spectroscopy. To confirm the molecular structure by reverse-phase (RP) HPLC, two types of chlorophyll (Chl) derivatives, 3-acetyl-Chl-a, having different phytadienyl groups to the 17-propionate were prepared: one had a Δ2, 10-phytadienyl group prepared by isomerization of the structurally determined BChl-b, and the other had a Δ2, 14-phytadienyl by oxidation of BChl-a from the other purple bacterium Rhodopseudomonas sp. Rits as reported previously. HPLC analyses of these derivatives showed their distinct retention times under reverse-phase conditions; the Δ2, 14-phytadienyl-type derivative was eluted more slowly than the Δ2, 10-phytadienyl type. The results clearly indicated that the positions of two C=C double bonds in the ester group affected RP-HPLC elution, which directly reflected the hydrophobicity in a molecule. RP-HPLC analyses thus serve as an aid for structural determination of (B)Chl molecules esterified with various long hydrocarbon chains in photosynthetic organisms, and also enable estimation of their hydrophobicity and hydrophilicity.

The 17-propionate esterifying variants of bacteriochlorophyll-a and bacteriopheophytin-a in purple photosynthetic bacteria

Most purple photosynthetic bacteria contain bacteriochlorophyll(BChl)-a (a magnesium complex) and bacteriopheophytin(BPhe)-a (its free base) as their photoactive pigments. These pigments are composed of two parts: a cyclic tetrapyrrole as the chromophore and a long hydrocarbon-chain as the propionate-type esterifying group at the 17-position. The hydrocarbon-chain is usually an isoprenoid-type C20 phytyl (Phy) group in both the pigments. In the ester group of BChl-a, several variants such as geranylgeranyl (GG), dihydrogeranylgeranyl (DHGG) and tetrahydrogeranylgeranyl (THGG) groups were found in the final stage of BChl-a biosynthesis. On the other hand, the esterifying variants in BPhe-a have not been studied as much due to the lower levels of this pigment relative to BChl-a. The esterifying group does not affect the electronic absorption properties of such pigments in the monomeric state, but drastically alters the hydrophobicity. In this study, BChl-a and BPhe-a in the six phylogenetically distinct classes of purple bacteria were analyzed in terms of their esterifying groups in the 17-propionate residues, using high-performance liquid chromatography. Both BChls-a and BPhes-a carrying GG, DHGG and THGG in addition to the usual Phy were found for all the bacterial species studied at measurable levels. In some of the species, the ratio of BPhes-a esterified with GG, DHGG and THGG over the total BPhe-a drastically decreased in comparison with that of the corresponding BChls-a. Especially, the relative content of BPhe-a with GG largely decreased. This observation might indicate that BPhe-a as a cofactor of reaction centers was preferentially esterified with partially reduced and flexible chains (THGG and Phy) rather than less reduced and rigid ones (GG and DHGG).

Structure-dependent demetalation kinetics of chlorophyll a analogs under acidic conditions.

Demetalation of chlorophyll (Chl) a and its analogs is an important reaction in oxygenic photosynthetic organisms, which produces the primary electron acceptors in photosystem II reaction centers and is crucial in the Chl degradation. From these viewpoints, demetalation reactions of four Chl a analogs, 3,8‐divinyl‐Chl a (DV‐Chl a), 3‐devinyl‐3‐ethyl‐Chl a (mesoChl a), 132‐demethoxycarbonyl‐Chl a (pyroChl a) and protochlorophyll a (PChl a), were kinetically analyzed under weakly acidic conditions, and were compared with that of Chl a. DV‐Chl a exhibited slower demetalation kinetics than did Chl a, whereas demetalation of mesoChl a was faster than that of Chl a. The difference in demetalation kinetics of the three chlorophyllous pigments originates from the electron‐withdrawing ability of the vinyl group as the peripheral substituent compared with the ethyl group. Removal of the electron‐withdrawing and homoconjugating 132‐methoxycarbonyl group in Chl a (Chl a → pyroChl a) accelerated demetalation kinetics by two‐fold. PChl a possessing the porphyrin‐type skeleton exhibited slower demetalation kinetics than Chl a. The structure‐dependent demetalation properties of Chl a analogs will be useful for understanding in vivo Chl demetalation reactions in oxygenic photosynthetic organisms.

Molecular Structures and Functions of Chlorophylls-a Esterified with Geranylgeranyl, Dihydrogeranylgeranyl, and Tetrahydrogeranylgeranyl Groups at the 17-Propionate Residue in a Diatom, Chaetoceros calcitrans

The 17-propionate ester group of chlorophyll(Chl)-a in some oxygenic phototrophs was investigated using HPLC. Chls-a esterified with partially dehydrogenated forms of a phytyl group were found in fully grown cells of a diatom, Chaetoceros calcitrans: geranylgeranyl (GG), dihydrogeranylgeranyl (DHGG), and tetrahydrogeranylgeranyl (THGG). Chls-a bearing such esterifying groups were reported to be found only in greening processes of higher plants, and thus these Chls-a have been thought to be biosynthetic precursors for phytylated Chl-a. Their molecular structures were unambiguously determined using 1H and 13C NMR spectroscopy and mass spectrometry. In particular, the positions of C═C double bonds in DHGG were identified at C2═C3, C6═C7, and C14═C15, and those in THGG were determined to be at C2═C3 and C14═C15. Notably, the present DHGG was different from the previously determined DHGG of bacteriochlorophyll-a in purple bacteria (C2═C3, C10═C11, and C14═C15). Moreover, thylakoid membranes as well as fucoxanthin-chlorophyll-a/c proteins called FCPs were isolated from the diatom, and their Chl-a compositions were analyzed. Chls-a esterified with GG, DHGG, and THGG were detected by HPLC, indicating that such Chls-a were not merely biosynthetic precursors, but photosynthetically active pigments.

Compositional Analysis of Carotenoids in a Purple Photosynthetic Bacteria, Rhodopseudomonas sp. Strain Rits, upon Irradiated Light Intensity

Recently We Isolated A Purple Photosynthetic Bacterium Rhodopseudomonas (Rps.) Sp. Strain Rits Which Is Phylogenetically Close To Rps. Palustris. Under Low-Light Conditions, This Strain Biosynthesized An Unusual Peripheral Antenna (Lh4) At The Expense Of The Usual Lh2. In This Study, We Analyzed The Composition Of Carotenoids From The Rits Strain Together With Rps. Palustris That Were Grown By Irradiation Of Different Light Intensity. All Seven Carotenoids Of Spirilloxanthin Biosynthetic Pathway (Lycopene, Rhodopin, 3,4-Didehydrorhodopin, Anhydrorhodovibrin, Rhodovibrin, Oh-Spirilloxanthin, Spirilloxanthin) Were Clearly Detected By High-Resolution Normal-Phase Hplc. In The Extract From The Cells, 3,4-Didehydrorhodopin Having 12 Conjugated Double Bonds As Well As One Terminal Hydroxy Group Was Amajor Carotenoid Under Normal- And High-Light Conditions, While Rhodopin Having 11 Conjugated Double Bond As Well As One Terminal Hydroxy Group Was A Major Component Under Low-Light Conditions. 3,4-Didehydrorhodopin Was Found To Be First Accumulated At A Significant Amount In Rps. Species Including The Rits Strain And Rps. Palustris Cga009/Dsm123.

Diversity and Localization of Bacteriochlorophylls Possessing Different 17-Propionate Groups in Purple Bacterial Antennae

In the final stage of biosynthesis of phytylated bacteriochlorophyll (BChl), the presence of BChl-a molecules possessing geranylgeranyl, dihydro- and tetrahydrogeranylgeranyl groups at the 17-propionate were reported, but the molecular structures of the latter two BChl-a intermediates were not determined in terms of the positions of C = C double bonds in the 172-ester. Recently, we isolated a purple photosynthetic bacterium, Rhodopseudomonas (Rps.) sp. strain Rits, which was phylogenetically close to Rps. palustris. This strain biosynthesized unusual peripheral antenna (LH4) at the expense of usual LH2 under low-light conditions, and accumulated up to 40% of total the BChls-a produced as BChl-a intermediates having different 17-propionate. In this study, we isolated significant amounts of BChls-a in pure form and determined their structures by both mass spectrometry and 1H− and 13C−NMR spectroscopy. We also analyzed the composition of the BChla intermediates in the isolated pigment-protein complexes (LH2, LH4 and RC-LH1) from Rps. sp. strain Rits, Rps. palustris CGA009 and Rps. palustris DSM123 by HPLC. The results indicate that the BChl-a intermediates are found in the RC-LH1 rather than in the peripheral antennae.