Cofactor-specific photochemical function resolved by ultrafast spectroscopy in photosynthetic reaction center crystals

Abstract

High-resolution mapping of cofactor-specific photochemistry in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides was achieved by polarization selective ultrafast spectroscopy in single crystals at cryogenic temperature. By exploiting the fixed orientation of cofactors within crystals, we isolated a single transition within the multicofactor manifold, and elucidated the site-specific photochemical functions of the cofactors associated with the symmetry-related active A and inactive B branches. Transient spectra associated with the initial excited states were found to involve a set of cofactors that differ depending upon whether the monomeric bacteriochlorophylls, BChl_A, BChl_B, or the special pair bacteriochlorophyll dimer, P, was chosen for excitation. Proceeding from these initial excited states, characteristic photochemical functions were resolved. Specifically, our measurements provide direct evidence for an alternative charge separation pathway initiated by excitation of BChl_A that does not involve P*. Conversely, the initial excited state produced by excitation of BChl_B was found to decay by energy transfer to P. A clear sequential kinetic resolution of BChl_A and the A-side bacteriopheophytin, BPh_A, in the electron transfer proceeding from P* was achieved. These experiments demonstrate the opportunity to resolve photochemical function of individual cofactors within the multicofactor RC complexes using single crystal spectroscopy.

In contrast to the diverse array of structures that have evolved for photosynthetic light-harvesting, a conserved feature of photosynthetic reaction centers (RCs) is the hexameric cofactor core that converts optical excited states to charge-separated states as the first chemical reaction in photosynthesis (1–3). Crystal structures from both oxygenic and nonoxygenic photosynthetic reaction center complexes show the hexameric cofactor core to be arranged with a pseudo twofold axis of symmetry consisting of the primary electron donor special pair (P), a dimer of (bacterio)chlorophyll molecules, and two branches, labeled A and B, that are equivalently positioned to serve as electron acceptor chains that extend across the membrane as depicted in the inset of Fig. 1 (4–6). The primary light-initiated electron transfer steps have been extensively investigated in bacterial Type II RCs and are considered to proceed from the excited singlet state P* to A side bacteriopheophytins (BPh_A) using A side bacteriochlorophyll (BChl_A) as a largely kinetically unresolved first electron acceptor (7–9) with the reaction directionality understood to be determined primarily by differences in local protein-site determined energetics for electron transfer along the two cofactor branches (10–12).

Fig. 1.

Polarized ground state optical absorption for a single R. sphaeroides R26 RC crystal (P2₁2₁2₁) at 100 K. Part (A) shows ground state absorption spectra measured with light polarized perpendicular (90°) and parallel (0°) to the long axis of the crystal. The inset in part (A) shows a representative photograph of the wild-type (WT) crystals used in the experiments. The (B) box shows transition moment projections along the crystal a (red) and c (black) axes, respectively. The inset in (B) illustrates the arrangement of the cofactors in RCs. Letters A and B denote branches in RC molecule.

The above view leaves in question the role for the conserved, but nominally inactive B-side pigments. Possible complexities in primary photochemistry for the bacterial RC multicofactor core have been suggested by findings of excitation energy-dependent variation photochemical pathways (13, 14), including those that do not involve P* (15, 16) that are analogous to the primary processes identified in Photosystem I and II reaction centers in oxygenic photosynthesis.(17–20). Analysis of a broad range of spectroscopic, excitation energy transfer, and electron transfer properties based on modeling the electronic structure of RCs have led to the concept that the hexameric cofactor core should be considered as a supermolecule with a ladder of exciton states composed of various contributions from each of the individual cofactors (21–23). Further, recent 2D electronic spectroscopic studies revealed surprisingly long-lived excited-state quantum coherence between cofactors preserved by correlated protein environments in photosynthetic light-harvesting and reaction center complexes (24–29). This intercofactor electronic coherence is significant as a conduit for energy transfer that is potentially relevant to electron transfer as well. Interpigment electronic coupling has been studied by ultrafast pump-probe anisotropy spectroscopy in solution (28, 30, 31). Interpigment couplings in these measurements are detected through delocalized excited-state absorption changes, including coherent wave-packet motion, (25, 31–33) and by polarization studies that resolve transition moment anisotropies that deviate from the individual molecular reference frames (30, 31, 34, 35). Limitations for photo-selection spectroscopy arise from the inability of excitation pulses to selectively isolate a single transition in regions of spectral congestion.

Ultrafast transient absorption spectroscopy on single crystals of the bacterial RC (36) and a cyanobacterial cytochrome b₆/f (37) has been used to resolve cofactor photochemistry directly related to crystal coordinate data. Further, in contrast to photo-selection spectroscopy in solution, polarized ultrafast spectroscopy on single crystals offers the additional opportunity to exploit the fixed orientation of cofactors for both pump and probe measurements. For example, the A-and B-branch cofactors have different projections along the unit cell axes of orthorhombic crystals, allowing both pump and probe to isolate individual transitions of the normally symmetry degenerate pairs (36).

In this paper, we report on polarization selective femtosecond transient absorption spectroscopy of cryogenically cooled (100 K) single reaction center crystals from Rhodobacter sphaeroides. Combinations of pump wavelength and polarization were used to selectively excite optical transitions of P_- (the lower exciton state of P), BChl_A and BChl_B in the Q_y spectral region. By isolating a single transition within the cofactor manifold at a time, we are able to map cofactor-specific charge separation pathways. This approach provides unique opportunities to exploit the fixed orientation of cofactors within crystals to achieve site-specific excitation and detection of cofactors, and to elucidate photochemical functions of cofactors associated with the active A branch and inactive B branch in bacterial RCs.

Results

Ground state polarized absorption spectra for cryogenically cooled (100 K) crystals are shown in Fig. 1. Extinction coefficient weighted projections for each of the RC cofactor optical transitions along the a and c crystal axes are plotted with wavelength positions according to cofactor peak assignments (23, 38, 39) in Fig. 1B. Magnitudes for polarized optical absorption for the RC P2₁2₁2₁ unit cell were calculated from the transition moment, the polarized light electric field, and unit vectors as described previously (36). Absorption spectra were measured with light polarized either parallel (0°) or perpendicular (90°) to the long axis of the needle-shaped crystal (inset of Fig. 1A). X-ray alignment measurements show that the unit cell directions lie parallel to the crystal axes (40, 41). The agreement between the measured spectra and calculated transition moment projections allows the experimental 0° and 90° directions to be identified as lying along the 4RCR crystal unit cell c and a axes, respectively (36). Further details are provided in the SI Text.

As illustrated in Fig. 1A, the ground state polarized absorption spectra show pronounced variation in both absorption peak positions and amplitudes as the polarization angle for the measuring beam is rotated from 0° to 90°. For instance, the BPh Q_x peak at 530 nm reflects predominate contribution from BPh_B at 0° polarization, while the spectrum recorded with 90° polarized light shows split absorption bands corresponding to the BPh_B (530 nm) and BPh_A (545 nm) contributions, respectively. The spectrum recorded with 90° polarized light shows a peak centered at 800 nm that reflects the greater contribution of BChl_A. With 0° polarized light, the BChl peak absorption shifts to 810 nm, and based on the agreement with transition moment projection onto the crystal axes can be understood to be contributed mainly by BChl_B. Note that the upper energy exciton component of P₊ is known to have an absorption band that overlaps with BChl_B around 810 nm but with only approximately 1/3 of the oscillation strength of the BChl_B (42). The red-most absorption band arises from the lower energy exciton component of P, P_- (43). Models have taken into account the effects of coupling between all of the cofactor transition dipoles within the RC and charge transfer states between BChl comprising P.(22, 23). The polarized spectra recorded for P_- is found to have a strong preferential absorption with 90° polarized light. The absorption spectra shown in Fig. 1 are from a R26 crystal. The experiments described below are performed on wild-type (WT) crystals. The ground state dichroism is similar for R26 and WT crystals and the absorption spectra for a typical WT crystal are shown in Fig. S1A.

Based on the ground state dichroism discussed above, the polarization angle can be combined with wavelength to selectively excite and probe otherwise degenerate optical transitions of cofactors associated with the A and B branches (36). The opportunity to resolve cofactor-specific photochemical function by polarization selective transient spectroscopy measurements in single crystals is illustrated in Fig. 2. Three pump wavelength and polarization combinations were utilized to selectively excite the Q_y transition of P_- (900 nm, bandwidth approximately 28 nm, 90° polarization Fig. 2A), BChl_A (790 nm, bandwidth approximately 20 nm, 90°polarization, Fig. 2B), and BChl_B (824 nm, bandwidth approximately 23 nm, 0° polarization, Fig. 2C). The spectra of the pump pulses are shown in Fig. S1B. The white-light probe polarization was set to be either 90° (top graphs) or 0° (bottom graphs). Probing at 0° and 90° produced dramatically different spectra, demonstrating the ability to selectively probe different transitions in the manifold (36). Specifically, bleaching of the Q_x transition of BPh_A around 545 nm is observed almost exclusively with the 90° polarized probe. This observation is consistent with the experimental polarized ground state spectra, in that the BPh_A Q_x transition moment is aligned preferentially with the a axis. Transient spectra for each pump and probe combination are compared at four delay times in Fig. 2. The corresponding 2D graphs illustrating the full time progression for the transient absorption measured during the experimental time frame are shown in Fig. S2.

Fig. 2.

Pump wavelength-dependent patterns of transient optical absorption changes in a single Rb. sphaeroides-wt crystal at 100 K. The pump is adjusted to 900 nm (90°), 790 nm (90°), and 824 nm (0°), in boxes (A, B, and C), to selectively pump Q_y optical transitions of P_-, BChl_A and BChl_B, respectively. The top spectra are for probing at 90° with respect to the crystal c axis and the spectra are offset for clarity. The vertical lines are placed at 545 and 595 nm. The bottom spectra are the spectra for probing at 0°.

The initial transient states and progression of subsequent energy and electron transfer events show clear differences that depend upon the cofactor-specific excitation. The position of the initial BChl Q_x transition bleach near 600 nm accompanying excitation in the near-IR Q_y region was found to shift characteristically depending upon the BChl cofactor selected for photoexcitation. For example, when the Q_y transition of P_- is selectively excited, the initial bleaching is at 604 nm corresponding to the Q_x transition of P (Fig. 2A, 0.1 ps trace). We note that the P Q_x transition makes only a minor projection onto the crystal axis probe by the 90° probe orientation. The bottom graph of Fig. 2A shows the transient spectrum measured with 0° probe following 900 nm, 90° excitation, and the larger P Q_x bleaching. When BChl_A is selectively excited, the initial bleaching occurs at 595 nm consistent with the Q_x transition of BChl_A (Fig. 2B, 0.1 ps trace). Finally, an initial bleaching at 609 nm is observed with an excitation wavelength of 824 nm and a polarization at 0° (Fig. 2C, 0.1 ps trace). This position is different from the Q_x transition of P at 604 nm and if the P₊ transition were excited, one would expect the bleach to be at 604 nm. Therefore, we assign the bleach observed at 609 nm to the Q_x transition of BChl_B. Even though there is significant spectral overlap between Q_y transition of BChl_B and P₊ state, the instantaneous bleach at 609 nm instead of at 604 nm indicates that the pump wavelength of 824 nm at 0° polarization excites mostly the Q_y transition of BChl_B instead of P₊.

In addition to the resolution of BChl Q_x bleaching components, the earliest transient spectra show initial excite states to involve a set of cofactors that differ depending upon whether BChl_A, BChl_B, or P_- was chosen for excitation. For example, with the selective excitation of P_-, a partial absorption decrease of the BPh_A Q_x transition at 545 nm is seen in the transient spectrum recorded with a 100 fs delay (Fig. 2A). This instantaneous BPh_A bleach recovers, and by 200 fs the transient spectrum reflects a localized P* state. When BChl_A is selectively excited by the 790 nm pump, a larger magnitude of bleach at 545 nm is observed to coincide with excitation. In addition to the instantaneous bleach of BPh_A, bleaching of carotenoid (Car) transition at 510 nm is also evident, suggesting that the initial excited-state perturbation extents to the Car cofactor. With BChl_B excitation by pump wavelength of 824 nm (Fig. 2C), instantaneous bleaching of both the BPh_A (545 nm) and Car (510 nm) cofactors is also observed. Remarkably, no involvement of BPh_B is observed upon excitation of any of the BChls, even the adjacent BChl_B. A small absorption loss at 530 nm is observed in the 100 fs transient spectra upon BChl_A and BChl_B excitation (e.g., Fig. 2 B and C) that corresponds to the position of the BPh_B Q_x absorption. However this feature is only detected with 90°, but not 0° probe polarization, Fig. 2. This polarization pattern matches that measured for the BPh_A Q_x, in the ground state spectrum, and is the opposite of that measured and predicted from the crystal coordinates for the BPh_B Q_x direction, Fig. 1.

Proceeding from these different initial excited states, distinguishable photochemical pathways are also resolved. When P_- (Fig. 2A) or BChl_B (Fig. 2C) are selectively excited, the initial excited states that include different amounts of BPh_A and Car contributions, relax in 250 fs to have characteristics of a localized P*. In both cases, the P* state in turn evolves to the state, marked by characteristic BPh_A and BChl_A Q_x bleaching at 545 nm and 595 nm, respectively and an absorption of at 670 nm. In contrast, when BChl_A is excited, the initial BPh_A Q_x bleach does not recover fully; i.e., the bleaching at 545 nm persists at 0.25 ps (Fig. 2B, 90° probe 0.25 ps trace), suggesting formation of a possible initial charge-separated state that forms directly, and does not evolve from P*. This interpretation is supported by the companion 0° probe spectrum (Fig. 2B, 0° probe), which measures the broad 650–700 nm bacteriochlorin anion absorption band at 0.25 ps (44). The absorption band at 650–700 nm at early delay time (0.12 ps and 0.25 ps traces) is broader than at 80 ps (Fig. 2B, 0° probe), which may reflect the biradical charge-transfer state formed initially upon BChl_A excitation. In contrast, upon excitation of either BChl_B or P_- , a BPh anion absorption band is not observed until charge separation is completed and the P⁺ state is formed.

Fig. 3 shows the single-wavelength kinetics of the BPh_A Q_x transition (545 nm - 540 nm, 90° probe), taken from the data shown in Fig. 2, and measured following selective excitation of P_-, BChl_A, and BChl_B, that also reflect the site-specific charge separation pathways. At the earliest observable time (approximately 100 fs), a dip with a width of the time resolution of the experiments (approximately 150 fs) is present in all three decay curves corresponding to the instantaneous bleach observed in the spectra. The magnitude of initial BPh_A transient is 10%, 45%, and 85% of the total absorption change following excitation of P_-, BChl_A, and BChl_B, respectively. The dynamics after the initial bleach are fitted with a single exponential function. When P and BChl_B are selectively excited, the initial BPh_A bleach fully recovers (i.e., ΔA recovers to 0 at approximately 0.25 ps) to a P* state that subsequently decays with a time constant of approximately 1.5 ps to the state. When BChl_A is selectively excited, the initial BPh_A bleach does not recover fully (i.e., ΔA does not recover to 0), which is attributed to a mixture of and P* states. The formation of following these excited states is fitted with a time constant of 1.9 ± 0.1 ps.

Fig. 3.

Pump wavelength-dependent kinetics for the BPh_A Q_x absorption (545 nm - 540 nm) in the same Rb. spheroides-wt crystal as in Fig. 2. The pump was adjusted to selectively excite different RC optical transitions P- (A), BChl_A (B) and BChl_B (C). The crystal temperature was 100 K. The red lines show single exponential decay fits.

The resolution of as an intermediate in the electron transfer from P* to BPh_A is also resolved in these measurements. Although resolvable in the spectra shown above, this is most clearly illustrated with a crystal that exhibited a blue shift of the BChl_A Q_x peak that allowed a very clear resolution of transient function shown in, Fig. S3. With excitation of P_-, the initial BChl Q_x bleach shows a single band with a peak at 604 nm associated with P*. During the subsequent electron transfer to form , an additional bleaching band builds in at 590 nm resulting in a final double peaked BChl Q_x absorption decrease, Fig. S3. The individual contributions of BChl_A and BPh_A can be tracked, in difference transient spectra using the 150 fs P* as a reference spectrum, Fig. 4A, and corresponding single wavelength kinetics are plotted in Fig. 4B. The 2.2 ± 0.2 ps single exponential bleaching kinetics associated with BPh_A reduction in this crystal lag behind the bleaching of BChl_A, which shows biexponential kinetics with decay times of 0.9 ± 0.2 ps and 2.3 ± 0.8 ps, as described Fig. 4. Similar lag time of approximately 0.9 ps for reduction of BPh_A compared to that of BChl_A is also found from difference transient spectra obtained for the crystal shown in Fig. 2, plotted in Fig. S4. We note that many experimental parameters can cause shifts in the absorption peaks for the RC cofactors (45, 46). In the case of the crystal used in Fig. 4, these perturbations allowed the transient kinetics for BChl_A to be more readily recognized, but comparable kinetics are observed in crystals without these perturbations, Fig. S4.

Fig. 4.

Difference transient absorption spectra following formation of P*. Part (A) shows difference transient spectra recorded with delays from 150 fs to 80 ps, and following subtraction of the spectrum recorded with a 150 fs delay from the pump pulse. This subtraction removes contributions of ground state bleaching of P and highlights the time-progression of the BPh and BChl Q_X transient components. Part (B) shows single wavelength BPh_A and BChl_A Q_X transient kinetics measured at 548 nm and 590 nm, respectively. The 590 nm transient was normalized to match the 548 nm amplitude. The BPh_A 548 nm kinetics were fit (solid line) using a single exponential 2.2 ± 0.2 ps decay. The BChl_A 590 nm kinetics were fit (solid line) using a biexponential with 0.9 ± 0.2 ps and 2.3 ± 0.8 ps decay times corresponding amplitudes of 0.0085 ± 0.002 and 0.004 ± 0.003, respectively. Excitation was provided by 890 nm pump pulse, and both pump and probe with 90° polarized, using the transient spectra shown in Fig. S3.

As shown here, polarization selective single crystal spectroscopy offers opportunities to resolve photochemical pathways within multicofactor photosynthetic complexes. For comparison, Fig. S5 provides an example of comparable wavelength selected excitation of detergent solubilized RCs in solution. A more complete analysis has been carried out earlier by van Grondelle and coworkers demonstrating charge-separation that did not initiate from P* (15, 16). Overall the transient absorption spectra and lifetimes measured in solution agree with those of single RC crystals, implying that the photochemical pathways are retained upon RC crystallization. The cofactor spacing between RCs within the crystal unit cell is greater than , and crystallographic packing is unlikely to alter primary photochemistry. Spectra recorded with 0.1 ps delay show initial excited-state spectra that include contributions from BPh_A and the carotenoid cofactors, along with those of the BChl or P cofactors used for excitation. The initial excited-state spectra relax to a more localized on P* by 0.25 ps. However, because of the absence of dichroic selection, 790 nm femtosecond pump pulses excite both BChl_A and BChl_B. The responses of BChl_A and BChl_B are convoluted, and the different photochemical functions of BChl_A and BChl_B are not readily resolved.

Discussion

Nature of the Initial Excited-States.

The instantaneous bleach of BPh upon excitation of BChl has been observed previously in decay associated spectral analysis of the spectra of RC solution (47). Here we are able to observe directly from the raw data of single crystal spectroscopy, and further discriminate between the involvement of BPh_A as part of a charge-transfer state created by excitation of BChl_A, and the different involvement of BPh_A as part of an excited state produced by excitation of either BChl_B or P.

The involvement of multiple cofactors upon single cofactor excitation can possibly be explained by strongly correlated electronic levels, possibly including charge-transfer mixing, among the individual cofactors in the RC (21, 28). Long-lived electronic coherence between the excited states of BPh and BChl was observed recently with 2-color photon echo experiments and was attributed to strong correlation between the protein-induced fluctuations in the transition energy levels of BChl and BPh (28). Electronic coherence between the cofactors allows the excitation to move coherently in space, enabling excited populations moving between different cofactors (24–29). Based on the observation of electronic coherence is much longer lived at low temperature (28), the fact that we observe multicofactor transitions in the initial excited states only at cryogenic temperature suggests that electronic coherence might play an important role. We also observe instantaneous bleaching of Car upon excitation of BChls and P, indicating Car participates in excitation energy transfer in the RC manifold. This observation is in accordance with the recent study of PSII reaction centers in which Car was suggested to act as an electronic coupling bridge and enhance energy transfer from the antenna to the reaction centers (48).

A possible alternative mechanism to explain the instantaneous bleaching of BPh_A and Car cofactors coupled to BChl excitation is through electrochromic Stark effects (49), associated with charge-transfer dipole character of initial BChl excited states. However, a Stark effect alone cannot satisfactorily explain our observations for two reasons. First, the Stark effect for an internal electric field would typically be expected to produce transient spectra with a derivative band shape, (50) but neither the instantaneous BPh_A nor Car transient bands appear to have such shapes. Secondly, a comparison of BPh_A and BPh_B Q_x directions shows that both cofactors have comparable alignments with respect to possible excited charge-transfer state directions, Table S1, Fig. S6. Measurements using internal and external electric fields show that the B-side cofactors have a markedly enhanced electrochromic response, indicative of a dielectric asymmetry in the RC (49). We find that the opposite effect, that only BPh_A, and not BPh_B, response to BChl initial excited states.

These considerations suggest that the instantaneous bleach of multiple cofactor transitions upon single cofactor excitation of BChl_A,B and P is most likely due to electronic coupling between the cofactor and not due to Stark effect. The lack of an involvement of the inactive BPh_B with the initial excited state formed upon excitation of BChl_A, BChl_B, or P implies an inherent asymmetry in electronic coupling between cofactors. Mutagenesis experiments have shown that directional electron transfer in RCs is largely determined by protein-tuned energies of the cofactor molecular orbitals (10, 12). Such energy level tuning can be expected to regulate the magnitudes of exciton, charge transfer, and coherent couplings between cofactors.

Photochemistry Initiated from P.

Following excitation of P, the consensus view is that BChl_A functions as the initial electron acceptor for P*, forming the transient state with a 3 ps risetime (room temperature), but is rapidly converted by electron transfer to BPh_A with a 0.9 ps time constant (7). However the overlap of the ground and excited-state optical absorptions of the cofactors has posed challenges for easy verification of this reaction scheme. The spectral resolution offered by polarized transient spectroscopy on crystals shown here provides a very clear resolution of biphasic transients of the BChl_A cofactor prior to BPh_A reduction. Pathway 1 in Scheme 1 summarizes the photochemistry initiated from P. These results are consistent with the kinetic scheme in which BChl_A functions as the initial electron acceptor (7), but also with a more complex function discussed below.

Scheme 1.

Cofactor-specific charge separation pathways revealed by single crystal spectroscopy. The brackets list the combination of cofactor optical transitions observed for the different initial excited-states, but not the levels of their contributions.

Photochemistry Initiated from BChl_A and BChl_B.

While selective excitation of BChl_B was found to create a delocalized excited state that uniformly decayed (< 200 fs) to form a localized excited state on P as described above (pathway 2 in Scheme 1), more complex, dual photochemical pathways were found to accompany BChl_A excitation (pathways 3a and 3b in Scheme 1). With up to 50% yield, BChl_A excitation was seen to create an initial charge-separated state, , that did not evolve from P*, that persists, apparently decaying by hole transfer to form (Pathway 3b). We note that 650 nm -670 nm BPh “anion band” absorption for this initial state differs from that detected during the transient state, possibly a reflection of the coupled biradical natural of this initial charge-separated state. The remaining portion of the BChl_A excitation appeared to decay to create P*, from which sequential electron transfer to BPh_A occurs with approximately 2 ps time constant. The presence of dual pathways suggests heterogeneity in the coupling of BChl_A and BPh_A that might add complexity to the sequential electron transfer pathway.

A component of primary charge separation occurring within the BChl_ABPh_A pair that does not evolve from P* was suggested previously based on decay associated spectral analysis of transients recoded from solutions at low temperature (15, 16). However, the broadness of ultrafast pump pulses and overlap of the BChl_A and BChl_B absorptions prevents exclusive cofactor excitation. The polarization selective spectroscopy in single crystals shown here resolves these cofactor-specific pathways with greater precision, and allows direct resolution of cofactor-specific photochemistry in the polarized transient spectra. The confirmation of alternative photochemical pathways in the bacterial RC, including those that do not involve P, provides a relevant model system for comparative analysis of photochemistry in RCs from oxygenic photosynthesis.

Single crystal ultrafast transient spectroscopy is shown to provide a means to deepen our understanding of the photophysical function of the multicofactor RC core, and establish the foundation for extending this approach to probe energy and electron transfer pathways in RC-light harvesting, PSI, PSII crystalline complexes.

Materials and Methods

Details regarding preparation of reaction center crystals, sample preparation for low temperature optical measurements, ground state absorption and transient absorption spectroscopies are presented in SI Text.