Carotenoid-to-Bacteriochlorophyll Energy Transfer in the LH1âRC

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Carotenoid-to-Bacteriochlorophyll Energy Transfer in the LH1-RC Core Complex of a Bacteriochlorophyll b-Containing Purple Photosynthetic Bacterium Blastochloris viridis Nikki Cecil Macasinag Magdaong, Dariusz M. Niedzwiedzki, Carrie Goodson, and Robert E. Blankenship J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b04307 • Publication Date (Web): 24 May 2016 Downloaded from http://pubs.acs.org on June 5, 2016

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The Journal of Physical Chemistry

Carotenoid-to-Bacteriochlorophyll Energy Transfer in the LH1-RC Core Complex of a Bacteriochlorophyll b-Containing Purple Photosynthetic Bacterium Blastochloris viridis Nikki Cecil M. Magdaong1,2,3, Dariusz M. Niedzwiedzki3, Carrie Goodson1 and Robert E. Blankenship1,2,3*

1

Department of Biology, Washington University in Saint Louis, One Brookings Drive, St. Louis, MO 63130 USA

2

Departments of Chemistry, Washington University in Saint Louis, One Brookings Drive, St. Louis, MO 63130 USA

3

Photosynthetic Antenna Research Center, Washington University in Saint Louis, One Brookings Drive, St. Louis, MO 63130 USA

*Corresponding author*: Robert E. Blankenship; Departments of Biology and Chemistry, Washington University in St. Louis, St. Louis, MO 63130-4899, USA. Tel.: +1 314 935-7971; fax +1 314 935-4432.

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Abstract

Carotenoid-to-bacteriochlorophyll energy transfer has been widely investigated in bacteriochlorophyll (BChl) a-containing light harvesting complexes. Blastochloris (B.) viridis utilizes BChl b, whose absorption spectrum is more red-shifted than that of BChl a. This has implications on the efficiency and pathways of carotenoid-to-BChl energy transfer in this organism. The carotenoids that comprise the light-harvesting reaction center core complex (LH1RC) of B. viridis are 1,2-dihydroneurosporene and 1,2-dihydrolycopene, which are derivatives of carotenoids found in the light harvesting complexes of several BChl a-containing purple photosynthetic bacteria. Steady-state and ultrafast time-resolved optical spectroscopic measurements were performed on the LH1-RC complex of B. viridis at room and cryogenic temperatures. The overall efficiency of carotenoid-to-bacteriochlorophyll energy transfer obtained from steady-state absorption and fluorescence measurements were determined to be ~27% and ~36% for 1,2-dihydroneurosporene and 1,2-dihydrolycopene, respectively. These results were combined with global fitting and target analyses of the transient absorption data to elucidate the energetic pathways by which the carotenoids decay and transfer excitation energy to BChl b. 1,2-Dihydrolycopene transfers energy to BChl b via the S2 → Qx channel with kET2 = (500 fs)-1 while 1,2-dihydroneurosporene transfers energy via S1→ Qy (kET1 = (84 ps)-1) and S2 → Qx (kET2 = (2.2 ps)-1) channels.


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Introduction The initial event of light-harvesting in photosynthesis is carried out by pigment-protein complexes with varying structures and pigment composition, depending on the organism from which they originate.1-2 In purple photosynthetic bacteria, there are two types of light-harvesting (LH) antennas: LH1 which is closely associated with the reaction center (RC) in a fixed stoichiometric ratio; and a peripheral LH2 antenna surrounding the LH1-RC complex that is variable in number, depending on environmental factors such as light intensity.3-5 The energy of light absorbed by LH2 is transferred to LH1 and finally to the RC where primary charge separation takes place.6-7 Crystal structures of various LH2 complexes8-10 have revealed the annular arrangement of alpha-helical polypeptides denoted α and β, which form the inner and outer surfaces, respectively. Bacteriochlorophylls (BChls) and carotenoids (Cars) are bound noncovalently within the α/β heterodimer, which together form an oligomeric assembly, the size of which depends on the bacterial species. A similar concept of assembly in LH1 is observed in the crystal structures of the LH1-RC core complex from Rhodopseudomonas (Rps.) palustris11 and Thermochromatium (T.) tepidum.12 The α/β pairs of LH1 surrounds the RC in an incomplete (Rps. palustris) or closed ring structure (T. tepidum). In Rhodobacter (Rb.) sphaeroides, the LH1 complex does not enclose the RC completely due to the presence of an additional PufX protein, which leads to the formation of a dimeric RC-LH1-PufX complex and facilitates quinone movement in the membrane.13 In photosynthesis, carotenoids perform light harvesting, photoprotective and structural functions.14-16 Carotenoids increase the absorption cross section of photosynthetic organisms by absorbing light in the region where (B)Chls are not efficient, and transfer the excitation energy to neighboring (B)Chls.17 In the presence of excess excitation energy in the photosynthetic unit,



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carotenoids carry out photoprotection by either quenching (B)Chl triplets to avoid forming harmful singlet oxygen species, or directly scavenging any singlet oxygen that forms.15-16, 18 This excess energy is then dissipated as heat. The unique photophysics of carotenoids necessary to carry out these functions arise from the polyene chain backbone having conjugated carboncarbon double bonds. There are three generally accepted electronic states that comprise the carotenoid energy level complexion, whose irreducible representations are based on an idealized C2h symmetry of all-trans linear polyenes.19-20 Selection rules in quantum mechanics dictate that in order for a transition to occur there must be a change in symmetry and pseudoparity sign such that one-photon absorption from the ground state S0 (11Ag˗) to the low-lying first excited singlet state S1 (21Ag˗) is forbidden while transition to the S2 (11Bu+) state is strongly allowed and accounts for carotenoid absorption in the visible region.21-25 Other “dark” states are also found within the vicinity of S1 and S2, which provide additional relaxation pathways and are involved in energy transfer to (B)Chl, but the nature of these states remain a matter of considerable debate.26 Blastochloris (B.) viridis27 (formerly Rhodopseudomonas (Rps.) viridis)28 is a BChl bcontaining purple non-sulfur bacterium with no peripheral LH2 antennas and instead has an LH1 complex closely associated with the RC, referred to as the core complex. B. viridis LH1 complex has main absorption maximum at ~1020 nm, far more red-shifted than its BChl a-containing counterparts. A non BChl-binding polypeptide, γ (MW = 4 kDa), postulated to have structural function,29-30 is present in addition to the BChl-binding α (MW = 6.8 kDa) and β (MW = 6.1 kDa) polypeptides. The availability of a crystal structure for the photosynthetic reaction center from B. viridis31-32 was vital in elucidating the mechanism of energy transfer and trapping processes in this organism. No high-resolution crystal structures are available for the LH1 or


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LH1-RC complex in B. viridis, however. Early structural and functional determination was based on electron microscopy and data modelling33-34 combined with biochemical analyses.29,

35-38

More recently, high-resolution imaging of the core complex in the native membrane was accomplished using atomic force microscopy,39 the results of which show a single reaction center surrounded by 16 LH1 subunits in a closed ellipsoid configuration. This structure is supported by results from preliminary X-ray crystallography studies.40 Previous spectroscopic investigations on the core complex of B. viridis have focused on the Qy near infrared (NIR) absorption region of BChl b to understand excited state properties and energy transfer and trapping dynamics between LH1 and RC.41-49 The basic spectroscopic characterization involving steady-state fluorescence spectroscopy (BChl b fluorescence excitation) demonstrated that carotenoids bound in the LH1 complex are able to transfer excitation energy to BChl b with ~40 % efficiency.50 However, an exact mechanism of Car → BChl b energy transfer has not been further investigated. The fact that Car → BChl b energy transfer occurs in this LH1 antenna system is not trivial. The major carotenoid that is incorporated into the antenna is 1,2-dihydroneurosporene, a simple derivative of the betterknown neurosporene. Spectroscopically, these two carotenoids are practically identical and it is expected that both will share other excited state properties like S1 state energy and lifetime. If so, simple arithmetic calculations show that there is a substantial mismatch between both the S2 and S1 excited states of the carotenoid and the Qx and Qy bands of BChl b. The energetic gaps between hypothetically interacting states is estimated to be ~3800 cm-1 (~110 nm) for S2–Qx and ~4500 cm-1 (320 nm) for S1–Qy. These gaps are so substantial that it is tempting to assume that Car → BChl b energy transfer will be nearly impossible. The experimental results shown here, however, demonstrate otherwise. 5 ACS Paragon Plus Environment


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In this work, the spectroscopic properties and energy transfer pathways of the carotenoids in purified LH1-RC complex from B. viridis have been investigated in detail. Steady-state and time-resolved absorption and fluorescence spectroscopic measurements were carried out at room and cryogenic temperatures. Global fitting and target analysis of transient absorption data were performed in order to elucidate the pathways by which carotenoids transfer energy to BChl b. Materials and Methods Sample preparation B. viridis strain DSM 133 cells were grown anaerobically in 1:1 YPS/RCV media.51-52 Cells were harvested by centrifugation and washed with 20 mM MES buffer containing 100 mM KCl, pH 8. Approximately 5 g of whole cells were resuspended to a volume of 30 mL with 20 mM Tris buffer, pH 8.0. A small amount of DNAse and MgCl2 were added to the sample before sonicating the cells using a Branson 450 sonifier. After sonication, the sample was centrifuged at 12,000 × g for 10 min at 4 °C using a Sorvall SS-34 rotor to pellet unbroken cells and cellular debris. The supernatant from this step was spun at 210,000 × g for 2 h, 4 °C using a Beckman Type 70 Ti (BT) rotor to collect the photosynthetic membranes. The pelleted membranes were resuspended with 20 mM Tris buffer, pH 8.0 to an optical density at 1010 nm, OD1010, of 100. Subsequently, 30% (v/v) lauryldimethylamine N-oxide (LDAO, Sigma) was added to a final concentration of 0.3% (v/v) at room temperature for ~5 min followed by centrifugation at 260,000 × g using a BT rotor for 1 h at 4 °C. The supernatant from this step was discarded. The pellet was resuspended with 20 mM Tris buffer, pH 8.0 to an OD1010 of 100, followed by a slow dropwise addition of dodecyl-β-D-maltoside (DDM, Calbiotech) to a final concentration of 5% (w/v). The mixture was stirred at room temperature for 2 h in the dark and centrifuged at 260,000 × g using a BT rotor for 1 h at 4 °C. Approximately 4 mL of the supernatant containing the


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solubilized LH1-RC core complex was loaded onto a tube containing 3 mL each of the following sucrose concentrations dissolved in 20 mM Tris buffer (pH 8.0), 0.02% DDM: 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8 M. The sucrose gradient tubes were spun for 16 h at 210,000 × g using a BT rotor at 4 °C. Two bands were obtained after centrifugation, which contained free pigments (top) and the RC-LH1 core complex (bottom). The bottom band was carefully collected and sucrose was removed using a PD-10 (GE Healthcare) column previously equilibrated with Tris buffer containing 0.02% DDM, pH 8.0 (buffer A). The sample was concentrated using centrifugal filters (Amicon 30,000 MW cutoff) and loaded onto a Sephacryl S-300 (GE Healthcare) gel filtration column (1.5 × 27 cm) previously equilibrated with buffer A containing 150 mM NaCl. Fractions with A280/A1010 = 0.79–0.82 were pooled, concentrated using Amicon filters, and desalted using PD-10 columns. The samples were flash frozen in liquid nitrogen before storage in the ˗80 °C freezer. Pigment composition analysis The pigment composition of the LH1-RC complex was analyzed using an Agilent 1100 series high-performance liquid chromatograph (HPLC) equipped with a Zorbax Eclipse XDB C18

column

by

applying

an

isocratic

mobile

phase

that

consisted

of

acetonitrile/methanol/tetrahydrofuran (58:35:7 v/v/v) at a flow rate of 1.5 mL/min. The pigments were extracted from the complex by mixing ~100 µL of concentrated LH1-RC with 2 mL of acetone/methanol (7:2 v/v). The sample was spun using a tabletop microcentrifuge for ~2 min. The acetone/methanol extraction was repeated until the pellet was colorless. Petroleum ether (~3 mL) was added to the supernatant followed by ~2 mL of warm salty water. The ether fraction was carefully collected using a glass pipet and transferred to another vial, then dried under a stream of argon gas while being kept on ice under dim light. The dried extract was then



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resuspended with acetonitrile and filtered before injection into the HPLC. Data analysis was performed using ChemStation software. Steady-state absorption and fluorescence spectroscopy Steady-state absorption spectra at room temperature (RT) and at 77 K were recorded using a Shimadzu UV-1800 spectrophotometer. For cryogenic temperature measurements, the buffer A – LH1-RC solution was mixed with glycerol in a 6:5 (v/v) ratio, transferred to a 1 cm square plastic cuvette and frozen in VNF-100 liquid nitrogen cryostat (Janis). Fluorescence and fluorescence excitation spectra were recorded at RT using a Horiba-Spex Nanolog fluorometer, on a sample with absorbance ≤0.1 at the emission wavelength (~1050 nm). The excitation and detection bandwidths were set to 2−4 nm, and fluorescence was measured at a right angle from the light source. 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 Spectra-Physics femtosecond laser system described previously.53 Excitation was preferentially set to excite the carotenoids at 484 nm (1,2-dihydroneurosporene) or at 525 nm (1,2-dihydrolycopene). The energy of the excitation beam was kept between 200 and 400 nJ at RT, corresponding to an intensity of ~0.5–1 × 1014 photons/cm2. For 77 K studies, the energy of the pump was decreased to 100 nJ (~3 × 1013 photons/cm2) in order to minimize permanent photobleaching of the sample. TA data processing, global and target analyses Dispersion in the LH1-RC TA datasets was corrected using Surface Xplorer software (Ultrafast Systems) by building a dispersion correction curve from a set of initial times of transient signals obtained from single wavelength fits of a few representative kinetics. Global 8 ACS Paragon Plus Environment

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and directed kinetic (target) analyses of the TA results were performed using a modified version of ASUFit software (provided by Dr. Evaldas Katilius of Arizona State University) and CarpetView software (Light Conversion), respectively. In principle, a temporal evolution of a TA signal, ∆A, at any delay time (t) can be decomposed to a superposition of nth Ci(t)⋅∆Ai(λ) products:54 n

∆ A( t , λ ) = ∑ C i ( t )∆ Ai ( λ )

(1)

i =1

The ∆Ai(λ) could be defined as a type of spectral component that will have temporal dynamics (rise and decay) defined by a concentration Ci(t) (kinetic function). Depending on how a fitting model is constructed (how mutual dependencies between Ci(t) are set), the ∆Ai(λ) components may either have a particular physical meaning or be some mathematical representations of the fitting results. In an ideal scenario, a fitting model mimics true, expected decay of an excitation (e.g. excitation energy decay/transfer after exciting S2 excited singlet state of the carotenoid in the LH1-RC) and in such case, the ∆Ai(λ) components are commonly called species associated difference spectra (SADS), and the fitting procedure is called target analysis.54 The SADS will spectroscopically comprise the transient absorption spectrum of a specific molecular species (e.g. carotenoid S1 state) and associated bleaching of the ground state absorption of the involved molecule. For complex multi-pigment systems like bacterial LH complexes, obtaining SADS might be challenging such that less complex global analyses of TA data are often used. The fitting model commonly used is one that assumes sequential decay of excitation in which the decay rates are slower (decay lifetimes are longer) in each subsequent step. The fitting results of TA using a sequential decay model are typically abbreviated as EADS — evolution associated difference spectra.54 Even though these typically do not represent SADS (unless sequence actually represents true excitation decay), EADS provide useful information about the temporal 9 ACS Paragon Plus Environment


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characteristics of the excited states of the molecules involved. The outcome could be further used to predict a realistic excitation decay path. Both fitting models have been applied to the TA results from the studied LH1-RC complex. The information obtained from EADS was subsequently used to perform target analysis. In both fitting procedures, the concentration of the initial spectro-kinetic component, EADS or SADS, associated with the S2 excited state of the carotenoid, has been convoluted by the temporal instrument response function (IRF). The IRF was assumed to have a Gaussian-like shape with the full width at half maximum (FWHM) of ~200 fs and was used as a fixed parameter.


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Results Pigment composition The pigment composition of the LH1-RC complex from B. viridis was analyzed using HPLC and the results are shown in Figure 1. The carotenoid composition of B. viridis has been previously reported55 to consist of 1,2-dihydrogenated derivatives of neurosporene and lycopene.

Figure 1. Pigment composition analysis of the LH1-RC complex from B. viridis. (Top) HPLC chromatogram at 470 nm detection and corresponding absorption spectra (inset) of the labeled



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peaks. A: BChl b, B: 1,2-dihydrolycopene, C: 1,2-dihydroneurosporene, D: 15,15’-cis-1,2dihydroneurosporene. (Bottom) Structures of the carotenoids. The corresponding absorption spectra (inset) of the labeled peaks in the sample chromatogram (top panel) confirm the presence of (B) 1,2-dihydrolycopene (1,2-dhlyco) and (C) 1,2-dihydroneurosporene (1,2-dhneuro) which are spectrally identical to those of lycopene and neurosporene, respectively.55-56 The major carotenoid in the complex is 1,2-dhneuro with the (0– 0) band (in the mobile phase solvent) at 470 nm (blue trace, inset). 1,2-dhlyco has similar absorption line shapes to 1,2-dhneuro except that its (0–0) band is red shifted to 505 nm (red trace, inset), consistent with the fact that these two molecules differ only in the number of conjugated carbon-carbon double bonds (N) as shown in Figure 1 (bottom panel). The small peak eluting at ~35 min is due to 15,15’-cis-1,2-dhneuro which is the carotenoid present in the reaction center.32 The presence of a “cis” band is indicated by the peak at ~330 nm.57 Other minor peaks in the chromatogram whose absorption spectra are not shown resemble those of neurosporene (~20 min) and lycopene (~24 min), whose spectra are identical to their 1,2-dihydro counterparts, as mentioned above. The early eluting components at 5–7 min have BChl-like spectra and are most probably BChl b oxidation products that are unavoidable during sample extraction and preparation. A relative estimate of 1,2-dhneuro:1,2-dhlyco was obtained from the peak area of each component divided by the extinction coefficient of the carotenoid57 and is roughly 4:1.

Steady-state absorption and fluorescence spectroscopy Absorption spectra of the B. viridis core complex recorded at room temperature (RT) and 77 K are shown in Figure 2. The spectra consist of multiple bands associated with BChl b bound


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to the LH1 antenna (Soret band at ~400 nm, Qx transition at ~600 nm and Qy transition ~1015 nm (RT) or ~1040 nm (77 K)).

Figure 2. Steady-state absorption spectra of the LH1-RC complex from B. viridis at room temperature (black trace) and 77 K (red trace).

The band at ~830 nm is associated with the Qy transition of monomeric accessory BChl b while the 790 nm peak is due to the bacteriopheophytins bound in the reaction center.58-59 The lower exciton state formed by the splitting of the Qy band of BChl b that serves as reaction center primary donor shows up at ~960 nm58, 60 and is hidden under the Qy band originating from LH1bound BChls. Upon freezing the sample to 77 K, the absorption lineshapes become sharper and narrower (red trace). In addition, the BChl b Qy band red shifts by ~25 nm from 1015 to 1040 nm. The absorption spectrum exhibits three distinct peaks at 453, 484 and 525 nm associated predominantly with the carotenoids in the LH1 complex.



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Figure 3 shows the fluorescence emission (em, blue trace) and excitation (exc, red trace) spectra of the LH1-RC complex from B. viridis recorded at room temperature.

Figure 3. Absorptance (1-T, where T is transmittance) (black trace) overlaid with fluorescence emission (blue trace) and fluorescence excitation (red trace) spectra. The profiles were used to determine the overall Car-to-BChl efficiency of energy transfer (EET) at positions indicated by dashed lines. The region between ~650–900 nm of the excitation spectrum was omitted for clarity.

The emission spectrum was recorded after the sample was excited at the BChl b Qx band at 600 nm. The fluorescence excitation spectrum was recorded by detection at the wavelength of maximum emission at 1040 nm. The 1˗T and exc spectra were normalized at the BChl b Qy and Qx bands. The overall Car-to-BChl efficiency of energy transfer (EET) was calculated from the ratio of the spectral amplitudes of the carotenoids in the excitation spectrum to that in the 1˗T


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spectrum at the regions indicated by dashed lines in Figure 3, where the contribution from BChl b is minimal. The LH1-RC core complex has an overall Car-to-BChl b EET of ~27% at 484 nm for 1,2-dhneuro, and ~36% at 525 nm for 1,2-dhlyco. Both carotenoids absorb at 484 nm, however by considering that the amount of 1,2-dhlyco is few times smaller compared to that of 1,2-dhneuro as shown by the pigment analysis results and steady-state absorption (Figure 2), the value of 27% EET for 1,2-dhneuro is a good approximation. The values are similar to those obtained in a previous publication.50 In comparison, the bacterial LH2 complexes that incorporate similar carotenoids like neurosporene and lycopene show substantially higher values of Car-to-BChl a EET. The EET for the neurosporene-containing LH2 from Rb. sphaeroides G1C was determined to be ~90%.61-62 LH2 complexes incorporating lycopene from Rhodospirillum molischianum62 and a genetically modified Rb. sphaeroides63 have an overall EET of ~54%, similar to those obtained for other LH2 complexes61, 64-65 that have carotenoids with N = 11.

Transient absorption spectroscopy In order to determine the excited state properties of each carotenoid in the LH1-RC complex, two excitation wavelengths were used to collect TA datasets. At 484 nm, both 1,2dhneuro and 1,2-dhlyco are excited while only 1,2-dhlyco is excited at 525 nm. The representative TA spectra recorded at room temperature and 77 K after excitation at 525 nm are shown in Figure 4.



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Figure 4. Transient absorption spectra of the LH1-RC complex from B. viridis recorded in VIS and NIR spectral ranges at RT (top panels) and 77 K (bottom panels) after excitation at 525 nm. The TA spectra probed in the visible region (Figure 4 A and C) show that aside from the narrowing of the peaks at 77 K, the observed trends are similar at both temperatures. Immediately after laser excitation (black traces), bleaching of the S0 (11Ag˗) → S2 (11Bu+) carotenoid ground state absorption occurs, seen as the negative signal between 400 and 540 nm. Concomitant with this is the presence of positive signal above 550 nm, which can be attributed to the S1 (21Ag˗) → Sn excited state absorption (ESA) of 1,2-dhlyco. At 500 fs (red traces), the S1 (21Ag˗) → Sn transition at 580 nm gains more intensity but becomes narrower at 1 ps (green traces) indicating vibrational cooling.66-69 At 5ps after laser excitation (blue traces), the intensities of the ground state bleaching and S1 (21Ag˗) → Sn transition are greatly diminished,


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which indicates that the S1 (21Ag˗) population has decayed to the ground state or has been transferred to BChl b. In addition, bleaching of the BChl b Qx band at ~600 nm becomes evident, and a peak at 540 nm that is blue-shifted from the main S1 → Sn band can be seen. The longevity of the transient absorption band at 540 nm beyond 100 ps (magenta traces) and its position on the blue side of the S1 → Sn ESA suggest that the band is associated with transient absorption of the carotenoid triplet state.70 The TA data were also recorded in the near infrared (NIR) region (Figure 4 B and D). Similar to the TA data in the visible region, the spectra recorded at 77 K are narrower and sharper compared to RT results. However, the TA spectra in the NIR are red shifted upon cooling to 77 K, which is not very evident in the TA data recorded in the visible region. The early 150 fs TA spectrum (black traces) shows a negative peak corresponding to the bleaching of BChl b Qy band. In addition, there are two positive peaks at ~1170 and ~1300 nm that decay within the IRF and could be assigned to the carotenoid S2 (11Bu+) → Sn transition.71-73 The presence of bleaching at such early time delay indicates either fast energy transfer from the carotenoid S2 state to BChl b or direct excitation of BChl b. Bleaching of the Qy band intensifies at later time delays and becomes more red-shifted. The recovery of the Qy band bleaching appears to be slightly faster at 77 K (Figure 4D) than at RT (Figure 4B). The RT and 77 K TA data of the LH1-RC complex excited at 484 nm are shown in Figure 5.



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Figure 5. Transient absorption spectra of the LH1-RC complex from B. viridis recorded in VIS and NIR spectral ranges at RT (top panels) and 77 K (bottom panels) after excitation at 484 nm.

Since excitation at 484 nm simultaneously excites both carotenoids, the 525 nm TA data (1,2dhlyco excitation only) was used to determine which spectral features belong to 1,2-dhneuro. At the early time delay of 150 fs (black traces) after laser excitation, the onset of carotenoid ground state bleaching occurs with the buildup of excited state absorption. The positive peak at 530 nm is due to the S1 (21Ag˗) → Sn transition of 1,2-dhneuro, while the peak at 580 nm is due to the S1 (21Ag˗) → Sn transition of 1,2-dihydrolyco as previously assigned in Figure 4. At 500 fs (red traces), the S1 (21Ag˗) → Sn excited state absorption gains more intensity but is broader compared to the S1 → Sn peak at 1 ps (green traces). The broader ESA can be attributed to a “hot” S1 state of the carotenoids. In going from 500 fs to 1 ps, the 1,2-dhneuro S1 → Sn peak (530 nm) gained 18 ACS Paragon Plus Environment

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intensity while that of 1,2-dhlyco (580 nm) is almost the same. At 5 ps (blue traces), the S1 → Sn transition of 1,2-dhlyco has decayed to almost half from its intensity at 1 ps while 1,2-dhneuro has only decayed slightly. In addition, the bleaching at 600 nm of BChl b Qx becomes apparent. At 100 ps time delay (magenta traces), the S1 → Sn peak has decayed and ground state bleaching is greatly diminished. Also at this time, peaks at 505 and 540 nm that are blue-shifted from the main S1 → Sn bands appear. The TA data recorded in the NIR at room temperature and 77 K are also shown in Figure 5 (B and D). At an early time delay after excitation of 150 fs, there are two positive peaks at ~920 and ~1170 nm due to the S2 → Sn transition of the carotenoids, as well as a negative peak at 1020 nm at room temperature and 1040 nm at 77 K, due to BChl b Qy bleaching. The peak at ~920 nm can still be observed at 500 fs (red traces), especially at 77 K and which is not present in the TA data at 525 nm excitation (Figure 4 B and D). This feature can therefore be assigned to 1,2-dhneuro. At longer time delays, the TA spectra mostly have features due to BChl b Qy bleaching, which become slightly red-shifted at both temperatures as time elapses but recovery of the bleaching is faster at 77 K than at room temperature. In order to determine the effective lifetimes of excited states, global fitting of the TA data recorded after excitation at 525 nm was performed using a sequential decay model54, which yielded evolution associated difference spectra (EADS) as shown in Figure 6 (A–D).



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Figure 6. Evolution associated difference spectra (EADS) obtained from global fitting of the TA data at 525 nm excitation recorded at RT (A, B) and 77 K (C, D). (E, F) are representative kinetic traces extracted at the specified wavelengths, where the black lines are raw data and colored lines are fits to the data. nd, non- decaying.

Representative kinetic traces at the indicated wavelengths are also provided in Figure 6 (E and F). In the visible region (Figure 6 A and C), five components were needed in order to obtain a 20 ACS Paragon Plus Environment

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good fit of the TA data. The first EADS (black traces) has a lifetime

Carotenoid-to-Bacteriochlorophyll Energy Transfer in the LH1âRC

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