Excitonic Energy Landscape of the Y16F Mutant of the Chlorobium

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B: Biophysical Chemistry and Biomolecules

On the Excitonic Energy Landscape of the Y16F Mutant of the Chlorobium tepidum FMO Complex: High Resolution Spectroscopic and Modeling Studies Anton Khmelnitskiy, Rafael G. Saer, Robert E. Blankenship, and Ryszard J Jankowiak J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b11763 • Publication Date (Web): 19 Mar 2018 Downloaded from http://pubs.acs.org on March 20, 2018

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The Journal of Physical Chemistry

On the Excitonic Energy Landscape of the Y16F Mutant of the Chlorobium tepidum FMO Complex: High Resolution Spectroscopic and Modeling Studies Anton Khmelnitskiy†, Rafael G. Saer§, Robert E. Blankenship§, and Ryszard Jankowiak†‡* †

Department of Chemistry and ‡Department of Physics, Kansas State University, Manhattan, KS, USA; §Departments of Biology and Chemistry, Washington University in St. Louis, Saint Louis, MO, USA

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

ABSTRACT: We report high-resolution (low-temperature) absorption, emission and nonresonant/resonant hole-burned (HB) spectra and results of excitonic calculations using a nonMarkovian reduced density matrix theory (with an improved algorithm for parameter optimization in heterogeneous samples) obtained for the Y16F mutant of the Fenna-MatthewsOlson (FMO) trimer from the green sulfur bacterium Chlorobaculum tepidum. We show that the Y16F mutant is a mixture of FMO complexes with three independent low-energy traps (located near 817, 821, and 826 nm), in agreement with measured composite emission and HB spectra. Two of these traps belong to mutated FMO subpopulations characterized by significantly modified low-energy excitonic states. Hamiltonians for the two major subpopulations (Sub821 and Sub817) provide new insight into extensive changes induced by the single point mutation in the vicinity of BChl 3 (where tyrosine Y16 was replaced with phenylalanine F16). The average decay time(s) from the higher exciton state(s) in the Y16F mutant depends on frequency and occurs on a picosecond time scale.

I. INTRODUCTION

The Fenna-Matthews-Olson (FMO) antenna complex from the green sulfur bacterium Chlorobaculum tepidum (C. tepidum) is an important model protein to study exciton dynamics and excitation energy transfer (EET) in photosynthetic complexes. Many theoretical parameter sets were reported for FMO over the years, each describing different linear and nonlinear optical 1 ACS Paragon Plus Environment

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spectra1–7. In this bacterium, light is harvested by chlorosomes (containing aggregated bacteriochlorophylls (BChl) c, d, or e, and carotenoids) enclosed by a lipid monolayer envelope, and a baseplate containing BChl a8. The energy harvested by chlorosomes is funnelled via the FMO complex and baseplate pigments to the reaction center (RC), where photochemistry takes place9. Originally, the structure of the FMO protein from C. tepidum was solved and revealed a homotrimeric complex containing seven BChl a per monomer10. More recently, an eighth molecule of BChl a was discovered to be present at the interface between adjacent FMO monomers11. Unfortunately, the amount of this BChl may vary from sample to sample, depending on the method of protein isolation/purification11,12. This is a potential problem, as the latter could lead to protein destabilization, as suggested by somewhat different intensities and positions of the absorption/emission band maxima reported in the literature1,2,13–15. 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 RC16,17. That is, energy could enter FMO via BChl 8 (whose site energy, based on theoretical calculations, was originally placed at 12600-12700 cm-1(793.7-787.4 nm)18,19, proceeding through either BChl 1 or 6 downward the energy ladder towards the energy sink. However, Ritschel et al.,20 showed that a relatively faster transfer is observed when initialization at BChl 1 or BChl 6 was considered. The latter is consistent with our current (vide infra) and recent modeling studies of the intact WT FMO, where simultaneous fits of multiple optical spectra suggested that BChl 6 might have the highest site energy2, especially that BChl 8 most likely contributes to the absorption spectrum near 803-805 nm7,12,21. Since no consensus regarding pigment site-energies exists as of yet, the site-directed mutagenesis approach was recently employed to further optimize the FMO Hamiltonian22. To provide more insight into the energy landscape of this important model protein complex, eight site-directed mutants of the FMO trimer were isolated and studied by 77 K absorption and 300 K circular dichroism (CD) spectroscopies15,23. That is, eight FMO mutants were constructed introducing changes in the environment of each of the BChl a within each FMO monomer to provide more insight into BChl site-energies. Earlier, two cysteine molecules at positions 49 and 2 ACS Paragon Plus Environment

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353 in the C. tepidum FMO complex, which reside near hydrogen bonds between BChls 2 and 3, and their amino acid binding partner serine 73 and tyrosine 15, respectively, were changed to alanine residues. The latter resulted in C49A, C353A, and C49A C353A double mutant, which were also analyzed by a combination of optical absorption and CD spectroscopies22. These modified complexes showed absorption properties that differ from the wild-type (WT) FMO protein. The authors argued that these altered absorption properties resulted from environmental changes near the site of the mutation. Here we focus on the Y16F mutant (in which tyrosine 16 was replaced with phenylalanine15,23), as indicated in Figure 1 for which the largest spectral changes were observed in the absorption and emission spectra.

Figure 1. Y16F mutant in which tyrosine 16 (Y16) was replaced with phenylalanine F16 (PDB ID: 3ENI).

We demonstrate below that our high-resolution 5 K optical spectra obtained for this mutant (in particular the shape of the emission spectrum, as well as the shapes of low temperature hole-burned (HB) spectra, do not support the original interpretation given for the Y16F mutant given in ref 15. This is supported by our modeling studies, where the excitonic structure of this mutant is investigated using Redfield theory24,25 to complement the recently published data obtained via 77 K time-resolved experiments.23 We argue below that this particular mutation (Y16 → F16) largely increased FMO heterogeneity and cannot be described by one subpopulation of trimers. Thus, to understand the nature of Y16F mutant it is important to use low-temperature high-resolution spectroscopy to demonstrate sample heterogeneity (if present), otherwise excitonic structure, the population dynamics, and excitation energy transfer (EET) cannot be properly described. New Hamiltonians and average decay times from the higher exciton state(s) in Y16F mutant (that depend on frequency) are discussed. 3 ACS Paragon Plus Environment


II. EXPERIMENTAL METHODS

2.1. Sample preparation. Site-directed mutations in the fmoA gene of Chlorobaculum tepidum, which encodes the FMO protein, were created as previously described22. Purification of the FMO complex was also performed according to previously published protocols22,26. Briefly, cells were lysed by sonication and their membranes were harvested by ultracentrifugation. The membranes were re-suspended in 20 mM Tris HCl, pH 8.0, and sodium carbonate was added to the stirring suspension up to a concentration of 400 mM. This mixture was left to incubate overnight at 4 oC, ultracentrifuged, and the supernatant containing the FMO complexes was dialyzed against 20 mM CAPS, pH 10.5. After dialysis, the protein complex was purified with a strong anion exchanger (Q sepharose fast flow, GE Healthcare, Pittsburgh PA) followed by gel filtration (Sephacryl S 200 HR, GE Healthcare). Fractions containing an A267/371 < 0.6 were pooled.

2.2. Experimental methodology. Details about the experimental setup were described elsewhere27. Briefly, absorption and hole-burned (HB) spectra were measured by a Bruker HR125 Fourier transform spectrometer. For all absorption and nonresonant HB (NRHB) spectra a spectral resolution of 4 cm-1 was used. Resonant HB spectra were obtained with 2 cm-1 spectral resolution. Fluorescence spectra were collected, with a resolution of 0.1 nm, by a Princeton Instruments Acton SP-2300 spectrograph equipped with a back-illuminated CCD camera (PI Acton Spec10, 1340 × 400). The laser source (λ = 488.0 nm) for fluorescence and (nonresonant) HB spectra was produced by a Coherent Innova 90 argon ion laser. Laser power was set by a continuously adjustable neutral density filter. Experiments were performed at 5 K inside Oxford Instruments Optistat CF2 cryostat. Sample temperature was read and controlled with a Lakeshore Cryotronic model 330 or Mercury iTC temperature controller for the former and latter cryostats, respectively.

III. THEORETICAL METHODOLOGIES

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Our modeling approach is described in ref 2; in brief, the disorder is introduced into the diagonal matrix elements (i.e., ‫ܧ‬଴௡ ) by a Monte-Carlo approach with normal distributions centered at ‫ܧ‬଴௡ (n labeling various pigments, i.e., n = 1-24) and with fwhm representing Γinh, which can be site-dependent or independent. Eigen decomposition of the interaction matrix provides eigen-coefficients (ܿ௡ெ ) and eigenvalues (߱ெ ). Phonon and vibrational Huang-Rhys (S) factors are used as free or fixed parameters and are optimized simultaneously against the experimental spectra. It is assumed that the phonon spectral density (weighted phonon profile) can be described by a continuous function, which is chosen to be a lognormal distribution, as illustrated in ref 28. We use experimentally determined phonon spectral density Jph(ω)28,29 for BChl 3, a broader spectral density shape for the remaining pigments (for details see ref 21), and vibrational spectral density Jvib(ω)29. Intramolecular vibrational modes (Jvib) are considered to be dynamically localized30. Sph = 0.4 while Svib = 0.19 and 0.3 for absorption and emission spectra, respectively. In simulations of optical spectra we use a non-Markovian reduced density matrix theory31 with a Nelder-Mead Simplex algorithm for parameter optimization32. Our in-house written software can simultaneously fit several experimental spectra at different temperatures, providing constraints on the pigment site energies of interest. NRHB spectra are modeled in the following way. After diagonalization of the Frenkel Hamiltonian, a pre-burn absorption spectrum is calculated from Redfield theory. The occupation numbers (squared eigenvector coefficients) of the lowest energy exciton state are used to determine the pigments to be burned. This corresponds to the low-fluence approximation, where only pigments contributing to the lowest energy state are burned. To fit NRHB spectra the post-burn site-energy distribution function (SDF) of the burned pigment is found from the pre-burn SDF, or if the energy landscape is modified the distribution is shifted to higher energies (vide infra), while all other diagonal elements of the Hamiltonian are unchanged. The Hamiltonian is again diagonalized and a postburn absorption spectrum is calculated. The resulting HB spectrum is calculated as the pre-burn absorption subtracted from the post-burn absorption spectrum. Each calculated curve is a result of 400,000 accumulated Monte-Carlo realizations and Gaussian smoothing (fwhm = 9 cm-1).

IV. RESULTS AND DISCUSSION



4.1 Absorption and fluorescence spectra. Frame A of Figure 1 shows the 5 K absorption spectra of wild type intact (WTI) FMO (curve a) and Y16F mutant (curve b), respectively. Both curves are normalized to the integrated absorbance in the Qy-region, assuming both trimers possess the same number of pigments, i.e. no pigments were lost during the purification of the Y16F mutant. Note that in contrast to the 77 K spectra reported in ref 15, the 5 K absorption (curve b) reveals two low-energy shoulders near 826 nm and 821 nm indicated by the solid arrows. Curve a′ is a scaled curve a, which fits well the weak lowest-energy feature near 826 nm in the mutant absorption spectrum. Note that a similar low-energy band with a maximum at 826 nm was previously observed in the intact WT FMO complex14,21, suggesting that curve b may have a minor contribution from the intact wild type (WTI) FMO or the mutated minor subpopulation with the 826 nm trap (vide infra) has very similar spectra as the spectra obtained for WTI FMO (vide infra). 0.5

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Figure 2. (Frame A). Absorption spectra of WTI FMO (a) and Y16F mutant (b) at T = 5 K. Spectra were normalized to the integrated absorbance in the Qy-region. Curve a′ is a scaled curve a (see text). Frame B: Y16F mutant minus WTI difference spectrum (b-a). Frames C and D show 5 K fluorescence spectra of the WTI FMO and Y16F mutant, respectively obtained with νex = 20492 cm-1 (i.e. 488.0 nm).

We hasten to add that comparison of WTI FMO absorption spectrum with mutant absorption spectrum is not straightforward, as reported WT FMO absorption spectra showed variable position of the lowest-energy band (i.e. ~825-826 nm); preparations with the lowestenergy absorption band near 825 nm (or 824.5 nm)14,33 were assigned to partly destabilized complexes, i.e. as a mixture of WTI and destabilized (WTD) trimers7,14. However, no matter which spectrum is used for comparison, it is obvious the single-point mutation in the Y16F mutant induces a very large blue shift of the lowest excitonic state and significantly changes the 6 ACS Paragon Plus Environment

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shape of the absorption, emission, and HB spectra. A detailed analysis of the data obtained for the Y16F mutant (including excitonic calculations) is reported below. Here we only note that the recent suggestion (based on 77 K absorption spectra15,23) that replacement of tyrosine 16 (Y16) with phenylalanine (F16) near BChl 3 leads to the loss of the entire 825 nm band is not accurate. We demonstrate that the blue-shift of BChl 3 by 149 cm-1 (as suggested in ref 15 using simple excitonic calculations) cannot explain the scaled difference spectrum (b – a) shown in frame B of Figure 2, nor the Y16F mutant’s absorption or our nonresonant holes (see Figure S1 in the Supporting Information, SI). The difference spectrum from frame B is modeled in Figure 11. The emission spectrum shown in frame D of Figure 2 (νex = 20492 cm-1), in agreement with the very complex absorption spectrum (curve b in frame A), suggests the presence of three fluorescence origin bands. The blue curve in Frame C is the 5 K fluorescence of WTI FMO sample from ref 2 and is shown here for comparison, further reinforcing the above suggestion that the mutant has three different subpopulations of FMO complexes (see below). From Figure 2 it is evident that replacement of Y16 with F16 results in drastic changes in the overall shape of the mutant absorption and emission spectra. The mutant’s absorption spectrum is less structured and reveals two distinct low-energy bands, manifested by the weak shoulders near 826 and 821 nm. The shape of the 5 K mutant fluorescence spectrum (Frame D) in turn suggests that mutant emission is contributed to by three independent bands with maxima near 818, 822 and 827 nm. Thus, there must be a third unresolved trap (i.e. emissive state) in the absorption spectrum. Based on the Stokes shift all three subpopulations must be also characterized by a small Huang-Rhys (S) factor that describes the strength of the electron-phonon coupling.

4.2. Low-energy holes – multiple energy traps. Recently, we have shown that the probing light intensity has to be considered in frequency- and time-domain experiments, as the probing white light can also contribute to the overall nonresonant bleach34 at liquid helium temperatures. Therefore, all spectra for modeling studies were obtained with attenuated FTIR white light intensity of ~13 µW/cm-2 to avoid eventual additional white light bleaching. That is, light intensity was restricted by adding colored and gray filters to the beam path before the sample holder. Again, three different low-energy states are distinguishable in the HB spectra (dashed curves) shown in Figures 3A (νB = 20492 cm-1) and Figure 3B (νB = 12407 cm-1). In both frames the broad low-energy holes, located near 817, 821, and 826 nm, are consistent with 7 ACS Paragon Plus Environment


the composite mutant fluorescence spectrum shown in frame D of Figure 2. HB spectra (1 to 5) in Figure 3A (partly saturated nonresonant holes) were obtained with increasing fluence (f = I∗ t) of 40, 215, 760, 2015 and 2440 J/cm2, where I is laser intensity (in W/cm2) and t burning time (in sec). HB spectra shown in Figure 3B were taken with f of 0.2, 1.5, 5, 11, 23, 60 J/cm2 respectively. Note that the shapes of these holes at low-f are very similar, as expected. The asterisk in Figure 3B indicates that hole #6 (in frame B) and holes 3-5 in frame A are already partly saturated and continuous burning (due to saturation of the low-energy holes) led to additional blue-shift at higher-f values (data not shown for brevity). The depths of the hole #5 in frames A and #6 in frame B are 28% and 35%, respectively. Absorption spectra are shown for comparison to relate the weak shoulders in the absorption spectra and the corresponding bleaches. HB spectra in Figure 3B show the zero-phonon holes (ZPHs) resonant with the burn wavelength (λB = 806.0 nm).

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Figure 3 (Frame A). Nonresonant HB spectra (curves 1-5) obtained with νB = 20492 cm-1 with increasing fluence (dashed colored curves) for curves 1-5, respectively. (Frame B) HB spectra obtained with νB = 12407 cm-1 as a function of increasing fluence (dashed curves, 1-6). The depths of the holes #1 in Frames A and B are 7.5% and 12% respectively. Absorption spectra are shown for comparison.

The three traps at located near 826, 821, and 817 nm represent three FMO subpopulations and are referred to as Sub826, Sub821, and Sub817, respectively. 4.3. Y16F mutant spectra corrected for the minor contribution from the Sub826. To properly model the two major (highly modified) subpopulations of the Y16F mutant, the minor contribution from Sub826 is subtracted. The corrected spectra (curves a-c) to be used below in our modeling studies are shown in Figure 4. Curves a′ and b′ in the inset correspond to mutant HB 8 ACS Paragon Plus Environment

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spectrum before correction for Sub826 and HB spectrum obtained for intact WTI FMO adopted from ref 21. Indeed, the lowest energy bleaches (indicated by the asterisk) are very similar. The corrected curve c in the main frame is the difference between spectra (a′ - b′). We hasten to add that the bleach in curve b′ with the minimum near 12107 cm-1 (826 nm) and emission maximum (near 827 nm; see Figure 2C), are very similar to that previously obtained data for intact FMO trimers14,21. Spectroscopically we cannot distinguish whether this small fraction of complexes is mutated without obvious spectral changes or it originates from a minor subpopulation of revertants. Since extensive analysis of the Y16F mutant at the DNA level did not indicate that any residual WT FMO is present15 we conclude that Sub826 is a small fraction of FMO complexes that did not change much after mutation. The small contribution from Sub826 was also

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Figure 4. Spectra a, b, and c are the corrected absorption, emission, and HB spectra for the Y16F mutant, i.e. the minor contribution from the intact WTI FMO was subtracted. Spectra a′ and b′ are described in text (c = a′ - b′). Thus, we assume that after correction for the minor contribution from Sub826 (i.e. the subpopulation very similar to that of WTI), spectra a, b, and c in Figure 4 are mostly contributed by two different subpopulations, i.e. Sub821 and Sub817 mentioned above. This assignment, so far based on experimental data, is further tested below by simulations where we use a nonMarkovian reduced density matrix theory31 with a Nelder-Mead Simplex algorithm for parameter optimization32. Our improved algorithm can model multiple spectra simultaneously in two different subpopulations of complexes (see section III). 9 ACS Paragon Plus Environment


4.4. Resonant holes burned into lowest energy states of Sub821 and Sub817. Frames A and B in Figure 5 show a series of HB spectra obtained with νB = 12150 cm-1 (823.0 nm) and νB = 12255 cm-1 (816.0 nm), respectively. These burnings occur preferentially into Sub821 and Sub817, respectively, though λB = 816.0 nm also excites a higher exciton state of Sub821 and higher-energy exciton states of the Sub826. That is, as mentioned above, in our experimental HB spectra there is a minor contribution from the low-energy 826 nm trap assigned to Sub826. In addition, the resonant holes are not only burned within the respective site distribution functions (i.e. zero-phonon action (ZPA) spectra of Sub821 and Sub817), but are also contributed to by the uncorrelated EET from higher to lower exciton(s) within each subpopulation. This is why the HB spectra reveal a weak contribution from the Sub826 (see the small double arrow in frames A and B of Figure 5). By the same token for λB = 816.0 nm there should be a broad bleach from Sub821, as observed (see the large double arrow in frame B). Note that no bleaching occurs near 817 nm (the maximum of the Sub817 indicated by the asterisk in frame A) for preferential burning at 823.0 nm into Sub821 and Sub826. Partly saturated HB spectra in Figure 5A/B show a pseudophonon sideband holes located ∼22 cm−1 to the red of the ZPH (marked in both frames by gray dashed arrows).

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Figure 5. Frames A and B show absorption (black curves) and hole burned spectra (curves 1-9 with increasing fluence) obtained with an excitation frequency of 12150 cm-1 and 12255 cm-1, respectively. 10 ACS Paragon Plus Environment

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In summary, a large fluence of 600 J/cm2 in frame B (νB = 12255 cm-1) in Figure 5 (curve 9) reveals only a bleach of two lower-energy traps, i.e. traps corresponding to Sub821 (near 821 nm; 12180 cm-1) and that of Sub826 (near 826 nm; 12107 cm-1). In contrast, large fluence burning at νB = 12150 cm-1 (frame A, curve 9) shows a narrow resonant hole at νB (within Sub821) and a small bleach (via EET) of the Sub826 (see double arrow). Thus, data shown in Figure 5A/B also support our above assignment that Y16F mutant spectra are contributed by three subpopulations of mutated complexes. The question is why there are two major subpopulations of trimers, i.e. Sub821 and Sub817? Based on our experience with FMO proteins (including various mutants) we believe that most isolated FMO trimers are mixtures of WTI and WTD trimers, with the typically observed contribution of ~30-40% and ~70-60%, respectively. The latter is most likely caused by minor protein conformational changes during isolation/purification procedures. Therefore, two conformations may be also observed in mutated FMO samples. This suggestion is further explored in section 4.5.

4.5. Simulations of various optical spectra using a non-Markovian reduced density matrix approach. The absorption (a), emission (b) and HB (c) spectra from Figure 4 are replotted in Figure 6A (thick lines) and compared with the simultaneously fitted spectra assuming two subpopulations of Y16F complexes mentioned above, i.e. Sub821 and Sub817. (We emphasize that the spectra in Figure 6A could not be properly described assuming just one modified subpopulation of mutated FMO trimers and parameters suggested in ref 15); see Figure S1 in the SI. We use two different phonon spectral densities J(ω) as in ref 21. That is, BChl 3 (contributing mostly to the lowest-energy exciton in WT FMO1,35–37 has a J(ω) measured experimentally via delta fluorescence line narrowing (delta FLN)29 with S = 0.4 and fitted by a lognormal function28, but for higher energy pigments, a broader spectral density shape is used, i.e., ωc = 45 cm-1, σ = 0.85 and S = 0.4, as the former J(ω) applied to all BChls (but with two different S-factors2) did not provide correct description of the temperature-dependent absorption spectra (data not shown). The parameters for the second J(ω) were determined from excitationdependent FLN calculations of the B777 complex38 (for a BChl a bound to a single α helix protein), while S was assumed to be site-independent. In our modeling studies we assume that the site distribution functions (SDFs) of BChls are identical in both conformations. Our best simultaneous fits (using our new algorithm) of the absorption (black), emission (blue), and low11 ACS Paragon Plus Environment


fluence nonresonant HB (red) spectra (for both subpopulations) are given by the thin dashed lines, and are labelled as (a + a′), (b + b′), and (c + c′), respectively. The root-mean-square-error (RMSE) for simultaneous fitting of absorption, fluorescence and NRHB spectra is 3.1 × 10–3. The individual contribution of Sub821 (curves a, b, and c) and Sub817 (curves a′, b′, and c′) are plotted in Figure 6B as dashed and solid lines, respectively.

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c 12000

Figure 6. (Frame A): 5 K experimental (thick solid lines) absorption (black), emission (blue) and HB (red) spectra. The dashed lines are the simulated spectra assuming a mixture model (see text). (Frame B): Individual components obtained for Sub821 (dashed lines) and Sub817 (solid lines). Spectra a/a′ (30%/70%), b/b′ (50%/50%), and c/c′ (55%/45%) correspond to absorption, emission, and HB spectra, respectively (T = 5 K); see text for details.

That is, to fit all spectra simultaneously we had to assume that the site-energy of BChl 3 in Sub821 and Sub817 (characterized by different low-energy traps) must be blue-shifted by 101 and 168 cm-1, in comparison to BChl 3 site energy in the intact WTI and WTD, respectively. Moreover, to obtain good fits, the site energies and inhomogeneous widths of other pigments had to be also adjusted. Among all pigments, BChl 4 undergoes the strongest shift of 208 cm-1 compared to the site energy in intact WT complexes. The source of such a large shift may be the proximity of the mutated Tyr 16 to BChl 4 (see Figure 7). It was already shown by Müh et al. that the hydroxyl group of tyrosine may have a significant effect on the site energy of neighboring chlorophyll molecules in CP29 and LHCII antenna complexes39. The modified pigment site energies, fwhm, and energy shifts in both subpopulations are summarized in Tables S1 and S2 in the SI. The large shifts are also observed for BChl 2, 5, 6, and 7, indicating that the loss of the H-bond between BChl 3 and Y16 also leads to other site energy shifts due to protein conformational changes (see Table S1). Table S1 summarizes the relative shifts of BChls in comparison to the new Hamiltonians for WTI and WTD complexes reported recently by our 12 ACS Paragon Plus Environment

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group in ref 7, where we argued that most FMO samples are mixtures consisting of contributions from the WTI and WTD; see also ref 14. Here we suggest that Sub821 (~30%) and Sub817 (~70%) originate from mutated fractions of WTI and WTD complexes, respectively. The latter is supported by a similar relative contribution in the mixture FMO model WTM (contributed to by both WTI (~30%) and WTD (~70%), as described in ref 7. Regarding the mutant data (see Figure 6B), we found that the contributions in Sub821 and Sub817 from absorption and emission are alike, indicating that fluorescence quantum yields in both subpopulations are similar. In contrast, the percent-hole depth is larger for the Sub821 in agreement with HB spectra obtained at very low fluence of 0.2 J/cm2.

Figure 7. Arrangement of BChl 3, 4 and Tyr 16 residue in the crystal structure of WT FMO complex from C. tepidum (PDB ID: 3ENI).

In order to calculate HB spectra one must assign a new site energy to pigment m after the HB process. The simplest approximation, used in refs 40-41, is to assume that after the HB transition, the protein can end up in any of the conformational substates that are present in the original inhomogeneous ensemble around a particular chromophore. This assumption worked well while modeling nonresonant HB spectra obtained for CP2942 and WSCP41. However, the average distribution of particular pigment contributing to the lowest-energy exciton (only the lowest exciton state is populated in an excitonically coupled system like FMO) may also change. That is, due to protein conformational flexibility and complexity of its energy landscape, it is possible that during the HB process conformational transitions occur shifting the average postburn site-energy distribution of a particular BChl contributing to the lowest-energy exciton. This is the case in WTI where, to properly describe the nonresonant hole, the site-energies of BChls 3 and 4 had to be shifted by 36 and 252 cm-1, respectively7. HB spectra for Y16F mutant shown in Figure 6 for Sub821 and Sub817 were best fit assuming blue shifts by 100 cm-1 of the post-burn site energy of BChl 3 (mostly contributing to the lowest energy exciton in both major 13 ACS Paragon Plus Environment


subpopulations; see Figure 8). The remaining BChls weakly contributing to the lowest exciton states (see inserts in Figure 8) in both Sub821 and Sub817 were placed randomly in the original inhomogeneous ensemble around a particular chromophore. The latter suggests that both subpopulations of the Y16F mutant have a similar protein energy landscape near BChl 4 and different energy landscape near BChl 3 in comparison with the WTI FMO. This clearly shows that energy tiers in the protein energy landscape are also modified by single point mutations. Similar energy tiers (about 5-20 cm-1, 30-70 cm-1, and 180-320 cm-1) have been observed in chromoproteins via single complex spectroscopy43. 4.6. The nature of the lowest energy exciton in Sub821 and Sub817. We have shown recently13,21 that both resonant and nonresonant HB spectra obtained for the so-called 825/826 nm band (depending on sample quality14) of the trimeric FMO of C. tepidum are consistent with the presence of a relatively slow (about 30-40 ps) uncorrelated EET between the lowest energy states of the monomers of the trimer (mostly localized on BChl 3), with a weak coupling between these states revealed via calculated emission spectra44. That is, the nature of the socalled 825/826 nm absorption band (at T~5 K) of the FMO trimer cannot be explained by a single transition. Although all three subunits in the trimer are identical (with identical site distribution functions (SDFs), calculations, due to diagonal energy disorder and uncorrelated EET between monomers, produces 24 different excitonic states. That is, trimeric organization leads to a triple splitting of each excitonic state of the monomer. Summation of excitonic energy “triplets” gives eight “effective” excitonic states of the isolated monomer. This is why the dashed spectra in Figure 8 consist of three states 1-3, plotted as black, blue, and red lines, respectively. The latter three bands correspond to: i) the lowest energy trap pigments with two donors (band 1); ii) pigments with one donor and one acceptor (band 2); and iii) the highest energy (band 3) with two donor pigments. Emission originates from bands 1 of Sub821 and Sub817, which play a role of the lowest-energy traps and emitters in both subpopulations. Insets in frames A and B show pigment contributions to the respective lowest-energy exciton states (1); see black dashed line). Excitons in both subpopulations are delocalized over several pigments, with the dominant contribution to the lowest energy exciton from BChl 3 (73%) in Sub821 and from BChl 2 and 3 (42% and 30% respectively) in Sub817. BChl 4 does not significantly contribute to the lowest energy excitons due to a large (208 cm-1) blue shift of its site energy. Other minor contributions 14 ACS Paragon Plus Environment

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Page 15 of 26

to the respective lowest energy excitons are illustrated in the insets of Figure 8. Note that the blue-shifted subpopulation (Sub817) has larger inhomogeneity in agreement with experimental

Distribution, dα (ν)

and calculated emission and HB spectra.

%

Pigment contributions to the exciton #1 of Sub821

A

80 60

3

40 20 0 1

2

3

4

5

6

7

8

max -1 2 ν1 = 12182 cm 1 fwhm = 72 cm-1

0

Distribution, dα (ν)

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


%

Pigment contributions to the exciton #1 of Sub817

B

80

3 30 -20

1

2

3

4

5

6

7

8

2 1

ν1max = 12242 cm-1 fwhm = 90 cm-1

0 12500

12000

Wavenumber (cm-1)

Figure 8. Lowest-energy exciton states (thin black dashed lines) and their contributions labeled as bands 1, 2 and 3 for Sub821 (frame A) and Sub817 (frame B) of the Y13F mutant, respectively. Insets shows pigment contributions to the respective lowest energy excitons (black dashed lines). Emission occurs from bands labeled as 1; see text for details.

4.7. Energy landscape in the Y16F mutant. An energy level diagram obtained for the two major subpopulations (Sub821 and Sub817) of the Y16F mutant is presented in Figure 9. The bands on the left correspond to the sum of scaled exciton states for the mixture of mutated intact/destabilized Y16F proteins. Averaged exciton band maxima (horizontal bars) for Sub821 and Sub817 are plotted in the center. In general, HB spectroscopy can be used to reveal inhomogeneous broadening parameters, i.e., the position and width of the SDF. This can be obtained via zero-phonon action (ZPA) spectroscopy45,46. In this approach, spectra are measured for fixed, low-irradiation doses (constant fluence) at various λB across the inhomogeneously broadened absorption profile. The ZPA spectrum is then obtained as a plot of resonant hole depth versus λB and thus provides a convenient means to directly measure the extent of static disorder. The latter, however, cannot be done for the excitonically coupled heterogeneous sample, like our Y16F mutant, with three different subpopulations of mutated FMO complexes. Nevertheless, we burned shallow holes (at constant fluence) at multiple wavelengths across the low-energy 15 ACS Paragon Plus Environment


absorption bands, i.e. from about 12285 cm-1 (814 nm) to 12420 cm-1 (805 nm). The results are plotted in Figure 9, which shows extracted ZPHs labelled from 1 to 10 with increasing burning frequency.

13000

Y16F mutant

Wavenumber (cm-1)

Sub821

13000

Sub817

12500

12500

10

Wavenumber (cm-1)

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 16 of 26

12280 cm-1 12225 cm-1

1 12000

12000

Figure 9. Sum of properly scaled (30%/70%) eight exciton states of both subpopulations (left) and corresponding exciton band maxima of Sub821 and Sub817 (center). The sharp peaks on the right correspond to the resonant holes obtained for constant fluence. Holes are labelled from 1 to 10 with increasing burning frequency.

That is, the sharp peaks on the right in Figure 9 correspond to the resonant holes obtained for a constant fluence of 6 J/cm2 (their Lorentzian fits are not shown for simplicity). However, the envelope of these ZPHs in such a complex sample (not shown here) does not reflect sample disorder (vide infra). Fast average relaxation time of 1.3 ps (excited state lifetime) is observed for burning into the main broad absorption band near 806 nm. Burning at longer wavelengths reveals slower (2-7 ps) excited-state decay times (T1); see Figure 10. T1 parameters (defined as T1 ≈ (2πcΓhom)-1, where c is the speed of light) were obtained from the measured (spectral resolution corrected) fwhm of Lorentzian fit of ZPHs (ΓZPH where ΓZPH = 2 Γhom). No resonant bleach is observed at λB ≤ 805 nm (in agreement with data obtained for the WTM FMO sample7) implying much faster (femtosecond) energy relaxation in this spectral range. Figure 10 shows excited-state decay time (T1) calculated from hole widths (ΓZPH) after correction for spectral resolution as a function of burn frequency (νB) for the mutant with corresponding error values.


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9.0 8.0 7.0 6.0

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


5.0 4.0 3.0 2.0 1.0 0.0

12100

12150

12200

12250

12300

Wavenumber

12350

12400

12450

(cm-1)

Figure 10. Frequency dependence of averaged excited-state decay times (T1) observed in Y16F mutant with corresponding error values (bars).

The absence of ZPHs at frequencies higher than about 12430 cm-1 is consistent with an ultrafast 77 K spectroscopic investigation of the Y16F mutan23, which showed a very fast component with a lifetime of < 230 fs (the latter was not fully resolved due to temporal limitations) in the evolution associated difference spectra (EADS). This feature had a main bleaching band near 790-805 nm, a region where HB cannot compete with extremely fast relaxation process(es). In fact, it is very likely that in that spectral region FMO complexes have even faster effective relaxation times (on the order of ~30-60 fs) as revealed via 77 K 2DES experiments for wild-type FMO samples47. As already mentioned above, similar behaviour was observed for both the L122Q mutant and WTM7, suggesting that the lifetime of the state(s) excited in this spectral region is(are) too short to allow for an observable resonant bleach. Thus, the envelope of these holes does not reflect the shape of pure SDFs. This is because these holes are also contributed to by the relaxation times (which change across absorption profile), i.e. they shorten towards the blue region due to fast transfer of excitation from the highest to lower energy excitons. As a result, the ZPH become broader and shallower towards the blue region. Some of these holes (in the long-wavelength region) must be also contributed by a weak resonant bleach into the SDF of individual subpopulations, but our resolution cannot disentangle such contribution from the exciton relaxation times. Thus, the values of T1 in Figure 10 should be 17 ACS Paragon Plus Environment


considered as the lower limit. Nevertheless, the average excited state lifetime obtained from holes 7-10 is about 2 ps, which increases to about 5-6 ps at lower frequencies (see holes 1-3). For example, ZPH #7 bleached at 12315 cm-1 revealed T1 of 2.4 ps that is also consistent with the third EADS bleach observed in Y16F mutant (with a minimum near 12330 cm-1) where a lifetime of 3.8 ps was observed23. The decay from the exciton state(s) near 12407 cm-1 (806.0 nm), which occurs on a picosecond time scale (~1.3 ps; see ZPH #10) is also consistent with the overlapping second and third EADS components with T = 77 K lifetimes of 420 fs and 3.8 ps, respectively.

4.8. Absorbance difference spectra between WTI FMO and Y16F mutant. Figure 11

compares 5 K experimental (curve a) and calculated (curve b) absorption difference spectra between the Y16F mutant and the WTI FMO. Calculated curves were obtained with the Hamiltonian presented in the SI (see Table S3). The agreement is significantly better than the simulated 77 K delta absorbance spectrum presented in ref 15 using simple excitonic calculations. 0.25

∆ 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

Page 18 of 26

a 0

b

-0.25

12750

12500 12250 Wavenumber (cm-1)

12000

Figure 11. Experimental (red curve a) and calculated (black curve b) absorbance difference spectra between Y16F mutant and WT FMO at T = 5 K.

V. CONCLUDING REMARKS

High-resolution spectroscopic and modeling studies have identified three independent low-energy traps in the Y16F mutant (Y16 → F16). We show that this mutant is a mixture of 18 ACS Paragon Plus Environment

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mutated FMO complexes with three independent low-energy traps (located near 817, 821, and 826 nm), in agreement with measured composite emission and HB spectra. The traps located near 826, 821, and 817 nm are assigned to three independent protein subpopulation, i.e. Sub826 (minor), Sub821, and Sub817. The last two fractions (assigned to two major subpopulations) correspond to mutated intact (~30%) and mutated destabilized (~70%) FMO complexes, respectively. We conclude that the presence of the two major subpopulations is not mutation induced, but most likely occurs during isolation/purification of FMO trimers, complicating analysis of a femtosecond transient absorption spectra when femtosecond (200 fs) excitation beam (i.e. broad band excitation with fwhm of about 270 cm-1) excites preferentially several high energy exciton(s) at ~800 nm. The very fast (femtosecond) dynamics of higher energy excitons cannot be resolved by HB spectroscopy, but the lack of hole-burning at frequencies higher than about 805 nm is consistent with proposed effective lifetimes of ~30-60 fs revealed by 2DES47 and sub-picosecond lifetimes (not well resolved due to temporal limitations) observed in recent pump-probe experiments23. The excited state lifetimes of about 1.3-6.6 ps revealed by ZPHs in the spectral range of 12140-12407 cm-1 (823.7-806.0 nm) are in agreement with the first three EADS components revealed by global fitting of transient absorption spectra reported recently by in ref.23. Sub821 and Sub817 have different BChl site energy shifts induced by single point mutation near BChl 3. The site energies of BChl 3 (due to H-bond loss with the BChl C3 acetyl group and protein conformational changes) are blue-shifted by 101 cm-1 and 168 cm-1 for Sub821 and Sub817, in comparison to BChl 3 site energy in the intact WTI and WTD, respectively. BChl 4 undergoes even larger blue shift (208 cm-1 compared to WTI) possibly due to the vicinity of mutated Tyr 16. Large shifts are also observed for BChls 2, 5, 6, and 7 (see the SI) indicating that this mutation led to significant changes in pigment-protein interactions. We hasten to add that Tyr residue near BChl 3 is very conservative and present in all known FMO complexes from various organisms. Most likely this residue is needed to provide the hydrogen bond to the acetyl group of BChl 3, lowering its site energy, and as a result, securing efficient energy transfer to the reaction center. We also suggest that this particular hydrogen bond to BChl 3 is important for the overall stability of the FMO complex. Although the hydrogen bond loss occurs in three other mutants (S73A, Y16F, W184F), a high degree of heterogeneity discussed in this work is observed only in the Y16F mutant. Hamiltonians obtained for both subpopulations in the Y16F mutant provide insight into extensive spectral changes. We show that in several ps the FMO 19 ACS Paragon Plus Environment


complexes in both subpopulations relax to the lowest energy exciton, while the average relaxation times depend on excitation wavelength, with faster relaxation revealed at higher energies. Effective lifetimes of highest energy excitons must be on the fs time, in agreements with absence of HB in this spectral range, and fs EET times revealed for Y16F mutant via ultrafast spectroscopic investigations23 and fs EET times observed in WT FMO in 77 K 2DES experiments47.

Appendix A. Supplementary data Supplementary data to this article can be found online at …… , which contain the following material: 1. Comparison of experimental and calculated absorption and HB spectra using the Hamiltonian and site energy changes suggested in Biochim. Biophys. Acta - Bioenerg. 2017, 1858 (4), 288– 296. 2. Comparison of site energies in cm-1 for the two subpopulations of the Y16F mutant with WT mixture model Hamiltonian). 3. Comparison of fwhm for site energies in cm-1 for the Y16F mutant with that of the WTI. 4. Hamiltonians for Y16F FMO mutant.

ACKNOWLEDGEMENTS 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 (Grants No. DESC0006678 to R.J.). We acknowledge Drs Adam Kell and Tonu Reinot for useful discussions. R.E.B and R.G.S acknowledge funding from the Photosynthetic Antenna Research Center, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Award DE-SC 0001035.

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Excitonic Energy Landscape of the Y16F Mutant of the Chlorobium

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