Molecular structures of (bacterio)chlorophylls [=(B)Chls] in photosynthetic apparatus are surveyed, and a diversity of the ester groups of the 17-propionate substituent is particularly focused on in this review. In oxygenic photosynthetic species including green plants and algae, the ester of Chl molecules is limited to a phytyl group. Geranylgeranyl and farnesyl groups in addition to phytyl are observed in (B)Chl molecules inside photosynthetic proteins of anoxygenic bacteria. In main light-harvesting antennas of green bacteria (chlorosomes), a greater variety of ester groups including long straight chains are used in the composite BChl molecules. This diversity is ascribable to the fact that chlorosomal BChls self-aggregate to form a core part of chlorosomes without any specific interaction of oligopeptides. Biological significance of the long chains is discussed in photosynthetic apparatus, especially in chlorosomes.
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All naturally occurring chlorophyll pigments are synthesized in photosynthetic organisms (1–4). A variety of chlorophyllous molecules, cyclic tetrapyrroles possessing an exo-five-membered ring, are found: typically, chlorophyll a (Chl a) in plants and bacteriochlorophyll a (BChl a) in photosynthetic bacteria (5–7). Moreover, a wider range of their biosynthetic intermediates and degradates (protochlorophyllide a, pheophorbide a, etc.) are detected in phototrophy (1) and their metabolites (purpurin 18, 132-oxopyropheophorbideetc.) are observed in nonphotosynthetic organisms including herbivores (8); many stable degraded products called geoporphyrins are also in sediments (9). All of them are categorized into a chlorophyllous family, but here photosynthetically active chlorophylls in matured phototrophs are considered which are engaged in light-harvesting (LH) and energy/electron-transferring processes at the antennas and reaction centers (RCs). Such chlorophyllous molecules have a propionate-type ester group except most Chls c, and the long chains in the ester are focused on in this review with regard to their distribution and biological implication. These esterifying substituents are not directly conjugated with the π-systems in a molecule and do not affect the electronic absorption spectra of their monomeric states. As a result, they have attracted little attention compared with other peripheral substituents (5), and here we spotlight the less remarked chains which occupy about one-third (or fourth) of (B)Chl molecules by weight.
Molecular structures of (bacterio)chlorophylls
The chlorophyllous pigments in this review have molecular structures as shown in Fig. 1. The nomenclature follows the IUPAC–IUB recommendation. Chls a/b/d are magnesium complexes (M = Mg) of chlorins (17,18-dihydroporphyrins) with a single C17–C18 bond (17S and 18S configurations) and characterized by their peripheral substituents (R3 and R7 groups). BChls a/b/g have bacteriochlorin π-systems with two single bonds at the C7–C8 and C17–C18. BChls c/d/e are called bacteriochlorophylls due to their occurrence in photosynthetic bacteria but possess the same chlorin moieties as Chls a/b/d. Chls c are a family of porphyrin compounds with complete π-conjugation (C7=C8 and C17=C18). The above (B)Chl molecules are classified as an ester (172-COOR), except most Chls c. The esterifying group (R in Fig. 1) is abbreviated as in P, F and M for phytyl, farnesyl and methyl groups, the letters being added just after (B)Chl x as the subscript form, for example, phytylated chlorophyll a = Chl aP.
The central magnesium ion of (B)Chls is substituted with two protons (M = H2) to give metal-free (bacterio)pheophytins, (B)Phes. The Mg is changed to other divalent metals to give the corresponding metal complexes, which are here called metal (bacterio)chlorophylls as in Zn-BChl a for zinc-substituted BChl a (M = Zn).
The 172-ester group of (B)Chls is cleaved to the corresponding acids, (bacterio)chlorophyllides (R = H). These compounds are abbreviated as (B)Chl xH, where the subscript H means a hydrogen atom. It is noteworthy that most Chls c refer to chlorophyllides c (Chls cH).
Esterification of (bacterio)chlorophyllides
At the final stage of the biosynthetic pathway in chlorophyllous pigments except most Chls c, (bacterio)chlorophyllides [(B)Chls xH] possessing a carboxy group (COOH) at the 172-position on the D-ring are esterified with various long chains including isoprenoids (10,11). The bioprocess proceeds between an unactivated acid component and an activated alcohol component catalyzed by an enzyme. Typically, chlorophyllide a (Chl aH) was esterified with geranylgeranyl (GG) diphosphate by a Chl synthase ChlG (produced by chlG gene) to give geranylgeranylated Chl a (Chl aGG). Such a biosynthesis is different from the conventional organic synthesis of esters, where an activated acid attacked a free alcohol; for example, acetyl chloride (MeCOCl) reacted with ethanol (EtOH) in a homogeneous organic solution to afford ethyl acetate (MeCOOEt). Usually, the diphosphates as activated alcohols are prepared from successive substitution of dimethylallyl diphosphate [Me2C=CHCH2OP(O)(OH)OP(O)(OH)2] with isoprenyl diphosphate [CH2=CMeCH2CH2OP(O)(OH)OP(O)(OH)2] and limited to diphosphated isoprenoid alcohols as the substrate for the above in vivo enzymatic esterification, except for synthesis of some BChl c/d/e molecules.
In some cases, a free alcohol, which is prepared by hydrolysis of phosphates, biosynthesized (or biodegraded) by other routes or incorporated from the outside of cells, is activated by kinase to produce the corresponding diphosphate as a substrate (12). Chlorophyllases, which hydrolyze the 17-propionate ester to the acidic chlorophyllide and the corresponding alcohol in biodegradation of chlorophyllous pigments, can catalyze the reverse esterification in vitro (13). The enzymatic reaction proceeds in the presence of a large excess of alcohol and is inactive in the in vivo synthesis of chlorophyllous pigments.
Chlorophylls in oxygenic phototrophs
Chl a and b molecules (see left and middle drawings of Fig. 2) are obtained from usual oxygen-evolving green plants (eukaryotes). Their derivatives including the 8-vinyl analogs are also detected in some species of prokaryote. Moreover, only Chl a species (not Chl b) are prepared in cyanobacteria. Chl a is used in LH antenna and electron-transferring RC apparatus and Chl b is situated exclusively in peripheral antenna systems. In RCs, other Chls function in the electron-transferring process (14): one Chl a′ (the 132-epimer of Chl a) molecule interacts with the other Chl a molecule to form the dimeric component in the primary electron donor (P700) of photosystem (PS) 1 and Phe a, a metal-free compound of Chl a, acts as the PS2 primary electron acceptor. It is noted that neither Chl b′ nor Phe b has been observed in any photoactive systems.
Both Chls a and b always have phytyl groups in their 172-ester in matured cells. In greening of angiosperms, GG, dihydrogeranylgeranyl (DHGG) and tetrahydrogeranylgeranyl (THGG) esters were also tentatively observed (15,16), but all three species quickly disappeared due to more hydrogenation to phytyl (hexahydrogeranylgeranyl) ester or hydrolysis to chlorophyllide. It is unknown whether these three species function photosynthetically or not; however, the observation provides information on the last stage of Chl biosynthesis (Scheme 1). In one pathway, Chl aH is esterified with GG diphosphate to give Chl aGG and successive hydrogenation by GG reductase yields Chl aP. In the other, GG diphosphate is first reduced and the resulting P diphosphate reacts with Chl aH by Chl synthase to give Chl aP. The actual biosynthetic pathway has not yet been determined: which way is active in vivo as well as whether both pathways proceed in natural systems. The positions of two double bonds in the THGG group of Chl aTHGG were determined from the mass spectroscopic analysis of its decomposed products to be C2=C3 and C14=C15 (17). The molecular structure of DHGG group has not been identified yet, but successive hydrogenation mentioned above suggests that either the C6=C7 or C10=C11 double bond of GG group should first be reduced (see Scheme 2).
The gene chlP encodes GG reductase. The chlP-inactivated mutant of a cyanobacterium Synechocystis sp. PCC 6803 grew slowly in the presence of glucose under low light conditions to accumulate exclusively Chl aGG as expected (18). The mutant could not be cultured photoautotrophically, indicating that Chl aGG is inactive as the alternative of Chl aP in the LH and RC proteins. Moreover, no other esterified Chls a including farnesylated Chl a (Chl aF) (12,13,19) could be found as naturally occurring and photoactive pigments. Chl aP molecules are nicely accommodated in the LH/RC proteins, which engage in LH and energy/electron-transferring processes. In matured photosynthetic species, all Chl b, Chl a′ and Phe a molecules are also phytylated in the ester at the 172-position. Thus, the phytyl ester is necessary and important for chlorophyllous pigments in oxygenic photosynthesis.
Recently, Chl d, the 3-CHO derivative of Chl a (see right drawing of Fig. 2) was recognized as a photofunctioning pigment in some cyanobacteria (20–22). The full molecular structure was determined to be phytylated Chl d (Chl dP) in Acaryochloris marina (23,24). All the other minor pigments, Chl d ′, Chl a and Phe a are also phytylated (14): Chl d ′P (PS1 primary electron donor), Chl aP (PS2 primary electron donor?) and Phe aP (PS2 primary electron acceptor). It is noteworthy that neither Phe d nor Chl a′ has been detected in the Chl d-dominant species.
Chlorophyll c is observed as antenna pigments in chromophyta. It is named for a family of 17,18-dedihydrogenated Chl derivatives possessing a fully conjugated porphyrin π-system. Chl c1, c2 and c3 are well known and have an acrylic acid moiety (–CH=CHCOOH) at the 17-position (see Fig. 3). Normally, Chl c has no ester group on the 17-position but is a free carboxylic acid. In Emiliania huxleyi, one of the haptophyte algae, nonpolar Chls c were detected in addition to usual polar Chls c. One of the new pigments was first reported to be a phytylated ester (Chl cP) but later revised based on the visible and mass spectral analyses of its carefully HPLC-separated sample to be monogalactosyldiacylglycerol (MGDG) ester of Chl c2 (see Fig. 3); the two acyl parts were primarily myristoyl (C14) and unsaturated stearoyl (C18:4) (25). In other haptophyte species (Chrysochromulina) Chl c2MGDG was observed with two myristoyl groups (26). The function of Chl c2MGDG is still unknown, but it might be a mediator for light-energy migration from polar Chl cH to nonpolar Chl aP. Chl c is always with Chl a in a photosynthetic cell as Chl b is beside Chl a, and Chl b and c molecules are not present in the same cell except a prasinophycean alga, Mantoniella squamata (27).
(Bacterio)chlorophylls in anoxygenic photosynthetic bacteria
Bacteriochlorophyll a (see left drawing of Fig. 4) is found in purple bacteria. Its 8,81-dedihydro-derivative, BChl b (see middle drawing of Fig. 4), possessing an ethylidene group at the 8-position is observed in some species of purple bacteria. Either BChl a or BChl b is a photosynthetically active pigment in a purple bacterium and no species has both of these in a single cell. As a minor component, the corresponding metal-free pigments BPhes a and b are present in BChl a- and b-dominant species, respectively. BPhes a and b act as the primary electron acceptor in PS2-type RC which is similar to Phe a in PS2-RC of oxygenic phototrophs. It is noted that BChl b molecules are observed in both LH and RC apparatus, while Chl b is seen in only peripheral LH; also, BPhe b is present in photosynthetic proteins but Phe b is not. Recently, zinc BChl a (Zn-BChl a) was found in a purple bacterium, Acidiphilium rubrum (28,29). Most acidophilic purple bacteria have Zn-BChl a as an alternative of BChl a and also possess BPhe a molecules in their PS2-type RCs. Moreover, a small amount of BChl a is observed in Zn-BChl a-dominant cells, but its function is not known.
Usually, (Zn-)BChl a, BChl b, BPhe a and BPhe b have a phytyl ester on the 17-position. From some batch cultures of purple bacteria, furthermore, GG, DHGG and THGG esters of BChl a were detected in HPLC (13) and the molecular structures of the latter two ester groups have not been fully determined yet. The biosynthetic pathway from BChls aH to BChl aP is still in debate as true in that from Chl aH to Chl aP (vide supra). In a purple bacterium Rhodospirillum (Rs.) rubrum, BChl aGG functions as an alternative of BChl aP but did not produce peripheral LH antennas (LH2) due to unstable complexation of LH2-oligopeptides with BChl aGG and/or poor assembly of the resulting complexes (30). In this species, BPhe aP was observed but not BPhe aGG. The enzyme produced from the gene bchP encoding GG reductase in this species could reduce metal-free BPhe aGG to BPhe aP but not the corresponding magnesium complex, BChl aGG. Rs. rubrum would be a strain where bchP in BChl aP-dominant purple bacteria was naturally mutated and its producing enzyme BchP enhanced the substrate specificity. In Ectothiorhodospira halochloris (one of purple bacteria), 6,7,14,15-tetrahydrogeranylgeranyl (10,11-dedihydrophytyl) ester of BChl b was found (31). Farnesyl ester of BChls a/b has never been isolated from natural purple bacteria (32), but the genetic mutant of an Rs. rubrum strain produced BChl aF (33). The mutant could not grow photoautotrophically, indicating that BChl aF must be inactive in LH/RC proteins as mentioned for Chl aGG (vide supra). Therefore, BChls in purple bacteria have a greater variety of ester groups, while Chls in oxygenic photosynthetic apparatus possess a phytyl group exclusively, showing that anoxygenic bacteria are more flexible and adaptable for growing photoautotrophically than oxygenic phototrophs.
Green bacteria have phytylated BChl a (BChl aP) molecules in their core LH and RC apparatus as in purple bacteria. In green filamentous (nonsulfur or gliding) bacteria, the RC is a PS2 type similar to that in purple bacteria and three BPhe aP molecules were observed in one RC unit. The RC of green sulfur bacteria is PS1 type, where two BChl a′P molecules form the special pair as the primary electron donor and 10,11,14,15-tetrahydrogeranylgeranyl (6,7-dedihydrophytyl) ester of Chl a is the primary electron acceptor (34). It is interesting that Chl a molecules were found in anoxygenic bacteria. The ester group is one of THGGs and different from the proposed structure in Chl aTHGG observed during the greening process mentioned above.
Most chlorophyllous pigments in green bacteria are BChls c, d and e (see right drawing of Fig. 4) in main peripheral antenna systems (chlorosomes). These molecules have chlorin π-systems but named as bacteriochlorophylls due to their occurrence in typical anoxygenic bacteria. BChls c, d and e self-aggregates without any significant help of proteins to form a core part of LH chlorosomes (35–41). This is completely different from other LH and RC systems which are constructed by specific interaction of (B)Chls with proteins (42). Free from the regulation, the esterifying alcohols of BChls c, d and e are not limited to isoprenoid alcohols and a variety of long straight chain alcohols including nonbranched alkanol and alkenol were observed. One of the green filamentous bacteria, Chloroflexus (Cf.) aurantiacus has stearyl, oleyl (C18) and cetyl (C16) esters of BChl c other than BChl cP and BChl cGG (43). Most green sulfur bacteria have farnesyl esters of BChls c, d and e in the main and other esters in a minor part. In some species of green sulfur bacteria, a greater variety of ester groups were detected: decyl (C10), lauryl (C12), myristyl (C14), pentadecyl and its monounsaturated (C15), cetyl and its monounsaturated (C16), heptadecyl and its monounsaturated (C17), stearyl and oleyl (C18), geranyl, di/tetrahydrofarnesyl, GG, 10,11,14,15-tetrahydrogeranylgeranyl, phytyl and (4-undecyl-furan-2-yl)methyl groups (44–50). Some of the esters might be artifacts during their separation, but most of them would be naturally occurring pigments.
Green bacteria have two types of (bacterio)chlorophylls possessing chlorin and bacteriochlorin π-systems in a cell. From Cf. aurantiacus, two genes encoding BChl synthase (bchG and bchG2) were found and recombinant BchG and BchG2 were examined for catalytic esterification of (bacterio)chlorophyllides (51,52). The BchG catalyzed esterification of BChl aH with diphosphates of GG and P to give BChl aGG and BChl aP but was not effective for the production of BChl c. On the other hand, the BchG2 catalyzed similar esterification of BChl cH but was not active for BChl aH. These results clearly indicate that BchG is BChl synthase for bacteriochlorin molecules and BchG2 is BChl synthase for chlorins. Such a substrate specificity was also observed in recombinant ChlG from Synechocystis sp. PCC 6803 (a Chl a-producing cyanobacterium) and BchG from Rhodobacter capsulatus (a BChl a-producing purple bacterium) (53). In Chlorobium (Cb.) tepidum (one of green sulfur bacteria), three genes were proposed to produce (B)Chl synthases from its genome sequence analysis (54): bchG (for BChl aP), chlG (for 6,7-dedihydrophytyl ester of Chl a) and bchK (for BChl cF). The last one was identified from the experiment in which the bchK-lacking mutant could not accumulate any BChl c molecules in the cell.
Bacteriochlorophyll g, the 3-vinyl derivative of BChl b (see middle drawing of Fig. 4) is the main chlorophyllous pigment in heliobacteria. The minor pigments, the 132-epimer of BChl g (BChl g′) and 81-hydroxylated Chl a (81-OH-Chl a), function as primary electron donor and acceptor, respectively, in its PS1-type RC similar to the RC of green sulfur bacteria. In the species Heliobacterium modesticaldum, all the (B)Chls have a farnesyl ester: BChl gF, BChl g′F and 81-OH-Chl aF (55).
Significance of long chains in ester of chlorosomal chlorophylls
In a chlorosome which is the main LH antenna of green photosynthetic bacteria, a large amount of BChl c/d/e molecules self-aggregate at the hydrophobic environment inside a micelle-like monolayer of lipid assembly. The photophysical properties of chlorosomal self-aggregates including electronic absorption bands and energy levels of excited states are regulated by changes of molecular structures, while those in other usual antenna systems are altered by change of oligopeptide scaffolds specifically interacting with structurally uniformed (B)Chl molecules. Chlorosomal BChls (BChls c/d/e) have a variety of substituents at the 7-, 8-, 12- and 20-positions as well as a mixture of the 31-epimers, besides many kinds of long chains at the 17-propionate ester as mentioned above (right drawing of Fig. 4). In Chloroflexus (a genus of green filamentous bacteria), BChl c molecule has methyl and ethyl groups at the 7/12/20- and 8-positions, respectively. The sole peripheral alkyl structures in Chloroflexus BChl cπ-moiety would lead to a ca 2:1 mixture of its 31R- and S-epimers and a diversity of its long chain ester groups (vide supra). In the Chlorobiaceae family (equal to green sulfur bacteria), multiple methylation occurs at the 82-, 121- and 20-positions of the chlorosomal BChls, resulting in various structurally different molecules in a chlorosome. The diversity in the peripheral substituents would lead to the major occurrence of farnesyl group at the propionate ester. How do the ester groups affect the supramolecular structures and photofunctions of chlorosomal self-aggregates? This is discussed below.
In vivo chlorosomal self-aggregates of BChls c
In culturing Cf. aurantiacus, some alcohols were added to the medium and the BChl c in the cells was analyzed after full growth (56). As exogenous alcohols, cetyl (C16), stearyl (C18) and phytyl alcohols were used whose esters were observed in natural BChls c; the cells then grew gradually and the corresponding esters of BChl c became the main pigment due to the successful incorporation of the added alcohols. When shorter straight chain decyl (C10) and lauryl alcohols (C12) were added, the corresponding esters of BChl c were produced instead of part of the conventional nonbranched C16/18 esters of BChl c. Under similar culturing conditions, slight incorporation of the longer H(CH2)20 group was observed and no substitution occurred with the H(CH2)22 group. All the cultured cells and isolated chlorosomes examined above gave the same Qy absorption maximum (the longest wavelength absorbing band) at about 740 nm and the same main fluorescence emission peak around 750 nm. These results indicate that the optical properties of Chloroflexus species are insensitive to such esterifying groups, probably because the species has a variety of ester groups in its ordinary cells.
In Cb. tepidum, the farnesyl group was exclusively observed for the ester of BChl c. Under culturing conditions similar to those above, longer branched phytyl and GG alcohols as well as other nonbranched stearyl and lauryl alcohols were partially incorporated to BChl c (57). The cultured cells supplemented by the first three alcohols gave the same Qy peak at 754 nm as the unsupplemented, but the lauryl-incorporated cells showed the 6 nm blueshifted Qy maximum. Shortening the ester chain would affect the optical properties of the Chlorobium strain which has no variation of the ester in its natural cells.
In vitro self-aggregates of chlorosomal BChls in aqueous micelle systems
The alteration of natural chlorosomes described above was a good examination for understanding the ester effect on chlorosomal self-aggregates, but other changes of composite substances could not be excluded due to the toxicity of exogenous alcohols supplemented. Therefore, we checked self-aggregates of artificially modified BChls c in an aqueous micelle (58).
First, BChl cF was extracted from Cb. tepidum to obtain a 31-epimeric mixture of the 8,12-homologs. The farnesyl ester of the isolated BChl c was substituted with methyl (M), propyl and hexyl groups without change of composition in the epimers and homologs. These BChls c were dissolved in an aqueous solution of a nonionic surfactant, Triton X-100 to give their self-aggregates inside the micelle where the hydrophobic environment surrounded by Triton X-100 assembly was formed. The absorption spectra of the resulting micelle solutions of BChl c were similar to those of natural chlorosomes, indicating that the artificial Triton X-100 micelle system was a good model for the natural. Qy peaks of farnesylated and hexylated BChls c in aqueous Triton X-100 solutions were situated at 743 and 740 nm, respectively, while those of propylated and methylated BChls c gave 752 and 751 nm. Shortening the ester chains from farnesyl and hexyl to propyl and methyl groups induced about 10 nm redshifts of the Qy peaks. The 17-propionate ester would affect the absorption spectra of chlorosomal self-aggregates in aqueous micelle systems, although this redshift is the reverse of the blueshift in altered Chlorobium cells (vide supra).
The above systems contained a mixture of composite BChls c and we next examined self-aggregates of structurally pure model compounds in an aqueous system (59). The cultured cells of Cf. aurantiacus gave 8-ethyl-12-methyl-BChl c exclusively. From the homologously pure BChl c, its major 31R-epimer possessing stearyl (S) ester was isolated by HPLC. The central magnesium was substituted with zinc to give a stable model compound, Zn-R[EM]BChl cS (see Fig. 5). This zinc complex self-aggregated in an aqueous α-lecithin solution similar to that in an aqueous Triton X-100 solution to show a typical chlorosomal absorption spectrum: λmax(Qy) =720 nm. The corresponding methyl ester (Zn-R[EM]BChl cM) in the same α-lecithin solution afforded a ca 10 nm redshifted Qy peak at 732 nm. This redshifted value is almost the same as that in Chlorobium BChls c in Triton X-100 micelles, showing that substitution of long ester chains in chlorosomal BChls with much shorter alkyl groups shifted the Qy peaks of their self-aggregates to longer wavelengths in aqueous micelle (or micelle-like) systems.
Considering the availability of model compounds, 31-demethylated BChl d models 1 (see left drawing of Fig. 6) lacking the 31-asymmetric carbon were prepared by modifying Chl a possessing 8-ethyl and 12-methyl groups exclusively (60). In an aqueous α-lecithin solution, all the methyl, hexyl, decyl and cetyl esters 1a–d self-aggregated to give Qy absorption peaks at 735–737 nm (61,62). The self-aggregated Qy peaks were independent of the length of ester chains. This situation is explained as follows. Self-aggregation of chlorosomal BChls occurred by specific interaction of their peripheral substituents: coordination of the 31-hydroxy group of a molecule to central metal of another molecule as well as hydrogen bonding of the coordinated hydroxy group with the 13-keto carbonyl group of the third molecule (36,39,60). The interactive hydroxy group of primary alcohols 1 is less sterically hindered than the secondary alcoholic OH in usual chlorosomal BChls possessing 1-hydroxyethyl group at the 3-position. Therefore, models 1 self-aggregated more strongly (60) to give more tightly packed oligomers which were insensitive to the change of ester chains.
Self-aggregates of chlorosomal BChls in nonpolar organic solvents
Hydrophobic environments inside a chlorosome apparatus were mimicked by aqueous micelles mentioned above, so as a simpler alternative system, nonpolar organic solvents were examined. The methyl, hexyl and farnesyl esters of epimeric mixtures of BChl c homologs isolated from Cb. tepidum (vide supra) self-aggregated in hexane containing 1% (vol/vol) dichloromethane similarly as in a chlorosome, to give almost the same Qy peaks at around 740 nm (T. Mizoguchi and H. Tamiaki, unpublished). In hexane, one of the nonpolar organic solvents, esterifying chains induced little change in the Qy peaks of self-aggregates. This behavior is different from the observation in Triton X-100 micelles described above.
In cyclohexane containing 0.1% (vol/vol) dichloromethane, homologously pure [EM]BChl dM (31R/S = 1/1, an epimeric mixture) gave a self-aggregated Qy peak at 718 nm which was almost the same as that (721 nm) of the corresponding phytylated ester, [EM]BChl dP (63). Both methyl and stearyl esters of epimerically and homologously pure Zn-R[EM]BChl d (see Fig. 5) in cyclohexane containing 1% (vol/vol) dichloromethane showed Qy peaks of their self-aggregates at around 700 nm (64). These results confirmed that Qy peaks of self-aggregates of chlorosomal BChls (secondary alcohol type) in nonpolar organic solvents were insensitive to the propionate ester groups.
Primary alcoholic model 1a possessing 3-CH2OH and 172-COOMe self-aggregated in hexane containing 1% (vol/vol) tetrahydrofuran (THF) to give a Qy peak at around 740 nm (60). The corresponding stearyl ester 1e also showed a similar value (738 nm) for its self-aggregated Qy peak. This insensitivity is the same as in Triton X-100 micelles. In self-aggregates of magnesium complexes 2a (methyl ester, see middle drawing of Fig. 6) and 2b (phytyl ester), almost the same Qy peaks were measured at around 750 nm (24,60). The same tendency was observed in cadmium complexes 3a/b (see right drawing of Fig. 6): λmax(Qy) = ca 740 nm (65,66).
Model compounds 4 possessing various isoprenoid-type esters were synthesized and dissolved in hexane containing 1% (vol/vol) THF (67). In the solution, all 4a–d (n = 1–4) gave the same absorption spectra as expected: λmax(Qy) = 734 nm. On the other hand, the circular dichroism (CD) spectra were changed by the length. In both natural and artificial chlorosomal self-aggregates, large CD signals are observed at the Qy region, which is typical of formation of structurally ordered chiral supramolecules. Increase in the length changed the CD shapes at the Qy region from S- to reverse S-types. CD spectra are normally more sensitive to the supramolecular structures than visible spectra. The above results indicated that the propionate ester of models possessing 3-CH2OH affected the supramolecular structure of self-aggregates.
Self-aggregates of synthetic models possessing various long chains
In natural chlorosomes, esterifying groups of their composite BChls are always single long-branched or nonbranched chains. We checked the effect of numbers of long alkyl groups on artificial self-aggregation (68). Compound 5a (see middle drawing of Fig. 7) having a stearyl amide was synthesized and self-aggregated in hexane containing 1% (vol/vol) THF to give a Qy peak at 734 nm, while doubly decylated 5b afforded a 735 nm Qy peak as its self-aggregates. Almost the same Qy peak positions are consistent with the observations described above. Both self-aggregates showed reverse S-type CD signals at the Qy absorption region and the intensity in oligomeric 5b was much larger than that in (5a)n. The enlargement in induced CD signals indicated that self-aggregates of double-chain types should have more ordinary supramolecules than those of single-chain types. This situation would be ascribable to intermolecular interaction among long chains in self-aggregates and/or interaction among the chains and environmental hexane.
Recently, Würthner and his colleagues reported that synthetic 6 (see right drawing of Fig. 7) possessing two lauryl groups self-aggregated in hexane containing <1% (vol/vol) THF to give a 742 nm Qy peak (69). The self-aggregates were deposited on highly ordered pyrolytic graphite to give some rod-shaped images (ca 6 nm ø × 300 nm) and other structures from atomic force microscopic analysis. They claimed that the multiple chains in a molecule would be important for the production of the rod-like supramolecules.
Self-aggregative 7 (see left drawing of Fig. 8) possessing (oligo)oxyethylene groups at the ester were synthesized as more hydrophilic models of chlorosomal BChls (70). These amphiphilic 7 were dissolved in water containing 1% (vol/vol) methanol and could not give Qy peaks at >700 nm which were characteristic of chlorosomal self-aggregates of this type chlorins. This aqueous system could not provide an environment for chlorosomal self-aggregation of 7. Addition of nonionic surfactants to the aqueous solutions of 7 changed the absorption spectra (71). When Triton X-100 (500 μM) was added to an aqueous solution of 7 (10 μM), a 734 nm peak was observed, indicating their chlorosomal self-aggregation. In an aqueous Triton X-100 solution, most molecules of 7a (n = 1) and 7b (n = 2) formed chlorosomal self-aggregates, while 7c (n = 3) and 7d (n = 4) produced large oligomers moderately and partially, respectively. Self-aggregation in Triton X-100 (m = 9 and 10, see middle drawing of Fig. 8) micelles occurred in the order of 7a = 7b > 7c > 7d. On the other hand, 7a and 7b in an aqueous Triton X-45 (middle drawing of Fig. 8) gave large oligomers partially and considerably, respectively, and almost all of 7c and 7d molecules formed such self-aggregates. The self-aggregativity of 7 in micelles of less hydrophilic Triton X-45 (m = 4 and 5) was the reverse of that in Triton X-100. The matching of lengths in (oligo)oxyethylene groups of 7 with those of nonionic Triton surfactants is important for chlorosomal self-aggregation.
Model compounds 8 (see right drawing of Fig. 8) having a charge group at the ester terminal were prepared (70). Positively charged 8a afforded a Qy peak at around 720 nm in an aqueous solution of an anionic surfactant (sodium 4-dodecylbenzenesulfonate or sodium dodecyl sulfate) and negatively charged 8b had little absorption in the region over 700 nm (71). In contrast, 8a gave no peaks at >700 nm, but 8b showed intense absorbance at 700–770 nm in an aqueous solution of a cationic surfactant (N-cetylpyridinium chloride or cetyltrimethylammonium bromide). As in nonionic systems, charge matching of terminal groups (cationic 8a and anionic micelles as well as anionic 8b and cationic micelles) is significant in the production of chlorosomal self-aggregates.
The importance in matching of esterifying groups with environments was observed in the following fluorous systems. Fluorinated and nonfluorinated nonyl esters of Zn-[EM]BChl d9a and 9b (see left drawing of Fig. 9) were synthesized (72,73). In hexane containing 1% (vol/vol) dichloromethane, both 9a and 9b self-aggregated to give Qy peaks at 703 and 705 nm. In a flon solvent, CClF2CF2CHClF, 9a afforded a 713 nm peak, but 9b gave no peak at >700 nm. Self-aggregation of 9a with 30% (wt/wt) fluorine atoms in a molecule occurred fluoro-specifically in the flon solvent (47% F). Moreover, synthetic 10 (50% F, see right drawing of Fig. 9) possessing four perfluorooctyl groups in the propionate ester self-aggregated in fluorous phases, perfluoro-2-butyltetrahydrofuran (73% F), perfluoro-N,N,N-tributylamine (76% F) and perfluorooctane (78% F) to give a 734 nm Qy band, while the monomeric form of 10 could not be dissolved in the fluorous solvents (74).
Supramolecular structural models for chlorosomal self-aggregates
In a chlorosome, BChl c/d/e molecules self-aggregate by specific intermolecular bonding of Mg⋯O(32)–H⋯O=C(131) as well as π–π interaction of their chlorin chromophores (vide supra). Such a local structure in the self-aggregates has been revealed by various works (36,39,60), but their whole supramolecular structures are still in debate. Freeze-fracture transmission electron microscopic images gave the rod structures as their supramolecular structures (75–77). The rods were parallel to the long axis of an ellipsoidal chlorosome and the diameters of Chloroflexus and Chlorobium chlorosomal rods were about 5 and 10 nm, respectively. Recent investigation of chlorosomes by cryoelectron microscopy and X-ray scattering suggested the presence of a 2 nm spacing lamellar structure in chlorosomal self-aggregates of Cb. tepidum (78). In the former rod-like supramolecule, a composite BChl molecule is partially overlapped with the neighboring BChls in the manner that the vectors of the C31 to C131 atom in a molecule are parallel. In the latter lamellar sheet, a BChl is proposed to interact with the neighbors in the antiparallel fashion. Therefore, any modification of the rod model could not lead to the proposed lamellar model, even if the tubular structure is cleaved. No detailed supramolecular structure based on the lamellar model is available at present and here we discuss how chlorosomal BChls form a rod structure.
Naturally occurring chlorosomal BChl molecules have a long chain group at the 17-propionate ester, which would affect the optical properties of their self-aggregates as mentioned above. Considering the fact that the chlorosomal surface is constructed of lipid monolayer containing proteins, carotenoids and others, the long chains of the composite lipid molecules directed to the chlorosomal interior interact with self-aggregates of chlorosomal BChls. As such an interacting moiety with hydrophobic parts of lipid self-assembly, the esterifying chains of the chlorosomal BChls are more favorable than other peripheral substituents (66,79). Hydrophobic interaction between these long chains would stabilize the relatively fragile lipid monolayer at the chlorosomal surface (67,69,80). Therefore, some ester chains of self-aggregates are directed to the outside of supramolecules and their chlorin π-moieties are located on the inside of the rods.
Taking account of the above proposal, two supramolecular models (b) and (c) as shown in Fig. 10 (81–83) are more probable for chlorosomal rods than model (a) where long esterifying chains make an inside core (84). Molecular modeling studies showed that 8-ethyl-12-methyl-BChl cS molecules made a monolayer B-type rod with a 5 nm diameter and 8-propyl-12-ethyl-BChls cF made a bilayer (c) type with about a 10 nm diameter (59). These models are in good agreement with observed rods (vide supra): Chloroflexus chlorosomes have 5 nm diameter rods by self-aggregation of 8-ethyl-12-methyl-BChls c and the Chlorobium have 10 nm rods by BChls possessing more sterically bulky alkyl groups at the 8- and 12-positions. The artificial chlorosomal self-aggregates described above indicated that long chain groups on the 17-substituents in the self-aggregates should interact with environmental molecules surrounding the supramolecules. These experimental observations also support the rod models (c) and (c). Therefore, the esterifying chains are important for the formation of highly ordered supramolecules (lacking any structural defects) which induce efficient energy migration in the chlorosomal self-aggregates.
Esterifying groups at the 17-propionate ester of (B)Chl molecules in photosynthetic proteins are limited to phytyl or sometimes to (tetrahydro)geranylgeranyl or farnesyl. The long branched chains interact hydrophobically with the oligopeptides and would play a role as the anchor of a (B)Chl molecule inside a protein. X-ray crystallographic analyses of (B)Chl–protein complexes (see references cited in Ref. 42) showed the presence of such interaction and also intermolecular interaction of phytyl groups under restricted environments. One reason why phytyl esters are exclusively used in (B)Chl molecules inside photosynthetic proteins might be that branched chains are superior to straight chains for the above hydrophobic interactions.
In chlorosomes, the ester groups of chlorosomal BChl molecules interact with flexible long chains of various lipid molecules including MGDG and/or of other BChl molecules in the same or a neighboring rod. Therefore, many types of ester groups including long straight chains are usable in chlorosomal BChls. As a result, other interactions (hydrophilic, electrostatic and fluorophilic interactions) were utilized to prepare chlorosomal models (vide supra) and the approaches considering these points are promising for the construction of novel photodriven nanodevices as efficient LH antenna systems.
Acknowledgements— We thank Drs. J. Harada and S. Sasaki, Ritsumeikan University, Dr. T. Miyatake, Ryukoku University and Dr. T. Oba, Utsunomiya University for their helpful discussions. This work was partially supported by Grants-in-Aid for Scientific Research (No. 17029065) on Priority Areas (417) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government and for Scientific Research (B) (No. 15350107) from the Japan Society for the Promotion of Science (JSPS) as well as by the “Academic Frontier” Project for Private Universities: matching fund subsidy from MEXT, 2003–2007.