Probing the Effect of the Binding Site on the Electrostatic Behavior of a

Jul 10, 2008 - Department of Life and Materials Engineering, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nag...
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J. Phys. Chem. B 2008, 112, 9467–9475

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Probing the Effect of the Binding Site on the Electrostatic Behavior of a Series of Carotenoids Reconstituted into the Light-Harvesting 1 Complex from Purple Photosynthetic Bacterium Rhodospirillum rubrum Detected by Stark Spectroscopy Katsunori Nakagawa,†,| Satoru Suzuki,‡ Ritsuko Fujii,‡,| Alastair T. Gardiner,§ Richard J. Cogdell,§ Mamoru Nango,†,| and Hideki Hashimoto*,‡,| Department of Life and Materials Engineering, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan, Department of Physics, Graduate School of Science, Osaka City UniVersity, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan, Glasgow Biomedical Research Centre, Institute of Biomedical and Life Sciences, UniVersity of Glasgow, 120 UniVersity Place, Glasgow, Scotland 8QQ, U.K., and CREST/JST, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan ReceiVed: February 29, 2008; ReVised Manuscript ReceiVed: April 22, 2008

Reconstitutions of the LH1 complexes from the purple photosynthetic bacterium Rhodospirillum rubrum S1 were performed with a range of carotenoid molecules having different numbers of CdC conjugated double bonds. Since, as we showed previously, some of the added carotenoids tended to aggregate and then to remain with the reconstituted LH1 complexes (Nakagawa, K.; Suzuki, S.; Fujii, R.; Gardiner, A. T.; Cogdell, R. J.; Nango, M.; Hashimoto, H. Photosynth. Res. 2008, 95, 339-344), a further purification step using a sucrose density gradient centrifugation was introduced to improve purity of the final reconstituted sample. The measured absorption, fluorescence-excitation, and Stark spectra of the LH1 complex reconstituted with spirilloxanthin were identical with those obtained with the native, spirilloxanthin-containing, LH1 complex of Rs. rubrum S1. This shows that the electrostatic environments surrounding the carotenoid and bacteriochlorophyll a (BChl a) molecules in both of these LH1 complexes were essentially the same. In the LH1 complexes reconstituted with either rhodopin or spheroidene, however, the wavelength maximum at the BChl a Qy absorption band was slightly different to that of the native LH1 complexes. These differences in the transition energy of the BChl a Qy absorption band can be explained using the values of the nonlinear optical parameters of this absorption band, i.e., the polarizability change Tr(∆r) and the static dipole-moment change |∆µ| upon photoexcitation, as determined using Stark spectroscopy. The local electric field around the BChl a in the native LH1 complex (ES) was determined to be ∼3.0 × 106 V/cm. Furthermore, on the basis of the values of the nonlinear optical parameters of the carotenoids in the reconstituted LH1 complexes, it is possible to suggest that the conformations of carotenoids, anhydrorhodovibrin and spheroidene, in the LH1 complex were similar to that of rhodopin glucoside in crystal structure of the LH2 complex from Rhodopseudomonas acidophila 10050. Introduction The first step in the photosynthetic processes is the capture of solar energy. In most species of purple photosynthetic bacteria light-harvesting is accomplished by corporation of two types of antenna complexes called LH1 and LH2. The light energy absorbed by the antenna system is then transferred to the reaction center (RC) where it is used to drive transmembrane charge separation. The efficiency of the light-harvesting process in most purple bacteria is rather high.1 These light-harvesting complexes contain both bacteriochlorophyll a (BChl a) and carotenoids. The light-harvesting apoproteins form a scaffold, which positions the pigments for optimal performance in energy transfer. Both the carotenoids and the BChls are involved in the lightharvesting process. In this study we concentrate on the role of carotenoids in the light-harvesting process and use a reconstitu* To whom correspondence should be addressed. Phone and Fax: +816-6605-2526. E-mail: [email protected]. † Nagoya Institute of Technology. ‡ Osaka City University. § University of Glasgow. | CREST/JST.

tion methodology to investigate the interaction of the carotenoids with their binding site. In the LH2 and RC complexes from purple photosynthetic bacteria, the conformations of the carotenoid pigments have been determined by both X-ray crystallographic studies2–11 and resonance Raman spectroscopy.12–22 These studies have been extensively reviewed in refs 23–28. In the LH2 complex the configuration of carotenoids is all-trans, whereas in the RC the carotenoid has a twisted 15,15′-cis conformation. These carotenoids are both able to participate in singlet-singlet energy transfer to BChl and to quench BChl triplet state in a triple-triplet exchange reaction. Carotenoids are, therefore, important photosynthetic pigments, and many studies have been carried out trying to define the mechanisms of these two energy transfer processes. Reconstitution of carotenoids into photosynthetic pigmentprotein complexes has proved a powerful technique with which to study their functions. In this regard using the LH1 complex is advantageous since LH1 complexes can be fully reconstituted in vitro from its constituent parts, i.e., LH1-R and LH1-β polypeptides and the pigments (BChl a and carotenoid).29–35

10.1021/jp801773j CCC: $40.75  2008 American Chemical Society Published on Web 07/10/2008

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Nakagawa et al. these protein-pigment complexes. We have used Stark spectroscopy in the present study to investigate the electrostatic properties of the different carotenoids reconstituted into the LH1 complexes from Rs. rubrum S1. Stark spectroscopy provides a very sensitive test of the faithfulness of the reconstitution process. Experimental Methods

Figure 1. Chemical structures of carotenoids used in this study: (a) spirilloxanthin, (b) anhydrorhodovibrin, (c) rhodopin, and (d) spheroidene. The CdC conjugated double bonds are colored in red, and the number, n, of these conjugated double bonds is also indicated. The first few carbon atoms in each carotenoid have been numbered 1-4 because of the discussion of their relative dihedral angles, which is important in the semiempirical molecular orbital calculations of their conformations.

With reconstitution it is possible to study the effects of adding series of carotenoids where such factors as the number of conjugated double bonds can be varied in a systematic way. Since many of a carotenoid’s photochemical properties change in a graded way depending on the number of conjugated double bonds, such studies can provide key insight into mechanism. In the present study, we have reconstituted a series of carotenoids into the LH1 complex of the purple photosynthetic bacterium Rhodospirillum (Rs.) rubrum. The chemical structures of the carotenoids used are shown in Figure 1. A similar series of reconstitutions using LH1 complex from Rs. rubrum has previously been reported.36–38 More recently, we reported the reconstitution of the LH1 complex with the carotenoid, spirilloxanthin.39 However, the resulting absorption and fluorescenceexcitation spectra of the reconstituted complex were not identical to those of the native LH1 complex as isolated from Rs. rubrum S1. Since the purity and reproducibility of the reconstituted LH1 complexes described previously were poor, we have developed the reconstitution protocol in order to overcome these problems. Stark spectroscopy is a well-established method with which to investigate the electrostatic properties of pigments. Measurement of nonlinear optical parameters such as polarizability change Tr(∆r) and static dipole-moment change |∆µ| upon photoexcitation can be important information about the electrostatic interaction of the pigments with their environment. Our group has previously described the use of Stark spectroscopy to probe the electrostatic environment around the BChl a dimer (special pair) in RC complex in the presence and the absence of the RC carotenoids.40 The electrostatic field surrounding the special pair was changed by about 10% when the carotenoid molecule was present. Stark spectroscopy has also been used to investigate the electrostatic field around the BChl a molecules in the native LH1 complex of Rs. rubrum S1 as a function of which detergent molecule was present in the solubilizing micelle surrounding the hydrophobic core of this complex.41 The measured Stark spectra showed a strong dependence upon which detergent was present. Analysis of these data has allowed quantitative investigation of the dependence of the electrostatic environments and conformations of carotenoids and BChl a in

Growth of the Cells of the Purple Photosynthetic Bacteria and Preparation of Chromatophores. Cells of Rs. rubrum wild-type strain S1, Rhodopseudomonas (Rps.) palustris, and Rhodobacter (Rb.) sphaeroides wild-type strain 2.4.1 used in this study were grown anaerobically in the light in C-succinate medium (modified from Cohen-Bazire et al.42) at 29 °C. These cells were harvested by centrifugation (18 800g × 10 min at 4 °C), resuspended in 20 mM Tris-HCl buffer (pH 8.0), and stored in freezer (-30 °C) until use. Chromatophores of Rs. rubrum S1 were prepared as described previously.41 The cells of Rps. palustris and Rb. sphaeroides 2.4.1 were just used as sources of the required carotenoids. Isolation and Purification of Native LH1 Complex from Rs. rubrum S1. The native LH1 complex was isolated and purified from the chromatophores of Rs. rubrum S1 as reported previously41 with the following small modifications. The isolated LH1 complex was loaded onto an ion-exchange (Whatman, DE52) column (1.5 cm i.d.) pre-equilibrated with 50 mM Tris-HCl buffer (pH 8.0) containing 0.03% LDAO. After the free carotenoids (which were not bound to the LH1 complex) were eluted with Tris-HCl buffer containing 0.1% Triton X-100 and 25 mM NaCl, the native LH1 complex was eluted with Tris-HCl buffer containing 0.03% LDAO and 200 mM NaCl. Then the NaCl was removed using a desalting column (GE Healthcare Bio-Sciences Corp., PD-10), and the concentration of LDAO was adjusted to 0.01% by dilution with cold 50 mM Tris-HCl buffer (pH 8.0). Isolation and Purification of Carotenoids (Spirilloxanthin, Anhydrorhodovibrin, Rhodopin, and Spheroidene). Figure 1 shows the series of carotenoids used in the present study. These carotenoids were extracted from the purple bacterial cells. Spirilloxanthin (Spx) was obtained from Rs. rubrum S1, anhydrorhodovibrin (Anh) and rhodopin (Rho) were obtained from Rps. palustris, and spheroidene (Sphe) was obtained from Rb. sphaeroides 2.4.1. Wet cells of Rs. rubrum S1 and Rps. palustris were suspended in methanol, stirred, and centrifuged (5000g × 10 min at 10 °C). Most of the BChl a was removed by this process. The centrifuged pellet was resuspended in benzene, stirred, and centrifuged again (5000g × 10 min at 10 °C). The benzene supernatant contained the carotenoids. This benzene extraction was repeated three times in order to collect all of the carotenoids. In order to remove contaminating lipids, the benzene fractions were washed with a saturated NaCl solution in a separating funnel. The pooled benzene solutions were dried over anhydrous Na2SO4 and then rotary evaporated to dryness. The all-trans isomers of the required carotenoids were obtained by alumina column chromatography (Merck, aluminum oxide 90) using mixtures of n-hexane and diethyl ether as eluents.39,43 Pure all-trans samples of these three carotenoids were recrystallized from solutions in tetrahydrofuran by addition of a small aliquot of n-hexane at -30 °C. Spheroidene was extracted from the cells of Rb. sphaeroides 2.4.1 as previously reported.44 The wet cells were suspended in an acetone/methanol mixture (7/2, v/v) and stirred for 30 min while bubbling with N2 gas. After centrifugation (5000g × 10 min at 10 °C), the supernatant, containing the carotenoids,

Carotenoids Reconstituted into the LH1 Complex was collected. This mixture of carotenoids was transferred to n-hexane. Then a mixture of 95% methanol/5% water was added in order to remove BChl. The carotenoid-containing n-hexane solution was dried over anhydrous Na2SO4 and then rotary evaporated to dryness. The carotenoids were then dissolved in n-hexane and separated by alumina chromatography. A fraction of pure all-trans spheroidene was eluted at a diethyl ether concentration of 10-15% in n-hexane. This fraction was rotary evaporated, and the spheroidene was redissolved in a minimal volume of n-hexane. Again this carotenoid was finally fully purified by crystallization at -30 °C in n-hexane. The purity and all-trans configuration of these carotenoids were confirmed by high-performance liquid chromatography (HPLC) analysis,45 electron impact (EI) mass spectroscopy, and 600 MHz 1H NMR spectroscopy. All these procedures were performed in dim light. Reconstitution of Carotenoids into the LH1 Complex. These reconstitutions were performed with the isolated carotenoids and the LH1 subunit-type complex prepared from Rs. rubrum S1, which contained LH1 polypeptides (LH1-R and LH1-β) and BChl a, as previously reported by Nakagawa et al.39 with minor modifications. Following reconstitution the LH1 complexes were purified by a combination of ion-exchange chromatography, using DEAE-cellulose DE52 (Whatman), and sucrose density gradient centrifugation. Following ion-exchange chromatography the reconstituted LH1 complexes were loaded onto a stepwise sucrose gradient (0.4 M/0.8 M/1.2 M) prepared in the presence of 20 mM Tris-HCl buffer (pH 8.0) containing 0.01% LDAO and 0.01% Triton X-100. After centrifugation at 165 000g × 16 h at 4 °C, the reconstituted LH1 complexes accumulated in 0.8 M sucrose layer. The reconstituted complexes were then loaded onto a DE52 column (1.5 cm i.d.) preequilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 0.03% LDAO, in order to exchange the detergent. The LH1 complexes were eluted from this column by the addition of 250 mM NaCl to this buffer. The NaCl was then removed by passing complex over a PD-10 column. Finally, the concentration of LDAO in the solution was adjusted to 0.01%, by dilution with cold 50 mM Tris-HCl buffer (pH 8.0). The final yield of each reconstituted LH1 complex was in the range of 50-60% with respect to the LH1 subunit-type complex. This value was similar with that reported by Fiedor et al.37 All these procedures were performed in dim light. Determination of the Ratio of BChl a to Carotenoid in the Reconstituted LH1 Complexes. The ratio of BChl a to carotenoids in both the native and the reconstituted LH1 complexes was determined as follows. The LH1 complex was loaded onto an ion-exchange (Whatman, DE52) column (0.5 cm i.d.), and the concentration of detergent was lowered by washing a column with a large volume of water. Then the residual water was removed by drying the DE52 with a N2 flow. The pigments were then extracted with an acetone/methanol mixture (7/2, v/v). This mixture was rotary evaporated to dryness, and the pigments were redissolved in n-hexane. The absorption spectrum of the pigments in the n-hexane solution was measured at room temperature. The concentration of the pigments was determined using a value of the following extinction coefficient of BChl a ε770 ) 71 500 (M-1 cm-1)37 and of carotenoids: spirilloxanthin, ε493 ) 151 000 (M-1 cm-1); anhydrorhodovibrin, ε485 ) 171 000 (M-1 cm-1); rhodopin, ε473 ) 181 500 (M-1 cm-1); spheroidene, ε454 ) 173 600 (M-1 cm-1). These values of their extinction coefficients were previously reported by Fujii et al.43 and Zhang et al.46

J. Phys. Chem. B, Vol. 112, No. 31, 2008 9469 Spectroscopic Measurements. The absorption and fluorescence-excitation spectra of the LH complexes were recorded, respectively, on a JASCO V-530 UV-vis spectrophotometer and a JASCO FP-6600 spectrofluorometer, using a 1 cm pathlength quartz cuvette.39,41 For the fluorescence-excitation spectra, the absorbance of the LH1 complexes at the peak of the Qy transition of BChl a was adjusted to an OD880 ) 0.1 and the emission was detected at 902 nm. The singlet-singlet energy transfer efficiency from carotenoid to BChl a in the LH1 complexes was determined by comparing the fluorescenceexcitation spectra with the fractional absorption spectra normalizing at the peak of the Soret transition of BChl a. These spectral measurements were all carried out at 4 °C. For the Stark spectroscopy, the LH1 complexes were concentrated by centrifugation in a centricon tube (MWCO ) 50 000). The solutions were centrifuged at 3000g × 10 min at 4 °C until an OD880 ) 10 was obtained. An amount of 50 mg of poly(vinyl alcohol) (PVA, Kuraray Co., Ltd. PVA-217) was dissolved into 1 mL of the sample solution of the LH1 complexes. A small part of this solution was dropped onto the surface of a glass substrate, on which gold electrodes (gap distance was 35-60 µm) had been installed. Any residual solvent was removed under reduced pressure at 4 °C. The Stark spectra were then recorded using a homebuilt setup, the details of which have already been reported.40,47 The Stark spectra were all recorded at room temperature. The nonlinear optical parameters, i.e., polarizability change Tr(∆r) and static dipole-moment change |∆µ| upon photoexcitation, of both the carotenoids and the BChl a molecules in the LH1 complexes were determined by a fitting the Liptay equation48 to the experimentally observed Stark spectra, as previously reported.41 Results Absorption and Fluorescence-Excitation Spectra of the Reconstituted LH1 Complexes. The LH1 complexes from Rs. rubrum S1 were reconstituted with the following carotenoids: spirilloxanthin (spx), anhydrorhodovibrin (anh), rhodopin (rho), or spheroidene (sphe). For simplicity, we will refer to these reconstituted LH1 complexes as “RECspx”, “RECanh”, “RECrho”, and “RECsphe”, respectively. Figure 2 compares the absorption spectra of the native LH1 complex isolated from Rs. rubrum S1 and those reconstituted with the carotenoids. The LH1 complexes were all dissolved in Tris-HCl buffer (pH 8.0) containing 0.01% LDAO. The dashed and solid lines in each panel show the absorption spectra at room temperature and at 4 °C, respectively. The dotted lines show the absorption spectra of the LH1 complexes dispersed in a PVA film on the glass substrate at room temperature. These absorption spectra were normalized at the Qy absorption band of BChl a (about 880 nm). The spectra show the well-known BChl a absorption bands at about 375 nm (Soret band) and about 590 nm (Qx band). The wavelength maximum of the BChl a Qy absorption band in the LH1 complexes is very temperaturedependent. The wavelength maximum of this band at 4 °C is 2-4 nm red-shifted compared to room temperature. When the LH1 complexes were dispersed in a PVA film, the maximum of the Qy absorption band was identical to that of the complex in buffer. Previously, we reported that the BChl a Qy absorption band of RECspx was shifted compared to the native LH1 complex.39 It was suspected that this was due to the presence of aggregated free carotenoids in the sample. In the present study contamination with aggregated carotenoids has been overcome with the modified purification protocol. The absorption spectrum of the

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Figure 2. Normalized absorption spectra of (A) the native LH1 complex from Rs. rubrum S1 and the LH1 complexes reconstituted with carotenoids (B) RECspx, (C) RECanh, (D) RECrho, and (E) RECsphe. The spectra were recorded at both room temperature (dashed black line) and at 4 °C (solid black line). The dotted lines show the normalized room temperature absorption spectra of these LH1 complexes dispersed in a PVA film on a glass substrate. The native LH1 complex was dissolved in 50 mM Tris-HCl buffer (pH 8.0) containing 0.01% LDAO, whereas the LH1 complexes reconstituted with carotenoids were dissolved in 20 mM Tris-HCl buffer (pH 8.0) containing 0.01% LDAO.

TABLE 1: Ratio of BChl a to Carotenoid and the Efficiency of the Singlet-Singlet Excitation Energy Transfer from Carotenoid to BChl a at 4 °C in the Native and the Reconstituted LH1 Complexes from Rs. rubrum S1 LH1 complex native RECspx RECanh RECrho RECsphe

BChl a/ carotenoida

excitation energy transfer efficiency (%)b

2.0:1.0 ( 0.1 2.0:1.0 ( 0.1 2.0:0.8 ( 0.1 2.0:0.8 ( 0.1 2.0:0.8 ( 0.1

27 ( 1 28 ( 1 41 ( 1 45 ( 1 65 ( 1

a The averages of three separate experimental determinations with three different reconstituted samples. b The averages of 15 separate experimental determinations with 3 different reconstituted samples.

Figure 3. Picture of the LH1 complexes reconstituted with carotenoids.

RECspx (Figure 2B) in the region of the BChl a Qy absorption is now the same as in the native LH1 complex (Figure 2A). The BChl a Qy absorption band in the reconstituted sample is shown in Table 2. The differences between these absorption maxima in the different reconstituted samples are small but very reproducible. In the reconstituted and native LH1 complexes the absorption bands observed in the region between 420 and 570 nm are due to the S0 f S2 transitions of the carotenoids. The position of these absorption bands depends on the number of CdC conjugated double bonds, the higher the number of conjugated double bonds the more the absorption maxima are shifted to

the red. This is illustrated in Figure 3, which shows a color of the LH1 complexes reconstituted with the different carotenoids. In the LH1 subunit-type complex, two BChl a’s were organized with one pair of LH1-R and LH1-β polypeptides.29,49,50 It was suggested that the ratios between LH1-R, LH1-β polypeptides, and BChl a in the reconstituted LH1 complexes were 1:1:2. In present study, the BChl a and carotenoids in the native and reconstituted LH1 complexes were extracted, and the BChl a-to-carotenoid ratios were determined. These ratios are shown in Table 1. In both of the native LH1 complex and the RECspx, the ratios were 2:1. In the other reconstituted

Carotenoids Reconstituted into the LH1 Complex

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TABLE 2: Nonlinear Optical Parameters (Tr(∆r) and |∆µ|) of Carotenoids and the BChl a Qy Absorption Band in the Native and the Reconstituted LH1 Complexes from Rs. rubrum S1 determined by Stark spectroscopya carotenoidb

BChl a (Qy)b

LH1 complex

Tr(∆r) (Å3/f2)

|∆µ| (D/f)

Tr(∆r) (Å3/f2)

|∆µ| (D/f)

BChl a Qy (nm)

native RECspx RECanh RECrho RECsphe

1600 ( 100 1400 ( 100 1500 ( 100 1100 ( 100 850 ( 50

8.1 ( 0.1 8.2 ( 0.1 8.4 ( 0.1 6.7 ( 0.1 6.0 ( 0.1

310 ( 10 290 ( 20 300 ( 10 260 ( 10 350 ( 10

3.3 ( 0.1 3.4 ( 0.2 3.4 ( 0.1 3.0 ( 0.1 3.6 ( 0.1

883 883 883 881 884

a f is the local electric field correction factor. b The averages of nine separate experimental determinations with three different reconstituted samples.

complexes the measured ratio was 2:0.8, suggesting that in these cases not all the possible carotenoid binding sites were fully occupied. There is, however, some uncertainty in these ratios because the extinction coefficients of the carotenoids are probably only accurate to (5%. Fiedor et al. described, previously, the carotenoid content in the reconstituted LH1 complexes was larger than in the native LH1 complex.37 A possible reason for the difference between these two results is that in the present study we took particular care to remove any nonbound, aggregated carotenoids. Figure 4 compares the fractional absorption spectra and the fluorescence-excitation spectra of the native and the reconstituted LH1 complexes. All these measurements were recorded at 4 °C. These spectra were normalized in the Soret absorption band of BChl a (∼376 nm). The singlet-singlet energy transfer efficiencies from carotenoid to BChl a in the LH1 complexes were evaluated by comparing the area under the curves in the region where the carotenoids absorb (420-570 nm). These efficiencies are summarized in Table 1. The carotenoid to BChl a energy transfer efficiency in the native LH1 complex and RECspx was the same. This provides strong evidence that the spirilloxanthin has been correctly reconstituted. Moreover, an efficiency of 27-28% is in good agreement with previously reported values.33,51,52 The energy transfer efficiencies were higher for the LH1 complexes reconstituted with carotenoids having fewer conjugated double bonds. Again this is in good agreement with previous studies.53 Stark Spectra of the Reconstituted LH1 Complexes. Figure 5 compares the Stark spectra of the native and the reconstituted LH1 complexes. All of these spectra were recorded at room temperature and normalized against the intensity of the applied electric field and the concentration of the samples. The Stark signals seen at 400-600 and 800-1000 nm were due to the S0 f S2 transitions of carotenoids and the Qy absorption band of BChl a, respectively. The Stark spectra obtained with both the native LH1 complex and RECspx were identical. This again shows the high quality of this reconstitution. The Stark signal from the BChl a Qy absorption band in RECanh is also very similar to that seen with the native LH1 complex. The Stark signal from the BChl a Qy absorption band in RECrho, however, is 5-7 nm blueshifted compared to the native complex, whereas in the case of RECsphe it is 1 nm red-shifted. There is a systematic shift in the peaks of the Stark shift from the S0 f S2 transitions of carotenoids that reflects the similar shifts that are seen in their absorption spectra, which depends on the number of CdC conjugated bonds (see Figure 2). The observed Stark spectra of the LH1 complexes were fitted by Liptay equation48 in both the region of the carotenoid

Figure 4. The 4 °C fractional absorption (1 - transmittance; solid black line) and fluorescence-excitation spectra (solid red line) of (A) the native LH1 complex from Rs. rubrum S1 and the LH1 complexes reconstituted with carotenoids (B) RECspx, (C) RECanh, (D) RECrho, and (E) RECsphe. These spectra were normalized at the BChl a Soret band (∼376 nm).

absorption and the BChl a Qy absorption. Since the absorption bands of carotenoids overlap with the red-edge of the Soret absorption band of BChl a (376 nm) and the blue-edge of the BChl a Qx absorption band (590 nm), fitting by the Liptay equation could only be achieved after the contributions in the absorption spectra from BChl a had been removed. This was achieved by subtracting the absorption spectrum of the native LH1 complex isolated from the carotenoidless strain of Rs. rubrum G9+ (data not shown). The difference spectra obtained in this way are shown in Figure 6. The results of fitting the Stark spectra with the Liptay equation are shown as the red dashed lines in Figure 5. It is clear that this fitting overlaps with the measured spectra very well indeed.

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Figure 6. Difference absorption spectra of the carotenoids in the LH1 complexes obtained by subtraction of the absorption spectrum of the carotenoidless LH1 complex isolated from Rs. rubrum strain G9+: (a) spirilloxanthin, (b) anhydrorhodovibrin, (c) rhodopin, and (d) spheroidene. The dashed red lines represent the results of fitting these difference spectra with Gaussians. This was done in order to facilitate the calculation of the derivative spectra needed to simulate the experimental Stark spectra.

of CdC conjugated double bonds. The two values of the two nonlinear parameters of the BChl a Qy absorption band in RECrho were smaller than those of the native LH1 complex. This probably reflects the 2 nm blue-shift of the wavelength maximum of the BChl a Qy absorption band in RECrho. The RECsphe complex has the BChl a Qy absorption maximum redshifted by 1 nm relative to the native complex, and in this case the value of its nonlinear optical parameters in this spectral region are larger than that of the native complex. These small but significant changes reflect some subtle differences in the electrostatic environment around carotenoid and BChl a molecules in these preparations. Figure 5. Normalized Stark spectra of (A) the native LH1 complex from Rs. rubrum S1 and the LH1 complexes reconstituted with carotenoids (B) RECspx, (C) RECanh, (D) RECrho, and (E) RECsphe. The spectra were normalized with respect to the intensity of the applied electric field and the concentration of the samples. The dashed red lines in each panel show the results of fitting to the Liptay equation.

On the basis of these fittings the nonlinear optical parameters, i.e., polarizability change Tr(∆r) and static dipole-moment change |∆µ| upon photoexcitation, of carotenoids and BChl a in the LH1 complexes can be determined. The values of these parameters are given in Table 2. Within experimental error the values of Tr(∆r) and |∆µ| of both the carotenoid and the Qy absorption band of BChl a in the native LH1 complex, RECspx, and RECanh are the same. This shows that the electrostatic environments around carotenoid and BChl a molecules in these complexes are the same. In the case of RECrho and RECsphe, however, the values of the nonlinear optical parameters of carotenoids were smaller than those of the others. This again reflects differences in the number

Discussion We have presented in this paper a technique that allows carotenoids to be reconstituted into LH1 complexes from Rs. rubrum S1 and shown that this reconstitution process accurately places added carotenoids into native carotenoid binding site. This improved technique is very reproducible. The quality of the reconstitution can be clearly seen by comparing the absorption, fluorescence-excitation, and Stark spectra of the native LH1 complex with the complex reconstituted with spirilloxanthin, the carotenoid which is present in the native complex. All of these spectra are essentially identical showing that the reconstitution has recreated a fully native LH1 complex. This technique was also applied to the reconstitution of the LH1 complex with three other carotenoids, anhydrorhodovibrin (n ) 12), rhodopin (n ) 11), and spheroidene (n ) 10), each having a different number (n) of CdC conjugated bonds. With each of these different carotenoids there are small differences in the local electric field around the BChl a

Carotenoids Reconstituted into the LH1 Complex

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molecules in comparison with the situation in the native LH1 complex. It is possible using the values determined for the nonlinear optical parameters of the BChl a Qy absorption bands in the reconstituted LH1 complexes to determine the magnitude of the local electric field in the binding site surrounding BChl a in the native LH1 complex. Also, based on the nonlinear optical parameters of carotenoids in each of the reconstituted LH1 complexes it is possible to use semiempirical molecular orbital calculations to suggest the conformation of the carotenoid. Determination of the Local Electric Field (ES) around the BChl a Molecules in the Native LH1 Complex from Rs. rubrum S1. As shown in Table 2, the wavelength maxima at the BChl a Qy absorption band and its nonlinear optical parameters in RECrho and RECsphe are different from those of the native LH1 complex from Rs. rubrum S1. In this case, due to these differences, it is possible, by a modification of a previous treatment described in refs 39 and 41, to determine the local electric field around BChl a molecules in the native LH1 complex from Rs. rubrum S1 (ES). The total electric field (En) around the BChl a molecules in the reconstituted LH1 complexes, i.e., RECrho or RECsphe, can be described by the sum of the externally applied electric field (Ea) and the difference of the magnitude of the local electric field felt the BChl a between the reconstituted and the native LH1 complexes (E∆), by the following equation.

En ) Ea + ES - E∆

(1)

The Qy absorption band of BChl a in the native LH1 complex, which was observed at 883 nm at 4 °C, is due to the transition from the ground state to the second lowest exciton sublevel (ε2) (due to the symmetry of the arrangement of BChl a molecules in LH1). We can then apply a second-order perturbation theory to derive the energy level of this exciton sublevel of the BChl a Qy absorption band in the reconstituted LH1 complexes. For this calculation, the interaction energy of -µ · E∆ was taken as a perturbation. Here, µ represents the transition dipole moment of the transition from the ground state to the exciton sublevel of the Qy band in the unperturbed system (the native LH1 complex). Then, the perturbed energy level (ε2E∆) can be described by eq 2.

εE2 ∆ ) ε2 - ∆µS · E∆

(2)

In the reconstituted LH1 complexes, the shift of the transition energy (Stark shift) of the BChl a Qy band due to the presence of E∆ can be described by eqs 3 and 4.

1 h∆νge ) -∆µrec · Ea - Ea · ∆RS · Ea 2 1 (∆µS + ES · ∆RS) · E∆ - E∆ · ∆rS · E∆ 2 1 ∆µS · ES - ES · ∆RS · ES (3) 2 ∆µrec ) ∆µi + ∆rrec · (ES - E∆) (4) In these equations, ∆rS equals the value of Tr(∆r) of the BChl a Qy band and ∆µS equals |∆µ| of the BChl a Qy band in the native LH1 complex. Also, ∆rrec and ∆µrec are, respectively, the values of Tr(∆r) and |∆µ| of the BChl a Qy band in the reconstituted LH1 complex, i.e., RECrho or RECsphe. h is the Planck constant. On the basis of eqs 3 and 4, the differences in the transition energy of the BChl a Qy absorption bands between the reconstituted and the native LH1 complexes can be calculated as shown in eq 5.

1 h∆νE∆ ) -∆µS · E∆ - E∆ · ∆rS · E∆ 2

(5)

From eq 2, the magnitudes of E∆ were determined to be -3.3 × 105 V/cm between RECrho and the native LH1 complex and 1.7 × 105 V/cm between RECsphe and the native LH1 complex. Also, by using the eq 5, the values of shift (ε2E∆) in the presence of E∆ can be calculated. The value of this shift is h∆νEge∆ ) 3.0 meV between RECrho and the native LH1 complex and h∆νEge∆ ) –1.7 meV between RECsphe and the native LH1 complex (see refs 54 and 55 for supportive information). For this calculation we have used a value of f ) 1.4 for the local-field correction factor.56 On the basis of these calculations it can be suggested that the presence of this field, E∆, is responsible for the shift of the transition energy of the Qy absorption band of BChl a in the reconstituted LH1 complexes (RECrho and RECsphe) compared with the native LH1 complex. In the case of RECrho a 2 nm blue-shift of the BChl a Qy absorption band is equal to a 3.2 meV shift in energy, and an 1 nm red-shift, seen in the case of RECsphe, is equal to an energy shift of -1.6 meV. Our calculations based solely on the Stark spectra, therefore, are good predictors of the direction and magnitude of the observed shifts of the BChl a Qy absorption bands. The difference between the values of |∆µ| of the BChl a Qy absorption bands in the reconstituted and the native LH1 complexes ∆(∆µ) are described by eq 6.

∆(∆µ) ) ∆µrec - ∆µS ) - ∆rrec · Ε∆ + (∆rrec - ∆rS) · ΕS Ε∆ ) -

(

)

∆rs ∆(∆µ) + 1· ES ∆rrec ∆rrec

(6) (7)

With the use of eq 7, we can determine the local electric field (ES) around BChl a molecules in the native LH1 complex from Rs. rubrum S1. With the use of the information obtained by comparing both RECrho and RECsphe with the native LH1 complex ES can be calculated to be 3.0 × 106 and 3.1 × 106 V/cm, respectively. These two independent calculations produce a very consistent answer. Prediction of the Carotenoid’s Conformation in the LH1 Complex, Using Semiempirical Molecular Orbital Calculations. In the reconstituted LH1 complexes, the values of the nonlinear optical parameters (Tr(∆r) and |∆µ|) of carotenoids were different depending on the type of carotenoid, as shown in Table 2. Figure 7A shows a plot of the values of these nonlinear optical parameters versus the number (n) of CdC conjugated double bonds. The change of the intrinsic dipole moment, ∆µi, of the carotenoid is given by eq 8, where ∆µ and ∆r are, respectively, the values of |∆µ| and Tr(∆r) of carotenoids in the reconstituted LH1 complexes as determined by Stark spectroscopy. Here, E is the local electric field around the carotenoid molecules in the reconstituted complexes.

∆µ ) ∆r · Ε + ∆µi

(8)

Since spirilloxanthin (n ) 13) is a symmetric molecule (see Figure 1), its ∆µi should be to zero. In this case, therefore, we can calculate E by simply assuming that ∆µ ) ∆r · E. Then using this value of E, we can calculate by difference the value of ∆µi of the other carotenoids. These values of ∆µi have been plotted in Figure 7B, see black circles, and they are linearly dependent on the number of conjugated double bonds.

9474 J. Phys. Chem. B, Vol. 112, No. 31, 2008

Figure 7. Analyzing the conformation of the carotenoids reconstituted into the LH1 complexes from Rs. rubrum S1. (A) A plot of Tr(∆r) (black circles) and |∆µ| (black triangles) of the carotenoids in the reconstituted LH1 complexes versus the number (n) of CdC conjugated double bonds. (B) The conformation of rhodopin glucoside in the LH2 complex from Rps. acidophila 10050 (PDB code ) 1NKZ). Again as in Figure 1 the first few carbon atoms have been numbered 1-4. (C) A comparison of the experimentally determined value of ∆µi (black circle) of the carotenoids when reconstituted into the LH1 complex and the calculated values of ∆µi with the torsion angle of C2-C3-C4 vs C1-C2-C3 set to either 65° (red squares) or -60° (black triangles).

Figure 7B shows the conformation of a carotenoid, rhodopin glucoside, present in the LH2 complex of the purple photosynthetic bacterium Rps. acidophila 10050, based on the X-ray crystal structure (PDB code ) 1NKZ, ref 10). The gray and red spheres depict the carbon and oxygen atoms, respectively. The dihedral angle θ between C2-C3-C4 and C1-C2-C3 in this carotenoid molecule is about 65.5°. In anhydrorhodovibrin, rhodopin, and spheroidene, geometry optimization was performed semiempirically using MOPAC 2000 with an AM1 Hamiltonian, with this torsion angle fixed at 65°. The reliability of the geometry optimization with the AM1 Hamiltonian in comparison with the crystallographic structures of various carotenoid molecules has already been confirmed in the previous reports.57–59 Then, based these structures the values of ∆µi were calculated using a MOS-F program.60–63 In Figure 7C, the results of these calculations are plotted as red squares. These calculations were repeated setting the torsion angle θ to be -60°. These results are plotted in Figure 7C by black triangles. As show in Figure 7C, the calculated values of ∆µi match those determined experimentally for both anhydrorhodovibrin and spheroidene when the torsion angle θ is set at 65°. However, this suggests that the conformation (helicity) of these two carotenoids when they have been reconstituted into the LH1 complex from Rs. rubrum S1 is very similar to that of rhodopin glucoside in the LH2 complex from Rps. acidophila 10050. The same is not true for rhodopin (Figure 7C). This may reflect the presence of a more polar group (hydroxyl group) in

Nakagawa et al. this molecule. In this case calculations of the values of ∆µi were made varying the torsion angles over a wide range. However, none of these calculations were able to accurately reproduce the experimentally determined value of ∆µi. This suggests that either rhodopin in the LH1 complex adopts a rather different conformation compared to that of rhodopin glucoside in LH2 or there is another factor involved that we have not been able to take account of. It is interesting to note that the local electric field around the BChl a molecules in RECrho is about 10% larger than that in the native LH1 complex. This might be due to a possible interaction between the polar hydroxyl group of rhodopin with the BChls in LH1. If this is the case, this interaction could cause an expansion of the local electric field around the BChl a. If the assumption is made that such a polar interaction between the carotenoids and BChls is not present in RECsphe and RECanh then it is possible to evaluate the interaction between the BChls and the CdC conjugated double bonds of these two carotenoids. Since the conjugation length in spheroidene is shorter than in anhydrorhodovibrin, it can be proposed that the interaction between the BChl a and spheroidene will be weaker than that with anhydrorhodovibrin. This would then explain the differences in the value of the electric field around the BChls in the presence of these two carotenoids. The present investigation that correlates the nonlinear optical parameters determined by Stark spectroscopy with those predicted by semiempirical molecular orbital calculations provides us with one of the promising strategies to discuss the conformation of the carotenoid molecules in the LH1 complexes. Conclusions The LH1 complexes of Rs. rubrum S1 were successfully reconstituted with a range of carotenoids having different numbers of conjugated double bonds. Great care was taken to remove any aggregated, surplus carotenoids from the preparations prior to any spectroscopic measurements. The reconstituted complexes were investigated using absorption, fluorescenceexcitation, and Stark spectroscopy. The major findings were the following: 1. The spectroscopic properties of RECspx were identical with those of the native LH1 complex from Rs. rubrum S1. 2. The local electric field around the BChl a molecules in the native LH1 complex from Rs. rubrum S1 was determined to be 3.1 × 106 V/cm. The local electric field around the BChl a in RECrho was ∼10% larger, and in RECsphe it was ∼5% smaller. 3. Semiempirical molecular orbital calculations based on the nonlinear optical parameters of carotenoids in the reconstituted LH1 complexes indicate that spirilloxanthin, anhydrorhodovibrin, and spheroidene adopt a conformation within the reconstituted LH1 complexes that is very similar to that of rhodopin glucoside in the LH2 complex from Rps. acidophila 10050. It was not possible to obtain a unique solution for the calculation of the conformation of rhodopin in RECrho. It is suggested that this may be due to a stronger interaction between the hydroxyl group in rhodopin with the BChl a molecules in the reconstituted complex. Acknowledgment. This work was supported by the Grantin-aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology (Grant Nos. 172004026 and 17654083). H.H., R.F., M.N., A.T.G., and R.J.C. thank SICP/JST for financial support. R.J.C. and A.T.G. thank the BBSRC for financial support.

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