VOLUME 101, NUMBER 37, SEPTEMBER 11, 1997
© Copyright 1997 by the American Chemical Society
Light-Harvesting Physics Workshop Bristonas, Lithuania September 14-17, 1996
Organizers R. van Grondelle L. Valkunas
Excitonic Interactions between the Reaction Center and Antennae in Purple Photosynthetic Bacteria† Gabrielle M. Owen and Arnold J. Hoff* Department of Biophysics, Huygens Laboratory, Leiden UniVersity, P.O. Box 9504, 2300 RA Leiden, The Netherlands
Michael R. Jones Department of Molecular Biology and Biotechnology, UniVersity of Sheffield, Western Bank, Sheffield S10 2UH, U.K. ReceiVed: October 29, 1996; In Final Form: January 6, 1997X
The interaction between the light-harvesting antenna (LH1) and the reaction center (RC) in the photosynthetic purple bacteria Rhodobacter sphaeroides R26 and strain 2.4.1 was studied with low-temperature (1.5 K) absorbance-detected magnetic resonance (ADMR) experiments combined with computer simulations. The triplet-minus-singlet (T-S) spectra of chromatophores of Rb. sphaeroides R26 and strain 2.4.1 show an extra positive component at approximately 884 and 887 nm, respectively, and an extra negative component at 897 and 901 nm, respectively, when compared to RCs of Rb. sphaeroides R26 and strain RCO1. Computer simulations using a simple model of the RC encircled by the antenna of LH1 show that the dipole strength and energies of the second-lowest exciton component of the antenna and the RC change when the coupling term between the RC and antenna is included in the Hamiltonian of the system. Thus the difference in the shape of the ADMR-detected T-S spectra of isolated RCs (or strain RCO1 chromatophores) and chromatophores is plausibly caused by a change in the excitonic interaction between the RC and antenna.
1. Introduction The antenna system of photosynthetic purple bacteria, which is responsible for collecting and transfering light energy to the † Abbreviations: ADMR, absorbance-detected magnetic resonance; RC, reaction center; T-S, triplet-minus-singlet; P, primary donor; LH1, B875 light-harvesting complex; LH2, B800-850 light-harvesting complex; BChl, bacteriochlorophyll; EPR, electron paramagnetic resonance; LHA, lightharvesting antennae; ESR, electron spin resonance. * Author to whom correspondence should be addressed at Leiden University. Telephone, +31 71 5275955; fax, +31 71 5275819; e-mail,
[email protected]. X Abstract published in AdVance ACS Abstracts, August 1, 1997.
S1089-5647(96)03375-5 CCC: $14.00
reaction center (RC), consists of one or more of the lightharvesting (LH) complexes: the core LH1 (B875) antenna surrounding the RC and the peripheral LH2 (B800-850) antenna. Recently the crystal structure of LH1 from the purple non-sulfur bacterium Rhodospirillum rubrum was determined to a resolution of 8.5 Å, revealing a ring, with an outer and inner diameter of 116 and 68 Å respectively, of 16 Rβ-subunits.1 A ratio of 32 molecules of bacteriochlorophyll (BChl) per LH1 complex was suggested,1 which agrees well with the ratio of 31-34 molecules of BChl per LH1 complex2 found in isolated © 1997 American Chemical Society
7198 J. Phys. Chem. B, Vol. 101, No. 37, 1997
Figure 1. Energy level diagram of the singlet and triplet manifold. S0, singlet ground state; S1 and S2, singlet excited states; T0 and T1, first and second excited triplet states; SA and TA, singlet and triplet absorption, respectively; F and DF, fluorescence and delayed fluorescence, respectively; P, phosphorescence; NR, nonradiative transition; IC, internal conversion. Enlarged T0 levels: X, Y, and Z, eigenenergies of the dipole-dipole interaction; D and E, zero-field splitting parameters. Filled circles, equilibrium populations. ν1, ν2, and ν3 are the frequencies corresponding to the (|D| ( |E|)/h and 2|E|/h transitions, respectively.
core complexes from a variety of purple non-sulfur bacteria, including R. rubrum. However, others have found a ratio of approximately 24 BChl molecules per LH1 complex.3,4 This discrepancy may be due to different sample preparation methods or growing conditions. The desire to know how the light-harvesting antenna function has stimulated both experimental and theoretical interest. Experimental spectra of purple-bacterial antenna from absorbance,5 hole-burning,6 picosecond absorbance difference,7 and femtosecond transient absorption experiments8 have been interpreted with the aid of exciton calculations in circular aggregates. In an excitonically coupled system the excitation is delocalized over a number of molecules. Only two exciton levels are dipole allowed in a homogeneous circular aggregate in which the transition dipole moments are oriented at the same angle to the ring. The two spectral bands of the LH1 antenna, B875 and B896, have been assigned to these two exciton levels by hole burning6 and picosecond absorbance difference spectroscopy.7 Alternatively some explain the LH1 antenna band as an inhomogeneously broadened spectral band9-12 that results in excitation localization. It has recently been found that site inhomogeneity in LH2 has practically no influence on exciton delocalization.13 In this paper we report the results of low-temperature absorbance-detected magnetic resonance (ADMR) studies of chromatophores of Rhodobacter sphaeroides R26 and strains wild-type 2.4.1 and RCO1, and RCs of Rb. sphaeroides R26. The new RC system, Rb. sphaeroides strain RCO1 chromatophores, has recently been constructed by deleting the pufBALM genes (coding for LH1 β and R-subunits) and reintroducing the pufLM genes into this double-deletion strain in trans.14 It preserves the RC as the sole pigment-protein complex in its natural membrane. An initial characterization of the strain RCO1 has demonstrated that the reaction center complex is assembled and possesses pigments in the same orientations as in Rb. sphaeroides type 2.4.1.14 ADMR is a branch of optical detection of triplet-state magnetic resonance (ODMR), which combines optical measurement (phosphorescence, fluorescence, absorption) with electron spin resonance (ESR) spectroscopy. In ADMR, changes in optical absorbance induced by magnetic resonance are monitored.15,16 In Figure 1 an energy level diagram of the singlet
Owen et al. and triplet manifold is shown for the case in which the sample is radiated with energy 2|E|, which equalizes the populations of the X and Y levels and induces a change in optical absorbance. In contrast to conventional absorbance difference spectroscopy, where one measures all the triplet states and radicals produced by the actinic flash, only the particular pigments connected to the radiated triplet state and not the other ones present in the sample are included in the ADMR-detected absorbance difference spectrum of the triplet state minus the singlet ground state. This is achieved by radiating the sample with the energy difference between two sublevels of the desired triplet, which is often designated by the two independent parameters D and E, also known as the zero-field splitting (ZFS) or fine structure parameters. The ZFS parameters are depicted in the magnified triplet sublevels in Figure 1. The relative order of the energy levels depends on the sign of D and E. The ZFS contain information about the probe’s environment since they are sensitive to local electric fields produced by nearby charges residues and electric dipoles. They additionally tell more about the structure of the probe because they are, by definition, averages over the spatial coordinates x12, y12, z12 of the distance vector r12 between the two unpaired electrons17
D)
〈
〉
2 2 2 2 3 g β µ0 r12 - 3z12 4 4π r5
E)-
〈
12
2 2 2 2 3 g β µ0 y12 - x12 4 4π r12
〉
(1)
(2)
The parameter E is a measure of the deviation from axial symmetry about the z-axis. For a planar molecule such as chlorophyll, one would expect D to be positive (the z12 component of r12 is on the average much smaller than r12). In Table 1 the |D| and |E| parameters are given for BChl and carotenoid (Car) triplets in various bacteria and in solution. In Table 2 the |D| - |E| and |D| + |E| values for different triplets in Rb. sphaeroides 2.4.1 are compared. The |D| - |E| transition of antenna 3BChl is the only energy level that overlaps with those of the primary donor triplet, 3P. Any possible interference of the antenna 3BChl signal with the 3P signal can be avoided by measuring in the |D| + |E| transition rather than the |D| - |E| transition. In this study ADMR of the triplet state of the primary donor, 3P, has shown that the presence of LH1 in chromatophores of Rb. sphaeroides R26 and strain 2.4.1 leads to different T-S spectra than those recorded for the antenna-lacking systems (Rb. sphaeroides R26 RCs and strain RCO1 chromatophores). The change can be due neither to the antenna triplet because of its different zero-field splitting parameters nor to the antenna absorption. The interaction between the antenna and reaction center has been examined previously in ref 18 from a purely theoretical point of view with emphasis on kinetics and probabilities of energy trapping. Here we perform an exciton calculation that emphasizes absorption properties of the RCLH1 system. Our simulations indicate that there is an excitonic interaction between the RC and the second-lowest excitonic component of LH1 and are supported by experimental evidence. 2. Materials and Methods RCs of the carotenoidless mutant Rb. sphaeroides R26 were prepared as described in ref 19. Chromatophores of Rb. sphaeroides strain RCO1, also referred to as RC-only chromatophores, were prepared as in ref 14. Chromatophores of
Excitonic Interactions in Photosynthetic Bacteria
J. Phys. Chem. B, Vol. 101, No. 37, 1997 7199
TABLE 1: ZFS parameters for the BChl and Carotenoid (Car) Triplet States in Various Bacteria and in Methyltetrahydrofuran (MTHF) species
triplet
preparation
|D| (10-4 cm-1)
|E| (10-4 cm-1)
ref
RC cell chrom. RC cells cells RC chrom. cells RC cells cells
160.3 ( 0.7 156.2 ( 0.7 156.2 ( 0.7 188.0 ( 0.4 186.8 ( 0.4 185.9 ( 0.6 290 ( 5 213 ( 10.7 323 ( 10 188.8 ( 0.4 187.8 ( 0.6 180 ( 4 230 ( 2 221 ( 2
39.7 ( 0.7 37.8 ( 0.7 37.8 32.0 ( 0.4 30.8 ( 0.4 32.4 ( 0.3 44 ( 6 66 ( 3.3 33 ( 10 33.8 ( 0.4 34.3 ( 0.3 40 ( 4 58 ( 5 57 ( 2
43 43 43 24 44 24 45 23 45 46 24 45 47 47
3
Rps. Viridis
P P 3P 3 P 3 P 3 P RC 3Car antenna 3BChl antenna 3Car 3P 3 P RC 3Car 3
Rb. sphaeroides R26 Rb. sphaeroides 2.4.1
R. rubrum S1 BChl a in MTHF BChl b in MTHF
TABLE 2: |D| - |E| and |D| + |E| Values for Various Triplets in Rb. sphaeroides 2.4.1 triplet
|D| - |E| (10-4 cm-1)
|D| + |E| (10-4 cm-1)
3 P antenna 3 BChl 3Car antenna 3 Car
153.5 ( 0.9 147 ( 14.0 246 ( 11 290 ( 20
218.3 ( 0.9 279 ( 14.0 334 ( 11 356 ( 20
Rb. sphaeroides R26 and type 2.4.1 were obtained by sonication of the cells for 10 min, followed by centrifugation and suspension in a buffer containing 10 mM Tris (pH 8.0). Glycerol (60-66% v/v) was added to prevent cracking upon freezing. The first acceptor quinone in the RC was reduced by adding excess sodium ascorbate and illuminating with continuous white light during cooling. All measurements were carried out at approximately 1.5 K to impede spin-lattice relaxation. The ADMR setup used for measurements was as described in ref 20 with the following adjustments. ADMR-detected triplet-minus-singlet (T-S) measurements were done using a helix connected to the microwave source with an output power of 17 dBm. The entrance and exit slits of the monochromator (Jobin-Yvon, 1200 lines/mm, blaze 750 nm) were both set at 1 mm. T-S spectra were obtained by monitoring the change in absorbance at a fixed microwave frequency while scanning the wavelength of detection. 3. Results In Figure 2 ADMR-detected triplet-minus-singlet (T-S) spectra of all samples are shown detected at various frequencies within their |D| - |E| transition bands. As the detection frequency is increased through the |D| - |E| band, the primary donor (P) band shifts to lower wavelengths. Similar behavior is seen in the |D| + |E| transition (Figure 3). The shifts with different applied microwave frequencies have been previously discussed in ref 21. In the |D| - |E| transition of isolated RCs and RC-only chromatophores (Figure 2A,B), the smooth shapes of the P bands are relatively constant with applied microwave frequency, but in the |D| + |E| transition (Figure 3A,B), they have a more visible long-wavelength shoulder, which decreases with increasing microwave frequency. The T-S spectra of chromatophores of R26 and strain 2.4.1 show the longwavelength shoulder in addition to two maxima, that is, one maximum and a shoulder in both the |D| - |E| (Figure 2C,D) and |D| + |E| transitions (Figure 3C,D). The T-S spectrum of R26 RCs could be recorded at higher microwave frequencies than the other samples because of its broader ADMR line width (not shown). Difference spectra of the T-S spectra of chromatophores and RCs reveal additional bands in the T-S spectra of chromatophores. Subtraction of the T-S spectrum at 655 MHz of RC-
Figure 2. T-S spectra at various applied microwave frequencies in the |D| - |E| transition for (A) Rb. sphaeroides R26 RCs, (B) Rb. sphaeroides RC-only chromatophores, (C) Rb. sphaeroides R26 chromatophores, and (D) Rb. sphaeroides type 2.4.1 chromatophores (all spectra normalized at maxima). In (A) the applied microwave frequencies are 455 (solid line), 465 (dotted line), 475 (dashed line), 485 (longdashed line), and 490 MHz (dot-dashed line). In (B)-(D) the applied microwave frequencies are 455 (solid line), 460 (dotted line), 465 (dashed line), 470 (long-dashed line), and 475 MHz (dot-dashed line).
only chromatophores from that of strain 2.4.1 chromatophores, with normalized areas between 860 and 930 nm, leads to two positive and one negative bands located at roughly 887, 919, and 901 nm respectively (Figure 4A). Subtraction of the T-S spectrum at 660 MHz of R26 RCs from the T-S spectrum of R26 chromatophores, with normalized areas between 860 and 930 nm, leads to two positive bands and one negative band
7200 J. Phys. Chem. B, Vol. 101, No. 37, 1997
Owen et al.
Figure 4. Difference (dotted line) between the P band of T-S spectra of (A) Rb. sphaeroides type 2.4.1 (dashed line) and Rb. sphaeroides strain RCO1 (solid line) chromatophores and (B) Rb. sphaeroides R26 chromatophores (dashed line) and RCs (solid line). Spectra were normalized by area between 10 753 and 11 628 cm-1 (860 and 930 nm) before subtraction.
between the transmitted light with the microwaves on and off, ∆ITr(λ), is given by
∆ITr(λ) ) Iexc exp[-{A(λ) + D(λ)}] × Figure 3. T-S spectra at various applied microwave frequencies in the |D| + |E| transition for (A) Rb. sphaeroides R26 RCs, (B) Rb. sphaeroides RC-only chromatophores, (C) Rb. sphaeroides R26 chromatophores, and (D) Rb. sphaeroides type 2.4.1 chromatophores (all spectra normalized at maxima). The applied microwave frequencies are 650 MHz (solid line), 655 MHz (dotted line), 660 MHz (dashed line), 665 MHz (long-dashed line), and 670 MHz (dot-dashed line).
located at roughly 884, 910, and 897 nm, respectively (Figure 4B). The frequencies 655 and 660 MHz were chosen because they were at the center of the ADMR transition band and thus had the best signal-to-noise. The |D| + |E| transition was chosen instead of the |D| - |E| transition to avoid interference from any BChl antenna triplet signal. 4. Discussion 4.1. Spectral Shape. The shape of the |D| + |E| T-S spectra of R26 and type 2.4.1 chromatophores is affected by singlet ground states that are correlated with the presence of 3 P and not by absorption from antenna or the BChl antenna triplet. In ref 22 the intensity of the light-transmitted ITr(λ) with no applied microwaves is given as
ITr(λ) ) Iexc[-{A(λ) + D(λ)}]
(3)
where A(λ) ) CS(λ)[S0] + CT(λ)[T0]+TCS(λ)[TS0], Ci are constants accounting for instrumental characteristics and RC concentration, D(λ) is the contribution of pigments unreactive to the presence of the triplet state, square brackets denote the fractional concentration; and TS0 represents those singlet ground states that are correlated with the presence of the triplet state 3P, such as new levels, shifted levels, etc. The difference
(1 - exp[-[∆S0]{CS(λ) - CT(λ) - TCS(λ)}]) (4) under the assumptions that the sum of the fractional concentrations of the singlet and triplet ground state is unity and [TS0] is equal to [T0]. Thus, in the recorded signal (∆ITr(λ)/ITr(λ)), there is no term due to D(λ), pigments unreactive to the presence of the triplet state, but indeed the term TCS(λ). The triplet states of antenna BChl, antenna carotenoids, and RC carotenoids do not influence the signals measured by radiating the sample in the |D| + |E| transition of 3P. The triplet state of the antenna BChl does definitely not contribute to the signal in the |D| + |E| transition because its |D| + |E| value is significantly different from that of 3P (Tables 1 and 2). The |D| + |E| and |D| - |E| parameters of the antenna BChl triplet state in chromatophores of Rb. sphaeroides 2.4.1 have been measured by FDMR to be (147 ( 14.0) × 10-4 cm-1 and (279 ( 14.0) × 10-4 cm-1,23 while the |D| + |E| and |D| - |E| values measured for 3P in Rb. sphaeroides 2.4.1 whole cells were (218.3 ( 0.9) × 10-4 cm-1 and (153.5 ( 0.9) × 10-4 cm-1.24 The |D| - |E| value of the antenna BChl triplet may be close to that of 3P, but the |D| + |E| values of the two triplets differ greatly. Reaction center and antenna carotenoids can also not be responsible for the signal since neither their |D| - |E| nor their |D| - |E| values overlap with the primary donor’s values, as shown in Tables 1 and 2, and furthermore, carotenoids do not absorb between 800 and 900 nm. No influence of antenna BChl or carotenoids is expected in the |D| + |E| transition of Rb. sphaeroides R26 since its ZFS values are very close to those of Rb. sphaeroides 2.4.1. 4.2. Excitonic Interactions. The possibility of excitonic interactions between the antenna pigments and the RC was
Excitonic Interactions in Photosynthetic Bacteria
J. Phys. Chem. B, Vol. 101, No. 37, 1997 7201 The xy-plane component of the transition dipole moment of each pigment is at an angle φ from its position vector and has a magnitude d|. The z-component of the antenna pigments transition dipole moment has a magnitude d⊥. The transition dipole moment unit vector µˆ n and unit position vector rˆn for antenna pigments n are
|
d| cos(nθ + φ) µˆ n ) d| sin(nθ + φ) d⊥
|
| |
cos(nθ) rˆn ) sin(nθ) 0
(9)
where d2| + d2⊥ ) 1. The unit position vector from antennapigments m to n is
rˆmn ) rˆn - rˆm )
|
( (
) )
(m + n) θ 2 (m + n) θ cos 2 0
- sin
|
(10)
The position vector between the RC and an antenna pigment n includes the vertical displacement between the LHA ring and the RC with the angle δ
Figure 5. Diagram of the model of the RC and antenna pigments used in exciton calculations. The RC is represented as a single dipole surrounded by a symmetric ring of N identical light-harvesting antenna pigments equally spaced at intervals of angle θ. Angle R is between RC and X-axis. Angle φ is between antenna position vector rn and antenna pigment. Angle δ is the arctangent of the antenna pigment’s component perpendicular to the XY-plane (d⊥) divided by component parallel to XY-plane (d|).
investigated using a Hamiltonian
Hˆ )
∑n |n〉 En 〈n| + n*m ∑ |m〉 Vmn 〈n|
(5)
which is the sum of a term containing the diagonal energies of the independent molecules and a term containing the couplings Vmn. In the case of point dipoles the interaction Vmn is equal to
Vmn ) Vnm )
5.04κmn|µm||µn| η2R3mn
(6)
where the dipole magnitudes |µm|, |µn| are given in D, the distance Rmn between the two dipoles is given in nm, and Vmn is given in cm-1, and η is the ratio of the permittivity to that in free space. The orientation factor κmn for a dipole-dipole interaction is
κmn ) (µˆ m ‚ µˆ n) - 3(µˆ m ‚ rˆmn)(µˆ n ‚ rˆmn)
(7)
where ˆ denotes unit vectors. The system of interacting molecules is taken here to be a single dipole representing the special pair of the RC, which is surrounded by a symmetric ring of N identical light-harvesting antenna (LHA) pigments equally spaced at intervals of θ ) 2π/ N, as shown in Figure 5. The RC is in the origin of the coordinate system and has its transition dipole moment oriented parallel to the xy-plane of the ring at an angle R from the x-axis. The transition dipole moment of the RC µˆ RC is
| |
cos R µˆ RC ) sin R 0
(8)
|
cos δ cos(nθ) rˆrca ) cos δ sin(nθ) sin δ
|
(11)
From eqs 7-9 and 11 the orientation factor κrca between the RC and antenna pigment n is found to be
κrca ) d| cos(nθ + φ - R) 3 cos δ cos(nθ - R)(d| cos δ cos φ + 2d⊥ sin δ) (12) From eqs 7, 9, and 10 the orientation factor κmn between antenna pigments m and n is found to be
κmn )
d2| [- cos(m - n)θ + 3 cos(2φ)] + d2⊥ 2
(13)
The interaction energies can now be found using eq 6 since κrca and κmn are known. The new values of the pigment energies can be determined by various matrix methods, including factorization into Hˆ ) Q C Λ C Q C T, with the orthonormal C and the eigenvectors Qk ) (Q1k, ..., QNk) in the k columns of Q eigenvalues along the diagonal of Λ C . The transition dipole moments νk of the eigenstates of the coupled system follow from the transition dipole moment vectors µn of the individual pigments and the eigenvector components Qnk as follows
νk )
∑n Qnk µn
(14)
A program was written in Matlab to simulate the interaction between the RC and the antenna pigments, using the variables described above. The inputs to the program were the number N of LHA, the angles φ, R, δ, the zero-point energies E°ant of the antenna pigments, the zero-point energy of the RC E°RC, d|, η, |µ|2, and |µRC|2. The orientation factors κrca and κmn were calculated using eqs 12 and 13. Subsequently the interaction energies were calculated using eq 6, using all antenna-antenna interactions and all interactions between the RC and the antenna pigments. The eigenvalues and eigenvectors of Hˆ were found through factorization into a Q C Λ C Q C T form. The exciton dipole strengths of the transitions were derived using eq 14. The starting parameters were chosen from published experimental results. The total number of BChls in the circular
7202 J. Phys. Chem. B, Vol. 101, No. 37, 1997
Owen et al.
aggregate was chosen to be 32,1,2 and the basic unit of LH1 was taken to be single BChl units with a transition dipole moment of 41 D2.25 The radius of the LH1 ring was taken within the range of values for purple bacteria: in the crystallographic study of ref 1 4.6 nm was measured for Rhodospirillum rubrum (average of outer and inner radii), and in electron microscopy studies 6.5 nm for Rs. rubrum,26 5.1 ( 0.15 nm for Rhodopseudomonas marina,27 and 5.35-6.6 nm for Rhodospirillum molischianum28 were measured. The angle φ was chosen to be π/2 or 7π/12, approximately analogous to the position of nine of the BChl molecules in LH2.29 Both the transition dipole moments of the RC as well as the LHA pigments were taken to be approximately parallel to the xyplane of the ring (d⊥ , 1), since the crystal structure of the RC,30 linear dichroism measurements,31 and polarized fluorescence studies32 have shown that P870, B875, and B896 have their Qy transition moments oriented approximately parallel to the membrane plane. The effect of the small out-of-plane component suggested by CD measurements32 is shown in the simulations. The Qy BChl a transition (E°ant was chosen to be the value measured in protein (12 500 cm-1). The value for η was taken to be equal to 1, as in ref 8, since it is not so well defined on the atomic level. All antenna-antenna interactions were included in the simulations. The primary donor was treated as a single entity with dipolar strength 82 D2 (twice the monomeric strength), rather than as two BChl molecules excitonically coupled through dipole-dipole interactions, because of the close proximity of its BChls. The interactions of P with the other accessory BChls in the RC were neglected. The input parameters were further constrained by the absorption energies (883 and 889 nm for B875 of R26 and strain 2.4.1 chromatophores at 6 K, respectively,33 and 896 and 898 nm for B896 of R26 at 4 K32 and strain 2.4.1 chromatophores at 77 K, respectively34). The P band in the T-S spectra was considered to be only from singlet-state absorbance. In a T-S spectrum of R26 RCs, the bleaching at 890 nm is due to the disappearance of the redshifted long-wavelength band of P, whose shift is partly due to the excitonic coupling between the two BChls of P. Exciton coupling is much weaker in the triplet state of 3 P; therefore, in a localized 3P state, part of the BChl absorption is bleached and part shifts back to the zero-point wavelength of BChl absorption in a protein matrix.15 A simulation in which only nearest-neighbor antennaantenna interactions were considered (no antenna-RC interactions) and the antenna pigments were assumed to be homogeneous was done to test that the computer program was working correctly. The eigenvalues of the eigenstates of the nearestneighbor coupled antenna system agreed with the theoretical values for the energies of the kth exciton level of the antenna ring
Ek ) E°ant + 2V cos(kθ)
(15)
where V is the nearest-neighbor interaction and E°ant is the zeropoint energy. Equation 15 can be derived from eqs 5, 6, and 13. The ratio of the dipole strengths of the k ) 0 and k ) (1 states agreed with those derived from equation 4 of ref 35, and only the k ) 0, k ) (1, and RC states had oscillator strength as in ref 35. The program was run using the parameters given in Table 3 and then run again setting the interaction between the RC and the antenna pigments (Vrca) to zero. The resulting energies and dipole strengths for simulations 1-3 are shown in Tables 4-6, respectively. The input parameters for simulations 1 and 2 differ only in the magnitude of the parallel (d|) and perpendicular (d⊥)
TABLE 3: Input Parameters for Exciton Calculations parameter
simulation 1
simulation 2
simulation 3
N φ R δ Rnm (nm) E°ant (cm-1) E°RC (cm-1) d| |µant|2 (D2) |µRC|2 (D2)
32 π/2 0 0 4.6 12 500 11 110 (0.99)1/2 41 82
32 π/2 0 0 4.6 12 500 11 110 (0.96)1/2 41 82
32 7π/12 0 0 4.6 12 500 11 100 (0.99)1/2 41 82
TABLE 4: Simulation 1, Wavelength (λ) in nm and Dipole Strength in D2, O ) π/2, d| ) (0.99)1/2, V ) -552.5 cm-1 k
λ
λ (Vrca ) 0)
|µk|2
|µk|2 (Vrca ) 0)
RC 0 (1 (1
900.3 895.0 889.8 889.6
900.1 895.0 889.8 889.8
32.7 13.1 649.4 698.8
82.0 13.1 649.4 649.4
TABLE 5: Simulation 2, Wavelength (λ) in nm and Dipole Strength in D2, O ) π/2, d| ) (0.96)1/2, V ) -527.2 cm-1 k
λ
λ (Vrca ) 0)
|µk|2
|µk|2 (Vrca ) 0)
RC 0 (1 (1
900.2 890.2 885.2 885.1
900.1 890.2 885.2 885.2
46.3 52.5 629.8 665.5
82.0 52.5 629.8 629.8
TABLE 6: Simulation 3, Wavelength (λ) in nm and Dipole Strength in D2, O ) 7π/12, d| ) (0.99)1/2, V ) -496.4 cm-1 k
λ
λ (Vrca ) 0)
|µk|2
|µk|2 (Vrca ) 0)
RC 0 (1 (1
900.2 884.3 879.8 879.7
900.1 884.3 879.8 879.8
60.9 13.1 649.4 670.6
82.0 13.1 649.4 649.4
components of the antenna pigments. The input parameters of simulations 1 and 3 differ only in the angle (φ) between the antenna dipoles and their position vector, that is, the acuteness of the angle that the antenna pigments make with the ring. These simulations were performed since the exact values of both the angle φ and the magnitude of the out-of-plane antenna pigment component are not yet known. The magnitude of Vrca was less than 4.2 cm-1 in simulations 1 and 2 and less than 4.6 cm-1 in simulation 3. The low coupling strengths agree with the slow trapping rate of excitations from the antenna pigments to the RC.36,37 In all simulations the energies and dipole strengths of all the k states except one of the k ) (1 states and the RC state are unchanged by the interaction Vrca, and all states have zero dipole strength except the k ) 0, k ) (1, and RC. Additionally the energies of the RC and the k ) (1 state change less than 1 nm when Vrca is changed to zero. The oscillator strength of the RC increases and that of the k ) (1 state decreases when Vrca is changed to zero.38 The change in RC oscillator strength is sensitive to the orientation of the antenna pigments (d|, d⊥, φ). Between simulation 1 and 2 the ratio of d⊥ to d| is increased from 0.1005 to 0.2041, and the RC oscillator strength of the coupled RCantenna system (Vrca * 0) increases from 32.7 to 46.3 D2. Between simulation 1 and 3 the angle φ is increased from π/2 to 7π/12, and the RC oscillator strength of the coupled RCantenna system (Vrca * 0) increases from 32.7 to 60.9 D2. The antenna pigments’ energies decrease between simulations 1 and 2 (from roughly 890 to 885 nm for the k ) (1 state) and simulations 2 and 3 (from roughly 890 to 880 nm for the k ) (1 state), but these can be adjusted by changing the radius R or the zero-point energy E°ant More precise calculations cannot be done until more structural information about LH1 is known.
Excitonic Interactions in Photosynthetic Bacteria The simulations have also been done including a Gaussian distribution of the site energies of the antenna pigments (inhomogeneous broadening was taken between 80 and 250 cm-1). The inhomogeneity causes all exciton levels of the antenna to have oscillator strength and results in a distribution to be seen around a central wavelength. The dipole strength of levels other than the |k| ) 1 and RC may change when setting Vrca to zero. These changes are in most cases negligible (less than a few D2) but could become significant for the k ) 0 level, depending on the amount of inhomogeneous broadening and the radius of the LH1 circle. Despite the differences the same general effect is still seen: the RC’s dipole strength decreases upon introducing RC coupling, and now both |k| ) 1 levels increase upon coupling. Thus dressing the optical stick spectra with a Gaussian band shape still is a good approximation. 4.3. T-S Spectra of RCs and Chromatophores. The difference in the shape of the T-S spectra of RCs and chromatophores are well explained by the simulation results in Tables 4-6. In the difference of the T-S spectra of chromatophores and RCs (Figure 4), an decrease is seen at 884 and 887 nm for R26 and strain 2.4.1, respectively, which corresponds well with the previously measured values for B875 (883 and 889 nm for R26 and strain 2.4.1 chromatophores, respectively, at 6 K33). The absorption of B875 has been shown by hole burning to correspond with the second-lowest exciton component state,6 and in Tables 4-6 a decrease was seen in the secondlowest exciton component when going from the RC-coupled to RC-uncoupled system. In the difference of the T-S spectra in Figure 4, an increase was located at 897 nm (R26) and 901 nm (strain 2.4.1), which corresponds well with the increase in RC intensity seen in Tables 4-6 when going from RC-coupled to RC-uncoupled system. In Figure 4 a band appears near the low-energy shoulder of P in the difference of the T-S spectra of RCs (or RC-only chromatophores) and chromatophores. The low-energy shoulder of both the primary donors of Rb. sphaeroides (P870) and Rhodopseudomonas Viridis (P960) absorption profiles has been shown to be intrinsic to the RC39 and to correspond with the origin band (ω0sp) of the marker mode,40 an intermolecular BChl vibrational mode of P. The theory of Hayes and Small,41 embellished with the marker mode progression,39 has been used to fit low-temperature absorption spectra of P870 and P960, even though they have different low-energy shoulders. It is possible that one of the parameters in the absorption spectrum theory of ref 39, such as the phonon frequency associated with the primary donor, changes when the RC is isolated or its antenna are removed, which may lead to the low-energy band in Figure 4. In all of the samples the shoulder in the T-S spectra is most prominent when the T-S peak position is shifted to longer wavelengths, which coincides with lower applied microwave frequencies in the transition band (Figures 2 and 3). The shoulder is present in the T-S spectra of all samples in both the |D| - |E| and |D| + |E| transition, revealed with the second derivative, but is weakest in the T-S spectra of the |D| - |E| transition of isolated RCs and RC-only chromatophores. At longer wavelengths the selected P molecules are closer together (their exciton interaction is stronger) and trapped in the most harmonic and uniform P intermolecular potential well. Consequently, a shoulder in the T-S spectrum may be visible because of less dispersion of the vibrational frequency associated with motion.42 5. Conclusions The triplet-minus-singlet (T-S) spectra of chromatophores of Rb. sphaeroides R26 and strain 2.4.1 measured with low-
J. Phys. Chem. B, Vol. 101, No. 37, 1997 7203 temperature ADMR show an extra positive component at 884 and 887 nm, respectively, and an extra negative component at 897 and 901 nm, respectively, when compared to RCs of Rb. sphaeroides R26 and strain RCO1. The difference in the shape of the ADMR detected T-S spectra of isolated RCs (or RConly chromatophores) and chromatophores has been shown via computer simulations using a simple model of the RC encircled by the antenna of LH1 to plausibly be a change in the excitonic interaction between the RC and antenna. Further experiments are currently being done to determine the oscillator strength in chromatophores and RCs. Acknowledgment. This work was supported by the Netherlands’ Foundation for Chemical Research (SON), financed by the Netherlands’ Organization for Scientific Research (NWO). We are grateful to S. Jansen for preparation of isolated RCs and R26 and 2.4.1 chromatophores. M.R.J. is a Biotechnology and Biological Sciences Research Council Senior Research Fellow. References and Notes (1) Karrasch, S.; Bullough, P. A.; Ghosh, R. EMBO J. 1995, 14, 631. (2) Gall, A. Doctoral Thesis, University of Glasgow, U. K., 1994. (3) Zuber, H.; Brunisholz, B. A. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton, FL, 1991; p 627. (4) Francke, C.; Amesz, J. Photosynth. Res. 1995, 46, 347. (5) Pearlstein, R. M.; Zuber, H. In Antennas and Reaction Centers of Photosynthetic Bacteria; Michel-Beyerle, M. E., Ed.; Springer-Verlag: Berlin, 1985; p 53. (6) Reddy, N. R. S.; Picorel, R.; Small, G. J. J. Phys. Chem. 1992, 96, 6458. (7) Novoderezhkin, V. I.; Razjivin, A. P. FEBS Lett. 1993, 330, 5. (8) Pullerits, T.; Chachisvilis, M.; Sundstro¨m, V. J. Phys. Chem. 1996, 100, 10787. (9) Pullerits, T.; Freiberg, A. Chem. Phys. 1991, 149, 409. (10) Visschers, R. W.; Van Mourik, F.; Monshouwer, R.; Van Grondelle, R. Biochim. Biophys. Acta 1993, 1141, 238. (11) Mourik, F. Van; Visscher, K. J.; Mulder, J. M.; Van Grondelle, R. Photochem. Photobiol. 1993, 57, 19. (12) Timpmann, K.; Freiberg, A.; Godik, V. I. Chem. Phys. Lett. 1991, 182, 617. (13) Dracheva, T. V.; Novoderezhkin, V. I.; Rajivin, A. P. FEBS Lett. 1996, 387, 81. (14) Jones, M. R.; Visschers, R. W.; Van Grondelle, R.; Hunter, C. N. Biochemistry 1992, 31, 4458. (15) Hoff, A. J. In AdVanced EPR: Applications in Biology and Biochemistry/; Hoff, A. J., Ed.; Elsevier: Amsterdam, 1989; p 633. (16) Maki, A. H. In Biological Magnetic Resonance; Berliner, L. J.; Reuben, J., Eds.; Plenum Press: New York, 1984; p 187. (17) Weil, J. A.; Bolton, J. R.; Wertz, J. E. Electron Paramagnetic Resonance: Elementary Theory and Practical Applications; John Wiley and Sons, Inc.: New York, 1994. (18) Novoderezhkin, V. I.; Razjivin, A. P. Photosynth. Res. 1994, 42, 9. (19) Feher, G.; Okamura, M. Y. In The Photosynthetic Bacteria; Clayton, R. K., Sistrom, W. R., Eds.; Plenum Press: New York, 1978; p 349. (20) Van der Vos, R.; Hoff, A. J. Appl. Magn. Reson. 1991, 2, 179. (21) Den Blanken, H. J.; Hoff, A. J. Chem. Phys. Lett. 1983, 98, 255. (22) Den Blanken, H. J.; Hoff, A. J. Biochim. Biophys. Acta 1982, 681, 365. (23) Angerhofer, A. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton, FL, 1991; p 966. (24) Hoff, A. J.; De Vries, H. G. Biochim. Biophys. Acta 1978, 503, 94. (25) Scherz, A.; Parson, W. W. Biochim. Biophys. Acta 1984, 766, 666. (26) Golubok, A. O.; Vinogradova, S. A.; Tipisev, S. Y.; Borisov, A. Yu.; Taisova, A. S.; Kolomytkin, O. V. Ultramicroscopy 1992, 42-44, 1228. (27) Meckenstock, R. U.; Krusche, K.; Brunisholz, R. A.; Zuber, H. FEBS Lett. 1992, 311, 135. (28) Boonstra, A. F.; Germeroth, L.; Boekma, E. J. Biochim. Biophys. Acta 1994, 1184, 227. (29) McDermott, G.; Prince, S. M.; Freer, A. A.; HawthornthwaiteLawless, A. M.; Papiz, M. Z.; Cogdell, R. J.; Isaacs, N. W. Nature 1995, 374, 517. (30) Allen, J. P.; Feher, G.; Yeates, T. O.; Komiya, H.; Rees, D. C. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5730.
7204 J. Phys. Chem. B, Vol. 101, No. 37, 1997 (31) Vermeglio, A.; Clayton, R. K. Biochim. Biophys. Acta 1976, 449, 500. (32) Kramer, H. J. M.; Pennoyer, J. D.; Van Grondelle, R.; Westerhuis, W. H. J.; Niederman, R. A.; Amesz, J. Biochim. Biophys. Acta 1984, 767, 335. (33) Otte, S. C. M.; Kleinherenbrink, F. A. M.; Amesz, J. Biochim. Biophys. Acta 1993, 1143, 84. (34) Shimada, K.; Mimuro, M.; Tamai, N.; Yamazaki, I. Biochim. Biophys. Acta 1989, 975, 72. (35) Novoderezhkin, V. I.; Razjivin, A. P. Biophys. J. 1995, 68, 1089. (36) Beekman, L. M. P.; Van Mourik, F.; Jones, M. R.; Visser, H. M.; Hunter, C. N.; Van Grondelle, R. Biochemistry 1994, 33, 3143. (37) Timpmann, K.; Freiberg, A.; Sundstro¨m, V. Chem. Phys. 1995, 194, 275. (38) There are some indications that the perfect ring structure seen in the electrograph pictures of reconstituted LH1 preparations1 in chromatophores is broken by the so called pufX gene product, which might function as a tunnel through which QBH2 can leave the RC. We have checked the effect of incomplete ring structure by calculating exciton spectra for partical rings of 8 or 16 monomers. Moving this ring segment around the RC, we still see the same effect of decreasing RC oscillator strength and correspondingly increasing antenna intensity as for complete rings, but the
Owen et al. intensity changes depend on size and position of the ring segment. The differences, however, were not very large, making it difficult to draw conclusions regarding the validity of the partial ring concept and the position of the purported ring-breaking pufX product. (39) Tang, D.; Johnson, S. G.; Jankowiak, R.; Hayes, J. M.; Small, G. J. In PerspectiVes in Photosynthesis; Jortner, J., Pullman, B., Eds., Kluwer Academic Publishers: Dordrecht, 1990; p 99. (40) Reddy, N. R. S.; Lyle, P. A.; Small, G. J. Photosynth. Res. 1992, 31, 167. (41) Hayes, J. M.; Small, G. J. J. Phys. Chem. 1986, 90, 4928. (42) Won, Y.; Friesner, R. A. In The Photosynthetic Bacterial Reaction Center: Structure and Dynamics; Breton, J. Vermeglio, A., Eds.; Plenum Press: New York, 1988; p 341. (43) Den Blanken, H. J.; Jongenelis, A. P. J. M.; Hoff, A. J. Biochim. Biophys. Acta 1983, 725, 472. (44) Den Blanken, H. J.; Van der Zwet, G. P.; Hoff, A. J. Biochim. Biophys. Acta 1982, 681, 375. (45) Frank, H. A.; Bolt, J. D.; De Costa, B.; Sauer, K. J. Am. Chem. Soc. 1980, 102, 4893. (46) Hoff, A. J.; Den Blanken, H. J.; Vasmel, H.; Meiburg, R. F. Biochim. Biophys. Acta 1985, 806, 389. (47) Den Blanken, H. J.; Hoff, A. J. Chem. Phys. Lett. 1983, 96, 343.
