Circular and Magnetic Circular Dichroism Studies of

In neat CCl4, a dispersion-type CD signal was observed for the Qy(0−0) ... Photosynthesis Research 2018 135 (1-3), 309-317 ... Anita Zupcanova , Jua...
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J. Phys. Chem. B 2002, 106, 3987-3995

3987

Circular and Magnetic Circular Dichroism Studies of Bacteriochlorophyll c Aggregates: T-Shaped and Antiparallel Dimers Mitsuo Umetsu, Ryoichi Seki, Zheng-Yu Wang, Izumi Kumagai, and Tsunenori Nozawa* Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku UniVersity, Aobayama 07, Aoba-ku, Sendai 980-8579, Japan ReceiVed: July 9, 2001; In Final Form: December 17, 2001

Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra have been measured on (31R)bacteriochlorophyll (BChl) c aggregates in organic solvents. BChl c exists as a monomer in acetone with a Qy(0-0) transition at 661 nm. In a mixed solvent system of methanol and dichloromethane (CH3OH/CH2Cl2 ) 1/4000), another Qy(0-0) transition appeared at 680 nm with a nondispersion-type CD signal. The ratio of MCD intensity to its dipole strength (B/D) for the Qy(0-0) transition is half the value of its monomer species, indicating a dimerization of BChl c. The apparent inconsistency between the CD and MCD results has been reconciled by assuming the formation of BChl c dimer with a T-shaped conformation. In neat CCl4, a dispersiontype CD signal was observed for the Qy(0-0) exciton components at 680 and 710 nm. MCD spectra of the exciton-type Qy(0-0) components suggest that two BChl c molecules are parallel with their macrocycle planes as judged by the mixing effect between the excited states of Qx(0-0) and Qy(0-0). The CD and MCD results are consistent with the fact that a BChl c dimer with an antiparallel conformation is dominant in the neat CCl4 as reported previously. Corresponding Qx(0-0) transitions were determined around 650 nm by the simultaneous deconvolution of absorption, CD, and MCD spectra. It is demonstrated that the antiparallel BChl c dimer shows two exciton components for both Qx(0-0) and Qy(0-0) transitions. Finally, we evaluated solvent dependence of the stability of the BChl c dimer by two-dimensional exchange spectroscopy (EXSY) experiments.

Introduction Bacteriochlorophyll (BChl) c is found as a major component in a special light harvesting entity, known as a chlorosome, of green photosynthetic bacteria.1,2 These antennae are attached to the cytoplasmic side of the inner cell membrane and have an elliptical shape.3,4 Since chlorosomes contain more than 50% pigment by weight, it is a general consensus that self-aggregation of BChl c plays an important structural role in the chlorosome. The Qy(0-0) transition of isolated BChl c monomer appears at 660-70 nm in hydrophilic organic solvents. BChl c forms high aggregates in hexane with a Qy(0-0) transition around 740 nm with a spectral shape similar to that of native chlorosome.5 A large number of studies have been made to elucidate the structure of the in vitro 740-nm aggregates. It becomes clear that the 31-hydroxyl and 131-keto groups in BChl c play important roles in ligation and hydrogen bonding in the high aggregates.6-14 However, the structure of the larger aggregates has not been solved due to the large molecular weight and structural inhomogeneity. Besides the 740-nm aggregates, BChl c is capable of forming various aggregates with different sizes and absorption maxima in organic solvents. The major homologue in the chlorosome, (31R)-BChl c with an R-type chiral center at the 31-position, is considered to form two small aggregates in CH2Cl2, CHCl3, CCl4, and benzene with absorption maxima at 680 and 70310 nm.15,16 The 710-nm transition is always accompanied by a 680-nm component in the absorption spectra.16-18 Olson and Cox attributed the aggregation state with an absorption maxi* To whom correspondence should be addressed. Telephone: +81-22217-7277. Fax: +81-22-217-7279. E-mail: [email protected].

mum at 710 nm to a tetramer species formed by association of two dimers on the basis of the concentration dependence of the absorption spectra.16 Causgrove et al. proposed a cyclic arrangement of three dimers for the 710-nm species of magnesiummethyl bacteriopheophorbide (Mg-BChlide) d based on the result of polarized fluorescence experiments.17 Recently, we have demonstrated that the 710-nm-rich species of (31R)-BChl c in neat CCl4 is predominated by dimers from a small-angle neutron scattering (SANS) experiment.19 The result is further supported by an antiparallel piggy-back dimer structure determined from detailed NMR measurements.20,21 At present, we suggest that the 710-nm transition should be assigned as a Qy(0-0) exciton transition of the BChl c dimer with an antiparallel conformation. However, the spectroscopic behavior is still not fully explained, especially for the absorption and circular dichroism (CD) spectra of the 680-nm species. Magnetic circular dichroism spectroscopy (MCD) has been applied to analyze electronic and aggregation states of photosynthetic pigments both in vivo and in vitro. MCD is generally expressed by a linear combination of three terms with different origins: the Faraday A-, B-, and C-terms, each of these terms showing different MCD properties.22 The Faraday A- and C-terms play an important role in elucidating the electronic structure of porphyrin.23-25 The lowest unoccupied molecular orbitals (LUMOs) and highest occupied molecular orbitals (HOMOs) are degenerate and show strong π-π* transitions from the HOMOs to the LUMOs in the near-ultraviolet region. The MCD of hemes with porphyrin backbones is often studied in vivo to analyze the function of the central metal and electron acceptor/donor because the MCD properties are dramatically

10.1021/jp012574b CCC: $22.00 © 2002 American Chemical Society Published on Web 03/21/2002

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Figure 1. Molecular structure of the intact (31R)-[E,E]BChl cF. Figure 2. Absorption spectra of (31R)-BChl c in acetone (solid line A), in the methanol-added CH2Cl2 solution (the volume of methanol to CH2Cl2 is 0.025%, dotted line B), in neat CH2Cl2 (dashed line C), and in neat CCl4 (solid line D). The concentration of BChl c was 0.05 mM.

varied with the change of electronic states of the hemes by ligation to the central metal or by electron transfer.26-29 Chlorophyll (Chl) and BChl with chlorin and bacteriochlorin backbones are known to show the MCD arising from the Faraday B-term because their HOMOs and LUMOs are not degenerate due to the reduction of the double bonds in macrocycle.30-33 The molecules with chlorin and bacteriochlorin backbones show two x- and y-polarized bands in the nearultraviolet region (Bx, By) and in the visible region (Qx, Qy), and MCD spectra can be utilized for detection of very weak electronic transitions such as Qx since the intensity is enhanced in MCD spectra by mixing with closely related states.34-37 Recently, we have reported a MCD approach to determining the coordination number of the Mg atom in Chl and BChl and to probing the aggregation state of the pigments.38-39 The correlation between the MCD intensity of the Qy(0-0) transition and the energy difference of Qx(0-0) and Qy(0-0) has proved to be a useful measure for estimating the type and number of the molecules ligating to the Mg atom and also for detecting the existence of aggregated pigments. In this study, we have consistently reassigned the Qx(0-0) and Qy(0-0) transitions in the absorption, CD, and MCD spectra of the (31R)-BChl c small aggregates with absorption maxima at 680 and 710 nm. The conclusion of this study can account for the SANS and NMR results of the 710-nm-rich species.19-21 The monomer-rich species with the 670- and 680-nm absorption components was measured in order to observe the electronic transitions under the condition that there exists no antiparallel dimer. The 710-nm-rich species in neat CCl4 was also measured at high concentration similar to that used for NMR measurements.20,21 The MCD spectrum is sensitive for detecting the Qx(0-0) transition of aggregated forms of BChl c as well as the monomeric state. Simultaneous deconvolution of absorption, CD, and MCD spectra reveals that the antiparallel BChl c dimer in neat CCl4 has a dispersion-type CD signal with two wellseparate exciton components for each of the Qx(0-0) and Qy(0-0) transitions. Further, we were able to quantitatively estimate the instability of the antiparallel dimer in deuterated dichloromethane (CD2Cl2) from two-dimensional exchange spectroscopy (2-D EXSY) experiments.

For absorption, CD, and MCD measurements, BChl c was dissolved in acetone, CH2Cl2, and CCl4. All the solvents used were spectral grade, and CH2Cl2 and CCl4 were treated with Na2CO3. Deuterated dichloromethane (CD2Cl2, D > 99.9%) purchased from Cambridge Isotope Laboratory Inc. was used for solution NMR experiment. Extinction coefficients used for BChl c were calculated to be 7.3 × 104 L/(mol×cm) in diethyl ether, based on values of the literature.38 Instruments. Absorption spectra were measured on a Beckman DU-640 spectrophotometer. CD and MCD spectra were measured on a Jasco J-720w spectropolarimeter. The conditions of CD and MCD measurements were as follows: bandwidth, 1.0 nm (200-800 nm); resolution, 0.5 nm; response, 1 s; scan speed, 20 nm/min; external magnetic field, 1.5T (only for MCD). Deconvolution of absorption, CD, and MCD spectra were performed using a nonlinear curve fitting tool GRAMS/32 V5.0 (Galactic) according to the following criteria: (1) each pair of the absorption, CD, and MCD spectra were deconvoluted into the same number of components; (2) each pair of associated components in the absorption, CD, and MCD spectra were constrained to give the same band center and bandwidth parameters; (3) a mixed Gaussian-Lorentzian function was used to fit each component and the weight factors were allowed to vary for different components but keep the same value for the same component of the associated absorption, CD, and MCD spectra; (4) the deconvolution continued until χ2 (the sum of squares of the deviations normalized by the variance of count) attained less than 3. NMR spectra were obtained with a Bruker Avance DRX400 spectrometer at 298 K. For the measurements in CCl4 solution, deuterated water (D2O) was infused with a 1-mm inner tube in the 5-mm NMR tube. Two-dimensional EXSY spectra were measured with a pulse sequence of NOESY and acquired using TPPI for phase sensitive detection. The 2-D spectra were collected with 256 t1 points, 2048 data point in t2, and 32 scans for each t1 points using a repetition time of 1 s.

Experimental Section

Results

Materials and Sample Preparation. Thermophilic green photosynthetic bacterium Chlorobium tepidum was grown as previously reported.40 BChl c was extracted from the dry cells with methanol and purified with a reversed-phase HPLC column as described previously.38 BChl c used in this study was (31R)[E,E]BChl cF in which the substituents at the 8- and 12-positions were ethyl groups, and the ester chain was farnesyl (Figure 1).

Absorption, CD, and MCD Spectra. Figure 2 shows the absorption spectra of (31R)-[E,E]BChl cF in various solvents. BChl c exists as a monomer in acetone with a Qy(0-0) transition at 661 nm (solid line A). The Qy(0-0) transition appeared at 670 nm with a shoulder around 680 nm in the methanol-added CH2Cl2 solution (v/v, methanol /CH2Cl2 )1 /4000; dotted line B). Aggregation of BChl c seems to cause another absorption

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Figure 4. MCD spectra of (31R)-BChl c in acetone (solid line A), in the 0.025% methanol-added CH2Cl2 solution (dotted line B), in neat CH2Cl2 (dashed line C), and in neat CCl4 (solid line D). The concentration of BChl c was 0.05 mM.

Figure 3. CD spectra of (31R)-BChl c in acetone (a), in the 0.025% methanol-added CH2Cl2 solution (b), in neat CH2Cl2 (c), and in neat CCl4 (d). The concentration of BChl c was 0.05 mM.

component of Qy(0-0) at 680 nm. In neat CH2Cl2, the Qy(00) was red-shifted to 679 nm with a tail extended beyond 710 nm (dashed line C), and there was a predominant absorption component at 710 nm in the nonpolar CCl4 solvent (solid line D). A small amount of the 710-nm species was observed in neat CH2Cl2. Hence, aggregation of BChl c depends on the polarity or nucleophilicity of the solvent. CD spectra are utilized to evaluate interaction between transition dipole moments in an aggregate of Chl and BChl. The CD spectra of BChl c in acetone and the methanol-added CH2Cl2 solution only showed a trough in the Qy region (Figure 3a,b). The 680-nm absorption component in the monomer-rich species of BChl c appears to contribute a very minor signal in the CD spectrum (Figure 3b). In contrast, a dispersion-type CD signal appeared with a maximum at 681 nm and a minimum at 709 nm in neat CH2Cl2 (Figure 3c) and with a maximum at 679 nm and a minimum at 707 nm in neat CCl4 (Figure 3d). Different from the monomer-rich species of BChl c (Figure 3b), the 680-nm absorption component showed a relatively sharp and intense peak in neat CH2Cl2 (Figure 3c) and CCl4 (Figure 3d). The rotational strength corresponding with the 680-nm component changed from negative to positive upon appearance of the 710-nm component (Figure 3b-d). Therefore, the origin of the 680-nm component in neat CCl4 is obviously different from that in the methanol-added CH2Cl2 solution. It suggests that the dispersion-type CD signal at 680 and 710 nm is related to the formation of a 710-nm species. Considering that the R-type BChl c forms an antiparallel dimer in neat CCl4,19-21 the antiparallel dimer of BChl c probably shows two Qy(0-0) exciton components at 680 and 710 nm. Figure 4 shows the MCD spectra of BChl c in acetone, methanol-added CH2Cl2 solution, neat CH2Cl2, and neat CCl4. The MCD intensity of the Qy(0-0) transition decreased as the absorption maximum of Qy(0-0) was red-shifted. The decrease of the Qy(0-0) MCD intensity indicates an increasing extent of aggregation of BChl c. One of the important characteristics in the MCD spectra is the enhanced Qx(0-0) and vibration bands of Qx or Qy (QV). Therefore, MCD spectra are useful for

the detection of a weak Qx(0-0) band which is not discernible in absorption spectra. In Figure 4, the assignment of the Qx(0-0) transition for the monomeric form is in agreement with our previous results.38 The Qx(0-0) transition was also redshifted from 627 to 655 nm, and the MCD intensity became weaker as the polarity of solvent decreased. Although a similar situation (red-shift from 592 to 610 nm) was observed for QV, the decrease of the QV MCD intensity appeared to be smaller than the Qx(0-0). The QV intensity (at 600 nm) is larger than that of the Qx(0-0) (at ∼655 nm) in neat CCl4. Deconvolution of Absorption, CD, and MCD Spectra. For the purpose of quantitative evaluation of transitions for the aggregated BChl c, we have performed a simultaneous deconvolution on the absorption, CD, and MCD spectra. The technique of coupled deconvolution has been very helpful for providing additional resolution of overlapping bands.41-44 In the deconvolution, the same parameters were used to fit pairs of associated components in the three spectra, i.e., the same number of components and the same band center and halfwidths. Figure 5 shows the results of deconvolution for the monomeric BChl c in acetone, and Table 1 summarizes the fitting parameters. Eight components were required for a satisfactory fit, which is the same number as previously reported.38 The bands 1 and 3 correspond to the Qy(0-0) and Qx(0-0) transitions of monomeric BChl c, respectively. In the CD spectrum (Figure 5b), the rotational strengths corresponding to bands 1 (Qy(0-0)) and 3 (Qx(0-0)) are negative and positive, respectively. For the monomer-rich species of BChl c in the methanol-added CH2Cl2 solution, nine components were required and the band marked by an asterisk (*) was included as the longest wavelength band for a satisfactory fit of the absorption and MCD spectra (Figure 6, Table 2). In comparison with the fitting components in Figure 5, bands 2 and 4 in Figure 6 are assigned to the Qy(0-0) and Qx(0-0) transitions of the monomeric BChl c, respectively. Band 1 with the absorption maximum at 680 nm can be assigned to the Qy(0-0) transition of the aggregated BChl c (Qy(0-0)agg). It gives rise to a very weak trough-type CD signal, and there is no dispersion-type CD signal from Qy(0-0) in the monomer-rich state of BChl c (Figure 6b). The wavelength of band 3 in the monomer-rich species is close to that of band 2 in a monomer state (Table 1). However, band 3 is the lowest energy component with a relatively strong negative MCD, while band 2 in Figure 5 shows a very weak positive MCD. Considering that Qx(0-0) and Qy(0-0) transitions should be of opposite MCD sign and Qx(0-0) is the lowest energy with negative MCD in a monomer

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Figure 5. Deconvolution of the absorption, CD, and MCD spectra of (31R)-BChl c in acetone. The concentration of BChl c was 0.05 mM.

TABLE 1: Fitting Parameters for BChl c in Acetone band

λ (nm)

a (103 L/ (mol‚cm))

∆b (cm2/ mmol)

∆Mc (cm2/ (mmol‚T))

* 1 2 3 4 5 6 7

677.0 661.2 647.1 626.7 609.2 592.7 576.2 551.2

4.56 66.1 7.03 11.3 8.08 5.49 4.28 3.51

2.18 18.6 1.04 2.35 1.47 2.08 1.11 0.56

6.20 76.6 3.83 29.7 2.25 21.7 7.32 1.31

assignment

Figure 6. Deconvolution of the absorption, CD, and MCD spectra of (31R)-BChl c in the 0.025% methanol-added CH2Cl2 solution. The concentration of BChl c was 0.05 mM.

TABLE 2: Fitting Parameters for the Monomer-Rich Species of BChl c in the 0.025 % MeOH-Added CH2Cl2 Solution band

λ (nm)

 (103 L/ (mol‚cm))

∆ (cm2/ mmol)

∆M (cm2/ (mmol‚T))

* 1 2 3 4 5 6 7 8

714.3 680.4 665.3 644.8 630.1 616.6 596.7 578.2 554.4

0.99 30.8 38.0 9.57 6.06 8.64 5.64 2.90 2.95

0.11 1.01 10.1 0.32 0.54 0.30 2.31 0.63 0.22

0.56 17.1 35.9 9.10 11.9 5.72 16.8 3.76 1.90

Qy(0-0)Md Qx(0-0)Md

a  is the extinction coefficient. b ∆ is the molar circular dichroic absorption. c ∆EM is magneto-molar circular dichroic absorption. d Qx,y(0-0)M represents the Qx,y(0-0) transition in a monomer state of BChl c.

state of BChl c,34,35,38 band 3 should be assigned as the Qx(0-0) transition (Qx(0-0)agg) from the aggregated BChl c with Qy(0-0)agg. For the neat CCl4 solution, we deconvoluted the spectra of the highly concentrated BChl c solution (2 mM) for a better comparison with the previous SANS and NMR results (Figure 7, Table 3).19-21 Ten fitting components were required for a satisfactory fitting. Bands 1 and 3 show a broad trough and sharp peak in the CD spectrum (Figure 7b), and both the bands have positive MCD (Figure 7c). Since bands 1 and 3 can constitute a dispersion-type CD signal and the MCD strength of the Qy(0-0) transition is expected to be positive, bands 1 and 3 can be assigned as two Qy(0-0) exciton components from the antiparallel BChl c dimer (Qy(0-0)D(1) and Qy(0-0)D(2), respectively).20,21 Therefore, the CD component of band 3 at 679 nm (Qy(0-0)D(2)) in neat CCl4 (Figure 7b) is essentially different from that of the Qy(0-0)agg component at 680 nm in the monomer-rich species of BChl c (the band 1 in Figure 6b).

assignment Qy(0-0)agga Qy(0-0)M Qx(0-0)agga Qx(0-0)M

a Q (0-0) x,y agg represents the Qx,y(0-0) transition for the aggregated BChl c.

The simultaneous deconvolution of the CD spectrum was very effective in identifying the band center and the half-width of band 3 in Figure 7a-c (Qy(0-0)D(2)), though the fitting parameters of band 4 are needed for a better fit of the absorption and MCD spectra. The band center of band 4 (671 nm) is comparable to the Qy(0-0) transition of monomeric BChl c (659∼670 nm),38 and the intensities of the CD and MCD are also similar to those of monomeric BChl c (see the values of ∆/ and B/D in Table 4). Therefore, band 4 may be assigned as the intrinsic Qy(0-0) of BChl c in the dimer. Because band 5 (663 nm) needs to be negative in MCD for a satisfactory deconvolution, bands 5 and 6 are the first and second lowest energy components with negative MCD, respectively (Figure 7c). They show a small minimum and maximum in the CD spectrum, the pair of which represents a dispersion-type CD signal similar to the Qy(0-0) signal (Figure 7b). The

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TABLE 3: Fitting Parameters for the 710-nm-Rich Species of BChl c in Neat CCl4 band

λ (nm)

 (103 L/ (mol‚cm))

∆ (cm2/ mmol)

∆M (cm2/ (mmol‚T))

1 2 3 4 5 6 7 8 9 10

711.6 702.6 678.8 670.6 663.3 643.2 626.7 608.6 578.0 556.5

22.1 1.71 7.55 1.65 0.34 3.35 3.27 2.55 1.22 1.65

9.63 0.71 26.3 0.49 5.82 4.30 1.72 2.91 0.33 1.12

10.5 0.47 6.05 1.86 2.38 4.78 3.30 9.66 1.00 1.31

assignment Qy(0-0)D(1)a Qy(0-0)D(2)a Qy(0-0)M Qx(0-0)D(1)a Qx(0-0)D(2)a

a

Qx,y(0-0)D represents the Qx,y(0-0) transition for the BChl c dimer with an antiparallel piggy-back conformation.

assignment of the Qx(0-0) transition to the first and second lowest energy components is consistent with the observation that the weak Qx(0-0) in Figure 4 shows a red shift to ∼655 nm by the change of solvent. In this case, bands 5 and 6 can be assigned to the Qx(0-0) transition according to the MCD results (Qx(0-0)D(1) at 663 nm and Qx(0-0)D(2) at 643 nm, respectively). Hence, the BChl c dimer with an antiparallel conformation in neat CCl4 has two exciton components for Qx(0-0) as well as Qy(0-0), and the two Qx(0-0) components also form a dispersion-type CD signal. Figure 8 shows the diagram representing the states energies for Qx and Qy transitions, according to the deconvolution results of monomer, aggregate with Qy(0-0)agg, and antiparallel dimer. The energies of both the Qx(0-0) and Qy(0-0) excited states decreases by aggregation of BChl c. One of the main advantages of the deconvolution analysis is the possibility to correlate the ratio of the MCD intensity to the absorption dipole strength (B/D) for the Qy(0-0) with the difference in energy (∆cm-1(Qx-Qy)) between Qx(0-0) and Qy(0-0). The correlation in a monomer state of BChl c has been found to be expressed with B/D ) -102 + 1.75 × 105/ ∆cm-1(Qx-Qy).38 For the Qy(0-0) transition of the monomer state in the methanol-added CH2Cl2 solution (band 2 in Table 2), the ratio of the molar circular dichroic absorption to the extinction coefficient (∆/) is close to that in acetone and the B/D value was similar to that of monomeric BChl c predicted from the ∆cm-1(Qx-Qy) (Table 4). In contrast, the B/D value

Figure 7. Deconvolution of the absorption, CD, and MCD spectra of (31R)-BChl c in neat CCl4. The concentration of BChl c was 2 mM.

for the Qy(0-0) of aggregated BChl c (Qy(0-0)agg) in the methanol-added CH2Cl2 solution was 47% of the predicted value from the energy difference. This indicates that band 1 (Qy(0-0)agg) in the methanol-added CH2Cl2 solution is originated from BChl c aggregate since the interaction among BChl molecules in an aggregate resulted in a decrease of the B/D value for Qy(0-0).38,39 The MCD property for Qy(0-0)agg together with the weak CD signal suggests that the BChl c aggregate with the Qy(0-0) component at 680 nm is a dimer with the planes of the macrocycles being not parallel.

TABLE 4: ∆E/E and B/D Values of the Qy(0-0) Transition of BChl c for the Monomer State in Acetone, the Monomer-Rich Species in the 0.025 % MeOH-Added CH2Cl2 Solution, and the 710-nm-Rich Species in Neat CCl4 wavelength (nm)

∆/ (104)

B/Dd (103β/cm-1)

∆cm-1 e (cm-1)

661.2

2.82

119.5

833

108

111

113 106

47 101

68.9 15.1 405 112 -

70 320 19 69 -

Monomer Statea Qy(0-0)M Monomer-Rich Speciesb Qy(0-0)agg Qy(0-0)M 710-nm-Rich Speciesc Qy(0-0)D(1)

680.4 665.3

0.33 2.66

53.4 107

811f 839g

711.6

4.36

48.3

Qy(0-0)D(2)

678.8

Qy(0-0)M

670.6

1024h 1495i 345j 816k -

a

34.8 2.96

77.3 108

predicted B/Dl (103β/cm-1)

ratiom (%)

This state corresponds to the BChl c in acetone (Figure 5). b This species corresponds to the BChl c in the 0.025% MeOH-added CH2Cl2 solution (Figure 6). c This species corresponds to the BChl c in neat CCl4 (Figure 7). d The MCD B-term and absorption dipole strength D were calculated from ∆Μ and  according to ref 38. e The ∆cm-1 is the difference in wavenumber between Qy(0-0) and the corresponding Qx(0-0) components. f These values are the ∆cm-1 between Qy(0-0)agg and Qx(0-0)agg in Table 2. g This value is the ∆cm-1 between Qy(0-0)M and Qx(00)M in Table 2. h This value is the ∆cm-1 between Qy(0-0)D(1) and Qx(0-0)D(1) in Table 3. i This value is the ∆cm-1 between Qy(0-0)D(1) and Qx(0-0)D(2) in Table 3. j This value is the ∆cm-1 between Qy(0-0)D(2) and Qx(0-0)D(1) in Table 3. k This value is the ∆cm-1 between Qy(0-0)D(2) and Qx(0-0)D(2) in Table 3. l The predicted B/D was calculated from the ∆cm-1 between Qy(0-0) and the corresponding Qx(0-0) as in ref 38: B/D ) -102 + 1.75 × 105/∆cm-1. m These are the ratios of the observed B/D values to those predicted from the energy difference.

3992 J. Phys. Chem. B, Vol. 106, No. 15, 2002

Figure 8. Diagram of the state energies for Qx and Qy transitions in a monomer state in acetone (Qx,y(0-0)), BChl c 680-nm aggregate in the methanol-added CH2Cl2 solution (Qx,y(0-0)agg), and antiparallel dimer in neat CCl4 (Qx,y(0-0)D(1), Qx,y(0-0)D(2)).

For BChl c in neat CCl4, the Qy(0-0) exciton components at 679 and 712 nm (Qy(0-0)D(1) and Qy(0-0)D(2)) may mix with corresponding Qx(0-0) components (Qx(0-0)D(1) and Qx(0-0)D(2)). However, comparison of the B/D values with those predicted from the energy difference shows only partial mixing between Qx(0-0)D(1) and Qy(0-0)D(2) since the MCD strength of Qy(0-0)D(2) is very large due to the small energy difference if the interaction exists (Table 4). The forbidden mixing between Qx(0-0)D(1) and Qy(0-0)D(2) supports that the planes of the BChl c macrocycles are parallel in the dimer and also indicates that there is no mixing between Qx(0-0)D(2) and Qy(0-0)D(1) (see discussion). Both of the B/D values of Qy(0-0)D(1) and Qy(0-0)D(2) were almost 70% of the predicted values from the energy difference between Qy(0-0)D(1) and Qx(0-0)D(1), and Qy(0-0)D(2) and Qx(0-0)D(2), respectively (Table 4). The similar behavior of the B/D values indicates that the B/D values for Qy(0-0)D(1) and Qy(0-0)D(2) are influenced by a similar aggregation effect and suggests that the two bands are derived from the same aggregate (the antiparallel dimer). Therefore, (31R)-BChl c predominantly forms the dimer with the antiparallel conformation in neat CCl4 at a high concentration of BChl c. Exchange of BChl c Molecules in the Antiparallel Dimer. In a previous study, we measured the absorption spectra of (31R)-BChl c in CH2Cl2 and CCl4 at various concentrations and observed that the ratio of the 680-nm absorption band to the 710-nm bands was varied greatly by the change of BChl c concentration in CH2Cl2, while the ratio was constant at various concentrations in CCl4.13 This means that some aggregate with the 680-nm absorption band is formed in CH2Cl2 besides the antiparallel dimer, and there is equilibrium between the 680nm aggregate and antiparallel dimer. Considering the CD and MCD results, the 680-nm aggregate probably corresponds to the dimer with the nonparallel planes of the macrocycles. Figure 9 shows the 5-H and 10-H proton resonances in the 1H NMR spectra of (31R)-[E,E]BChl cF in CCl4 and CD2Cl2 at the concentration of 2 mM. In CCl4, the 5-H proton shows two resonances at 6.7 and 7.7 ppm, a characteristic of the antiparallel dimer.20 The 5-H proton also resonated at 6.5 and 7.5 ppm in CD2Cl2; however, there was no 5-H proton resonance for the 680-nm aggregate. This may be due to a fast exchange for the aggregated BChl c in the 680-nm aggregate. For the 10-H resonance, split of the signal was not observed in CD2Cl2 (Figure 9b). This also may be due to the fast exchange in the antiparallel dimer in CD2Cl2. To compare the stability of the antiparallel dimer formed in CD2Cl2 and CCl4, we estimated the exchange rate between two

Umetsu et al.

Figure 9. 1H NMR spectra of (31R)-BChl c (2mM) in CCl4 (a) and CD2Cl2 (b) at a temperature of 298 K.

Figure 10. Negative-phase contour plot of the 2-D EXSY spectrum for (31R)-BChl c (2 mM) in CD2Cl2 with a mixing time of 60 ms at a temperature of 298 K.

BChl c molecules in the dimer by 2-D EXSY spectra (Figure 10). In CD2Cl2, cross-peaks due to exchange were observed for 32-CH3, 5-H, 71-H, 181-H, 201-CH3, and 31-H resonances. These proton resonances did not show cross-peaks in the EXSY spectra with a very short mixing time (τm) of 5 ms. Taking into account the structure of an antiparallel dimer,20 the results strongly indicate that the intensities of these cross-peaks are enhanced through exchange rather than either nuclear Overhauser enhancement (NOE) or J leakage. The calculated exchange rate k from a series of EXSY spectra follows the equation of k ) ln{(r + 1)/(r - 1)}/τm, where r ) (IAA + IBB)/(IAB + IBA) with the assumption that the exchange system of BChl c in CD2Cl2 can be approximated as a two-site exchange between two uncoupled systems of spin A and B.20,45 The values of ln{(r + 1)/(r - 1)} calculated from the 5-H intensities in CD2Cl2 were proportional to the τm time over 5-75 ms, while the exchange rate in CCl4 was not changed in the range of 75-400 ms (Figure 11). This indicates a much faster exchange rate in CD2Cl2 than in CCl4. Actually, the exchange rate in CD2Cl2 estimated from the least squares line in Figure 11 was about 15 times that in CCl4. The high exchange rate in the antiparallel dimer can account for the instability of the dimer in CD2Cl2 (CH2Cl2), and would result in coexistence of the transient dimer

CD Studies of Bacteriochlorophyll c Aggregates

Figure 11. Correlation of ln{(r + 1)/(r - 1)} with mixing time τm. r is (IAA + IBB)/(IAB + IBA), where IAA and IBB are the intensities of diagonal peaks and IAB and IBA are the intensities of cross-peaks. The 5-H proton resonances at 6.7 and 7.7 ppm (6.5 and 7.5 ppm in CD2Cl2) were applied for the calculation. The data for the CCl4 and CD2Cl2 solutions are plotted with filled and unfilled circles, respectively, and each of the least squares lines for the CCl4 and CD2Cl2 solutions was calculated in the range of 75-400 and 10-75 ms, respectively. k is the slope value of the least squares line.

with only a 680-nm-absorption component (680-nm aggregate) and the antiparallel dimer. Discussion In this study, we have assigned the Qx(0-0) and Qy(0-0) transitions for the (31R)-BChl c small aggregates with absorption maxima at 680 and 710 nm. The results indicate that the origin of the 680-nm component is different between the monomerrich species and the 710-nm-rich species. We proposed in this study that (31R)-BChl c forms two types of dimer with different conformation, i.e., a transient dimer (680-nm aggregate) in which the planes of the BChl c macrocycles are not parallel and a dimer with an antiparallel conformation. The transient dimer has a Qy(0-0) absorption band at 680 nm and does not show a dispersion-type CD signal, while the antiparallel dimer reveals a dispersion-type CD signal with two Qy(0-0) exciton components at 680 and 710 nm. A similar aggregation behavior has been observed for Chl a dimer.46 Chl a is well-known to form a dimer called a “T-shaped dimer” with a Qy(0-0) absorption maximum at 667 nm in CCl4, in which only one C131 keto carbonyl group in the dimer ligates on the Mg atom of the adjacent Chl a molecule so that the planes of the macrocycles are not parallel.30,47 It has been suggested in the exciton theory that the two transition moments forming a skewed orientation should give rise to an intense absorption with higher energy and a weak absorption with lower energy and these absorption bands should show very weak CD signals.48 In toluene, Chl a forms the T-shaped dimer with a Qy(0-0) absorption band at 668 nm, but the Qy(0-0) transition is redshifted to 702 nm at low temperature.46 The red shift is considered to be due to the formation of a dimer where the planes of the macrocycles are parallel.46 In the proposed Chl a dimer model with a parallel conformation, the Qy(0-0) dipole vectors form an angle of about 180° with each other, and it is in consistency with the exciton theory that there should appear two Qy(0-0) exciton components, one at 672 nm without dipole strength and another at 700 nm with the Qy(0-0) dipole strength from both monomers.46,49 In the case of the antiparallel BChl c dimer, there are two Qy(0-0) exciton components at 680 nm with relatively small dipole strength (Qy(0-0)D(2)) and at 710 nm with large dipole strength (Qy(0-0)D(1)). This may be

J. Phys. Chem. B, Vol. 106, No. 15, 2002 3993 relevant to the fact that the Qy(0-0) dipole vectors form an angle of about 150° with each other.20 Recently, formation of two dimers with different conformations has been reported for Chl a′.50 Chl a′ shows two Qy(0-0) transitions at 690 and 715 nm in aqueous methanol solution, and the presence of two types of aggregates (a T-shaped dimer and a stacked dimer with an antiparallel conformation) has been identified by principal multicomponent spectral estimation (PMSE) analysis.50 The T-shaped dimer of Chl a′ shows a broad spectrum with an absorption maximum at 670-672 nm and a tail expanding to 700 nm. The absorption bands contribute little to the CD spectrum, whereas the stacked dimer has two exciton components at ∼694 and 715 nm which constitute a dispersion-type CD signal. The aggregation behavior and CD properties of (31R)-type BChl c essentially resemble those of Chl a/a′, and this supports that (31R)-BChl c forms two different (transient and antiparallel) dimers and shows two Qy(0-0) exciton components at 680 and 710 nm by the formation of the antiparallel dimer. Assignment of the 680-nm component in the 710-nm-rich species as one of the two Qy(0-0) exciton components from the antiparallel dimer has been supported by Causgrove et al.17 From a comparison between the absorption, fluorescence excitation, and emission spectra of Mg-BChlide d, they assigned the 680-nm shoulder in the mixture of CCl4 and hexane as a higher energy level of exciton transitions from the 710-nm species. They proposed a cyclic arrangement of three antiparallel dimers for the 710-nm species of Mg-BChlide d. Our investigation by SANS and NMR provides evidence for an antiparallel dimer for (31R)-BChl c.19,20 Considering the absence of an ester long chain in Mg-BChlide d, the long side chain in BChl c might prevent the formation of a larger aggregate from the dimer. Electronic transitions of the 710-nm Mg-BChlide d species may not change from the dimer by formation of the cyclic aggregates. Although the 710-nm species has been described as highly fluorescent,17,51 the lifetime of the BChl c 710-nm species is comparable to that of the Chl a dimer with an absorption maximum at ∼700 nm (BChl c, 3.1 ns; Chl a, 3.7 ns).51-53 This similarity may support that the electronic transitions for the 710-nm species of BChl c are essentially due to an antiparallel dimer. The two Qy(0-0) exciton components (Qy(0-0)D(1) and Qy(0-0)D(2)) from the antiparallel dimer make a dispersion-type CD signal, but the rotational strength of Qy(0-0)D(2) is 1.5times greater than that of Qy(0-0)D(1). Scherz et al. calculated the spectral properties for dimers of bacteriochlorophyll and bacteriopheophytin in consideration of exciton interaction between nondegenerate states and perturbation of the ground state besides degenerate interaction.54 Their calculation results showed that the degenerate interaction gives a substantial splitting of the Qy transition and nonconservative CD signal can be rationalized by considering the nondegenerate interaction. Therefore, the difference in rotational strength between Qy(0-0)D(1) and Qy(0-0)D(2) is accounted for by exciton interaction between nondegenerate states in neighboring molecules. Warshel et al. made a quantum mechanical calculation for reaction center with quantum-mechanical-consistent-forcefield/π-electron (QCFF/PI) method at the self-consistent-field/ configuration-interaction (SCF/CI) level55,56 and suggested that charge-transfer (CT) transition can have an influence on the absorption spectrum if intermolecular distance is less than 5 Å.55 Considering the antiparallel BChl c dimer has plane-toplane distances of 3.2-3.4 Å, the CT transition might influence the dipole strength of Qy(0-0)D(1) and Qy(0-0)D(2).

3994 J. Phys. Chem. B, Vol. 106, No. 15, 2002 MCD is useful for assignment of Qx(0-0) transition and has been applied for detecting various aggregation forms of pigments.38,57,58 The MCD intensity of the Qy(0-0) transition can be expressed as a mixing between the dipole moments of Qx(0-0) and Qy(0-0) divided by the difference in energy between Qx(0-0) and Qy(0-0) transitions.22 For the monomer state of Chl and BChl, we have reported that the MCD intensity of the Qy(0-0) transition is inversely proportional to the ∆cm-1(QxQy) (where ∆cm-1(Qx-Qy) is the difference in energy between Qx(0-0) and Qy(0-0)).38,39 Mixing between the dipole moments of Qx(0-0) and Qy(0-0) tends to decrease by aggregation of Chl and BChl, and we can estimate the degree of the decrease in the mixing from comparison with the B/D value predicted from the ∆cm-1(Qx-Qy).39 For the Qy(0-0) component of the transient dimer (Qy(0-0)agg), the B/D value was about half the predicted value from the energy difference due to a decrease of the mixing quantity between the dipole moments of Qx(0-0) and Qy(0-0). This MCD result supports the formation of a dimer which has no distinct rotational strength for Qy(0-0). In the case of the antiparallel BChl c dimer, the Qy(0-0) component at 679 nm (Qy(0-0)D(2)) seems to mix little with the Qx(0-0) component at 663 nm (Qx(0-0)D(1)). Assuming that the Q transition dipole moments for a dimer (Qx,y(0-0)D(1) and Qx,y(0-0)D(2)) can be expressed as a linear combination of two Q dipole moments of monomer states, the mixing between Qx(0-0)D(1) and Qy(0-0)D(2) (Qx(0-0)D(2) and Qy(0-0)D(1)) should be forbidden in the condition that the planes of the macrocycles are parallel, since the Qx(0-0)D(1) and Qy(0-0)D(2) (Qx(0-0)D(2) and Qy(0-0)D(1)) is in a parallel direction. Hence, the fact that each of Qy(0-0) exciton components mixes only with the corresponding Qx(0-0) component suggests that the planes of the BChl c macrocycles are almost parallel in the dimer and supports our previous NMR results.20 The decrease in ratio of the B/D value is different from that for Qy(0-0)agg, whereas there is a similarity for the decrease in the ratio of the B/D values between Qy(0-0)D(1) and Qy(0-0)D(2) (Table 4). These results indicate that the mixing quantity between dipole moments is influenced by the structure of the dimer. From the EXSY spectra, we are able to estimate the exchange rate for the antiparallel dimer in solutions. An increased exchange rate in CD2Cl2 indicates that the polarity of solvent strongly influences the bonding forces that bind two BChl c molecules in the dimer. The facts that in CD2Cl2 the exchange system of the antiparallel BChl c dimer could be approximated as the two-site exchange and no 5-H proton resonance was observed for the transient BChl c dimer suggest that the transient dimer is formed in the exchange process of the antiparallel dimer. Considering the structure of the antiparallel BChl c dimer, the transient dimer is possibly T-shaped by only a ligation of the 31-hydroxyl group to Mg (Figure 12a). The T-shaped structure is supported by the very weak CD signal at 680 nm (Qy(0-0)agg) without dispersion-type from the deconvolution result (Figure 6). In the antiparallel BChl c dimer, each of the 31-CH(OH) hydroxyl groups coordinates to the Mg atom in the adjacent molecule (Figure 12b),8,9,17,20 while the transient dimer has an uncoordinating hydroxyl group (Figure 12a). The polarity of CD2Cl2 might weaken the binding force of the coordination in the antiparallel dimer and stabilize the free hydroxyl group in the T-shaped dimer by solvation. Consequently, BChl c may tend to interchange in the dimer, and form the two types of dimer (the transient and antiparallel dimers) in CD2Cl2 (CH2Cl2). The transient dimer cannot be formed in CCl4 since it is difficult to stabilize the uncoordinating hydroxyl group in the T-shaped dimer.

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Figure 12. Possible models for the 680-nm transient dimer (a) and an antiparallel dimer model with a piggy-back comformation (b). The antiparallel dimer has been determined by NMR results.20

In conclusion, the simultaneous deconvolution of absorption, CD, and MCD spectra demonstrates that the origin of the 680nm absorption component in a monomer-rich species is different from that of 710-nm-rich species. For the 680-nm dimer, a weak CD signal and the decreased B/D value for the Qy(0-0) transition lead us to a aggregation model of T-shaped conformation. In the case of highly concentrated BChl c in neat CCl4, the spectroscopic properties of Qx(0-0) and Qy(0-0) transition can be interpreted in terms of an antiparallel dimer that shows two exciton components for each of the Qx(0-0) and Qy(0-0) transitions. Further, the 2-D EXSY experiments reveal that the polarity of CD2Cl2 (CH2Cl2) destabilizes the antiparallel BChl c dimer and results in a transient dimer with a T-shape. Acknowledgment. The authors would like to express our heartfelt thanks to Prof. Huub J. M. de Groot of Leiden University for his hospitality and discussions and are grateful to Kees Erkelens, Fons Lefeber, and Johan Hollander for technical assistance with NMR measurements. This work was supported by the JSPS research fellowships for young scientists and JSPS postdoctoral fellowships for research abroad. References and Notes (1) Olson, J. M.; Gerola, P. D.; van Brakel, G. H.; Meiburg, R. F.; Vasmel, H. In Antennas and Reaction Centers of Photosynthetic Bacteria; Michel-Beyerle M. E., Ed.; Springer Series in Chemical Physics 42; Springer: Berlin, 1985; p 67. (2) Blankenship, R. E.; Olson, J. M.; Miller, M. In Anoxygenic Photosynthetic Bacteria; Blankenship, R. E., Madigan, M. T., Bauer, C. E., Eds.; Kluwer: Dordrecht, The Netherlands, 1995; p 399. (3) Staehelin, L. A.; Golecki, J. R.; Fuller, R. C.; Drew, G. Arch. Mikrobiol. 1978, 119, 269. (4) Staehelin, L. A.; Golecki, J. R.; Drew, G. Biochim. Biophys. Acta 1980, 589, 30. (5) Blankenship, R. E.; Brune, D. C.; Freeman, J. M.; Trost, J. T.; King, G. H.; McManus, J. H.; Nozawa, T.; Wittmershaus, B. P. In Green Photosynthetic Bacteria; Olson, J. M., Ormerod, J. G., Amesz, J., Stackebrandtt, E., Tru¨per, H. G., Eds.; Plenum Press: New York, 1988; p 57. (6) Mimuro, M.; Nozawa, T.; Tamai, N.; Shimada, K.; Yamazaki, I.; Lin, S.; Knox, R. S.; Wittmershaus, B. P.; Brune, D. C.; Blankenship, R. E. J. Phys. Chem. 1989, 93, 7503. (7) Uehara, K.; Olson, J. M. Photosynth. Res. 1992, 33, 251.

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