Article pubs.acs.org/JPCB
Ultrafast Spectroscopic Investigation of Energy Transfer in SiteDirected Mutants of the Fenna−Matthews−Olson (FMO) Antenna Complex from Chlorobaculum tepidum Nikki Cecil M. Magdaong,†,‡,§ Rafael G. Saer,†,§ Dariusz M. Niedzwiedzki,§ and Robert E. Blankenship*,†,‡,§ †
Department of Biology, ‡Department of Chemistry, and §Photosynthetic Antenna Research Center, Washington University in Saint Louis, One Brookings Drive, St. Louis, Missouri 63130, United States S Supporting Information *
ABSTRACT: Ultrafast transient absorption (TA) and time-resolved fluorescence (TRF) spectroscopic studies were performed on several mutants of the bacteriochlorophyll (BChl) acontaining Fenna−Matthews−Olson (FMO) complex from the green sulfur bacterium Chlorobaculum tepidum. These mutants were generated to perturb a particular BChl a site and determine its effects on the optical spectroscopic properties of the pigment−protein complex. Measurements conducted at 77 K under both oxidizing and reducing conditions revealed changes in the dynamics of the various spectral components as compared to the data set from wild-type FMO. TRF results show that under reducing conditions all FMO samples decay with a similar lifetime in the ∼2 ns range. The oxidized samples revealed varying fluorescence lifetimes of the terminal BChl a emitter, considerably shorter than those recorded for the reduced samples, indicating that the quenching mechanism in wild-type FMO is still present in the mutants. Global fitting of TA data yielded similar overall results, and in addition, the lifetimes of early decaying components were determined. Target analyses of TA data for select FMO samples generated kinetic models that better simulate the TA data. A comparison of the lifetime of excitonic components for all samples reveals that the mutations affect mainly the early kinetic components, but not that of the lowest energy exciton, which reflects the flexibility of energy transfer in FMO.
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INTRODUCTION Photosynthetic organisms convert light into chemical energy by utilizing pigment−protein complexes that perform light harvesting and electron transfer functions.1 Chlorobaculum tepidum2 is a widely studied green sulfur photosynthetic bacterium which contains a unique light harvesting antenna called a chlorosome, which enables the organism to carry out photosynthesis under extremely low photon fluxes.3 Chlorosomes are made up of large, self-assembled aggregated bacteriochlorophylls (BChl c, d, or e) and carotenoids enclosed by a lipid monolayer envelope and a baseplate protein that contains BChl a.4−6 The energy absorbed by the chlorosomes is funneled to the site of primary charge separation, a type I reaction center (RC), through a molecular wire known as the Fenna−Matthews−Olson (FMO) complex.7,8 FMO has a homotrimeric quaternary structure (Figure 1A), where each monomer (Figure 1B) contains seven BChl a pigments enclosed by mostly β pleated sheet polypeptides.9 An eighth BChl a molecule has been shown to be located between the monomeric interfaces, the amount of which has been shown to vary with the method of protein purification used.9−11 The relative simplicity of and availability of a high-resolution crystal structure for FMO have rendered it a model system for various computational and spectroscopic studies14−36 that aim to understand the structure−function and energy transfer mechanism in this pigment−protein complex. The goals of © XXXX American Chemical Society
Figure 1. (A) Structure of the trimeric FMO complex showing the arrangement of the various BChl a molecules (cyan) enclosed by the polypeptide backbone (gray). (B) Mutants of the FMO complex used in this study were generated using the indicated amino acid residues (various colors). The numbering of BChls is according to Fenna and Matthews.12 For clarity, only the macrocyclic ring of each BChl a is shown in a protein monomer. Figure was generated using Visual Molecular Dynamics (VMD) software13 and PDB ID 3ENI.9
deducing the site energy of each BChl a molecule, as well as the energies of the excitons formed by electronic coupling of the Received: February 8, 2017 Revised: April 7, 2017 Published: April 19, 2017 A
DOI: 10.1021/acs.jpcb.7b01270 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry B
prior to removal from the chamber and use in the experiments. All samples for spectroscopic measurements at 77 K contained glycerol (6:5 glycerol/FMO solution, v/v). Steady-State Absorption Spectroscopy. Steady-state absorption spectra at 77 K were recorded using a Shimadzu UV-1800 spectrophotometer. Samples were frozen in a VNF100 liquid nitrogen cryostat (Janis, Woburn, MA). Time-Resolved Fluorescence Spectroscopy. Timeresolved fluorescence (TRF) measurements were carried out at 77 K using a universal streak camera system (Hamamatsu Corporation, Middlesex, NJ), which consists of a cooled N51716-04 streak tube, a C5680 blanking unit, an Orca2 digital CCD camera, and an A6365-01 spectrograph from Bruker Corporation (Billerica, MA) described in detail previously.43 The laser repetition rate was set to 4 MHz, corresponding to ∼250 ns between subsequent pulses. The wavelength of the excitation beam, depolarized and focused on the sample in a circular spot of ∼1 mm diameter, was set to 590 nm, with very low photon flux of ∼1010 photons/cm2 per pulse. Note that this formula is a broadly used modification of a strict physical formula of photon flux that corresponds to numbers of photons/(m2 s). The sample emission was measured at a right angle to the excitation beam with a long-pass 665 nm filter placed at the entrance slit of the spectrograph. The integrity of the samples was examined by observing the photon counts in real time over the time course of the experiment. These were constant, indicating the absence of sample photodegradation. The data obtained from the TRF experiments were fitted using DecayFit-Fluorescence Decay Analysis Software 1.4, FluorTools, which fits the kinetic traces using iterative reconvolution with least-squares analysis. The goodness of fitting was confirmed by minimizing the χ2 parameter and checking the distribution of the weighted residuals. Transient Absorption Spectroscopy. Time-resolved pump−probe absorption experiments were carried out using Helios, a femtosecond transient absorption (TA) spectrometer (Ultrafast Systems LLC, Sarasota, FL) coupled to a SpectraPhysics femtosecond laser system described previously.44 The wavelength of the excitation beam was preferentially set to excite the highest energy exciton at ∼800 nm (Figure 2). White light probing continuum was generated using a 4 mm sapphire plate. Excitation energy was kept between 8 and 20 nJ, corresponding to a photon flux of (0.4−1.0) × 1013 photons/ cm2 per pulse, in order to minimize permanent photobleaching of the sample and minimize exciton annihilation effects that can be generated at high excitation intensities.35 TA Data Processing and Global Analysis. Dispersion in the FMO TA data sets was corrected for using Surface Xplorer software (Ultrafast Systems) by building a dispersion correction curve from a set of initial times of transient signals obtained from the single wavelength fits of a few representative kinetics. Global analysis of the TA results was performed using a modified version of ASUFit software (provided by Dr. Evaldas Katilius of Arizona State University) according to a sequential decay model that yielded evolution associated difference spectra (EADS).45 The temporal instrument response function (IRF) with a full width at half-maximum (fwhm) of ∼200 fs, assumed to have Gaussian-like properties, was set as a fixed parameter. Directed kinetic analysis of the TA results was performed using the software CarpetView (Light Conversion Ltd., Lithuania). The fitting procedures used models with probable excitation decay pathway following excitation of the higher excitons of FMO. The results of target modeling of TA data sets are
BChls, have also been the subject of these investigations. The assignment of site and excitonic energies is not trivial because the absorption spectrum of FMO, particularly at room temperature, shows a broad Qy band at ∼805 nm that lumps all contributions from the individual BChls. Upon lowering the temperature to 77 K, this band resolves into three peaks at ∼805, 815, and 825 nm. According to previous studies,8,15,17,18,37 the lowest energy exciton resides mostly on BChl a3, which interfaces with the cytoplasmic membrane where the RC is embedded. Still, the issue of which particular BChls contribute to the absorption bands remains to be fully resolved. Assigning spectral peaks to specific BChls and excitonic components has an implication to the energy transfer pathways in FMO as well. In a previous study, data from two-dimensional electronic (2DE) spectroscopy and theoretical computations19,32 were used to determine the excitonic energies and extent of exciton localization on the various BChls of FMO. The results showed that excitons 3 and 7 are delocalized between BChls a1 and a2, excitons 5 and 6 are between BChls a5 and a6, and excitons 2 and 4 are between BChls a4 and a7. Spatial overlap between interacting excitons is important in the two proposed pathways of excitation decay within FMO: exciton 7 → 3 → 2 → 1 and exciton 6 → 5, 4 → 2 → 1. Recent 2DE spectroscopic results,36 however, revealed a more convoluted branching of relaxation steps that do not support the previous model of two pathways. Rather, each exciton state transfers its excitation into multiple states. In addition, new insights into the population dynamics and energy transfer in the FMO complex enabled the calculation of the transfer rates between excitons.36 The ability to generate site-directed mutants of FMO in Chlorobaculum tepidum has been reported previously.38 The cysteine residues (49 and 353) were mutated to alanine in order to confirm that the observed redox-dependent quenching mechanism of FMO39,40 is in fact due to the formation of a thiyl radical.41 Interestingly, these mutations also perturbed the optical absorption spectrum of FMO, and this was expanded in a subsequent study,42 through mutation of amino acid residues near specific BChls. A few of these mutants that have the most dramatic effects on the steady-state absorption and are pertinent to the energy transfer pathways are used in this work. In this study, the dynamics of energy transfer in the Fenna− Matthews−Olson complex from Chlorobaculum tepidum were investigated by applying time-resolved fluorescence and ultrafast pump−probe transient absorption spectroscopic methods on site-directed mutants of FMO. The results reveal differences in the spectral shapes and excited-state decays between the mutants and wild-type (WT) protein. These results are utilized to decipher the complex energetic landscape of the FMO protein and how each mutation affects the dynamics of the various excitonic components.
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EXPERIMENTAL METHODS Sample Preparation. The generation of the FMO mutants is described elsewhere.42 Both WT and mutant FMOs were purified according to the protocol published previously35,38 and are herein referred to as “oxidized” samples. Reduced samples were prepared by placing the FMO samples in an anaerobic chamber (80% N2, 10% H2, 10% CO2) for 30 min, followed by addition of sodium dithionite to a final concentration of 10 mM. The samples were incubated for another 30 min in the anaerobic chamber, transferred to plastic cuvettes, and sealed B
DOI: 10.1021/acs.jpcb.7b01270 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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Y345F mutant has peaks at 815 and 811 nm that are more pronounced than those in the Y16F spectrum, and the lowest energy band is at 824 nm. The mutation of tryptophan 184 (W184) to F near BChl a6 mostly affects the blue edge of the absorption spectrum, with the 815 nm band gaining more intensity and the 805 nm peak decreasing in intensity and blueshifting to ∼800 nm. These differences in spectral profiles are discussed in more detail by Saer et al.,42 in conjunction with circular dichroism data and simulation of excitonic spectra. Overall, the effects on the absorption spectra of each BChl a site mutation confirm the contribution of particular BChls to excitonic bands as previously assigned.32,36,46 Time-Resolved Fluorescence Spectroscopy. Timeresolved fluorescence data were recorded at 77 K for the WT FMO and mutants, which are shown in Figure 3. The TRF spectra for all samples show a major peak at ∼825 nm, except for the Y16F mutant, which has a major peak at ∼816 nm and a small shoulder at ∼826 nm. The decay lifetimes of the oxidized samples (Table 1, top panels) obtained from the fitting of the kinetics data yielded two components, the first of which has a higher amplitude and is faster in the 150−465 ps range. The second component is in the 670 ps−2.2 ns time range. Y16F, which is the mutation near BChl a3, has the shortest fluorescence decay lifetime, followed by the mutation near BChl a6 (W184F). On the other hand, V152N (near BChl a1) has the longest fluorescence decay in the series. The lifetimes of S73A (BChl a2) and Y345F (BChl a4) are comparable to that of the WT FMO. It is evident that perturbations in key BChl sites have implications in the excited-state decay process. Energy from the chlorosome baseplate enters the FMO complex at the level of BChl a8, proceeding through either BChl a1 or 6 and subsequently to BChl a3, the terminal BChl a acceptor in FMO that transfers the excitation energy to the RC. Whereas differing fluorescence lifetimes were obtained for the oxidized FMO samples, all of the reduced mutant samples behaved similarly as the WT, in that satisfactory fitting of the TRF data was obtained with one monoexponentially decaying component with a lifetime of 2.1−2.6 ns (Figure 3, bottom panels). This result implies that the redox-dependent quenching mechanism in WT FMO that has been previously attributed to cysteine residues41 is still present in the mutant samples, albeit to slightly varying degrees. The steady-state optical spectrum of the Y16F mutant (Figure 2) does not show the peak corresponding to the lowest energy exciton at 825 nm, yet a small shoulder in the TRF spectra (Figure 3, oxidized and reduced middle panels) is apparent in this region. The decay profiles for the Y16F mutant (oxidized) extracted at the two band positions reveal different kinetic lifetimes, with the 816 nm band decaying faster (∼88 ps) than the 826 nm band (∼140 ps). The shorter lifetime of the 816 nm band compared with that of the 826 nm band is possibly due to energy transfer between the 816 and 826 nm exciton states. It suggests that the exciton band at ∼825 nm is not completely abolished in the Y16F mutant, such that it still shows up in the fluorescence spectrum, even though the absorption spectrum has little intensity in this region. Extensive analysis of the Y16F mutant at the DNA level does not indicate that any residual WT FMO is present.42 Transient Absorption Spectroscopy. The ultrafast kinetics of the FMO samples were investigated using pump− probe transient absorption spectroscopy. The TA spectra recorded in the near-infrared (NIR) region at various time delays after laser excitation are shown in Figure 4. The TA
Figure 2. Steady-state absorption spectra of FMO wild-type (WT, black trace) and various mutants recorded at 77 K. The numbers in parentheses refer to the BChl a number near the mutation. All spectra were normalized at their maximum and offset for clarity. The cyan trace corresponds to the spectrum of laser excitation, approximated by a Gaussian curve that was used in the TA experiments. The excitonic bands of the WT FMO as given in the accompanying table were simulated by seven Gaussians (thin black traces) whose peak positions were adopted from Thyrhaug et al.,36 and the sum is given by the magenta trace.
commonly abbreviated as SADS (species associated decay spectra).45
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RESULTS Steady-State Absorption Spectroscopy. The absorption spectra of the FMO samples recorded at 77 K are shown in Figure 2. At cryogenic temperature, the overlapping bands in the Qy region due to the various excitonic components are resolved into three main bands peaking at 805, 815, and 825 nm as can be seen in the spectrum of the WT FMO (Figure 2, black trace). The 825 nm band represents the lowest energy exciton that is mostly localized on BChl a3, in accord with previous studies.15,31,37 Mutation of selected residues near the acetyl group of the different BChl a molecules leads to perturbations in the absorption profile. It is evident that each mutation affects more than one excitonic component. For instance, the mutation of valine 152 (V152) to asparagine (N), which is near BChl a1, leads to a blue-shift of the peak maximum to ∼803 nm and a decrease in intensity of the peak at 815 nm (violet trace). Changing serine 73 (S73) to alanine (A) does not shift the peak positions at 805, 815, and 825 nm, but the peak ratios are different from the WT FMO, particularly due to a decrease in the 815 nm band intensity. The tyrosine 16 (Y16) to phenylalanine (F) mutation near BChl a3 shows the most dramatic effect with a blue-shift of the 815 nm band to 811 nm and the loss of the 825 nm band (red trace), which further confirms the assignment of the 825 nm band to BChl a3. The C
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Figure 3. continued
D
DOI: 10.1021/acs.jpcb.7b01270 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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Figure 3. Time-resolved fluorescence data for the various FMO samples obtained at 77 K under oxidizing (top panels) and reducing (bottom panels) conditions. For each FMO sample, the top panel is the contour plot of the TRF data (inset contains the scale of photon counts), the middle panel shows the representative spectra at the indicated time delays, and the bottom panel shows kinetic traces (black lines) integrated over the wavelength range shown in the labels with the corresponding fits (green and orange) and instrument response function (IRF, blue). Results of the fitting are summarized in Table 1 (top panels). The color labels in each TRF profile correspond to photon counts.
state absorption peaks (Figure 2). Positive peaks at ∼790 nm, between 815−825 nm and ∼830 nm, correspond to ESA, while SE contributes mostly to the lowest energy 825 nm band. Although the band positions shift in the various mutant FMO samples, the TA spectra show that all samples behave similarly, in that the bleaching of the lowest energy band
signals can be interpreted as a convolution of several photophysical processes: photobleaching (PB) or depopulation of the ground state, excited-state absorption (ESA), and stimulated emission (SE).28,29,46 For example, in the WT TA spectra (Figure 4A,B), there are three prominent PB bands at ∼805, 815, and 825 nm, which also correspond to the steadyE
DOI: 10.1021/acs.jpcb.7b01270 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry B
In the TA spectra of the V152N mutant (Figure 4C,D), the 805 nm band (WT TA) shifts to ∼803 nm, and the onset of bleaching is immediately apparent and intense at ∼150 fs (black trace), persisting longer than in the WT sample (compare the red, green, and blue traces in Figure 4A,C). The bleaching of the 815 nm band compared to that of the 825 nm band is slightly different, particularly in the early time delays of 500 fs (red trace) to 1 ps (green trace). The initial TA profile of the S73A mutant (Figure 4E,F, black trace) also shows an immediate onset of bleaching of the 805 nm band, similar to the 803 nm band of the V152N mutant. The bleaching ratios between the 805, 815, and 825 nm bands are slightly different in the early time delays of 500 fs and 1 ps (Figure 4E, red and green traces). The TA spectral profile of the Y16F mutant (Figure 4G,H) is dominated by a major PB feature at ∼811 nm and an ESA feature between 780 and 800 nm. A shoulder at ∼805 nm is apparent immediately after laser excitation but persists for less than 500 fs, implying ultrafast transfer to the lower energy components. There is a fast recovery of the main bleaching band at 811 nm compared with the WT and the other FMO mutants (see the cyan and violet traces). The TA spectrum of the Y345F mutant has immediate onset of bleaching at 804, 811, and 823 nm and ESA band between 815 and 820 nm at ∼150 fs after laser excitation (Figure 4I,J, black trace). At 500 fs (red trace) and 1 ps (green trace) time delays, the 804 nm band merges with the 811 nm band such that the bleaching of these two bands dominate the TA spectra, together with an ESA feature between 790 and 800 nm. The 150 fs TA spectrum of the W184F mutant (Figure 4K,L, black trace) has a broad bleaching feature centered at ∼800 nm, whose intensity is considerably less than the 815 nm band, in contrast with those of the WT, V152N, S73A, and Y345F TA spectra at the same time delay (black traces). The bleaching bands at 815 and 825 nm at 1 ps (green trace) have similar intensities, as can also be seen in the WT TA spectra at 1 ps. For all samples, the spectra recorded under oxidizing (Figure 4, left panels) and reducing (Figure 4, right panels) conditions show overall similar spectral line shapes, and the recovery of bleaching intensities differs mainly at later time delays (∼200 ps, cyan traces and 2 ns, violet traces). In WT FMO, this is due to the presence of a redox-dependent quenching mechanism that has been attributed to cysteine residues near the terminal BChls.38,41 At room temperature, this quenching occurs with an ∼60 ps lifetime, based on previous spectroscopic studies.27,35,39 It is evident that this quenching mechanism is still present in the mutant samples studied here. In order to determine the kinetic lifetimes from the TA data, global fitting was performed using a sequential decay model that yielded evolution-associated difference spectra (EADS),45 as shown in Figure 5. The goodness of fitting results was verified by minimization of the χ2 parameter and checking the residual distribution. Sample fits to selected kinetic traces of the main TA peaks are shown in Figure 6 for the oxidized (A, C, E, G, I, and K) and reduced (B, D, F, H, J and L) samples, and the lifetimes obtained are summarized in Table 1 (bottom panels). Global fitting of the TA data required six (WT, Y16F, W184F) or seven (V152N, S73A, Y345F) spectro-temporal components for the samples under oxidizing conditions (Figure 5, A, C, E, G, I, and K). The first EADS is a very fast component with a lifetime of
