Impact of Single-Point Mutations on the Excitonic Structure and

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Spectroscopy and Photochemistry; General Theory

Impact of Single Point Mutations on the Excitonic Structure and Dynamics in Fenna-Matthews-Olson Complex Anton Khmelnitskiy, Tonu Reinot, and Ryszard J Jankowiak J. Phys. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

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The Journal of Physical Chemistry Letters

Impact of Single Point Mutations on the Excitonic Structure and Dynamics in Fenna− −Matthews− −Olson Complex Anton Khmelnitskiy,† Tonu Reinot,# and Ryszard Jankowiak†‡* †

Department of Chemistry and ‡Department of Physics, Kansas State University, Manhattan, Kansas, United States of America

*Corresponding Author: Ryszard Jankowiak, Department of Chemistry, Kansas State University, Manhattan, KS, USA; Tel: 785-532-6785; E-mail: [email protected]

TOC

ABSTRACT: Hole burning (HB) spectroscopy and modeling studies reveal significant changes in the excitonic structure and dynamics in several mutants of the FMO trimer from the Chlorobaculum tepidum. The excited state decay times (T1) of the high energy excitons are significantly modified when mutation occurs near bacteriochlorophyll (BChl) 1 (V152N mutant) or BChl 6 (W184F). Longer (averaged) T1 times of highest energy excitons in V152N and W184F mutants suggest that site energies of BChls 1 and 6, believed to play an important role in receiving excitation from the baseplate BChls, likely play a critical role to ensure the femtosecond (fs) energy relaxation observed in wild-type FMO. HB spectroscopy reveals preferentially slower T1 times (about one picosecond on average) since fs times prohibit HB due to extremely low HB quantum yield. Uncorrelated (incoherent) excitation energy transfer times between monomers, the composition of exciton states, and average, frequency dependent, excited-state decay times (T1) are discussed.

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The Fenna− −Matthews− −Olson (FMO) antenna complex from the green sulfur bacterium Chlorobaculum (C.) tepidum is an important model protein to study exciton dynamics and excitation energy transfer (EET) in photosynthetic complexes. Single point mutations provide an excellent opportunity for the study of exciton structure and interexciton level relaxation. Many theoretical parameter sets were reported for FMO over the years, each describing different linear and nonlinear optical spectra.1–7 In this bacterium, the energy harvested by chlorosomes is funnelled via the baseplate pigments and FMO trimers (made of three identical subunits) to the reaction center (RC), where photochemistry takes place.8 Originally, the structure of the FMO protein from C. tepidum was solved and revealed a homotrimeric complex containing seven BChl a per monomer (Figure 1A, B).9 More recently, an eighth molecule of BChl a was discovered to be present at the interface between adjacent FMO monomers.10 While there is a consensus that BChl 3, which interfaces with the cytoplasmic membrane in which the RC is located, mostly contributes to the lowest-energy sink (trap), the initial state, i.e., which BChl molecule(s) is(are) excited first, is still a matter of debate. Because of the orientation of FMO in relation to the RC, it was suggested that BChl 8 is the entry point of excitations from the chlorosome/baseplate system, with BChl 3 being the energy sink that transfers this energy to the P840 dimer of the RC.11,12 However, site energy of BChl 8 near 803-804 nm (12453-12438 cm1 2,7 ) most likely excludes such scenario. Moreover, calculations of Ritschel et al.,13 showed that a relatively faster transfer is observed when initialization at BChl 1 or BChl 6 is considered. Thus, it is likely that BChls 1 and/or 6 collect excitation from baseplate pigments, implying the existence of one or two pathways (depending on modeling studies)4,14,15 of EET. However, a single pathway with BChl 6 being the highest energy pigments is most consistent with our recent modeling studies of the intact WTI and WTM (a mixture of intact and destabilized complexes)7 FMOs, where simultaneous fits of multiple optical spectra suggested that BChl 6 has the highest site energy2 followed by similar site energies of BChls 1 and 2.7 The mounting evidence suggests that BChls 3, 4, 5 and 7 are lower in energy than BChls 1, 2 and 6 (with the lowest-energy of BChl 3).11,12 Eight FMO mutants were constructed recently in the Blankenship laboratory and described using simple excitonic calculations.16 The latter work assumed, for simplicity, that only the site energy of the pigment near mutation undergoes a spectral shift. However, we have shown recently,17 based on high-resolution 5 K optical spectra and modeling studies that single point mutation in the vicinity of BChl 3 (where tyrosine Y16 was replaced with phenylalanine F16) induced extensive spectral changes in the Y16F mutant which led to formation of three independent low-energy traps (located near 817, 821, and 826 nm), in agreement with measured composite emission and HB spectra.17 The two major traps were assigned to mutated FMO subpopulations characterized by significantly modified low-energy excitonic states. Hamiltonians for both subpopulations (Sub821 and Sub817) were provided shedding more light into extensive changes induced by the single point mutation in the vicinity of Y→F mutation.17 In contrast, mutation induced changes in spectra obtained for the L122Q mutant (where leucine 122 near BChl 8 is replaced with glutamine) were minor, in comparison with spectra of the typically studied WTM FMO, contributed 70 and 30% of intact (WTI) and partly destabilized (WTD) proteins, respectively.7 Resonant HB spectra showed that the internal energy relaxation times in the WTM and L122Q mutant were similar with no resonant (narrow) bleach at νB >12450 cm-1 (i.e. λB ≤ 803 nm, implying femtosecond (fs) energy relaxation 12450 cm-1) spectral region. In order to obtain information on the excited state lifetimes (T1) and interexciton level relaxation in the remaining six mutants (see Figure 1) we measured low temperature absorption/emission spectra and performed high-resolution resonant HB experiments. The main objective of this letter is to explore the mutation induced changes of T1, in particular in the high energy excitons region, i.e. the region where the largest spectral changes are typically observed.16 It is well known that the excited state lifetimes, that are related to the so-called homogeneously broadened lines (Γhom), are easily obtained via hole-burning spectroscopy19,20 via Eq. 1

1 1 1 , = π cΓhom = + T2 2T1 T2 *

(1)

where T2 is the total dephasing time. T2* is the pure dephasing time that strongly depends on temperature since dephasing is caused by phonon scattering; c is the velocity of light (cm s-1).19,20 At T = 4 K Γhom, obtained for excitonically coupled systems are strongly dominated by the exciton level decay times (T1), with T2* being just the minor contribution. Therefore T1 = (2πcΓhom)-1, where Γhom = ½ ΓZPH.19,20 Below we discuss several FMO mutants, focusing on the frequency dependent excited-state decay times (T1) revealed experimentally. A comparison with calculated T1 values is also provided. In this letter we focus on three mutants: 1) V152N (here mutation occurs near BChl 1, i.e. valine is replaced by asparagine); 2) S73A (here serine near BChl 2 is replaced with alanine); and 3) W184F (where tryptophan 184 near BChl 6 is replaced with phenylalanine). The effect of single point mutations on the dynamics in the remaining three FMO mutants (Y345F, F243Y and Q198V), where single point mutation occurred near BChls 4, 5, and 7, respectively, is briefly addressed in the Supporting Information. As mentioned above we discussed the Y16F and L122Q mutants in refs 7,17.

Figure 1. A: Crystal structure of FMO trimer from C. tepidum (PDB ID: 3ENI). The polypeptide chain is shown as a yellow ribbon representation. The BChl pigments are colored in red. B: Translucent representation of an FMO monomer showing the eight labeled BChl molecules. The phytyl tails of the BChls are removed for clarity. Frames C-E: Mutation induced changes near BChls 1, 2, and 6, corresponding to V152N, S73A, and W184F mutants, respectively.

In the modeling studies we apply a recently developed algorithm7 to simultaneously fit multiple optical spectra. While intra-monomer interactions are modeled using non-Markovian Redfield theory,21,22 the incoherent EET occurring between lowest energy molecules of various 3 ACS Paragon Plus Environment


monomers is accounted for by the generalized Förster theory.23 Calculations are performed for the FMO trimer. To fit NRHB spectra, at first a set of pigment energies is obtained (diagonal elements of the Hamiltonian) and a pre-burn absorption spectrum for a single trimer is calculated. Next, for the same set of pigment-energies, thermally and quantum mechanically scaled occupation numbers are found. Using occupation numbers and Monte-Carlo approach, one pigment’s energy is changed (randomly within its pre-burn SDF, or if the energy landscape is modified the distribution is shifted to higher energies) and a post-burn absorption spectrum is calculated. Then pre- and post-burn spectra are subtracted to give NRHB spectrum of a trimer. Each calculated curve is a result of 400,000 accumulated Monte-Carlo realizations and Gaussian smoothing (fwhm = 9 cm-1). We foresee that high resolution HB studies, and modeling of the optical spectra of site-directed mutants reported in this letter will shed more light on mutation induced changes in both excitonic structure and relaxation dynamics, complementing the recently published data obtained by ultra-fast spectroscopic investigation.24 The revealed exciton level decay times (via frequency-domain HB spectroscopy) are briefly compared with those obtained via time-resolved experiments.18,24 We also discuss the EET times between monomers of the FMO trimer and the composition of exciton states. First, we have determined uncorrelated (incoherent) EET between the three monomers of the WT FMO trimer. Namely, due to disorder and EET between BChl 3 pigments (and in part BChl 4 molecules, which have small probability to be the lowest energy pigments) in the three FMO monomers, the lowest energy absorption band near 825 nm is contributed by three subbands (for more details see Figure S1 and Table S1 in the Supporting Information). One subband corresponds to the lowest energy pigment (band 1) in the trimer (that cannot transfer energy), with the second sub-band (shifted blue; band 2) corresponding to pigments that serve as an acceptor and donor, and the last sub-band (band 3) corresponding to the highest energy pigments that efficiently transfer energy to the two lower energy acceptors (the two lowest energy subpopulations of BChls) within trimer. That is, band 1 is lowest in energy and band 3 highest in energy. The calculated average uncorrelated EET times between states 3→2, 2→1, and 3→1 in WT FMO are 17, 16, and 47 ps, i.e. the disorder-averaged inter-monomer transfer time across the 825 nm band is about 27 ps. For perfect EET to the lowest energy exciton band, long-lived emission originates only from sub-state 1. Also, only these pigments are bleached as revealed by modeling of the nonresonant HB spectrum (vide infra). The previously observed time-dependent redshift of the FMO emission spectrum, where the ~30 ps time constant was related to energy transfer in the protein trimer between monomer subunits25 is consistent with our calculations. Moreover, other studies that focused on slower processes taking place in FMO complexes, both at room and 77 K temperatures, also found that a complete spectral equilibration in FMO proteins occurs in about 26 ps,26 which we interpret as equilibration within the trimer caused by the uncorrelated EET between monomers. Note that these calculations for the entire FMO trimer are consistent with our earlier simplified models27 where only incoherent EET between BChls 3 of FMO trimer was taken into account. Figure 2A compares the extracted resonant holes (four black narrow spikes) burned in the spectral region of 12250-12600 cm-1 obtained for the typically studied WT FMO with holes burned in the V152N mutant (ten red holes shown below), where valine (V) near BChl 1 (see Figure 1C) is replaced with asparagine (N). Spectra a (black) and b (red) are the absorption spectra obtained at 4 K for typical WT FMO and V152N mutant samples, respectively, and are 4 ACS Paragon Plus Environment

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WTM V152N

A ×0.06 0.05

T=4 K

a

Absorbance

Absorbance

shown here for an easy comparison. The sharp holes (if present) are always accompanied by broad spectral changes caused by the modified excitonic interaction due to a nonresonant bleach of the lowest energy exciton state (vide infra). The shape of the broad bleach in all HB spectra obtained at νB within the 12,250-12,600 cm-1 spectral range is nearly indistinguishable from the nonresonant bleach obtained for νB = 20,490 cm-1. (Compare HB spectra in Figure 2C and in Figure 3 (curve c)). An example, showing both contributions, i.e. the sharp hole and the broad spectral changes, obtained for νB = 12,498 cm-1 (red curve), is shown in Figure 2C. Absorption spectrum (black) is shown for a comparison. Inset shows the extracted ZPHs with a corresponding Lorentzian fit (blue dashed curve) with the fwhm of 12.5 ± 1.0 cm-1. The latter, corrected for the spectral resolution of 2 cm-1, corresponds to the excited-state decay time of 0.9 ± 0.1 ps. All resonant holes in Figure 2A are obtained with a constant fluence f = 6 J/cm2.

b

0

0.05

C

12525

×0.06

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∆ Absorbance

0 2 1.8 1.6 1.4 1.2 1 0.8

B

12750

12500

12250

12000

12500

12475

×0.2

0.025

T1 (ps)

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


×10

0

12750

Wavenumbers (cm-1)

12500

12250

12000

Wavenumbers (cm-1)

Figure 2. Frame A: Spectra a and b are the 4 K absorption spectra obtained for a typical WT FMO (black curve a) and V152N mutant (red; curve b). Spectra a and b are normalized by the integral intensity in the Qy region. The top four holes (black curves) and the ten red holes (at the bottom) correspond to the extracted resonant holes observed in a typical WT FMO and V152N mutant, respectively. All frequency dependent resonant holes were obtained using constant burning fluence. Frame B: The excited-state decay times (T1) calculated from hole widths (ГZPH) obtained as a function of burn frequency (νB) for WT (black squares) and V152N mutant (red circles) with corresponding error values (bars). Frame C: HB spectrum (lower red curve) obtained for νB = 12498 cm-1 and its corresponding absorption spectrum (black curve). Inset shows the extracted ZPH at νB with the corresponding Lorentzian fit (blue dashed curve). The fwhm and depth of the ZPH are 12.5 ± 1.0 cm-1 and 3 %, respectively.

Note that V→N mutation only weakly affects the site energies of BChls 3 and 4, as the lowest energy band is similar to that observed in a typical WT FMO sample. (See also calculated spectra discussed below). Mostly the absorption band observed in WT near 12420 cm-1 (~805 nm) shifts to higher energy in the V152N mutant. We hasten to add that the WT sample was recently assigned to mixture of two subpopulations of proteins, i.e. ~70% of intact wild-type (WTI) FMO and ~30% of destabilized complexes (WTD).7 The latter is most likely induced by protein conformational changes during the isolation/purification procedures. However, the changes between WTI and WTD are minor affecting only the site energies of BChls 3 and 4 contributing to the 1st and 2nd lowest energy exciton states (see ref 7 for details). The absence of 5 ACS Paragon Plus Environment


resonant bleach in WT FMO sample at νB ≥ 12450 cm-1, as illustrated in Figure 2A/B, implies very fast (femtosecond) energy relaxation in this spectral range in agreement with ultrafast pump-probe and 2DES data.18,28 That is, the lifetimes of the states excited in this region (i.e. at νB ≥ 12450 cm-1) are too short to allow for an observable resonant bleach (i.e. observation of the broad ZPH), although very fast relaxation to the lowest state occurs as reflected by the associated broad nonresonant bleach. Similar behaviour was observed in L122Q mutant where leucine near BChl 8 was replaced with glutamine.7 That is, the internal energy relaxation times in the WTM and L122Q mutant, of excitons at νB ≥ 12450 cm-1, are similar.7 This is not surprising, as the latter mutation weakly affects the shapes of absorption, emission, and nonresonant HB spectra, which were very similar to those measured for WT.7 Interestingly very different dynamics occurs in the V152N mutant for which resonant holes can be burned at much higher energies (see the ten resonant holes shown in red in frame A of Figure 2). The ZPH widths, % hole depths (all holes are very shallow, 12400 cm-1 (λB < 806.5 nm). In summary, although the averaged T1 time in W184F mutant is about 1.5 ps, T1 values vary from about 1.1 - 1.7 ps. Thus, this mutation also affects (i.e. slows down) the fast relaxation observed in WT FMO for the highest energy excitons. Simulated spectra of W184F mutant are shown in frame C of Figure 4. Curves a and a′ are the experimental (solid) and calculated (dashed) absorption spectra, respectively. The blue curves are the measured (solid curve b) and calculated (dotted curve b′) fluorescence spectra obtained with λex = 488.0 nm. Note that this mutant has an additional weak emission band near 12230 cm-1 (indicated by an asterisk). This band most likely originates from a small subpopulations of complexes that most likely miss BChl 3 (calculated spectra not shown). The solid red spectrum (curve c) is the experimental HB spectrum obtained with νex = 12500 cm-1. The red dashed-dotted spectrum (curve c′) is the calculated nonresonant HB spectrum. The only difference between spectra c and c′ is the presence of a narrow ZPH (with a fwhm of ~8 cm-1) at νB = 12500 cm-1 in curve c, which is not accounted for in our simulated nonresonant hole. The very good agreement between the broad spectral changes in spectra c and c′ clearly suggests that the broad bleach is caused by the blue shift of burned pigments contributing to the lowest energy sub-state. The excitonic states, pigment contributions, and pigment contributions to different excitonic state are shown in Figures S4, S5 and Table S6, respectively; see the Supporting Information. NRHB spectra for W184F are best fit assuming a 36 cm-1 and 290 cm-1 blue-shift of the post-burn site energy distributions for BChls 3 and 4, respectively. Finally, the calculated average uncorrelated EET times between states 3→2, 2→1, and 3→1 in W184F mutant are 12, 11, and 20 ps (see Table S1), which (on average) are somewhat faster than those observed in WT and V152N mutant (see Table S1 for details). The latter is most likely caused by slightly modified site energies and larger disorder (see Table S5 and S4). Figure 5A-C shows a similar series of spectra to those presented in Figure 4A-C but obtained for the S73A mutant. Again, absorption spectrum of S73A mutant (spectrum b) is compared with a typical absorption curve of WT FMO (curve a). In this case, the absorption spectra were normalized by the amplitude of the lowest-energy absorption band intensity in the Qy region. Note that the main difference between the ZPHs widths is in the region where absorption spectra differ most, i.e. between 12270-12400 cm-1 energy range; see frame B in Figure 5. The sharp holes below the dashed line, as above, represent the extracted ZPHs; their hole widths and corresponding T1 values are summarized in Table S2. Note that in this mutant the T1 decay times calculated from hole widths (ГZPH) as a function of burn frequency have very different frequency dependence than those obtained for mutant V152N and W184F mutants.



Interestingly, no sharp holes can be burned in S73A mutant at frequency higher than about 12450 cm-1, resembling the behavior observed in WT FMO and mutant L122Q discussed in ref 7.

Figure 5. Frame A: Curves a and b are the absorption spectra of WT and S73A mutant, respectively. The six narrow spikes below the dotted line are ZPHs obtained for S73A mutant (red curves). Frame B: T1 decay times calculated from hole widths (ГZPH) as a function of burn frequency for WT (black squares) and S73A FMO (red circles) obtained for constant fluence with corresponding error values (bars). Frame C: Experimental (solid curves) and calculated (dashed curves) absorbance (black), emission (blue) and NRHB (λex = 488.0 nm) (red) obtained for S73A mutant. Inset in frame C shows the arrangement of BChl 2 and two neighboring residues: Ser 73 and Asn 79. The RMSE for simultaneous fitting of absorption, fluorescence and NRHB spectra is 2.3 × 10-3.

However, the exciton relaxation times are significantly modified in the valley between the second and third absorption bands (i.e., near 12270-12400 cm-1) in agreement with the different bleaching ratio between the 805 nm (12422 cm-1) and 815 nm (12270 cm-1) bands observed in the transient absorption (TA) experiments for the early time delays of 500 fs, 1 ps, and 10 ps reported in ref 24. The experimental 4 K absorption (a), emission (b) and HB (c) spectra shown in Figure 5C. Curves b and c are obtained with νex/νB of 20,490 cm-1, respectively. The calculated spectra are given by curves a′, b′, and c′, respectively. The agreement with experiment is very good. However, to obtain very good agreement with experiment we had to assume that 2/3 of BChl 2 are converted to bacteriopheophytin (BPheo). This is supported by the measured absorption spectrum (curve a) which shows a significant absorption increase near 13168 cm-1, which definitely corresponds to absorption of BPheo. Note that BChl 2 is ligated to a solvent molecule3 and the R-OH group of Serine (S)73 (see Figure 1D) forms H-bond with the N of the amide group of Asparagine (Asn), fixing it in a favorable position for binding H2O molecule to the Mg ion. We suggest that substitution of S73 with Alanine (Ala) allows the amide group of Asn 79 to rotate and assume various positions relative to the Mg ion, which likely leads to the loss of Mg. The inset in frame C illustrates this possible scenario showing the arrangement of BChl 2 and two neighboring residues Ser 73 and Asn 79. The best fit of NRHB spectrum for S73A is obtained assuming a 65 cm-1 and 230 cm-1 blue-shift of the post-burn site energy distributions for BChls 3 and 4, respectively, suggesting again that the energy landscape is differently modified. The calculated average uncorrelated EET 10 ACS Paragon Plus Environment

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times between states 3→2, 2→1, and 3→1 in S73A mutant are 13, 10, and 25 ps (see Table S1), which (on average) are faster than those observed in WT and V152N mutant (see Table S1 for details) and closer to transfer times obtained for W184F mutant. The excitonic states, pigment contributions, pigment contributions to different excitonic state and Hamiltonian for the S73A mutant are shown in Figures S6, S7 and Table S7, S8 and S9 in the Supporting Information. Finally, Figures S8-S10 in the Supporting Information show for completeness the best fits obtained for absorption, emission, and nonresonant HB spectra for Y345F, F243Y, and Q198V mutants that are only briefly discussed in this work. Pigment site energies for the latter three mutants can be easily extracted from Table S10. In summary, this work focused on modified excitonic structure and dynamics induced by single point mutation near BChl 6 (W184F mutant) and BChl 1 (V152N mutant) that appear to play a very important role in very fast exciton transport as the primary entering point for the excitation. While no resonant bleach is observed at λB ≤ 803-804 nm in WT FMO and mutated FMO complexes near BChls 2 (vide supra), BChl 3,17 BChl 8,7 and BChls 4, 5, and 7 (see Supporting Information, Figures S11-S13), implying very fast (fs) energy relaxation in that spectral region, a slower dynamics takes places upon mutation in the vicinity of BChls 1 and 6. That is, single point mutations near BChls 1 and 6 significantly modify the lifetimes of the highest-energy excited states. The latter is in a good agreement with the calculated excited-state lifetimes in similar energy range (see Tables 1 and S2). For example, the 0.6-0.7 ps values obtained for the four highest energy excitons in WT FMO and S73A mutant are similar and consistent with our HB data, as no ZPHs were bleached in that region. In turn, the slower experimentally estimated T1 values for V152N (~1 ps) and W184F (~1.2 – 1.9 ps) mutants for high energy excitons are also in good agreement with the averaged calculated values of 1.1 and 1.3 ps, respectively. Table 1. T1 lifetimes for seven excitonic states of a monomer of WT FMO and its several mutants calculated using non-Markovian Redfield theory (see main text for details). Exciton# 1 2 3 4 5 6 7 Average T1 for the four highest-E excitons

WT 1.3 1.1 0.9 0.7 0.5 0.7

V152N 1.0 1.0 1.9 1.0 0.4 0.9

S73A 1.0 1.7 1.0 0.8 0.4 0.6

W184F 1.0 0.8 0.8 0.9 1.2 2.2

0.7

1.1

0.7

1.3

Thus, it appears that BChls 1 and 6 and protein environment in their vicinity are critical to ensure a very fast (< 1ps) energy relaxation timescale from the highest energy excitonic state located around 12,500 cm-1 (~800 nm) to the lower energy state(s). Recall that femtosecond excited-state decay times prohibit HB process in this spectral range due to extremely low HB quantum yield and decreased peak absorption cross-section. This is consistent with 2DES data where very fast (< 100 fs)40 exciton dynamics was observed in WT FMO complexes at both 11 ACS Paragon Plus Environment


room-temperature and 77 K. Interestingly, although the absorption spectra of V152N, W184F, and S73A mutants are drastically different, shapes of the corresponding nonresonant HB spectra are similar, indicating that contributions (dm(ω)) of BChls 3 and 4 to the lowest-energy sub-state within the FMO trimer (bleached via HB), and their site energies are weakly affected by mutation. This is why in recent 2DES experiments (77 – 300 K),28 where pump excitation was in resonance only with the lowest excitonic state, no changes in the beating frequencies were observed in mutants when compared to the WT FMO. It is feasible that the observed oscillations stemmed from the ground-state vibrational packet, revealing frequencies assigned to the intramolecular BChl vibrations,28 which apparently are not affected by mutation. In contrast, significant absorption changes and/or changes in the excited state decay times (T1) are clearly observed in V152N, W184F, and S73A mutants, when compared with the WT FMO. This indicates that multiple BChls site-energies change in agreement with our modeling studies (see Table S10 that summarizes all site energies). It is evident that mutations V152N and W184F lead to significant red shifts of the site energies of pigments 1, 6 (V152N) and 2, 6 (W184F), while mutation S73A, apart from pheophytinization of 2/3 of BChl 2, is not accompanied by large red shifts for other pigments. We suggest that the site-energy shifts of the BChls 1, 2 and 6 in particular, shared by several high-energy excitons, are responsible for the observed changes in higher energy excited-state decay times. As a result, those states shift to higher energies and overlap between them decreases (Figures S2 and S4). Similarly, increase of the gap in the absorption spectra between the major ~805 nm and ~815 nm bands results in slower relaxation times for excitonic states in that region for V152N and S73A mutants, as clearly demonstrated by the resonant HB spectroscopy (compare data in Figures 2B and 5B). The calculated average excited-state lifetimes of the four highest-energy excitonic states for Y345F, F243Y and Q198V mutants (details of calculations not shown) are 0.7, 0.7 and 0.8 ps, respectively, which are similar to those observed for WT FMO complex (see Table 1). The lifetimes are consistent with the absence of ZPH at λB ≤ 802 nm in these mutants (Figures S11-S13). Finally, we suggest that the W184F mutant contains a minor contribution (~5%) from complexes that most likely lack BChl 3 characterized by the blue shifted emission band near 12200 cm-1, complicating examination of the energetic relaxation of its excitons. The V152N mutant shows significantly red-shifted emission, while S73A mutant reveals significant conversion of BChl 2 into BPheo, reflecting complex changes in structure, dynamics, and protein conformations induced by these single point mutations. Figure 6 depicts exciton energy levels scheme (including the band from the localized BChl 8) and dominant pigment contributions for WT FMO and its V152N, S73A, W184F mutants.


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WT

S73A

V152N

W184F

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Wavenumbers (cm-1)

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12800 6

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2,1,6

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8 7,5 7,5 2,1,7

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12200

Figure 6. Exciton energy level scheme for monomers of WT and three mutants based on our calculations. Dominant pigment contributions (>15%) for each exciton are indicated by corresponding BChl numbers.

Note that not only energetics but also composition of various excitonic states changes upon mutation. Thus, the excitonic structure and dynamics are very sensitive to the protein environment. In summary, our data indicate that optical spectra of the mutants described in this work cannot be explained by single site-energy shifts of BChls where single-point mutation occur, as suggested in ref 16 (see Table S10, which summarizes the site energy shifts of all mutants in comparison to the WTI FMO, whose Hamiltonian was recently reported in ref 7). The latter is not surprising since mutations can induce changes in the energetics, heterogeneity, dynamics, and protein flexibility. For example, it was demonstrated that a small alteration in the local structure of the Escherichia coli cyclic AMP receptor protein (CRP), via amino acid substitution, dramatically changes overall protein dynamics which plays an important role in modulating the allosteric behavior of CRP.41 It was also shown that (M)L214G mutation in the WT bacterial reaction center (bRC) modified total electron-phonon coupling strength of the special pair BChl a and significantly increased inhomogeneous broadening.42 A presence of different subpopulations was also observed in the (M)L214G mutant, where after mutation a large fraction (~50%) of the HA chromophores shifted red (with the significantly broadened absorption band), while the remaining fraction of HA cofactors remained nearly the same site energy as HA in WT bRC42. The latter, however, did not affect the electron transfer time in the (M)L214G mutant, which was similar to that observed in the WT bRC (~1 ps), though quantum efficiency of P+QA− state formation in mutant has been altered.

ASSOCIATED CONTENT: Supporting Information contains description of methodologies used, Hamiltonians, Redfield excited-states lifetimes, detailed data regarding the excitonic structure of Y345F, F243Y and



Q198V mutants, including fits of their optical spectra, as well as summary of Förster EET times between monomers for WT FMO and its mutants. AUTHOR INFORMATION Corresponding Author

*Email: [email protected] Notes

The authors declare no competing financial interests ACKNOWLEDGMENTS This work was supported by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (Grant No. DESC0006678 to R.J.). We acknowledge Dr. Adam Kell for useful discussions and Drs. Rafael Saer and Robert Blankenship for providing the WT and FMO mutant samples. REFRENECES (1)

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