Pressure-Induced Red Shift and Broadening of the Qy Absorption of

Nov 26, 2008 - ... from the green nonsulfur bacterium (Chloroflexus aurantiacus), which has a single homologue with 8-ethyl and 12-methyl groups...
0 downloads 0 Views 147KB Size
J. Phys. Chem. B 2008, 112, 16759–16765

16759

Pressure-Induced Red Shift and Broadening of the Qy Absorption of Main Light-Harvesting Antennae Chlorosomes from Green Photosynthetic Bacteria and Their Dependency upon Alkyl Substituents of the Composite Bacteriochlorophylls Tadashi Mizoguchi,† Tae-Yeun Kim,† Seiji Sawamura,‡ and Hitoshi Tamiaki*,† Department of Bioscience and Biotechnology and Department of Applied Chemistry, Faculty of Science and Engineering, Ritsumeikan UniVersity, Kusatsu, Shiga 525-8577, Japan ReceiVed: June 5, 2008; ReVised Manuscript ReceiVed: September 16, 2008

When pressure was applied to the main light-harvesting apparatus (chlorosomes) isolated from several green photosynthetic bacteria (up to 128 MPa), the Qy-absorption band in an aqueous solution was shifted to longer wavelengths. The shift, ∆ν, was completely reversible for (de)compression and also showed a linear relation as a function of the applied pressure. The pressure-sensitivity in the ∆ν was dependent upon the bacterial species. The pressure coefficient, ∆ν/∆P, was -565 to -535 cm-1GPa-1 for the chlorosomes from several green sulfur bacteria (Chlorobium species), which have several bacteriochlorophyll(BChl) homologues at the 8- and 12-positions as the antenna pigments. In contrast, a smaller value (-445 cm-1 GPa-1) was estimated for the chlorosomes from the green nonsulfur bacterium (Chloroflexus aurantiacus), which has a single homologue with 8-ethyl and 12-methyl groups. These results were confirmed by the similar pressure dependency of in vitro self-aggregates of isolated BChls-c having various alkyl substituents at the 8- and 12-positions. The present pressurization study enables us to discuss a physiological meaning of a variety of antenna pigments in green photosynthetic bacteria. Introduction “Chlorosomes” are main extramembraneous light-harvesting complexes of green sulfur (Chlorobium (Chl.) species) and green nonsulfur bacteria (Chloroflexus (Cfl.) species) [see refs 1-3 for comprehensive reviews and references cited therein]. The chlorosomes of both species consist of a core and an envelope. The core is made up of light-absorbing bacteriochlorophyll (BChl)-c, d, or e molecules, depending on organisms, in a highly aggregated state, whereas the envelope is a monolayer of mostly glyco/phospholipids containing proteins.4,5 The chlorosomes are believed to be an excellent antenna system in terms of capturing extremely low light irradiation, since a particular green sulfur bacterium found in a deep-sea (>2000 m) hydrothermal vent is assumed to use dim light from geothermal irradiation for photosynthesis.6 Moreover, chlorosomes are suitable for preparation of an artificial antenna system, since they are constructed on a simple structural principle, that is, self-aggregation of antenna pigments (BChls-c/d/e) without any assistance of proteins.7,8 According to freeze-fracture electron micrography on both Chl. and Cfl. species, chlorosomes from Chl. species are 70-260 nm long, 30-100 nm wide, and ∼25 nm high;9,10 whereas those from Cfl. species were considerably smaller with lengths ∼100 nm, widths 20-40 nm, and heights 10-20 nm.10,11 Furthermore, the presence of rod-like architectures, often called “rodelements”, in both species was reported.9-12 In contrast to the above features, Psˇencˇík et al. and Oostergetel et al. have claimed that the structure of the core should be lamella organization.13-15 The actual supramolecular structure is still under discussion. * To whom correspondence should be addressed. Phone: +81-77-5612765; fax: +81-77-561-2659; e-mail: [email protected]. † Department of Bioscience and Biotechnology. ‡ Department of Applied Chemistry.

The envelope of both Chl.- and Cfl.-type chlorosomes contains BChl-a molecules associated with a specific protein, CsmA.16,17 The BChl-a molecules mediate excitation energy from chlorosomes to other antenna complexes including B808-866 antenna and FMO protein (based on the discovery by Fenna, Matthews, and Olson). Interestingly, the molar ratio of antenna BChls inside chlorosomes to the energy-accepting BChl-a in Chl. species, depending on the species and growth conditions, was ∼90/1,18 whereas in Cfl. species it was only ∼25/1.19 These values correspond to the size of chlorosomes as mentioned above. Therefore, efficiency of the excitation energy transfer is modulated by stoichiometry of the light-harvesting BChls and energy-accepting BChls-a and/or the overall size of chlorosomes. In addition to the above distinctiveness of Chl. and Cfl. species, the composition of antenna BChls inside chlorosomes is strongly dependent on the species: the BChls in Chl. species are additionally methylated at the 82- and 121-positions (see X1, X2, X3, and X4 in Figure 1), whereas those in Cfl. species have invariant substituents at the 8- and 12-positions.1,2,7,8 The effects of these substituents of the BChls on the self-assembly were studied by the following in vivo experiments. Growth of green sulfur bacteria (Chl. species) was strongly dependent upon intensity of irradiation light, and their absorption peaks were shifted to a longer wavelength with lowering of the intensity.20-23 As the intensity decreased, relatively more 82-methylation proceeded. The in vivo results indicated that the methylation of the antenna BChls in Chl. species gave more red-shifted Qy peaks of chlorosomes.24 In the biosynthesis of BChl-c, the enzyme for 82-methylation was proposed to be BchQ by analysis of the mutant lacking bchQ in the green sulfur bacterium, Chl. tepidum.25 This mutant gave about 15 nm blue-shifted Qy absorption of the chlorosomes compared with that of the wild type. Therefore, the variety of BChls found in Chl. species may play a key role in fine-tuning the absorption properties in order

10.1021/jp804990f CCC: $40.75  2008 American Chemical Society Published on Web 11/26/2008

16760 J. Phys. Chem. B, Vol. 112, No. 51, 2008

Figure 1. Molecular structures of antenna BChls found in chlorosomes from green photosynthetic bacteria.

to capture extremely low-light intensities and to achieve efficient energy transfer to energy-accepting BChls-a situated at chlorosomal envelopes. In this study, we have tried to understand the physiological meaning of a variety of antenna BChls having different alkyl substituents inside chlorosomes on their modulation of the absorption properties. For this purpose, we isolated several types of chlorosomes from both Chl. and Cfl. species and examined the pressure-dependence of their absorption spectra, since pressure-dependence spectroscopy provides useful information on the elucidation of internal structures of natural materials, especially with the strongly coupled self-assembly of BChl molecules. The high-pressure experiments were often combined with hole-burning spectroscopy and were applied to several photosynthetic protein complexes.26-31 The results obtained led to new insights into excitation energy transfer dynamics and the Qy electronic structures of BChl molecules on lightharvesting complexes (LH) in photosynthesis. These studies have also shown that increasing the pressure causes a substantial red shift and broadening of the long-wavelength-absorbing Qy band in the aggregated BChls. The pressure-induced red shift is generally much larger than that observed for monomeric BChl molecules. Therefore, aggregated and red-shifted BChl molecules appear to show a greater pressure sensitivity than monomeric pigment molecules. According to the previous report on green bacterial antennae (chlorosomes) at high pressure,26 the shift and broadening of the Qy-absorption were comparable to those of the strongly coupled BChl-a molecules in LH2 and LH1 from purple bacteria and were much larger than those of the monomeric molecules in the LH2.29-31 When the pressure applied to chlorosomes increases, local stacking of antenna BChls in the self-assembly is expected to be modulated by compression. On the basis of pressure-induced red shift and broadening of the Qy absorption of several types of chlorosomes, we will discuss the physiological meaning of a variety of antenna BChls having different alkyl substituents at the 8- and 12positions in green photosynthetic bacteria. Experimental Methods 1. General Methods. Visible absorption spectra under atmospheric pressure were measured with a Hitachi U-3500 spectrophotometer (Hitachi, Ltd., Tokyo, Japan). Dynamic light

Mizoguchi et al. scattering (DLS) profiles were measured with a Malvern Zetasizer Nano-ZS (Malvern Instruments, Ltd., Worcestershire, UK). Liquid chromatography-mass spectrometry (LCMS) of extracted BChls was performed using a Shimadzu LCMS2010EV system (Shimadzu, Kyoto, Japan) comprising a liquid chromatograph (SCL-10Avp system controller, LC-10ADvp pump, and SPD-M10Avp photodiode-array detector) and a quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) probe as described previously.32 HPLC was performed using reverse-phase chromatography under the following conditions: column, Cosmosil 5C18AR-II (4.6 × 150 mm, Nacalai Tesque, Kyoto); eluent, 10% H2O in methanol; flow rate, 0.5 mL/min; and detection wavelength, 435 nm. 2. Bacterial Strains and Growth Conditions. Chl. tepidum strain ATCC49652,33 Cfl. aurantiacus strain OK-70-fl,34 Chl. Vibrioforme strain NCIB8327,35 and Chl. phaeobacteroides strain 154936 were cultured anaerobically as described previously. All the bacteria were used after their single-colony isolation. 3. Isolation of Chlorosomes from Green Bacteria. Chlorosomes were prepared from the harvested cells of green bacteriasChl. tepidum, Cfl. aurantiacus, Chl. Vibrioforme, and Chl. phaeobacteroidessin 2 M sodium thiocyanate, 10 mM potassium phosphate (pH 7.4), and 10 mM sodium ascorbate by the method of Gerola and Olson.18 The fraction that banded at 15% (wt/vol) sucrose after ultracentrifugation was used. Optical density was about 1.0/10 mm at the Qy absorption maximum for spectroscopic measurements. 4. Extraction of BChls from Chlorosomes. Each composite BChl was extracted from the corresponding chlorosomes as described previously.37 The extracts were subjected to HPLC in order to determine the composition of BChls in chlorosomes. Simultaneously, each component was characterized by APCILCMS. 5. Preparation of Micelle-like Aggregates of BChl-c in an Aqueous Solution. A homologous mixture of BChls-c was extracted from the cells of Chl. tepidum and purified as described previously.38 R[E,M]BChl-c, (31R)-epimer with 8-ethyl and 12methyl groups in Figure 1, was isolated from the above homologous mixture by reverse-phase HPLC. The homologous mixture of BChls-c and HPLC-isolated R[E,M]BChl-c was dissolved in methanol containing Triton X-100 (Nacalai Tesque, Kyoto) at the concentration of 2.0 mM, and 50 µL of this solution was injected to 5.0 mL of 50 mM Tris-HCl buffer (pH 8.0) ([BChl] ) 20 µM).39 The concentration of Triton X-100 in the final solution was adjusted to 0.025% (wt/vol) for preparation of the aggregate solution and 0.10% (wt/vol) for the monomer solution. The suspension was homogenized for 10 s using a vortex mixer and kept in the dark for more than 60 min at room temperature. The size distribution of the micelle-like aggregates was examined by DLS. 6. Measurements of Absorption Spectra under High Pressure. The samples in an aqueous solution were subjected to hydrostatic compression using a high-pressure optical system as described previously.40 The change in the optical path length upon compression was determined to be only +0.1 mm (under 300-400 MPa compression). As the path length used in the present study was 13.0 mm, the change was estimated to be less than 1%. Thus, the change in the optical path length upon compression up to 128 MPa was negligible. Absorption spectra were measured at a 32 MPa interval in order to remove the thermal effect by a Shimadzu UV-3100Rd spectrophotometer with spectral resolution of 0.05 nm at room temperature. Several

Pressure Dependency of Qy Band of Chlorosomes

J. Phys. Chem. B, Vol. 112, No. 51, 2008 16761

Figure 2. A representative HPLC profile of pigment extracts from chlorosomes of Chl. tepidum.

measurements were performed with different samples in order to check the reproducibility of the data. Results and Discussion 1. A Variety of BChls in Chlorosomes and Their Molecular Structures. Figure 1 depicts the molecular structures of BChls found in green bacterial antennae, chlorosomes. These BChl molecules are defined by their peripheral substituents at the 7- and 20-positions (R7 and R20 in Figure 1). BChl-c, -d, and -e molecules are found in Chl. tepidum/Cfl. aurantiacus, Chl. Vibrioforme, and Chl. phaeobacteroides, respectively (these four bacteria were used in this study). Each type of BChl consists of a mixture of epimers at the chiral 31-position (indicated by an asterisk in Figure 1). Additionally, several homologues, which differ by the degree of methylation at the 82- and 121-positions (X1, X2, X3, and X4 in Figure 1), are present in green sulfur bacteria (Chl. tepidum, Vibrioforme, and phaeobacteroides). In contrast, the BChls in the green nonsulfur bacterium (Cfl. aurantiacus) have a single molecular structure at those positions (8-ethyl and 12-methyl homologue), although they have several long chain esterifying alcohols on the 17-propionates (R17 in Figure 1). Figure 2 shows a representative HPLC profile of the pigments extracted from the chlorosomes of Chl. tepidum as an example. The assignment of each peak labeled in Figure 2 was confirmed by APCI-LCMS analysis and was consistent with the literature.41 We were able to detect five different epimers and homologues at the 31-, 82-, and 121-positions. The (31R)epimers having ethyl (E), propyl (P), and isobutyl (I) at the 8-position and methyl (M) and ethyl (E) at the 12-position are termed R[E,M], R[E,E], R[P,M], R[P,E], R[I,M], and R[I,E], respectively. The (31S)-epimers are represented by use of “S” just before the bracket. The effect of these alkyl substituents on self-assembly has been well studied by in vitro experiments using homologously and epimerically pure BChls.34,36-38,42,43 However, there have been few attempts to elucidate the substituent effect on natural chlorosomal supramolecules as mentioned in the Introduction. 2. Absorption Spectra of Chlorosomes under Atmospheric Pressure. Figure 3a shows the electronic-absorption spectra of chlorosomes from Chl. tepidum (solid) and Cfl. aurantiacus (dotted) in 50 mM Tris-HCl buffer (pH 8.0) at room temperature under atmospheric pressure (0.1 MPa). The absorption spectra of both chlorosomes were characterized by their redshifted Qy-absorption bands at around 740 nm compared to those of the monomeric BChl-c, which is due to the selfaggregation. The monomeric form of BChl-c prepared in an aqueous solution is also shown for comparison (the broken line in Figure 3b) (vide infra). The Qy absorption of chlorosomes from Chl. tepidum [full-width at half-maximum (fwhm) ) 1012 cm-1] was much broader than that from Cfl. aurantiacus [fwhm

Figure 3. Electronic-absorption spectra of chlorosomes from Chl. tepidum (solid) and from Cfl. aurantiacus (dotted) (a) and the micellelike aggregates containing 0.025% (wt/vol) Triton X-100 (solid) and monomer containing 0.10% (wt/vol) Triton X-100 (broken) prepared by a homologue mixture of BChl-c from Chl. tepidum (b) in 50 mM Tris-HCl buffer (pH 8.0) at room temperature under atmospheric pressure.

) 694 cm-1]. The absorption from energy-accepting BChl-a embedded in chlorosomal envelopes (∼800 nm) was clearly seen in Cfl. aurantiacus, whereas the absorption in Chl. tepidum was hidden in the red-side of the aggregated BChl-c band due to its broadening. According to earlier “single-molecular” spectroscopic studies on chlorosomes,33,34,44,45 the distribution of emission peak positions of a single chlorosome was dependent upon bacterial species. In Cfl. aurantiacus, fluorescence peak positions of BChl-c aggregates were distributed from 742 to 752 nm.34 In contrast, the peak positions of Chl. tepidum were distributed in a wider range from 750 to 768 nm.33 The fluorescence spectroscopy of chlorosomes at the single-unit level revealed that BChl-c aggregates in a single chlorosome from Chl. tepidum were more heterogeneous than those from Cfl. aurantiacus, which was consistent with the present observation. 3. Pressure-Dependency of Chlorosomes. 3.1. PressureDependency of Chlorosomes from Chl. tepidum and Cfl. aurantiacus. Because the red-shifted Qy transition of chlorosomes is parallel to the long axis of ellipsoidal chlorosomes, it is sensitive to molecular orientation in the self-assembly.46,47 Hereafter, we focus our attention on this band. Figure 4 shows the pressure-dependency of absorption spectra of chlorosomes from Chl. tepidum (a) and Cfl. aurantiacus (b) in an aqueous buffer solution. When applied pressure on the aqueous chlorosomal solutions increased (from 0.1 to 128 MPa), their Qy absorption bands were shifted to longer wavelengths (thin solid to thick solid lines in Figure 4). A red (low-energy) shift of 72 cm-1 (from 13 464 to 13 392 cm-1) for Chl. tepidum and 53 cm-1 (from 13 526 to 13 473 cm-1) for Cfl. aurantiacus were observed. These shifts were completely reversible for compression and decompression of the chlorosomal solution and showed a linear relation as a function of the applied pressure (see the fitted lines in Figure 5a). The reversibility of pressure effects was checked by recording the atmospheric pressure spectrum after releasing the applied pressure.

16762 J. Phys. Chem. B, Vol. 112, No. 51, 2008

Figure 4. Changes in the absorption spectra of chlorosomes from Chl. tepidum (a) and from Cfl. aurantiacus (b) upon increasing pressure from 0.1 (thin solid) to 128 MPa (thick solid) in 50 mM Tris-HCl buffer (pH 8.0) at room temperature.

Figure 5. Pressure-induced red (low-energy) shift (a) and broadening (b) of the Qy absorption of chlorosomes from Chl. tepidum (b), Cfl. aurantiacus (O), Chl. Vibrioforme (0), and Chl. phaeobacteroides (9) in 50 mM Tris-HCl buffer (pH 8.0) at room temperature.

Figure 5 shows the pressure-induced red shift (a) and broadening (b) of the Qy absorption of several chlorosomes as a function of the applied pressure. Over the whole investigated pressure range, the pressure coefficients, ∆ν/∆P, determined as the slopes of a single straight line fitted to the data points of the band peak position were found to have the following values: -555 cm-1 GPa-1 for Chl. tepidum (b) and -445 cm-1 GPa-1 for Cfl. aurantiacus (O). The standard deviation of the coefficients was determined to be ( 22 (for Chl. tepidum) and ( 8 cm-1 GPa-1 (for Cfl. aurantiacus) from three independent measurements. The pressure-induced broadening of the Qy absorption showed a similar relationship for (de)compression shown in Figure 5b. Band broadenings of 47 cm-1 (1012 f 1059 cm-1) for Chl. tepidum and 7 cm-1 (694 f 701 cm-1) for Cfl. aurantiacus were observed with increasing pressure (0.1 f 128 MPa), and these fitted slopes gave 36 ( 4 cm-1 GPa-1 for Chl. tepidum and 7 ( 2 cm-1 GPa-1 for Cfl. aurantiacus.

Mizoguchi et al. In an earlier high-pressure study on chlorosomes from Chl. tepidum,26 the pressure-induced red shift and broadening of the Qy absorption was determined to be -440 and 12 cm-1 GPa-1, respectively, at 100 K under 752 MPa compressed conditions. These values were smaller than those found in the present investigation (-555 and 36 cm-1 GPa-1). The inconsistency is ascribed to the different experimental conditions, that is, temperature and pressure range. As shown in Figure 3 of ref 26, the initial slopes of the fitted line (from 0.1 to ∼100 MPa) were steeper than those fitted to the entire investigated range (from 0.1 to 752 MPa). Thus, the pressure parameters reestimated in the range from 0.1 to ∼100 MPa were considered to be almost identical to those found in the present study. The pressure-induced red shift and broadening of the Qy absorption were remarkably different for the two types of chlorosomes, indicating that the self-aggregates in chlorosomes from Chl. tepidum exhibit a higher sensitivity to compression than those from Cfl. aurantiacus. Therefore, the local stacking of antenna BChls in chlorosomes is expected to be modulated by compression in a different way. This might be ascribable to the different composition of their antenna pigments (vide supra). When a solution is compressed, its volume decreases corresponding to the applied pressure. In absorption spectra, this concentration effect is confirmed by the corresponding increase of absorbance. The effect on compression had been demonstrated by the monomeric (B)Chl-a in Et2O, where the intermolecular interaction among (B)Chl-a molecules was not considered.48,49 Upon compression of chlorosomes, an increase of absorbance was only found in Cfl. aurantiacus (see Figure 4). The pressure-induced broadening of the Qy absorption of Chl. tepidum was considerably larger than that of Cfl. aurantiacus. Therefore, the concentration effect could not be observed in Chl. tepidum. These results also support the idea that the local stacking of antenna BChls in chlorosomes is modulated in a different way under compression. Solvatochroism on increased pressure was also estimated using the in vitro micelle system as described below. 3.2. Pressure-Dependency of Chlorosomes from Other Chl. Species. We also examined the pressure-dependency of other Chl. species having various homologues at the 8- and 12positions. One is Chl. Vibrioforme containing exclusively BChld, and the other is Chl. phaeobacteroides containing BChl-e as their antenna BChls (see Figure 1). Their pressure-dependencies are depicted in Figure 5. The pressure-induced red shifts [broadenings] were estimated to be -565 [12] cm-1 GPa-1 for Chl. Vibrioforme (0) and -535 [38] cm-1GPa-1 for Chl. phaeobacteroides (9). The sensitivities were quite similar to those found in Chl. tepidum, except the pressure-induced broadening of the Qy absorption in Chl. Vibrioforme containing BChl-d, which was one-third smaller than those of the other two Chl. species and twice the value of that in Cfl. aurantiacus. This exception might be ascribable to there being no methylation at the 20-position in any of the BChl homologues. These results also indicate that the 7-formyl group in BChl-e did not affect the supramoelcular structure of the chlorosomes from Chl. phaeobacteroides.50 Table 1 summarizes the pressure coefficients of four different chlorosomes thus obtained. The pressure-dependency was clearly divided into two categories: one was Chl. species containing various homologues at the 8and 12-positions, and the other was Cfl. aurantiacus containing a single BChl homologue at those positions. The pressuredependencies of the corresponding living cells showed a similar tendency for those found in the isolated chlorosomes (data not shown).

Pressure Dependency of Qy Band of Chlorosomes

J. Phys. Chem. B, Vol. 112, No. 51, 2008 16763

TABLE 1: Pressure Parameters of in Vivo and in Vitro Self-Aggregates in 50 mM Tris-HCl Buffer (pH 8.0) at Room Temperature (cm-1 GPa-1) Chl. tepidum Chl. Vibrioforme Chl. phaeobacteroides Cfl. aurantiacus BChls-c mixturea R[E,M]BChl-ca monomerb

shift

fwhm

-555 -565 -535 -445 -520 -455 -135

36 12 38 7 19 9 9

a In vitro micelle-like self-aggregates were prepared using an aqueous solution containing 0.025% (wt/vol) Triton X-100. b In vitro micelle-like monomer was prepared using an aqueous solution containing 0.10% (wt/vol) Triton X-100.

Figure 6. Size distribution of the micelle-like aggregates composed of the mixture of five BChl-c homologues.

4. Pressure-Dependency of Micelle-like Aggregates. Selfaggregation of BChl molecules inside chlorosomes occurs in the micelle-like hydrophobic environment made by a lipid monolayer. Therefore, in vitro self-aggregates as a model for chlorosomes were prepared using an aqueous solution containing Triton X-100.39 Figure 2 shows an HPLC chromatogram of the extracts of chlorosomes from Chl. tepidum. The first eluted band was the same as BChl-c homologue, R[E,M]BChl-c, found in Cfl. aurantiacus, although in Cfl. aurantiacus the 17-propionate is different (see Figure 1). We prepared two kinds of micellelike aggregates: one was prepared using the mixture of five homologues as a model for Chl.-type chlorosomes and the other using the single and epimerically pure homologue, R[E,M]BChlc, as a model for Cfl.-type chlorosomes. Figure 3b shows the electronic-absorption spectrum of the micelle-like aggregates (solid) prepared by the homologue mixture as a model for Chl.-type chlorosomes. In comparison with the intact absorption spectrum of chlorosomes from Chl. tepidum (the solid line in Figure 3a), the micelle-like aggregates well-reproduced the absorption properties. Furthermore, we estimated the size distribution of the micelle-like aggregates using DLS. Figure 6 shows the DLS profile of the aggregates composed of the mixture of five BChl-c homologues. The micelle-like aggregates gave a hydrodynamic diameter centered at around 160 nm. The sizes of the aggregates were roughly parallel to the length of the long axis of chlorosomes determined by electron micrography.9,10 The micelle-like aggregates prepared using a single BChl gave almost identical absorption and size distributions (data not shown). The hydrodynamic diameters of natural chlorosomes isolated from Chl. tepidum had been already reported to be 53.651 and 105 nm52 by different researchers, whereas the value of chlorosomes used in the present study was estimated to be ∼160 nm. The discrepancy in the diameters would be mainly ascribed to the difference in

Figure 7. Pressure-induced red (low-energy) shift (a) and broadening (b) of the Qy absorption of the micelle-like aggregates and the monomer in 50 mM Tris-HCl buffer (pH 8.0) containing Triton X-100 as a surfactant at room temperature: the micelle-like aggregates were prepared by a mixture of five BChl-c homologues (b) and HPLCisolated R[E,M]BChl-c (O) as shown in Figure 2. The micelle-like monomer corresponding to the homologues mixture was shown by closed squares (9) (the monomer to the isolated R[E,M]BChl-c was omitted for simplicity).

samples, because chlorosomes were quite changeable in terms of their pigment composition as well as overall sizes under different culturing conditions. Therefore, the micelle-like aggregates prepared by Triton X-100 are a good structural model for chlorosomes in terms of both absorption and morphological properties. Figure 7 shows the pressure dependency of the micelle-like aggregates thus obtained. With increasing pressure (0.1 to 128 MPa), their Qy absorption bands were shifted to longer wavelengths as seen in chlorosomes. The pressure-induced red shifts [broadenings] were estimated to be -520 [19] cm-1 GPa-1 for the micelle-like aggregates prepared by a homologous mixture of BChls-c (b) and -455 [9] cm-1 GPa-1 for those prepared by a single BChl-c (O). These coefficients were in fair agreement with those of the corresponding chlorosomes (see Table 1). In addition, we prepared the micelle-like aggregates of another single BChl-c, R[E,E]BChl-c, and its pressureinduced red shift was determined to be -470 cm-1 GPa-1 (data not shown). Therefore, it was confirmed that the pressure coefficients depended on the variety of BChl homologues having different alkyl substituents at the 8- and 12-positions. The results also indicated that the 17-priopionate in BChls did not affect the pressure parameters, since the parameters obtained by Cfl.type chlorosomes (several esters, seen in Figure 1) and those by the micelle-like aggregates composed of a single farnesylated BChl-c showed a similar tendency. The results were also consistent with the previous report on the effect of the 17-propionate substituent on the self-assembly of BChl-c derivatives.39 To estimate solvatochroism on increased pressure, we examined the pressure-dependency of monomeric BChl-c prepared by the Triton X-100 micelle-system containing a higher concentration of the surfactant. Its absorption spectrum is shown

16764 J. Phys. Chem. B, Vol. 112, No. 51, 2008 by the broken line in Figure 3b. The monomer solution exhibited little CD signal at around its Qy absorption region; this means there were no intermolecular interactions among BChl-c molecules (data not shown). The pressure-induced red shift [broadening] of the monomer solution was estimated to be -135 [9] cm-1 GPa-1 (closed squares (9) of Figure 7). The shift value is compatible with that determined to the BChls-a in a FMOprotein (about -100 cm-1 GPa-1).53 Therefore, the solavtochroism in the present oligomeric BChls would be similar to the above, and the difference in pressure coefficients found in Chl.- and Cfl.-type chlorosomes mainly reflects the difference in intermolecular interactions among their composite BChls over the investigated pressure range. 5. Mixing of Various Antenna BChl Homologues is Essential for Construction of Pressure-Sensitive Supramolecules. In purple photosynthetic bacteria, a regulation of the antenna system for decreasing light intensity incident to the bacteria is achieved by modulation of their antenna peptides at the amino acid level.54,55 For example, in Rhodopseudomonas palustris under low light conditions, unique LH4 having different absorption properties from usual LH2 was biosynthesized as the major peripheral antenna system.55 The difference in the present pressure coefficients between Chl.- and Cfl.-type chlorosomes might be ascribable to the fact that only the green sulfur bacteria (Chl. species) were found in extremely low-light environments. Typically, one green sulfur bacterium (Chl. phaeobacteroides strains) has been found at a depth of around 100 m in the Black Sea (light intensity, less than 0.25 µE m-2 s-1),56,57 where organisms experience a pressure of about 1.0 MPa. A particular species of green sulfur bacteria was reported to be found in a deep-sea (>2000 m) hydrothermal vent and assumed to use a dim light from geothermal radiation for photosynthesis;6 such organisms experience a pressure of about 20 MPa. In natural chlorosomes, BChl-a molecules in baseplates and FMO-protein situated at chlorosomal envelopes, which absorb at around 800 nm, are energy-accepting parts. At a low light intensity (compressed environments), rapid and efficient energy transfer in a chlorosome would be advantageous for growth of organisms, and longer wavelength-absorbing self-aggregates of BChls are favorable for the energy-donating part.58 The pressure-induced red shift of the energy-accepting parts was determined to be about -100 cm-1 GPa-1 for BChl-a in FMO-protein at 4.2 K.53 This observation indicated that the energy-accepting parts in green bacteria are less sensitive to applied pressure. To achieve the efficient energy transfer between energy-donating and energy-accepting parts, it was essential to increase the spectral overlapping between the donor and acceptor. Therefore, a large pressure-induced red shift and broadening of the Qy absorption of energy-donating parts (chlorosomes), mostly due to mixing of antenna BChl homologues having different alkyl substituents, would occur in an extremely low light (compressed) environment. According to previous freeze-fracture electron microscopic studies, the rod-like architectures were estimated to be ∼10 nm in diameter for Chl. species9,10 and to be 5-6 nm for Cfl. species.10,11 Mixing of various antenna BChl homologues would be useful for the efficient construction of large size core parts as well as enhancement of the pressure-sensitivity described above. The mixing would induce steric hindrance among composite BChls in their self-assembly, and as a result, the size of the self-assembly is expected to be larger at the expense of the stability of supramolecules. The effects of the pressureinduced red shift upon the size of supramolecules were also seen in structurally well-defined purple bacterial antennae, LH1

Mizoguchi et al. and LH2. Reddy et al. demonstrated that the pressure parameters of the aggregated BChls-a in LH1 and LH2 on absorption spectra were estimated to be -500 and -300 cm-1 GPa-1, respectively.53 Because the LH1 ring (∼11 nm) was larger than the LH2 ring (∼6 nm),28 these results clearly indicated that largesize supramolecules exhibited a higher pressure sensitivity. Conclusions In the present investigation, we demonstrated the peripheral alkyl substituents effects upon the self-assembly of antenna BChls in light-harvesting chlorosomes from several green photosynthetic bacteria (Chl. tepidum, Chl. Vibrioforme, Chl. phaeobacteroides, and Cfl. aurantiacus) using absorption spectra in combination with high pressure. The effects were evaluated using pressure-induced red shift and broadening of the Qy absorption band. The pressure parameters obtained for the chlorosomes were clearly divided into two categories: one was the chlorosomes isolated from the green sulfur bacteria (Chlorobuim species) having various alkyl substituents at the 8- and 12-postions as their composite antenna BChls, and the other was the chlorosomes from the green nonsulfur bacterium (Chloroflexus species) having a single molecular structure at the positions (no homologous distribution for the alkyl substituents). The substituent effect was confirmed using an in vitro model system that differs only by the substituents at the 8- and 12-positions. The present pressurization results enabled us to discuss a physiological meaning of a variety of antenna pigments having different alkyl substituents at the 8- and 12-positions in green photosynthetic bacteria. Consequently, the mixing of the various antenna BChls inside chlorosomes is essential for construction of both a pressure-sensitive energy donating part for fine-tuning the absorption properties and a large size antenna system for efficient capturing of low light irradiation. Acknowledgment. We thank Mr. Hiroyoshi Inose and Mr. Shinsuke Inomata at Ritsumeikan University for their experimental assistance. This work was partially supported by the Academic Frontier Project for Private Universities: a matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of the Japanese Government, 2003-2007; and also by Grants-in-Aid for Scientific Research (B) (No. 19350088) from the Japanese Society for the Promotion of Science (JSPS) (to H.T.) and for Young Scientists (B) (No. 17750167) (to T.M.) from MEXT. References and Notes (1) Blankenship, R. E.; Olson, J. M.; Miller, M. In Anoxygenic Photosynthetic Bacteria; Blankenship, R. E., Madigan, M. T., Bauer, C. E., Eds.; Kluwer Academic Publishers: Dordrecht, 1995; pp 399-435. (2) Olson, J. M. Photochem. Photobiol. 1998, 67, 61. (3) Blankenship, R. E.; Matsuura, K. In Light-HarVesting Antennas in Photosynthesis; Green, B. R., Parson, W. W., Eds.; Kluwer Academic Publishers: Dordrecht, 2003; pp 195-217. (4) Oelze, J.; Golecki, J. R. In Anoxygenic Photosynthetic Bacteria; Blankenship, R. E., Madigan, M. T., Bauer, C. E., Eds.; Kluwer Academic Publishers: Dordrecht, 1995; pp 259-278. (5) Sørensen, P. G.; Cox, R. P.; Miller, M. Photosynth. Res. 2008, 95, 191. (6) Beatty, J. T.; Overmann, J.; Lince, M. T.; Manske, A. K.; Lang, A. S.; Blankenship, R. E.; van Dover, C. L.; Martinson, T. A.; Plumley, F. G. Proc. Natl. Acad. Sci. USA 2005, 102, 9306. (7) Balaban, T. S.; Tamiaki, H.; Holzwarth, A. R. Top. Curr. Chem. 2005, 258, 1. (8) Miyatake, T.; Tamiaki, H. J. Photochem. Photobiol. C 2005, 6, 89. (9) Staehelin, L. A.; Golecki, J. R.; Drews, G. Biochim. Biophys. Acta 1980, 589, 30. (10) Saga, S.; Tamiaki, H. J. Biosci. Bioeng. 2006, 102, 118.

Pressure Dependency of Qy Band of Chlorosomes (11) Staehelin, L. A.; Golecki, J. R.; Fuller, R. C.; Drews, G. Arch. Mikrobiol. 1978, 119, 269. (12) Huber, V.; Katterle, M.; Lysetska, M.; Wu¨rthner, F. Angew. Chem., Int. Ed. 2005, 44, 3147. (13) Psˇencˇík, J.; Ikonen, T. P.; Laurinma¨ki, P.; Merckel, M. C.; Butcher, S. J.; Serimaa, R. E.; Tuma, R. Biophys. J. 2004, 87, 1165. (14) Psˇencˇík, J.; Arellano, J. B.; Ikonen, T. P.; Borrego, C. M.; Laurinma¨ki, P. A.; Butcher, S. J.; Serimaa, R. E.; Tuma, R. Biophys. J. 2006, 91, 1433. (15) Oostergetel, G. T.; Reus, M.; Chew, A. G. M.; Bryant, D. A.; Boekema, E. J.; Holzwarth, A. R. FEBS Lett. 2007, 581, 5435. (16) Sakuragi, Y.; Frigaard, N.-U.; Shimada, K.; Matsuura, K. Biochim. Biophys. Acta 1999, 1413, 172. (17) Pedersen, M. Ø.; Pham, L.; Steensgaard, D. B.; Miller, M. Biochemistry 2008, 47, 1435. (18) Gerola, P. D.; Olson, J. M. Biochim. Biophys. Acta 1986, 848, 69. (19) Schmidt, K. Arch. Microbiol. 1980, 124, 21. (20) Bobe, F. W.; Pfenning, N.; Swanson, K. L.; Smith, K. M. Biochemistry 1990, 29, 4340. (21) Borrego, C. M.; Garcia-Gil, L. J. Photosynth. Res. 1995, 45, 21. (22) Borrego, C. M.; Gerola, P. D.; Miller, M.; Cox, R. P. Photosynth. Res. 1999, 59, 159. (23) Ishii, T.; Kimura, M.; Yamamoto, T.; Kirihara, M.; Uehara, K. Photochem. Photobiol. 2000, 71, 567. (24) Tamiaki, H. Photochem. Photobiol. Sci. 2005, 4, 675. (25) Frigaard, N.-F.; Chew, A. G. M.; Li, H.; Maresca, J. A.; Bryant, D. A. Photosynth. Res. 2003, 78, 93. (26) Wu, H.-M.; Ra¨tsep, M.; Young, C. S.; Jankowiak, R.; Blankenship, R. E.; Small, G. J. Biophys. J. 2000, 79, 1561. (27) Freiberg, A.; Ellervee, A.; Kukk, P.; Laisaar, A.; Tars, M.; Timpmann, K. Chem. Phys. Lett. 1993, 214, 10. (28) Ra¨tsep, M.; Wu, H.-M.; Hayes, J. M.; Blankenship, R. E.; Cogdell, R. J.; Small, G. J. J. Phys. Chem. B 1998, 102, 4035. (29) Reddy, N. R. S.; Wu, H.-M.; Jankowiak, R.; Picorel, R.; Cogdell, R. J.; Small, G. J. Photosynth. Res. 1996, 48, 277. (30) Wu, H.-M.; Ra¨tsep, M.; Jankowiak, R.; Cogdell, R. J.; Small, G. J. J. Phys. Chem. B 1997, 101, 7641. (31) Wu, H.-M.; Ra¨tsep, M.; Jankowiak, R.; Cogdell, R. J.; Small, G. J. J. Phys. Chem. B 1998, 102, 4023. (32) Mizoguchi, T.; Harada, J.; Tamiaki, H. FEBS Lett. 2006, 580, 6644. (33) Saga, Y.; Wazawa, T.; Mizoguchi, T.; Ishii, Y.; Yanagida, T.; Tamiaki, H. Photochem. Photobiol. 2002, 75, 433. (34) Saga, Y.; Wazawa, T.; Nakata, T.; Ishii, Y.; Yanagida, T.; Tamiaki, H. J. Phys. Chem. B 2002, 106, 1430.

J. Phys. Chem. B, Vol. 112, No. 51, 2008 16765 (35) Saga, Y.; Oh-oka, H.; Hayashi, T.; Tamiaki, H. Anal. Sci. 2003, 19, 1575. (36) Saga, Y.; Matsuura, K.; Tamiaki, H. Photochem. Photobiol. 2001, 74, 72. (37) Mizoguchi, T.; Saga, Y.; Tamiaki, H. Photochem. Photobiol. Sci. 2002, 1, 780. (38) Mizoguchi, T.; Hara, K.; Nagae, H.; Koyama, Y. Photochem. Photobiol. 2000, 71, 596. (39) Mizoguchi, T.; Tamiaki, H. Bull. Chem. Soc. Jpn. 2007, 80, 2196. (40) Sawamura, S. Pure Appl. Chem. 2007, 79, 861. (41) Balaban, T. S.; Holzwarth, A. R.; Schaffner, K. Biochemistry 1995, 34, 15259. (42) Nozawa, T.; Ohtomo, K.; Takeshita, N.; Morishita, Y.; Osawa, M.; Madigan, M. T. Bull. Chem. Soc. Jpn. 1992, 65, 3493. (43) Steensgaard, D. B.; Wackerbarth, H.; Hildebrandt, P.; Holzwarth, A. R. J. Phys. Chem. B 2000, 104, 10379. (44) Shibata, Y.; Saga, Y.; Tamiaki, H.; Itoh, S. Biophys. J. 2006, 91, 3787. (45) Saga, Y.; Wazawa, T.; Ishii, Y.; Yanagida, T.; Tamiaki, H. J. Nanosci. Nanotech. 2006, 6, 1750. (46) Matsuura, K.; Hirota, M.; Shimada, K.; Mimuro, M. Photochem. Photobiol. 1993, 57, 92. (47) Shibata, Y.; Saga, Y.; Tamiaki, H.; Itoh, S. Biochemistry 2007, 46, 7062. (48) Ellervee, A.; Linnanto, J.; Freiberg, A. Chem. Phys. Lett. 2004, 394, 80. (49) Ellervee, A.; Freiberg, A. Chem. Phys. Lett. 2008, 450, 386. (50) Tamiaki, H.; Kubo, M.; Oba, T. Tetrahedron 2000, 56, 6245. (51) Montan˜o, G. A.; Bowen, B. P.; LaBelle, J. T.; Woodbury, N. W.; Pizziconi, V. B.; Blankenship, R. E. Biophys. J. 2003, 85, 2560. (52) Wang, Z.-Y.; Marx, G.; Umetsu, M.; Kobayashi, M.; Mimuro, M.; Nozawa, T. Biochim. Biophys. Acta 1995, 1232, 187. (53) Reddy, N. R. S.; Jankowiak, R.; Small, G. J. J. Phys. Chem. 1995, 99, 16168. (54) McLuskey, K.; Prince, S. M.; Cogdell, R. J.; Isaacs, N. W. Biochemistry 2001, 40, 8783. (55) Tadros, M. H.; Waterkamp, K. EMBO J. 1989, 8, 1303. (56) Overmann, J.; Cypionka, H.; Pfennig, N. Limnol. Oceanogr. 1992, 37, 150. (57) Manske, A. K.; Glaeser, J.; Kuypers, M. M. M.; Overmann, J. Appl. EnViron. Microbiol. 2005, 71, 8049. (58) Causgrove, T. P.; Brune, D. C.; Blankenship, R. E. J. Photochem. Photobiol. B 1992, 15, 171.

JP804990F