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Mutation-Induced Changes in the Protein Environment and Site Energies in the (M)L214G Mutant of the Rhodobacter Sphaeroides Bacterial Reaction Center Ryszard J Jankowiak, Olga Rancova, Jinhai Chen, Adam Kell, Rafael Saer, J. Thomas Beatty, and Darius Abramavicius J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b06151 • Publication Date (Web): 26 Jul 2016 Downloaded from http://pubs.acs.org on July 29, 2016

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The Journal of Physical Chemistry

Mutation-Induced Changes in the Protein Environment and Site Energies in the (M)L214G Mutant of the Rhodobacter sphaeroides Bacterial Reaction Center Ryszard Jankowiak1,2,*, Olga Rancova3, Jinhai Chen1, Adam Kell1, Rafael G. Saer4,%, J. Thomas Beatty4, and Darius Abramavicius3,* 1

Department of Chemistry and 2Department of Physics, Kansas State University, Manhattan, KS 66506, USA; 3Department of Theoretical Physics, Vilnius University,10222 Vilnius, Lithuania; 4 Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada Abstract

This work focuses on the low-temperature (5 K) photochemical (transient) hole-burned (HB) spectra within the P870 absorption band, and their theoretical analysis, for the (M)L214G mutant of the photosynthetic Rhodobacter sphaeroides bacterial reaction center (bRC). To provide insight into system-bath interactions of the bacteriochlorophyll a (BChl a) special pair, i.e., P870, in the mutated bRC, the optical lineshape function for the P870-band is calculated numerically. Based on the modeling studies, we demonstrate that (M)L214G mutation leads to a heterogeneous population of bRCs with modified (increased) total electron-phonon coupling strength of the special pair BChl a and larger inhomogeneous broadening. Specifically, we show that after mutation in the (M)L214G bRC a large fraction (~50%) of the bacteriopheophytin (HA) chromophores shifts red and the 800 nm absorption band broadens, while the remaining fraction of HA cofactors retains nearly the same site energy as HA in the wild-type bRC. Modeling using these two subpopulations allowed for fits of the absorption and nonresonant (transient) HB spectra of the mutant bRC in the charge neutral, oxidized, and charge-separated states using the Frenkel exciton Hamiltonian; providing new insight into the mutant’s complex electronic structure. Although the average (M)L214G mutant quantum efficiency of P+QA− state formation seems to be altered in comparison with the wild-type bRC, the average electron transfer time (measured via resonant transient HB spectra within the P870-band) was not affected. Thus, mutation in the vicinity of the electron acceptor (HA) does not tune the charge separation dynamics. Finally, quenching of the (M)L214G mutant excited states by P+ is addressed by persistent HB spectra burned within the B-band in chemically oxidized samples. *Corresponding authors % Current address: Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA

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I. Introduction The Rhodobacter (Rb.) sphaeroides bacterial reaction center (bRC) is the simplest and most thoroughly studied of all photosynthetic RC complexes.1–3 For a recent and comprehensive overview of the electronic structure and charge separation (CS) mechanisms in bRCs, see ref 4. The photosystem core in Rb. sphaeroides is a bRC complex dimer surrounded by the lightharvesting 1 complex (LH1) and the PufX protein.5–7 The RC of this bacterium is a type-II RC, which contains a ‘special pair’ bacteriochlorophyll a (BChl a) dimer (called “P”) bound by RC M and L proteins on the periplasmic side of the membrane.8 The cofactors in the bRC form two branches with pseudo-C2 symmetry conventionally named A and B, where the A branch is mainly associated with the L protein, and the B-branch with the M protein. The special pair is flanked by two accessory BChls called BA and BB, with the subscript referring to the two cofactor branches (see Figure 1). Two bacteriopheophytin a molecules (in the HA and HB sites) are located between the accessory BChls and two quinones (QA and QB), which are bound near the cytoplasmic side of the bRC. An iron (Fe2+) atom is located between QA and QB, and a carotenoid is bound near the special pair on the B-branch side.9 The electron transfer (ET) reaction is initiated when light is absorbed by P; i.e., PA/PB chromophores, or by energy transfer to P from the surrounding LH1. Electrons are transferred through the A-branch pigments, from the special pair through BA to HA,10 before passing to QA and finally the QB quinone.11 The back reactions at each step are 2-4 orders of magnitude slower than the forward reactions, resulting in a quantum yield (QY) near unity in wild-type (WT) bRC. Figure 1 shows the cofactor arrangement in the bRC of Rb. sphaeroides and its (M)L214G mutant.12 In this mutant, leucine (L) is replaced with glycine (G) in the vicinity of the HA cofactor. It has been shown that structural heterogeneity (dynamic and/or static) at the HA site can be increased upon decreasing the volume of the (M)214 amino acid side chain, although its origin has not yet been elucidated.13 Thus, the structural and/or functional role of the protein environment in the (M)L214G mutant is investigated below via hole-burning (HB) spectroscopy, which can provide insight into sample heterogeneity, inhomogeneous broadening, electronphonon (el-ph) coupling strength, as well as ET rate.14,15

Figure 1. (Left) Structure of the mutant Rb. sphaeroides bRC cofactors (based on the PDB code 2J8C) with cofactors labeled (PA, PB, BA, BB, HA, HB, QA, QB). The G residue in the (M)214 position replacing L in the mutant bRC is shown in light blue, and labeled (M)G214. Forward ET follows the pathway: PA/PB


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→ BA → HA → QA → QB. (Right) Configuration of the PA and BA phytyl tails near (M)214. Magenta: native bRC BChls; green: (M)L214G BChls; red: PA tail of the native bRC; yellow: PA tail of the (M)L214G bRC; blue: (M)L214 in the native bRC structure; and grey: (M)G214 in the mutant structure. The HA molecule from the (M)L214G structure is shown as a transparent grey chlorin.

Recent two-dimensional electronic spectroscopy (2DES) experiments at 77 K revealed a range of vibrational modes involved in the excitation dynamics16 and possibly in the CS process in the bRC. The 2DES of bRCs with point mutations in the vicinity of the special pair showed that energy transfer dynamics remains largely unaffected while there are changes in the beating frequencies of mutants.17 Long-lived coherences have been also observed in the Photosystem II RC, suggesting that they may reflect underlying electronic–vibrational resonances that could play a functional role in enhancing energy transfer.18 The ultrafast energy transfer (on a timescale of ~100 fs) between two states resolved within the B-band of the oxidized bRC was obtained by polarization-dependent 2DES,19 suggesting that excited states localized on two bRC branches interact. These data demonstrate the complexity of el-ph interactions, which can be resolved at low-temperatures (5 K) by high-resolution frequency-domain spectroscopies.14,20 For example, resonant holes obtained via transient HB spectra can directly provide ET times, since these proteins show negligibly small persistent HB. In this work, we hypothesize that: i) in analogy to the WT bRC21 the upper exciton component of the special pair (PY+) in the (M)L214G mutant bRC cannot be assigned to the shoulder observed near 12,250 cm-1; and ii) the different bandwidth and spectral position of the lower excitonic band of the special pair (PY−) in the mutant, in comparison with the WT bRC,21 may be caused by changes in el-ph coupling strength and/or altered inhomogeneous broadening. Finally, although complete mutation may occur, we will show that only a subpopulation of pigments may change site energy due to disorder and spatial constraints. To address the above we compare low-temperature (5 K) absorption and various types of HB spectra obtained for the Rb. sphaeroides (M)L214G mutant bRC. We focus on the lineshape parameters for the P-band and experimentally determined information on the B and H chromophores in order to provide constraints for the modeling of the entire bRC, including the B and H cofactors. We calculate the lineshape function of P870 numerically without introducing the mean-phonon approximation.22 Parameters obtained from the theoretical fits of P870 and resonant HB spectra for the (M)L214G mutant are compared with recently revised parameters obtained for the WT bRC,22 and further used to model absorption and nonresonant (transient) HB spectra in a broad spectral range. To support the conclusions derived from the experiments, we present a multimer model of the bRC pigment aggregate. We show that the L → G mutation near HA induces significant changes in the el-ph coupling parameters and inhomogeneity of the mutated bRC. II. Experimental Methods. The generation of the (M)L214G mutation via site-directed mutagenesis and subcloning of the pufM gene encoding the M subunit of the bRC was previously described.23 bRCs were expressed as RC-LH1 core complexes in trans in Rb. sphaeroides strain ∆RCLH, which lacks chromosomal copies of the genes coding for the bRC, LH2, and LH1 complexes.24 Purification of the bRCs was carried out according to ref 23. Briefly, cells from 5.6 L of culture in RLB medium (E. coli medium LB supplemented with MgCl2 and CaCl2)25 were harvested by


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centrifugation and resuspended in 10 mM Tris, 150 mM NaCl, 2 mM MgCl2, pH 8.0. A few crystals of lyophilized DNase I (Sigma-Aldrich) were added to the cells prior to disruption at 18,000 psi in a French press. The cell debris was pelleted by centrifugation and chromatophore membranes were obtained by ultracentrifugation at 183,960 x g for 2 hr. The membranes were suspended in 10 mM Tris, 100 mM NaCl, pH 8.0, and lauryldimethylamine oxide (LDAO, Sigma-Aldrich) was added to an optimal concentration derived by titrating the LDAO concentration on a small sample of the membranes. Insoluble material was removed from the membrane suspension by ultracentrifugation at 183,960 x g for 30 min. Imidazole was added to the supernatant containing the solubilized bRCs up to 5 mM concentration before applying the suspension to 5 mL of Ni-NTA affinity chromatography resin (Qiagen) equilibrated in 10 mM Tris, 100 mM NaCl, 0.1% LDAO, pH 8.0. The resin was washed with the equilibration buffer until no trace of LH1 absorption at 875 nm was observed by absorption spectroscopy. bRCs were eluted in 10 mM Tris, 100 mM NaCl, 300 mM Imidazole, 0.1% LDAO, pH 8.0, and dialyzed against 10 mM Tris, 0.1% LDAO. All bRC samples were stored at –80 oC. For HB experiments, samples were diluted 50:50 (v/v) with a buffer:glass solution. The glass-forming solution was 55:45 (v/v) glycerol:ethylene glycol. Details about the measurement setup were described elsewhere. 26 A Bruker HR125 Fourier transform spectrometer was used to measure the absorption and HB spectra with resolutions of 4 and 1-4 cm-1, respectively. The laser source for nonresonant HB at 496.5 nm was produced from a Coherent Innova 200 argon ion laser. Tunable wavelengths came from a Coherent CR899 Ti:Sapphire laser (line width 0.07 cm-1) pumped by a Spectra-Physics Millenia Xs diode laser (532 nm). Laser power in the experiments was precisely set by a continuously adjustable neutral density filter. All experiments were performed at 5 and 300 K in an Oxford Instruments Optistat CF2 cryostat, with sample temperature read and controlled by a Mercury iTC temperature controller. To avoid CS induced by white light interrogation during absorbance measurements, i.e., to ensure the correct intensity of the P-band in comparison with the B- and H-bands, extremely low light intensity (~650 nW/cm2) was used to measure absorption and HB spectra. III. Theoretical methodologies 3.1. Calculations of the P870 absorption band and transient resonant holes. In the lowtemperature limit the HB spectrum of a single site (transition) is defined by ∆A = A(Ω, t) – A(Ω, 0), where Ω, 0 = − Ω is the pre-burn absorption spectrum, and Ω, = − Ω

(1)

(2)

Describes the post-burn absorption spectrum.27,28 In eqs 1 and 2, is the burn/excitation frequency, is the photon flux, is the burn time, σ is the absorption cross-section, is the HB QY, is the pre-burn SDF that describes the probabilities of encountering different zerophonon transition frequencies in the statistical ensemble, and is the single site absorption profile. In the low-photon fluence limit, the exponent in eq 2 can be expanded in a Taylor series to obtain


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Ω, ≈ Ω, 0 − − Ω −

(3)

The absorption lineshape function was calculated as reported in ref 22. This approach is used to describe resonant holes of the P-band of the bRC. 3.2. Simulation of mutant spectra for charge neutral, charge separated, and oxidized bRCs. In the case of several coupled optical transitions, as is the case with the bRC, the postburn relaxed state has to be identified. In the case of the WT bRC the post-burn state is usually associated with the CS state. However, this is not so obvious for mutants, since the QY of CS may be affected. We therefore focus on absorption spectra of various states of the mutant and start from the site basis, i.e., we characterize properties of various pigments (chromophores) and their interactions. The site representation allows one to take into account that the binding pocket of each pigment molecule in the protein complex might have different solvation properties and pose geometrical restraints on the chromophore. The microscopic model describing the bRC consists of six coupled molecular transitions (for simplicity we ignore optically dark charge transfer states). Simulations are based on the Frenkel exciton model with coupling to a harmonic environment constituting the thermal bath and enabling relaxation and dephasing processes. The excitation of each pigment is parametrized by its transition dipole and the site energy . The excitations of different sites within the bRC are excitonically coupled by matrix elements !" of the Frenkel exciton Hamiltonian. The fluctuations of surrounding protein and vibrational degrees of freedom of the chromophore (thermal bath) in each site are described by the spectral density #′′. Each site's coupling to the bath is scaled by parameter % . The conformational disorder inherent to proteins is included by the ensemble averages obtained by sampling the site energies in the Frenkel exciton Hamiltonian from Gaussian distribution with its own standard deviation & . The Frenkel exciton Hamiltonian is diagonalized to give the exciton energies and eigenvectors (the latter reflect the contributions of the particular pigments to the observed excitonic band). It then becomes possible to calculate spectral lineshapes for eigenstates (excitons). It is important to note that the exploration of the properties of P870 band corresponds to the exciton basis while the parameters for the simulations of the whole spectra are set in the site basis. Consequently, the absorption spectrum is obtained in the exciton representation by a cumulant expansion approach when each exciton is described by the spectral lineshape as implemented in the package Spectron.29,30 A detailed description of the approach is available in ref 21 where it has been applied to the WT bRC. More details on the setup of the site-related parameters and simulations of the absorption spectrum are given in the Supporting Information. This approach is used to describe absorption spectra of the mutant bRC and its nonresonant HB spectra. IV. Experimental Results and Modeling Studies 4.1. Absorption and transient HB spectra of WT and mutant (M)L214G bRCs. Curves a and a′ in Figure 2 are the WT bRC absorption and P+QA− transient (photochemical) HB spectra (i.e., difference absorption spectra with the laser on and off) obtained at 5 K. The same spectra obtained for the (M)L214G mutant are shown as curves b and b′, respectively. A transient bleach


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in the Qx region (data not shown) was observed near 600 nm in both samples, which most likely corresponds to the Qx-band of the special pair BChls. Note that the L → G mutation induces significant changes across the absorption spectrum.

Absorbance (a.u.)

1

BA/BB

T= 5 K

P-band

HB/HA

b a

∆ A (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0

a′′ b′′

-0.5 750

800

850

900

Wavelength (nm)

Figure 2. Absorption (curves a and b) and transient P+QA− HB spectra (curves a′ and b′) obtained for the WT (blue) and (M)L214G mutant (red), respectively. λB = 496.5 nm and T = 5 K. All spectra were normalized to fit the low-energy range near 910 nm, i.e., near the maxima of the site-distribution functions (SDFs) of the mutant and WT bRCs (see solid arrow and Table 1).

Some changes in the Qy-absorption region (700-850 nm) were expected, as a recent X-ray structure of the (M)L214G mutant confirmed that G replaced L and revealed that the phytyl tail of the accessory BChl a in the BA site of some of the bRCs adopted a different conformation; that is, it was thought that the bRCs could be a mixture of proteins containing either of the two conformations.23 However, the distribution of protein conformations and their spectral differences, as well as the origin of the large changes observed in the P870 band (the broad absorption band with a maximum near 890-900 nm; labeled as P-band) are not well understood (vide infra). 4.2. Absorption spectra of ferricyanide-treated WT and mutant (M)L214G bRCs. A comparison of the 5 K absorption spectra in charge neutral (CN) bRCs shown in Figure 2 reveal, in agreement with ref 23 that the H-band in the Qy-region shifts blue after mutation (compare curve b with curve a near 760 nm). Other spectral changes difficult to interpret are also observed. This is likely caused by modified inter-cofactor electronic coupling and different delocalization induced by excitonic interactions. Therefore, an assignment of pigment site-energies in the (M)L214G mutant can be obtained only from modeling studies using excitonic calculations. More information on the difference of B- and H-cofactor properties in WT and mutant bRCs is available from optical spectra obtained for oxidized bRCs, i.e., without the P-band contribution. However, in this case one has to consider possible electrochromic shifts induced by P+. This is shown in Figure 3, which compares the room-temperature absorption spectra (normalized to the B-band) of the chemically oxidized (ferricyanide-treated) WT (curve a) and (M)L214G (curve b) bRCs. Note that the H-band region in Figures 2 and 3 cannot be directly compared, as in Figure 3 we compare WT and mutant bRCs in oxidized state.


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T = 295 K

T=5K 0.2

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


H

* 0.1

B

a

750 800 Wavelength (nm)

b

0 600

700

800

900

Wavelength (nm)

Figure 3. Room-temperature (295 K) absorption spectra of ferricyanide-treated WT bRC of Rb. sphaeroides (black curve, a) and the (M)L214G mutant (blue curve, b). The inset shows the absorption of the same samples at 5 K normalized at the B-band. The downward-pointing red arrow shows an increase in absorption in the (M)L214G mutant, and the red asterisk indicates a shoulder on the H cofactor absorption of this mutant bRC (see also the black asterisk below curve b in Figure 4). The integrated area of both spectra is conserved.

The major spectral difference observed at room temperature (main frame of Figure 3) is in the vicinity of the absorption region assigned to the H-band (low-energy part of this band), and high-energy part of the B-band. A clearer picture emerges from the 5 K spectra shown in the inset, which suggest that most likely the HA band (near the L → G mutation site) shifts red (see the red arrow) and broadens by a factor of ~2 (FWHM ~ 310 cm-1; the difference spectrum not shown), while the contribution from the HB-band (on higher energy side of the H-band) is largely unaffected. Note that the integrated area of both absorption spectra shown in the inset of Figure 3 is conserved. The red thick arrow indicates the red-shifted absorbance of a subpopulation of HA chromophores in the (M)L214G mutant, while the red asterisk suggests that not all HA pigments are shifted red. This result suggests that there are two subpopulations of (M)L214G bRC mutant complexes with different arrangements of the cofactor and site energies, and, as a result, different delocalization. We call these two subpopulations MI and MII. Composition of the modified B- and H-bands, i.e., the excitonic structures of these two subspecies is obtained below by excitonic calculations (see Table 2). 4.3. Persistent holes burned in the ferricyanide-treated (M)L214G mutant. Figure 4 (curves a and b) shows the expanded absorption of the ferricyanide-oxidized (M)L214G bRC mutant obtained at 295 and 5 K, respectively. This chemical modification blocks CS and significantly reduces the P-band absorption. However, excitation energy transfer (EET) to P+ can still be present as observed previously in the WT bRC.31 Note that the location of one possible P+ state (based on ref 32) is indicated by the downward red dashed arrow. Spectrum c is the persistent (nonphotochemical) HB spectrum burned resonantly into the B-band at ωB = 12,561 cm−1 (λB = 796.1 nm), indicated by the upward-pointing red solid arrow. This spectrum (hole depth ~ 2%) was obtained with a fluence (f) of ~250 J/cm2, where f = I·tB (with I and tB corresponding to laser intensity and burn time, respectively). The main bleach has a minimum near 12,430 cm-1 with a FWHM of ~130 cm-1. Curve d (the inverted/expanded curve c) may be


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∆ Absorbance

a

ωB

interpreted as the exciton state contributed to by BA and BB (see calculated contributions in Table 2), due to exciton delocalization. The value of Γhom = 3.4 cm-1, obtained from the FWHM of the zero-phonon hole (ZPH) (corrected for spectral resolution) corresponds to a decay time of 1.6 ps. Its possible origin is discussed in section 5.3, however, due to different subpopulations MI and MII mentioned above, only modeling studies (using excitonic calculations) provide more insight into composition of these bands (see section 4.5). 0

b -0.005

0.6

FWHM ~7.4 cm -1

-0.01

T= 5K

12500

∆ Absorbance

a

12550

0.4 1600

Wavenumber (cm-1 )

0.01

*

b

0.2

0

0

d

Oxidized bRC (M)L214G

-0.01

c 12000

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ωB 12500

13000

13500

14000

Wavenumber (cm-1) Figure 4. Room-temperature (295 K; curve a) and 5 K (curve b) absorption spectra of oxidized (M)L214G bRC mutant. Spectrum c is the persistent HB spectrum obtained with λB = 796.1 nm (ωB = 12,561 cm-1). Curve d is the inverted and somewhat expanded curve c to fit the low-energy side of the BBband. The inset shows the 5 K ZPH (curve b) and its Lorentzian fit (curve a) with FWHM of ~7.4 cm-1. The latter, after correction for spectral resolution of 2 cm-1, corresponds to ~6.8 cm-1, hence Γhom = 3.4 cm1 . The black asterisk indicates a shoulder within the H-band (see text and Figure 3).

Figure 5 shows the absorption (curve a) and persistent HB (curve b) spectra of the chemically oxidized mutant. Spectrum b is obtained by selective Qy-excitation into the B-band at lower energy than spectrum c in Figure 4, i.e., at 12,398 cm-1 (~806.6 nm). As mentioned above, the main absorption band is contributed to by both BA and BB chromophores. The downward dashed red arrow (shown in Figure 4 as well) indicates the location of one of the possible P+ states.32 The inset shows curve b in a smaller spectral range with the bleach fitted by broad and narrow Lorentzian curves (i.e., curves c and d, respectively). The anti-hole peaks near 12,500 cm-1. The FWHM of curve d (~56 cm-1) corresponds to ~190 fs, while the narrow hole is resolution-limited. A possible origin of holes c and d is discussed in section 5.3.


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ωB

b

B B/B A

1

T=5K

d

c

Absorbance

Oxidized bRC (M)L214G

FWHM ~ 56 cm − 1

12300

0

12400

12500

a

12600

ω (cm-1)

b

ω B = 12398 cm-1

10000

11000

12000

13000

∆ Absorbance (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


14000

Wavenumber (cm-1)

Figure 5. 5 K absorption (curve a) and persistent HB (curve b) spectra obtained for ferricyanide-treated (M)L214G mutant using selective excitation into the B-band at 12,398 cm-1 (~806.6 nm). The HB spectrum was obtained with f ~ 250 J/cm2. The inset shows the bleach fitted with two Lorentzian curves c and d (see text for details). ωB means the burn/excitation frequency.

4.4. Resonant transient (photochemical) HB spectra burned within the P870 band of the (M)L214G mutant. Figure 6 shows the 5 K P870 absorption band (curve b; frame A) and resonant (transient) HB spectra (curves a-d in frame B; noisy solid lines) in the P-band region. Note that generation of transient HB spectra requires the presence of a third, relatively long-lived state, i.e., the P+QA− state. That is, the excited state evolves into a CS state, leaving a transient hole in the absorption spectrum with a ZPH at the frequency of the original excitation (resonant HB), and with shape defined by the strength of el-ph coupling. In this case, the ground state is depopulated for the lifetime of the long-lived state and the spectral hole can be observed only for the duration of the P+QA− lifetime. The transient holes discussed below are acquired as the difference between the absorption spectra measured while the excitation is on and off (post-burn absorption). The latter allows one to eliminate a possible contribution from a persistent hole, which in the case of bRC (due to very efficient CS) is negligibly small. The absorption band maximum near 890 nm is the same as curve b in Figure 2 (CN mutant bRC; i.e., no ferricyanide present), but the absorption spectrum in Figure 6 exhibits somewhat weaker absorption near 850 nm (sample from a different batch; although a small baseline difference for the above mentioned P870 absorption spectra cannot be entirely excluded). Spectra a-d in frame B were obtained at λB/ωB of 908.8/11,004, 906.0/11,038, 903.0/11,074, and 890.0/11,236 nm/cm-1, respectively. Note that transient holes are not affected by the baseline as well because holes correspond to difference absorption spectra with the laser on and off. All spectra shown in Figure 6 were numerically reconstructed using eqs 1-3 (see ref 22 for details), utilizing the lognormal shape of spectral density, J(ω).33 As before, the inclusion of the special pair marker mode ωsp with a Lorentzian shape (ΓLsp) is necessary to fit the 870 nm band of HB spectra excited at different wavelengths.22,34,35 The fits in both frames A and B are shown as dashed lines.


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0.1 4 3

0.05

b

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ZPL

Abs. (arb. u.)

A

SSA

0 500 Wavenumber (cm-1)

2 1

a

c

0

∆Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Absorbance


0

T=5K

a c b

-0.05

B

d

850

900

950

Wavelength(nm)

Figure 6. Frames A and B show the P870 absorption band (curve b; solid line) and transient HB spectra (noisy solid curves a-d) obtained for the (M)L214G mutant at T = 5 K, respectively. Curve c (yellow area in frame A) is the SDF with FWHM (~200 cm-1) representing inhomogeneity of the system (Γinh). Burning wavelengths in frame A are indicated by the dashed arrows labeled 1-4; the inset shows the extracted single site absorption (see text). The simulated curves (using a lognormal J(ω)) in both frames are plotted as dashed lines. Transient holes shown in Frame B (spectra a-d) were obtained with λB of 908.76, 905.96, 903.02, and 890.0 nm corresponding to burning frequencies (ωB) of 11,004, 11,038, 11,074 and 11,236 cm-1, respectively. The parameters obtained from simulations are reported in Table 1.

The simulated holes were obtained for selected burning frequencies (only four holes are shown in frame B for brevity) using the same parameters as those used in the fit of the P870 band. All simulated spectra match the experimental curves (both overall shape and width) very well across the entire P870 band. The shape of the SDF is shown by curve c in frame A, with a maximum near 10,988 cm-1 and FWHM of 22 cm-1 (corresponding to inhomogeneous broadening Γinh). The single site absorption spectrum extracted from the simultaneous fits is shown in the inset of Figure 6. Figure 7 shows, as an example, three ZPHs (obtained with 2 cm-1 spectral resolution) burned from left to right at 10,925 cm-1 (915.3 nm; black curve), 10,956 cm-1 (912.5 nm; green curve), and 10,994 cm-1 (909.6 nm; red curve) with I = 210 mW/cm2. After correction for spectral resolution, the ZPHs have FWHM of 11.1 ± 0.8, 9.8 ± 1.0, and 9.0 ± 0.9 cm-1, i.e., the ZPH width depends slightly on excitation wavelength, reflecting a small dispersion in the CS time. The average Γhom ~5.0 ± 0.5 cm-1 corresponds to an average CS time of 1.1 ± 0.1 ps (recall that 2Γhom = ΓZPH). The latter value is similar to that observed previously for the WT bRC and Zn-bRCs, which were also about 1 ps.21,36


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0

∆Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-0.0025

11.5 ± 1.0

9.5 ± 1.0 10.2 ± 1.0

-0.005

10900

10950

11000

Wavenumber (cm-1)

Figure 7. ZPHs for the (M)L214G mutant of Rb. sphaeroides. The blue dashed arrows indicate FWHM of the ZPHs. From left to right holes were obtained with burning frequencies of 10,925 cm-1 (black solid curve), 10,956 cm-1 (green solid curve), and 10,994 cm-1 (red solid curve) with I = 210 mW/cm2. The dashed lines correspond to Lorentzian fits (see text for details).

The fitting parameters obtained from the simultaneous fit of the P870 absorption band and several (resonant) transient HB spectra of (M)L214G bRC mutant along with analogous parameters obtained for WT bRC (from ref 22) are listed in Table 1. The parameters include: the lognormal phonon spectral density (ωc and σ, cutoff frequency and standard deviation, respectively), el-ph coupling strength (Sph), ωsp is the frequency of the special pair marker mode (with a Lorentzian lineshape and FWHM = ΓLsp), reorganization energy (Eλ), homogeneous linewidth (Γhom), maximum of the SDF (ωSDF), Ssp is the Huang-Rhys factor for the special pair marker mode, and Stot represents total el-ph coupling strength of the P870 band, while Γinh describes inhomogeneous broadening. The most significant differences are the el-ph (Sph) coupling and increased inhomogeneity (larger Γinh) in the mutant. A similar ωSDF is consistent with data shown in Figure 2, as the low-energy part of the P870 bands and 496.5 nm excited transient P+QA− HB spectra are very similar for WT and mutant bRCs. These extracted parameters are then used in modeling studies reported in section 4.5. Table 1. El-ph coupling parameters for lognormal phonon spectral density and Lorentzian special pair marker mode in the WT and (M)L214G mutant bRCs from Rb. sphaeroides. #Γinh is a FWHM of a Gaussian shape. (The fitted value of the Ssp factor depends on the quality of baseline of the P870 band). Rb. sphaeroides

ωc

σ

Sph

ωsp

ΓLsp

Ssp

Stot

Eλ

Γhom

ωSDF

Γinh

WT22

35

0.47

1.7

125

30

1.5

3.2

254

5.75

10,975

150

(M)L214G mutant

25

0.4

3.05

120

25

1.752.05

4.85.1

293329

6.0 ±1.0

10,988

220

#

All values in units of cm-1 except S and σ (dimensionless).


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4.5. Multimer model of the mutant bRC. The above presented lineshape analysis revealed lineshape parameters of the lowest energy absorption band. In order to support the assumptions derived from the experimental data, we additionally perform simulations of the mutant’s optical spectra starting from the site representation. Having reliable parameters describing the couplings to the phonon bath in the P870 band, we can focus now on the entire absorption spectrum, including the B- and H-cofactors as described in the section 3.2. To provide more insight into the mutant’s electronic structure, we perform simulations of the 5 K absorption spectra of original (M)L214G mutant (CN) and its ferricyanide-oxidized state. The simulated (nonresonant) transient HB spectrum corresponds to a difference absorption based on the CS state. In the simulations we also incorporate the experimental suggestion that the (M)L214G mutant is comprised of two subpopulations with different HA site properties (see section 4.2 and Figure 3); that is, 50% of bRC complexes retain spectroscopic characteristics similar to the WT bRC (subpopulation MI), whereas in the remaining 50% the Qy-transition of HA chromophores shifts red and the corresponding band broadens (subpopulation MII). The 50/50 ratio of the subpopulations is consistent with the 0.5/0.5 occupancy of two BA phytyl tail conformations in the crystal structure of the (M)L214G mutant.23 Additionally we note that the structure of the Bband in the absorption spectrum of the CN bRC (Figure 2) exhibits at least three peaks but the positions of two lowest precisely coincide with the positions of peaks in the WT bRC. This suggests that some fraction of the B pigments in the mutant retained very similar properties as in the WT case. However, the H-band also shows some structure (asymmetry on the low energy side) suggesting that it is not composed of one excitonic band. Thus, to properly escribe our data we find two sets of site-related parameters for two subpopulations MI and MII. The optimal set of simulation parameters is given in the Supporting Information. The obtained absorption and transient HB spectra are shown in Figure 8. The simulation results shown in Figure 8A confirm that the absorption spectrum of the mutant bRC in the CN state indeed can be described as a sum of two ensemble-averaged spectra )** of equally contributing subpopulations: '( = )* '( + '( . The MI subpopulation is modeled based on parameters obtained for the WT bRC (model A) presented in ref 21 with very minimal changes (% − 0.05, %. ± 0.15, and &. − 15 123). In the MII subpopulation the site energy of HA is red shifted towards the B-band region and the static energy disorder of this site increased substantially from &. = 40 123 to &. = 160 123, as suggested by the spectroscopic data discussed above. The fitting procedure provided equal site energies of pigments HA and BA in the MII subpopulation. The total simulated absorption spectrum '( obtained with these parameters is shown in Figure 8A. The absorption spectra of MI and MII and their excitonic bands are shown in the Supporting Information (frames B and C of Figure S1, respectively). The ensemble: averaged components 〈789 7 〉?@ d1, suggesting that the lifetime of the P+QA− state in subpopulation MII has a longer lifetime due to larger static heterogeneity, leading to a deeper hole for this subpopulation bleached during the corresponding bottleneck state, i.e., the longer-lived P+QA− state. Here we hasten to add that the QY in the MI subpopulation is also unknown and it does not have to be the same as that in the WT bRC. This is, consistent with data reported in ref 13 where, based on evolution-associated difference spectra, it was shown that this mutant bRC has more efficient recombination between HA− and P+, leading to a QY of P+QA− state formation at room temperature of 10%;13 the latter value, however, could be model-


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dependent and its room temperature value might be different at low temperature. Though our simple model seemingly confirms that there are at least two subpopulations with different properties in the (M)L214G mutant, the dichotomy might be not as simple. As already shown for other photosynthetic pigment-protein complexes the dichotomy can be temperature dependent and responsible for non-linear temperature-dependent band shifts and broadenings.57,58 These results demonstrate that straightforward comparison of the data obtained at different temperatures for the pigment-protein complexes is hardly possible. V. Concluding remarks. It has been shown that structural heterogeneity (dynamic and/or static) can be increased upon decreasing the volume of the (M)214 amino acid side chain, although its origin has not been elucidated.13 In this work, we demonstrate that changes in static disorder and el-ph coupling of the special pair dimer are responsible for the observed spectral modifications in the P870 band, leading to a deeper understanding of the mutation-induced changes in the protein environment. Parameters were revealed from simultaneous fits of P-band absorption and resonant (transient) HB spectra obtained at several ωB of the (M)L214G mutant. Spectra were fitted assuming a lognormal distribution for the phonon spectral density J(ω),33 providing a more physically meaningful representation of Stot for P870. Notably, the value Stot = 4.7–5.1 for the (M)L214G mutant (including Ssp, i.e., the Huang-Rhys factor for the PA/PB special pair) is ~50% larger than the Stot = (Sph + Ssp) obtained for the WT bRC.22 Another major difference between the WT and mutant bRCs is a significantly increased (by ~50%) Γinh. Interestingly, the value of ωsp (the special pair marker mode) for P870* in both the WT and mutant bRCs is similar (i.e., 120-125 cm-1), indicating that the coupled librational motion of the two monomers of the special pair (a likely significant contributor to the dynamics) does not change upon mutation. Although the mutation site of (M)214 is close to HA, which is the primary electron acceptor, and the presence of two distinct bRC subpopulations was demonstrated, it appears that the L → G mutation only weakly altered the primary CS time, which at low temperatures is still on the order of ~1 ps. Similar ET times in WT, (M)L214G and the Zn-bRC (containing six ZnBChls36) of Rb. sphaeroides indicate that the CS process is robust in spite of these mutations. This is consistent with recent mutations of the special pair of BChls (within 12 Å) in bRCs that did not perturb the energy transfer rates.17 Therefore, it appears that the protein primary structure (amino acid sequence) does not necessarily finely tune the rates of ET and/or EET processes. Although transient HB spectroscopy cannot yield information on secondary ET rates, i.e., P+HA− → P+QA−, it appears that the (M)L214G mutation led to two different subpopulations of bRCs with different decay times of the respective P+QA− states. Agreement between experimental and simulated optical spectra was achieved by including Frenkel exciton Hamiltonian parameters for both subpopulations, which provides new insight into the electronic structure of the (M)L214G mutant. The best fits were obtained with two subpopulations characterized by different HA site properties, i.e., 50% of bRC complexes retain spectroscopic characteristics similar to the WT bRC21 (subpopulation MI), whereas in the remaining 50% the Qy-transition of HA chromophores shifts red and the corresponding band broadens leading to a different mixing and composition of excitonic states in subpopulation MII. We anticipate that parameters provided in this work could be further tuned in more advanced theoretical calculations, where a complete set of optically dark charge transfer states is also taken into account.


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ASSOCIATED CONTENT Supporting Information Description of theory, excitonic coupling matrix elements, site energies, static energy disorder, and exciton band shapes. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; Phone: 785-532-6785 (R.J.) *E-mail: [email protected]; Phone: 00370-5-236-62-81 (D.A.).

Notes The authors declare no competing financial interests. Acknowledgements R.J. and his group acknowledge the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy through Grant DE-SC0006678 for support. O.R. and D.A. acknowledge support from the Lithuanian Science Council (grant No: MIP-090/2015). J.T.B. thanks NSERC Canada for funding through the Discovery Grants system; R.G.S. thanks NSERC for a postgraduate fellowship. References (1) Hoff, A. J.; Deisenhofer, J. Photophysics and Photosynthesis. Structure and Spectroscopy of Reaction Centers of Purple Bacteria. Phys. Rep. 1997, 287, 1–247. (2) Zinth, W.; Wachtveitl, J. The First Picoseconds in Bacterial Photosynthesis—Ultrafast Electron Transfer for the Efficient Conversion of Light Energy. ChemPhysChem. 2005, 6, 871– 880. (3) Parson, W. W.; Warshel, A. Mechanism of Charge Separation in Purple Bacterial Reaction Centers. In The Purple Phototrophic Bacteria; Hunter, C. N., Daldal, F., Thurnauer, M. C., Beatty, J. T., Eds.; Advances in Photosynthesis and Respiration, Volume 28; Govindjee, Ed.; Springer: Dordrecht, The Netherlands, 2009; pp 355–377.


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(4) Savikhin, S.; Jankowiak, R. Mechanism of Primary Charge Separation in Photosynthetic Reaction Centers. In The Biophysics of Photosynthesis; Golbeck, J., van der Est, A., Eds.; Springer: New York, 2014; pp 193–240. (5) Scheuring, S.; Francia, F.; Busselez, J.; Melandri, B.A.; Rigaud, J.-L.; Lévy, D. Structural Role of PufX in the Dimerization of the Photosynthetic Core Complex of Rhodobacter sphaeroides. J. Biol. Chem. 2004, 279, 3620–3626. (6) Scheuring, S.; Busselez, J.; Lévy, D. Structure of the Dimeric PufX-Containing Core Complex of Rhodobacter blasticus by in Situ Atomic Force Microscopy. J. Biol. Chem. 2005, 280, 1426–1431. (7) Qian, P.; Hunter, C. N.; Bullough, P. A. The 8.5 Å Projection Structure of the Core RC– LH1–PufX Dimer of Rhodobacter sphaeroides. J. Mol. Biol. 2005, 349, 948−960. (8) Blankenship, R. E. Molecular Mechanisms of Photosynthesis; Blackwell Science: Oxford, 2002. (9) Lancaster, C. R. D.; Ermler, U.; Michel, H. The Structures of Photosynthetic Reaction Centers from Purple Bacteria as Revealed by X-Ray Crystallography. In Anoxygenic Photosynthetic Bacteria, Blankenship, R. E., Madigan, M. T., Bauer, C. E., Eds.; Kluwer Academic: Dordrecht, The Netherlands, 1995; pp. 503–526. (10) Arlt, T.; Schmidt, S.; Kaiser, W.; Lauterwasser, C.; Meyer, M.; Scheer, H.; Zinth, W. The Accessory Bacteriochlorophyll: A Real Electron Carrier in Primary Photosynthesis. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11757–11761. (11) Jones, M. R. The Petite Purple Photosynthetic Powerpak. Biochem. Soc. Trans. 2009, 37, 400–407. (12) Koepke, J.; Krammer, E.-M.; Klingen, A. R.; Sebban, P.; Ullmann, G. M.; Fritzsch, G. pH Modulates the Quinone Position in the Photosynthetic Reaction Center from Rhodobacter sphaeroides in the Neutral and Charge Separated States. J. Mol. Biol. 2007, 371, 396–409. (13) Pan, J.; Saer, R. G.; Lin, S.; Guo, Z.; Beatty, J. T.; Woodbury, N. W. The Protein Environment of the Bacteriopheophytin Anion Modulates Charge Separation and Charge Recombination in Bacterial Reaction Centers. J. Phys. Chem. B 2013, 117, 7179–7189. (14) Jankowiak, R.; Reppert, M.; Zazubovich, V.; Pieper, J.; Reinot, T. Site Selective and Single Complex Laser-Based Spectroscopies: A Window on Excited State Electronic Structure, Excitation Energy Transfer, and Electron–Phonon Coupling of Selected Photosynthetic Complexes. Chem. Rev. 2011, 111, 4546–4598. (15) Jankowiak, R. Probing Electron-Transfer Times in Photosynthetic Reaction Centers by Hole-Burning Spectroscopy. J. Phys. Chem. Lett. 2012, 3, 1684–1694.


22

Page 23 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(16) Westenhoff, S.; Palecek, D.; Edlund, P.; Smith, P.; Zigmantas, D. Coherent Picosecond Exciton Dynamics in a Photosynthetic Reaction Center. J. Am. Chem. Soc. 2012, 134, 16484– 16487. (17) Flanagan, M. L.; Long, P. D.; Dahlberg, P. D.; Rolczynski, B. S.; Massey, S. C.; Engel, G. S. Mutations to R. sphaeroides Reaction Center Perturb Energy Levels and Vibronic Coupling but Not Observed Energy Transfer Rates. J. Phys. Chem. A 2016, 120, 1479–1487. (18) Fuller, D. F.; Pan, J.; Gelzinis, A.; Butkus, V.; Senlik, S. S.; Wilcox, D. E.; Yocum, C. F.; Valkunas, L.; Abramavicius, D.; Ogilvie, J. P. Vibronic Coherence in Oxygenic Photosynthesis. Nat. Chem. 2014, 6, 706–711. (19) Schlau-Cohen, G. S.; de Re, E.; Cogdell, R. J.; Fleming, G. R. Determination of ExcitedState Energies and Dynamics in the B Band of the Bacterial Reaction Center with 2D Electronic Spectroscopy. J. Phys. Chem. Lett. 2012, 3, 2487–2492. (20) Jankowiak, R. Fundamental Aspects of Fluorescence Line-Narrowing Spectroscopy. In Shpol’skii Spectroscopy and Other Site-Selection Methods: Applications in Environmental Analysis, Bioanalytical Chemistry, and Chemical Physics; Gooijer, C., Ariese, F., Hofstraat, J. W., Eds.; Chemical Analysis: A Series of Monographs on Analytical Chemistry and Its Applications, Volume 156, Wiley-Interscience: New York, 2000; pp 119–145. (21) Rancova, O.; Jankowiak, R.; Kell, A.; Jassas, M.; Abramavicius, D. Band Structure of the Rhodobacter sphaeroides Photosynthetic Reaction Center from Low-Temperature Absorption and Hole-Burned Spectra. J. Phys. Chem. B 2016, 120, 5601–5616. (22) Reppert, M.; Kell, A.; Pruitt, T.; Jankowiak, R. Comments on the Optical Lineshape Function: Application to Transient Hole-Burned Spectra of Bacterial Reaction Centers. J. Chem. Phys. 2015, 142, 094111. (23) Saer, R. G.; Hardjasa, A.; Rosell, F. I.; Mauk, A. G.; Murphy, M. E. P.; Beatty, J. T. Role of Rhodobacter sphaeroides Photosynthetic Reaction Center Residue M214 in the Composition, Absorbance Properties, and Conformations of HA and BA Cofactors. Biochemistry 2013, 52, 2206–2217. (24) Tehrani, A.; Beatty, J. T. Effects of Precise Deletions in Rhodobacter sphaeroides Reaction Center Genes on Steady-State Levels of Reaction Center Proteins: A Revised Model for Reaction Center Assembly. Photosynth. Res. 2004, 79, 101–108. (25) Jun, D.; Saer, R. G.; Madden, J. D.; Beatty, J. T. Use of New Strains of Rhodobacter sphaeroides and a Modified Simple Culture Medium to Increase Yield and Facilitate Purification of the Reaction Centre. Photosynth. Res. 2014, 120, 197–205. (26) Feng, X.; Neupane, B.; Acharya, K.; Zazubovich, V.; Picorel, R.; Seibert, M.; Jankowiak, R. Spectroscopic Study of the CP43′ Complex and the PSI–CP43′ Supercomplex of the Cyanobacterium Synechocystis PCC 6803. J. Phys. Chem. B 2011, 115, 13339−13349.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 28

(27) Hayes, J. M.; Small, G. J. Photochemical Hole Burning and Strong Electron–Phonon Coupling: Primary Donor States of Reaction Centers of Photosynthetic Bacteria. J. Phys. Chem. 1986, 90, 4928–4931. (28) Hayes, J. M.; Gillie, J. K.; Tang, D.; Small, G. J. Theory for Spectral Hole Burning of the Primary Electron Donor State of Photosynthetic Reaction Centers. Biochim. Biophys. Acta 1988, 932, 287–305. (29) Zhuang, W.; Abramavicius, D.; Hayashi, T.; Mukamel, S. Simulation Protocols for Coherent Femtosecond Vibrational Spectra of Peptides. J. Phys. Chem. B 2006, 110, 3362–3374. (30) Abramavicius, D.; Palmieri, B.; Voronine, D. V.; Šanda, F.; Mukamel, S. Coherent Multidimensional Optical Spectroscopy of Excitons in Molecular Aggregates; Quasiparticle versus Supermolecule Perspectives. Chem. Rev. 2009, 109, 2350–2408. (31) Pan, J.; Lin, S.; Woodbury, N. W. Bacteriochlorophyll Excited-State Quenching Pathways in Bacterial Reaction Centers with the Primary Donor Oxidized. J. Phys. Chem. B 2012, 116, 2014–2022. (32) Jordanides, X. J.; Scholes, G. D.; Shapley, W. A.; Reimers, J. R.; Fleming, G. R. Electronic Couplings and Energy Transfer Dyanmics in the Oxidized Primary Electron Donor of the Bacterial Reaction Center. J. Phys. Chem. B 2004, 108, 1753–1765. (33) Kell, A.; Feng, X.; Reppert, M.; Jankowiak, R. On the Shape of the Phonon Spectral Density in Photosynthetic Complexes. J. Phys. Chem. B 2013, 117, 7317–7323. (34) Lyle, P. A.; Kolaczkowski, S. V.; Small, G. J. Photochemical Hole-Burned Spectra of Protonated and Deuterated Reaction Centers of Rhodobacter sphaeroides. J. Phys. Chem. 1993, 97, 6924–6933. (35) Reddy, N. R. S.; Kolaczkowski, S. V.; Small, G. J. Nonphotochemical Hole Burning of the Reaction Center of Rhodopseudomonas viridis. J. Phys. Chem. 1993, 97, 6934–6940. (36) Neupane, B.; Jaschke, P.; Saer, R.; Beatty, J. T.; Reppert, M.; Jankowiak, R. Electron Transfer in Rhodobacter sphaeroides Reaction Centers Containing Zn-Bacteriochlorophylls: A Hole-Burning Study. J. Phys. Chem. B 2012, 116, 3457–3466. (37) Breton, J. Orientation of the Chromophores in the Reaction Center of Rhodopseudomonas viridis. Comparison of Low-Temperature Linear Dichroism Spectra with a Model Derived from X-Ray Crystallography. Biochim. Biophys. Acta 1985, 810, 235–245. (38) Tang, D.; Johnson, S. G.; Jankowiak, R.; Hayes, J. M.; Small, G. J.; Tiede, D. M. Structure and Marker Mode of the Primary Electron Donor State Absorption of Photosynthetic Bacteria: Hole Burned Spectra. In Perspectives in Photosynthesis, Proceedings of the 22nd Jerusalem


24

Page 25 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Symposium on Quantum Chemistry and Biochemistry, Jerusalem, Israel, May 15–18, 1989; Jortner, J., Pullman, B., Eds.; Kluwer Academic: Dordrect, The Netherlands, 1990; pp 99–120. (39) Jordanides, X. J.; Scholes, G. D.; Fleming, G. R. The Mechanism of Energy Transfer in the Bacterial Reaction Center. J. Phys. Chem. B 2001, 105, 1652–1669. (40) Steffen, M. A.; Lao, K.; Boxer, S. G. Dielectric Asymmetry in the Photosynthetic Reaction Center. Science 1994, 264, 810–816. (41) Guo, Z.; Lin, S.;Woodbury, N. W. Protein Dielectric Environment Modulates the ElectronTransfer Pathway in Photosynthetic Reaction Centers. J. Phys. Chem. B 2013, 117, 11383– 11390. (42) Ryu, I. S.; Dong, H.; Fleming, G. R. Role of Electronic-Vibrational Mixing in Enhancing Vibrational Coherences in the Ground Electronic States of Photosynthetic Bacterial Reaction Center. J. Phys. Chem. B 2014, 118, 1381–1388. (43) Jia, Y. W.; Jonas, D. M.; Joo, T.; Nagasawa, Y.; Lang, M. J.; Fleming, G. R. Observation of Ultrafast Energy Transfer from the Accessory Bacteriochlorophylls to the Special Pair in Photosynthetic Reaction Centers. J. Phys. Chem. 1995, 99, 6263–6266. (44) Jackson, J. A.; Lin, S.; Taguchi, A. K. W.; Williams, J. C.; Allen, J. P.; Woodbury, N. W. Energy Transfer in Rhodobacter sphaeroides Reaction Centers with the Initial Electron Donor Missing. J. Phys. Chem. B 1997, 101, 5747–5754. (45) Vulto, S. I. E.; Streltsov, A. M.; Shkuropatov, A. Y.; Shuvalov, V. A.; Aartsma, T. J. Subpicosecond Excited-State Relaxation of the Accessory Bacteriochlorophylls in Native and Modified Reaction Centers of Rb. Sphaeroides R26. J. Phys. Chem. B 1997, 101, 7249–7255. (46) King, B. A.; McAnaney, T. B.; de Winter, A.; Boxer, S. G. Excited-State Energy Transfer Pathways in Photosynthetic Reaction Centers: 5. Oxidized and Triplet Excited Special Pairs as Energy Acceptors. Chem. Phys. 2003, 294, 359–369. (47) Zazubovich, V.; Tibe, I.; Small, G. J. Bacteriochlorophyll a Franck–Condon Factors for the S0 → S1(Qy) Transition. J. Phys. Chem. B 2001, 105, 12410–12417. (48) Small, G. J. On the Validity of the Standard Model of Primary Charge Separation in the Bacterial Reaction Center. Chem. Phys. 1995, 197, 239–257. (49) Finkele, U.; Lauterwasser, C.; Zinth, W. Role of Tyrosine M210 in the Initial Charge Separation of Reaction Centers of Rhodobacter sphaeroides. Biochemistry 1990, 29, 8517–8521. (50) Nagarajan, V.; Parson, W. W.; Gaul, D.; Schenck, C. Effect of Specific Mutations of Tyrosine-(M)210 on the Primary Photosynthetic Electron-Transfer Process in Rhodobacter sphaeroides. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 7888–7892.


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(51) Nagarajan, V.; Parson, W. W.; Davis, D.; Schenck, C. C. Kinetics and Free Energy Gaps of Electron-Transfer Reactions in Rhodobacter sphaeroides Reaction Centers. Biochemistry 1993, 32, 12324–12336. (52) Jia, Y.; DiMagno, T. J.; Chan, C. K.; Wang, Z.; Popov, M. S.; Du, M.; Hanson, D. K.; Schiffer, M.; Norris, J. R.; Fleming, G. R. Primary Charge Separation in Mutant Reaction Centers of Rhodobacter capsulatus. J. Phys. Chem. 1993, 97, 13180–13191. (53) Shochat, S.; Arlt, T.; Francke, C.; Gast, P.; van Noort, P. I.; Otte, S. C. M.; Schelvis, H. P. M.; Schmidt, S.; Vijgenboom, E.; Vrieze, J.; Zinth, W.; Hoff, A. J. Spectroscopic Characterization of Reaction Centers of the (M)Y210W Mutant of the Photosynthetic Bacterium Rhodobacter sphaeroides. Photosynth. Res. 1994, 40, 55–66. (54) van der Vos, R.; Franken, E. M.; Sexton, S. J.; Shochat, S.; Gast, P.; Hore, P. J.; Hoff, A. J. Optically Detected Magnetic Field Effects on Reaction Centers of Rhodobacter sphaeroides 2.4.1 and its Try M210 → Trp Mutant. Biochim. Biophys. Acta 1995, 1230, 51–61. (55) van Brederode, M. E.; van Mourik, F.; van Stkkum, I. H. M.; Jones, M. R.; van Grondelle, R. Multiple Pathways for Ultrafast Transduction of Light Energy in the Photosynthetic Reaction Center of Rhodobacter sphaeroides. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 2054–2059. (56) Pawlowicz, N. P.; van Stokkum, I. H. M.; Breton, J.; van Grondelle, R.; Jones, M. R. An Investigation of Slow Charge Separation in a Tyrosine M210 to Tryptophan Mutant of the Rhodobacter sphaeroides Reaction Center by Femtosecond Mid-Infrared Spectroscopy. Phys. Chem. Chem. Phys. 2010, 12, 2693–2705. (57) Meldaikis, J.; Zerlauskiene, O.; Abramavicius, D.; Valkunas, L. Manifestation of Protein Conformations in the B850 Absorption Band of Light-Harvesting Complex LH2. Chem. Phys. 2013, 423, 9–14. (58) Rancova, O.; Abramavicius, D. Static and Dynamic Disorder in Bacterial Light-Harvesting Complex LH2: A 2DES Simulation Study. J. Phys. Chem. B 2014, 118, 7533–7540.


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