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Understanding the Spectroscopic Properties of the Photosynthetic Reaction Center of Rhodobacter sphaeroides by a Combined Theoretical Study of Absorption and Circular Dichroism Spectra Yanliang Ren,† Wei Ke,† Yongjian Li,† Lingling Feng,† Jian Wan,*,† and Xin Xu*,‡ Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education and Department of Chemistry, Central China Normal UniVersity, Wuhan 430079, China, and State Key Laboratory for Physical Chemistry of Solid Surfaces, College for Chemistry and Chemical Engineering, Center for Theoretical Chemistry, Xiamen UniVersity, Xiamen 361005, China ReceiVed: April 13, 2009; ReVised Manuscript ReceiVed: May 23, 2009
In the present study, we calculate eight low-lying (1.3-1.7 eV energy region) electronic excited states in well accordance with the absorption and CD spectroscopic properties of PSRC from Rb. shpaeroides by using time-dependent density functional theory (TDDFT). Our present calculations demonstrate that, only when the interactions among the prosthetic groups have been taken into account, a set of satisfactory assignments for both absorption and CD spectra of PSRC from Rb. sphaeroides can be achieved simultaneously. Photosynthesis in green plants, algae, and many species of bacteria is one of the most important biochemical processes on earth. Although photosynthesis can occur in different ways in different species, some features are common. For example, the process always begins when energy from light is absorbed by hydrophobic pigment-protein complexes called photosynthetic reaction centers (PSRCs), in which transmembrane charge separation and stabilization processes occur, driving all the subsequent chemistry of photosynthesis. The purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides is often used as a model system for studies of PSRCs.1,2 X-ray crystallographic study of the reaction centers from Rb. sphaeroides3 has shown the detailed organization of the prosthetic groups (four bacteriochlorophylls a, two bacteriopheophytins a, two ubiquinones, and one nonheme iron) anchored to the two poplypeptide L and M subunits. Two subunits as well as the prosthetic groups are organized with an approximately macroscopic C2 symmetry. Two bacteriopheophytins a (HL and HM) are located on either side of a “special pair” (denoted P or PL and PM) which is a dimer of bacteriochlorophylls a, and two additional bacteriochlorophylls a (BL and BM) are positioned in between HL or HM and P (see Figure 1 for a reduced model). Excitation of the primary electron donor P to its lowest excited singlet state P* elicits electron transfer exclusively down the L-side of the native pigment-protein complex,4 in spite of the symmetrical arrangement of the L and M sides. This highly directional electron transfer has been reported by the experimental studies,5,6 but the underlying mechanism of the electron transfer is still not exposed completely. The possible explanations range from electronic factors to geometric factors such as a difference in the redox potentials of the cofactors,7,8 charge * To whom correspondence should be addressed. Phone: +86-2767862022. Fax: +86-27-67862022. E-mail:
[email protected] (J.W.);
[email protected] (X.X.). † Central China Normal University. ‡ Xiamen University.
10.1021/jp903386z CCC: $40.75
Figure 1. Schematic of a reduced chromophores cluster model of RC from Rb. sphaeroides.
separation favored by large electrostatic field along the L-branch,9,10 and asymmetric distances between chromophores, B and H,11 etc. To make clear the elementary processes of electron transfer in PSRCs, knowledge of the electronic ground states and excited states of the prosthetic groups in PSRCs is a prerequisite. Reed and co-workers did a thorough spectroscopic study12 and reported detailed absorption and circular dichroism (CD) spectra (see Figure 2a and c) for PSRC from Rb. sphaeroides. This sets up a solid experimental basis with which theoretical results can be compared to challenge the theoretical model and methodology employed, while the theoretical results, in turn, provide a sophisticated understanding of the experimentally observed spectroscopic properties of PSRCs. Several theoretical studies11,13-19 have been carried out to make qualitative or quantitative assignments of the experimental spectroscopic properties of PSRCs. Zerner and co-workers16 did a pioneer calculation of the excited states of the prosthetic groups 2009 American Chemical Society
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Figure 2. Absorption and circular dichroism (CD) spectra of PSRC from Rb. sphaeroides: (a) experimental absorption spectrum,12 dashed line represents the experimental absorption spectrum in the oxidation state of P; (b) TDDFT excitation spectrum of model I calculated at the B3LYP/ 6-31G level; (c) experimental CD spectrum, dashed line represents the experimental CD spectrum in the oxidation state of P; (d) TDDFT CD spectrum of model I calculated at the B3LYP/6-31G level.
in PSRC from Rhodopseudomonas (Rps.) Viridis by using a semiempirical method of INDO/S, which qualitatively reproduced the features of the experimental spectra. Hasegawa and co-workers11,18,19 employed the SAC-CI method to calculate the excited states of each individual chromophore in PSRC first, using a reduced model (substituents on the individual chromophores were cut off and terminated by hydrogen atoms or methyl groups), and then made a resultant description (i.e., put them together) of the spectroscopic properties of the prosthetic groups in PSRCs of Rps. Viridis and Rb. sphaeroides, respectively. The individual chromophores model of PSRC is, however, unable to take the effect of chromophore-chromophore interactions on the excited states into account. To our knowledge, no previous first principle study is available for both absorption and CD spectra of PSRC of Rb. sphaeroides, based on a proper chromophores’ cluster model of the prosthetic groups in PSRC. It is generally agreed that electron transfer starts from the singlet excited state 1P*. The characteristics of the singlet excited states occurring in a “special pair” are unfortunately not yet clearly clarified. This can be one of the key factors to understand the driving force that elicits electron transfer down the L-side instead of the M-side. In the present study, eight low-lying (1.3-1.7 eV energy region) electronic excited states with oscillator strengths larger than 0.1 were calculated in well accordance with the absorption and CD spectroscopic peaks of PSRC from Rb. shpaeroides, by using time-dependent density functional theory (TDDFT).20-23 In spite of its possible deficiency, TDDFT has been shown to be reliable for calculations of the low-lying excited states of chlorophylls.24-26 Our present calculations demonstrate that, only when the interactions among the prosthetic groups have been taken into account, a set of satisfactory assignments for both absorption and CD spectra of PSRC from Rb. sphaeroides can be achieved simultaneously. The calculations of the low-lying singlet electronic excited states of the prosthetic groups in PRSC from Rb. sphaeroides were performed at two different levels of model chemistry. Model I was a reduced supermolecular cluster model, which includes P, BM, BL, HM, and HL chromophore (see Figure 1).
Model II was a simplified individual chromophore model (see Figure S1 of the Supporting Information). Considering the balance between computational cost and calculation accuracy, two ubiquinones and nonheme iron were ignored in model I, since their distance to P, BL, and BM is too long to influence the low-lying excited states. In addition, the most low-lying singlet electronic excited states of the prosthetic groups are of conjugated π f π* transition in nature. The saturated side chain of chromophores was, therefore, truncated with a methyl group in models I and II. The initial geometric parameters of models I and II were built up according to a data set of the crystal structure of Rb. sphaeroides (PDB ID: 1PCR). The hydrogen atoms were added by using Gaussview 3.07,27 and their positions were subsequently optimized by using PM328 as implemented in the Gaussian 03 program package.27 The eight low-lying vertical singlet excited states of model I were calculated by using the TDDFT method at the 6-31G level. The important molecular orbitals (MOs) related to the electron transition configuration analysis were illustrated in Figure 3 and Figures S2 and S3 of the Supporting Information. The calculated excited states, including excitation energies, oscillator strengths (>0.1), and rotatory strengths, together with the experimental CD spectroscopic peak are listed in Table 1 and Tables S1 and S2 of the Supporting Information. The theoretical and experimental absorption and CD spectra in the 1.3-1.7 eV energy region are compared with each other and quantitatively assigned in Figure 2. As can be seen from Table 1, the present TDDFT calculated results share a discrepancy of about 0.3 eV excitation energy for all the low-lying singlet excited states of model I as compared with the experimental absorption and CD spectroscopic peaks of PSRC from Rb. sphaeroides. This systematic discrepancy is most likely attributed to the effects of basis set and surrounding protein moiety. For a convenient comparison of theoretical absorption and CD peaks with those of the experimental spectra, the horizontal axis in Figure 2 for the calculated excitation energies is, therefore, shifted downward by 0.3 eV.
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Figure 3. Contour maps of some important molecular orbitals of model I calculated at the B3LYP/6-31G level.
TABLE 1: Excited States of Models I and II for PSRC from Rb. sphaeroides Calculated at the TD-B3LYP/6-31G Level model I a
state
chro.
1
PM f P*M PL f P*M BM f B*M, PL f B*M BL f B*L, PL f B*M PL f B*M, BM f B*M PM f P*L HL f H*L HM f H*M
14 A 181A 191A 201A 211A 251A 261A 271A a
b
exptl12
model II c
Eex
osc.
1.717 1.819 1.863 1.871 1.877 1.947 1.955 1.959
0.421 0.154 0.222 0.464 0.285 0.143 0.209 0.205
d
R (10
-40
cgs)
302.0 -64.3 -1021.0 848.9 497.5 234.1 -252.3 -203.1
chro.
a
b
c
Eex
osc.
PM f P*M PL f P*M BM f B*M BL f B*L
1.729 1.822 1.895 1.895
0.330 0.121 0.370 0.358
PM f P*L HL f H*L HM f H*M
1.963 1.952 1.957
0.203 0.293 0.236
d
R (10
-40
cgs)
163.0 -28.5 -7.0 8.3 52.8 -0.145 -6.371
b
Eex
CD peak
1.45 1.53 1.53 1.54 1.54 1.54 1.66 1.66
i ii ii iii iii iii iv iv
Chromophore. b Excitation energy in eV. c Oscillator strength in au. d Rotatory strengths.
It is actually difficult to quantitatively assign the nature of the low-lying singlet excited states by comparing with the experimental absorption peaks based only on the information of excitation energies and oscillator strengths, because there are so many low-lying excited states located in this small energy region. Fortunately, with the complementary information of rotatory strengths versus the experimental CD peaks, a satisfactory assignment is achieved simultaneously in the present study for both absorption and CD spectra of the corresponding lowlying excited states of PSRC. The experimental spectroscopic study (see dashed line in Figure 2) showed that both peak i in the CD spectrum and peak I in the absorption spectrum almost vanished when the “special pair” P was oxidized to P+. This experimental fact suggests that the nature of peak i or peak I should be mainly attributed to the electron excitation within P. Our calculations confirm this observation, showing that the nature of the first lowest singlet excited state is mostly due to the electron transition from the 877th molecular orbital (MO-877) to MO-883. MO contour maps (Figure 3 and Figure S2 of the Supporting Information) clearly demonstrate that both of them are located on the molecular backbone of PM. Previously, Hasegawa and coworkers used the SAC-CI method11 to calculate the first excited state 141A at 1.32 eV, using a simplified model of each chromophore, and assigned it to the electron excitation of P f P*. The present study clearly suggests that such an excitation, associated with experimentally observed peak i and I, occurs within the PM unit (i.e., PM f P*M). While MO-883 is the lowest unoccupied molecular orbital (LUMO), MO-877 is an occupied orbital fifth below the highest occupied molecular orbital (HOMO, i.e., MO-882 in model I). This reveals the limitation for model study of the spectroscopic properties of PSRC based only on orbitals of HOMO and LUMO.29 The present study assigns the second and third lowest singlet excited states 181A and 191A to peak ii of the experimental CD spectrum, as both of them possess a negative ellipticity (see rotatory strengths in Table 1) in accordance with the experimental observation (see Figure 2). The electronic transition configuration analysis (see Table S1 of the Supporting Information) shows that the nature of the second lowest singlet excited
state 181A mainly results from the electron transition from MO875 to MO-883, which suggests a PL f P*M electron transition in nature. Similarly, the third lowest singlet excited state 191A is attributed to BM f B*M and charge transfer PL f B*M. The latter provides an explanation for why a strong bleaching of the second CD peak (peak ii) was observed when P was oxidized to P+. SAC-CI study11 computed the second and third excited state 181A and 191A at 1.39, and 1.48 eV, respectively, and assigned them to the electron excitation of BL f B*L and BM f B*M, respectively. They attributed these two excited states to the experimental absorption spectroscopic peak II without a calculation of the rotatory strengths. Their separate chromophore models did not recognize the contribution from P. Comparison with the experimental CD spectrum suggests that the excited states 201A, 211A, and 251A from model I correspond to the third experimental CD peak (peak iii in Figure 2c), as they are calculated with positive rotatory strengths. The excited states 261A and 271A are calculated with negative rotatory strengths and therefore should be assigned to the fourth experimental CD peak (peak iv in Figure 2c). The electronic transition configuration analysis and the detailed assignment on the natures of these excited states are provided in Tables S1 and S2 and Figures S2 and S3 of the Supporting Information. The experimental absorption spectroscopic peaks are therefore assigned in detail as shown in Figure 2b. The present study clearly demonstrates that a proper understanding of the experimental spectroscopic properties of the photosynthetic reaction center of Rhodobacter sphaeroides can be achieved by a combined theoretical study of absorption and circular dichroism spectra. To examine the effects of chromophore-chromophore interactions on the excited states, we also calculate the vertical singlet excited states of each individual chromophore with model II. The calculated results were listed in Table 1 and Tables S1 and S2 of the Supporting Information. The corresponding contour maps of some important MOs are illustrated in Figures S2 and S3 of the Supporting Information. As can be seen from Table 1, the CD spectrum is more sensitive to the chromophore- chromophore interactions than the absorption spectrum. Thus, the chromophore-chromophore
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interactions have a small influence on the excitation energy if such an excitation is within each individual chromophore. On the other hand, the chromophore-chromophore interactions significantly influence the rotatory strengths. Certainly, charge transfer states involving different chromophores are absent in model II calculations. Generally, the rotatory strengths calculated by using model I are much more close to the experimental CD spectroscopic profile than those of model II. This is especially true for the 191A and 201A excited states, where the rotatory strengths calculated by using model II are too small to be well compared with the corresponding experimental data. Therefore, the present study signifies the importance to take explicitly into account the chromophore-chromophore interactions. In conclusion, our present assignments of the low-lying singlet excited states of model I can give a generally satisfactory interpretation of the observations in the experimental absorption and CD spectroscopic studies in the energy region of 1.3-1.7 eV for the PSRC from Rb. sphaeroides. The first and second lowest singlet excited states occuring in the “special pair” are quantitatively assigned to PM f P*M and PL f P*M, while it is known that PM is closely connected with the L-branch from the unoccupied MOs’ point of view (see Figure S4 of the Supporting Information). This provides a valuable addition to the current understanding for the factors that is most likely responsible for the start driving force for the electron transfer along the L-side exclusively. Acknowledgment. This work was supported by the National Basic Research Program of China (Nos. 2007CB116302 and 2007CB815206), the Natural Science Foundation of China (Nos. 20672041, 20873049, 20525311, and 10774126), the Program for New Century Excellent Talents in University of China (NCET-06-0673), and China National Technology Platform (No.2005DKA64001). Supporting Information Available: The detailed computational models and calculated results. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Rees, D. C.; Komiya, H.; Yeates, T. O.; Allen, J. P.; Feher, G. Annu. ReV. Biochem. 1989, 58, 607.
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