Excitation Energy-Transfer Dynamics of Brown Algal Photosynthetic

Letter pubs.acs.org/JPCL

Excitation Energy-Transfer Dynamics of Brown Algal Photosynthetic Antennas D. Kosumi,*,†,‡ M. Kita,‡,§ R. Fujii,†,⊥ M. Sugisaki,‡,§ N. Oka,# Y. Takaesu,# T. Taira,# M. Iha,# and H. Hashimoto*,†,‡,§ †

The Osaka City University Advanced Research Institute for Natural Science and Technology (OCARINA), 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan ‡ JST/CREST, 4-1-8 Hon-chou, Kawaguchi, Saitama 332-0012, Japan § Department of Physics, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan ⊥ JST/PRESTO, 4-1-8 Hon-chou, Kawaguchi, Saitama 332-0012, Japan # South Product Co. Ltd., 12-75 Suzaki, Uruma-shi, Okinawa 904-2234, Japan S Supporting Information *

ABSTRACT: Fucoxanthin−chlorophyll-a/c protein (FCP) complexes from brown algae Cladosiphon okamuranus TOKIDA (Okinawa Mozuku in Japanese) contain the only species of carbonyl carotenoid, fucoxanthin, which exhibits spectral characteristics attributed to an intramolecular charge-transfer (ICT) property that arises in polar environments due to the presence of the carbonyl group in its polyene backbone. Here, we investigated the role of the ICT property of fucoxanthin in ultrafast energy transfer to chlorophyll-a/c in brown algal photosynthesis using femtosecond pump−probe spectroscopic measurements. The observed excited-state dynamics show that the ICT character of fucoxanthin in FCP extends its absorption band to longer wavelengths and enhances its electronic interaction with chlorophyll-a molecules, leading to efficient energy transfer from fucoxanthin to chlorophyll-a. SECTION: Spectroscopy, Photochemistry, and Excited States

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properties are strongly influenced by their surrounding environment such as solvent polarity and are thought to result from an intramolecular charge-transfer (ICT) state, which is stabilized in polar environments.6,9 Peridinin (Per)−chlorophyll (Chl)-a protein (PCP) complexes from dinoflagellates contain the carbonyl carotenoid Per and show highly efficient (>95%) excitation energy transfer (EET) from Per to Chla.13−15 An EET channel via S2 of Per accounts for 20−30% of the total efficiency in PCP.16 A highly efficient EET reaction results from the ICT state strongly coupled with S1 in polar environments, forming the S1/ICT state, which increases the S0−S1/ICT transition dipole moment.13 Consequently, the EET yield via S1/ICT is greater than 80%.13,17,18 Despite the intensive investigations of PCP in terms of its crystal structure and excited-state dynamics, an EET mechanism of FCP complexes both from brown algae and diatoms has been poorly understood. In the present study, we investigated the ultrafast EET dynamics in FCP from brown algae. As Fx is reported to exhibit an ICT character in polar environments,8,19,20 we examined the roles of ICT in ultrafast Fx →

iatoms and brown algae are major players in biochemical cycles and primary biomass production.1 Photosynthetic light-harvesting antenna, fucoxanthin (Fx)−chlorophyll-a/c protein (FCP) complexes, associated with these organisms bind the carbonyl carotenoid Fx, which is the most abundant marine carotenoid (Figure 1A).2 In a primary process of algal photosynthesis, light-harvesting pigment−protein complexes such as FCP collect light energy in the blue to green region of the spectrum and transfer the captured energy to reaction centers, where primary electron-transfer reactions convert the energy into an electrochemical gradient.3 The electronic states of all-trans-carotenoids are described by analogy to linear polyenes with C2h symmetry as they share a similar π-electron conjugated backbone.4 The S0 ground state is denoted as 11Ag−, and a strong absorption band in the visible region is attributed to the S0−S2 (the optically allowed 11Bu+ state) transition. The lowest-lying excited singlet state, S1 (21Ag−), is optically forbidden because S0 and S1 have the same symmetry. Due to the forbidden nature of the S0−S1 transition, carotenoids exhibit extremely weak S1 fluorescence.3,4 Carbonyl carotenoids display higher S1 emission compared to analogues without a carbonyl group5 and have several unique excited-state properties, such as characteristic transient absorption and stimulated emission bands.6−12 These © XXXX American Chemical Society

Received: May 15, 2012 Accepted: August 31, 2012

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The Journal of Physical Chemistry Letters

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Figure 1. (A) The chemical structure of Fx and (B) the steady-state absorption spectrum of isolated FCP complexes. Steady-state absorption spectra of each pigment bound to FCP measured in the indicated solvents are also shown.

Chl EET in brown algal photosynthetic antennas. Brown algal FCP has an advantage over that from diatoms, as previously investigated by femtosecond spectroscopy,21−23 because brown algae lacks the carotenoids, diadinoxanthin (Ddx), and/or diatoxanthin (Dtx) involved in diatoms (see below). This situation makes it easier for us to clarify EET processes in FCP and can resolve the open question in the previous studies.22,23 Figure 1B shows steady-state absorption spectra of FCP and its four principal pigments, Fx, Chl-a, and Chl-c1/c2, in different solvents. The pigment composition of FCP from diatoms has previously been analyzed by binary HPLC,24 whereas that from brown algae in the present study was analyzed by combined binary HPLC and 1H NMR spectroscopy,25 revealing that minor carotenoids, including Ddx/Dtx, are only found in FCP from diatoms. In the spectra obtained here, all absorption bands of the pigments bound to FCP had shifted to lower energy compared to those measured in organic solvents. In particular, the absorption band of Fx exhibited a pronounced red shift of greater than 0.2 eV, suggesting that Fx molecules strongly interact with other pigments and/or surrounding proteins in FCP. Figure 2A shows the photoinduced absorption (PIA) spectra of FCP taken after the excitation of Fx into its S2 state. Previous spectroscopic studies have demonstrated that Fx molecules bound to different protein sites in FCP from diatoms possess the different S2 absorption energies owing to heterogeneous environments surrounding Fx molecules.22,23,26−28 To avoid convolution in the analysis of excited-state dynamics due to Fx molecules in different protein environments, we first selected an excitation energy of 2.25 eV, corresponding to the red edge of the Fx absorption band. In the PIA spectra, negative absorbance changes of around 1.85 and >2.25 eV are assignable to the bleaching of Chl-a and Fx, respectively. A broad transient absorption band ranging from 1.6 to 2.2 eV also appeared immediately after excitation. Upon comparison with the transient absorption of isolated Fx molecules in a polar solvent, the broad absorption band can be assigned to the S1/ICT state of Fx (see also the Supporting Information).8,19,20 The bleaching and transient absorption signals of Fx decreased with increasing delay times, while the Chl-a bleaching

Figure 2. (A) PIA spectra of FCP obtained by excitation at 2.25 eV and (B) their corresponding kinetic traces. (C) EADS for excitation at 2.25 eV obtained by a global fit.

correspondingly increased, suggesting the involvement of ultrafast Fx → Chl-a energy transfer. The kinetic traces of Fx and Chl-a following excitation at 2.25 eV were also determined (Figure 2B). Probe energies of 1.83, 2.14, and 2.45 eV were selected as these correspond to the Chl-a bleaching, S1/ICT transient absorption, and Fx bleaching, respectively. In the traces, the solid lines represent the best-fit curves for the rise and decay phases convoluted with the instrument response function assuming a Gaussian temporal profile. The obtained kinetic traces were analyzed globally, and five exponential components were required to fully fit the traces. The time constants of each exponential component were determined to be 40 fs, 450 fs, 2.1 ps, 37 ps, and >400 ps (see Table 1). Evolution-associated difference spectra (EADS) corresponding to each component were also determined by a global fit (Figure 2C and see also the Supporting Information). The first component (40 fs) is assignable to S2 of Fx. The 2660



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Figure 2B includes not only the Qy bleaching but also the S1/ ICT transient absorption, which makes it difficult to determine the Fx → Chl-a EET dynamics. In order to extract the pure Chl-a bleaching kinetics, the difference in the traces probed at 1.83 and 1.91 eV was examined (see Figure S2 in the Supporting Information). The relative amplitudes of each component involved in the Chl-a bleaching are presented in Table 1. From the obtained relative amplitudes of each excited state, the EET efficiencies via the Fx excited states were determined to be 26 (S2), 38 (hot S1/ICT), and 31% (S1/ICT) (Table 1). We were also able to calculate the EET rates and intrinsic lifetimes of each excited state of Fx bound to FCP for excitation at 2.25 eV based on the obtained decay time constants and EET efficiencies (Figure 3). The estimated EET yield via S2 is lower than the 40% previously reported for FCP from diatom,21 and our results also suggest that the Fx → Chl-a energy transfer via S1/ICT is more efficient. Further, our result shows an efficient EET via hot S1/ICT, whereas such an EET pathway has been reported to be a minor channel in the antenna systems containing carbonyl carotenoids.13,21 Polı ́vka et al. observed a large contribution of hot S1/ICT in the Chl-a bleaching kinetics in the LHC from dinoflagellates, which is closely related to FCP because it contains Chl-a/c2, Per (major), and Ddx/Dtx (minor). However, they concluded that a separate hot S1/ICT state was not necessary to explain the data because the fitted species-associated different spectra corresponding to the hot and relaxed S1/ICT states were quite similar.31 Therefore, we illustrate the different EET models with (Figure 3A) or without (Figure 3B) the pathway via hot S1/ ICT. In these models, the intrinsic S1/ICT lifetime was determined to be 15 or 31 ps, which agrees well with that in methanol (18−23 ps).8,19,20 However, comparison of the S1/ ICT transient absorption spectra measured in FCP and methanol suggests the weaker ICT character of Fx bound to FCP than that in methanol because the S1-like band is dominant in the PIA spectra (see the Supporting Information). It is generally accepted that S1-rich S1/ICT has a longer lifetime than ICT-rich S1/ICT.8,20 Therefore, the S1/ICT lifetime in FCP should be longer than that in methanol, suggesting that the model presented in Figure 3B is favorable. Nevertheless, we cannot entirely exclude the EET pathway via hot S1/ICT because the hot S1/ICT component becomes more predominant in the Qy bleaching kinetics when FCP is excited at higher energy (see below). In most photosynthetic antennas, EET via S2 is the dominant channel, although a few species can efficiently transfer captured

Table 1. Time Constants and Relative Amplitudes of the Exponential Components of Each Excited State Probed at 1.83 eV for eExcitation at 2.25 eVa S2

hot S1/ICT

S1/ICT

Qy

time constant

40 ± 10 fs

450 ± 20 fs

2.1 ± 0.1 ps

amplitude energy transfer efficiencyb (yield)

−27% 26% (26%)

−40% 38% (52%)c

−33% 31% (89%)c

37 ± 1 ps, >400 ps 100%

a

EET efficiencies for each state determined by relative amplitudes and the overall efficiency are also shown. bOverall energy-transfer efficiency from Fx to Chl-a is assumed to be 95%. cFor the model represented in Figure 3B, the EET efficiency and yield via S1/ICT were determined to be 69 and 93%, respectively.

second (450 fs) and third (2.1 ps) EADS are quite similar to each other. Both of them exhibit the Fx bleaching and the S1/ ICT transient absorption, while the S 1 /ICT transient absorption band of the third EADS is slightly blue-shifted compared to that of the second EADS. These components have been ascribed to the vibrational hot (unrelaxed) and relaxed S1/ ICT states, respectively,21−23 as has been reported for Per in PCP.13 The fourth EADS (37 ps) was reported to represent excitation annihilation between Chl-a molecules.13 The fifth EADS (>400 ps), which has the longest lifetime, is assignable to Qy of Chl-a. EET yields via the excited states of carotenoids to (bacterio)Chls in photosynthetic light-harvesting complexes (LHCs) are often estimated by comparing the excited-state lifetimes of carotenoids in solution to those of carotenoids bound to protein complexes, assuming that the same internal conversion rates are applicable under both conditions.3 However, as the S1/ICT lifetimes of carbonyl carotenoids are strongly modulated by surrounding environments,6,7 we estimated the EET yields based on the relative amplitudes of the exponential components involved in the kinetic trace of Qy bleaching and the overall EET efficiency determined by fluorescence excitation measurements, as reported previously.13,29 The overall Fx → Chl-a EET efficiency is reported to be ∼80% for diatom FCP containing the minor carotenoid Ddx/Dtx,21 whereas that of brown algal FCP exceeds 90% (especially that of Fx corresponding to the red-most area of the steady-state absorption is more than 95%).30 Therefore, we assumed the overall EET efficiency to be 95% for excitation at 2.25 eV. It should be noted that the kinetic trace at 1.83 eV shown in

Figure 3. Schematic representation of EET dynamics for FCP excited at 2.25 eV. (A) A model for EET including the pathway via hot S1/ICT and (B) without the pathway via hot S1/ICT. 2661



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light energy to (bacterio-)Chl via S1.32 In the case of a Förstertype weak dipole interaction limit, an EET rate is proportional to both the Coulomb interaction and spectral overlap between donor fluorescence and acceptor absorption.33 The S2 energies of carbonyl carotenoids are significantly lower than those of their noncarbonyl counterparts, but the S1 energy remains almost unaffected.8 Thus, S1/ICT is sufficiently high to maintain a favorable spectral overlap with Qy of Chl-a, while S2 is shifted to lower energies to allow the efficient capture of green light, which is critical for the survival of marine organisms. The lower S2 energy of carbonyl carotenoids causes a narrower S2−S1/ICT energy gap, leading to rapid internal conversion of S2.34 In fact, the S2 lifetimes of carbonyl carotenoids were reported to be 50 fs in Per7 and Fx19,20 and 35 fs in siphonaxanthin,35 which are much shorter than those of noncarbonyl carotenoids with equivalent conjugation lengths (100−200 fs36). The extremely fast S2 decay may suppress energy transfer via S2 in algal photosynthetic antennas. In FCP from brown algae, 69% of the total energy absorbed by Fx is transferred to Chl-a via S1/ICT. S1 of carotenoids intrinsically has an optically forbidden character, leading to inefficient energy transfer via S1. However, the S1/ICT−S0 transition dipole moment of carbonyl carotenoids can be enhanced by the coupling of S1 and ICT, as demonstrated by measurements of steady-state fluorescence and stimulated emission in PIA in polar solvents.5,6,8 The large S1/ICT−S0 transition dipole moment enables Fx to efficiently transfer excitation energy to Chl-a. Recent investigations have revealed that at least two different types of Fx molecules give rise to the different spectroscopic properties of FCP as a result of the heterogeneous environments surrounding Fx molecules.22,26−28 To investigate the heterogeneous environments surrounding Fx molecules and their light-harvesting function, we measured excitation energydependent PIA of FCP (Figure 4). Excitation energies were

selected at 2.25, 2.48, and 2.69 eV as these correspond to the lower, middle, and higher energies, respectively, of the S2 absorption of Fx, in addition to an energy of 1.85 eV, corresponding to the Qy band of Chl-a. Comparison of the PIA spectra excited at 2.25 and 2.48 eV shows that the Fx bleaching shifts to higher energy with increased excitation energy, whereas the S1/ICT transient absorption remained almost unaffected. These results clearly demonstrate the selective excitation to the Fx molecule, which possesses different S2 energy when bound to different protein sites. The excitation energy dependence of the PIA spectra were previously reported for the FCP from diatoms and LHC from dinoflagellates, both containing Ddx/Dtx. These results showed the different S1/ ICT transient absorption spectra when the complexes were excited at a lower (2.25 or 2.30 eV) or a higher energy (2.48 eV). Consequently, the difference in the excitation-energydependent PIA spectra of FCP from diatoms originates from Ddx/Dtx, as reported in LHC from dinoflagellates.31 Therefore, the present study for FCP from brown algae unequivocally supports this idea. The excited-state dynamics induced by the different excitation energies into S2 were essentially identical, whereas the lifetimes of Fx depended strongly on the excitation energy. The excitation-energy-dependent lifetimes and relative amplitudes of each Fx state involved in the kinetic traces of the Qy bleaching (difference in kinetic traces at 1.91 and 1.83 eV) are shown in Table 2. With increasing excitation energy, the lifetimes of each state became longer, whereas the relative amplitudes of S1/ICT in the Qy bleaching kinetics decreased. The observed increase in EET efficiency to Chl-a via S1/ICT upon excitation at 2.25 eV (see Table 2) suggests that Fx corresponding to the red-most area of the steady-state absorption exhibits a strong ICT character of the S1/ICT state, which may result from an interaction between Fx and amino acid residues of FCP through their hydrogen bond.37,38 As the crystal structure of FCP has not yet been determined to date, it is difficult to accurately attribute properties and functions to individual Fx molecules bound to different protein sites. Despite this limitation, our results shed light on the influence of the ICT character of Fx on the efficient capabilities of brown algal photosynthetic antennas. On the basis of our present findings, we would like to discuss the possibility that Chl-c1/c2 participates in the ultrafast EET dynamics of FCP. Fluorescence excitation measurements by Papagiannakis et al. suggest that the EET efficiency from Chl-c2 to Chl-a is 100% and occurs on a time scale of 100 fs in FCP from diatoms.21 On the other hand, Polı ́vka et al. have reported that the Chl-c2 → Chl-a EET is 1.4 ps after excitation to the Qy band of Chl-c2 in LHC from dinoflagellates.31 However, it remains to be clarified whether the Fx → Chl-c1/c2 energytransfer reaction is involved in the singlet EET of FCP. In the PIA spectra shown in Figure 4, a negative absorbance change following excitation to S2, which is clearly distinct from Fx bleaching, is seen at 2.65 eV and corresponds to the Soret band

Figure 4. Excitation energy-dependent PIA spectra of FCP taken at 0.5 ps. The spectra were normalized at the Qy bleaching.

Table 2. Time Constants and Relative Amplitudes of the Exponential Components of Each Excited State of Fx Monitored at the Qy Bleaching excitation energy

S2

hot S1/ICT

S1/ICT

2.25 eV 2.48 eV 2.69 eV

40 ± 10 fs (27%) 40 ± 10 fs (35%) 50 ± 10 fs (47%)

450 ± 20 fs (40%) 590 ± 20 fs (52%) 670 ± 20 fs (42%)

2.1 ± 0.1 ps (33%) 3.1 ± 0.1 ps (13%) 4.2 ± 0.1 ps (11%)

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of Chl-c1/c2. As the observed negative change was rather pronounced after excitation at 2.69 eV, which excites both S2 of Fx and the Soret band of Chl-c1/c2, the negative absorption at around 2.6 eV can be assigned to the bleaching of Chl-c1/c2. In the PIA spectra excited at 2.25 eV, the negative change was also observed and remained at a longer delay (>2 ps) (Figure 2A), even though the Fx bleaching translates to a positive phase due to the Qy transient absorption of Chl-a (see the Supporting Information). As Chl-c1/c2 is not directly excited at 2.25 eV, our findings demonstrate the involvement of the Fx → Chl-c1/c2 EET. Unfortunately, as the Fx and Chl-c1/c2 bleaching and the Qy transient absorption of Chl-a (Chl-c1/c2) strongly overlapped in the spectral region above 2.6 eV, the details of the Fx → Chl-c1/c2 EET dynamics remain unclear. However, the appearance of the Chl-c1/c2 bleaching immediately after excitation does not exclude the possibility of Fx → Chl-c1/c2 EET processes at an ultrafast time scale. In summary, we investigated the ultrafast Fx → Chl EET dynamics in brown algal FCP by means of femtosecond pump−probe spectroscopic measurements and demonstrated that the highly efficient energy transfer from Fx to Chl-a via the S1/ICT state is due to the ICT character of Fx. Fx molecules bound to different protein sites exhibited distinct spectroscopic properties, leading to efficient light-harvesting capabilities. The energy-transfer mechanism from carbonyl carotenoids, such as Fx and Per, to Chl-a involving the ICT state represents an important light-harvesting strategy of algal photosynthetic systems. An energy strategy using oxygenated carotenoids and the local protein environment to extend their absorption bands into the green region of the visible spectrum may be a necessary development for plants living in aquatic habitats.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (D.K.); hassy@ocarina. osaka-cu.ac.jp (H.H.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS H.H. thanks the Nissan Science Foundation and Human Frontier Science Program for financial support. REFERENCES

(1) Falkowski, P. G.; Barber, R. T.; Smetacek, V. Biogeochemical Controls and Feedbacks on Ocean Primary Production. Science 1998, 281, 200−206. (2) Büchel, C. Fucoxanthin-Chlorophyll Proteins in Diatoms: 18 and 19 kDa Subunits Assemble into Different Oligomeric States. Biochemistry 2003, 42, 13027−13034. (3) Polívka, T.; Sundström, V. Ultrafast Dynamics of Carotenoid Excited States From Solution to Natural and Artificial Systems. Chem. Rev. 2004, 104, 2021−2072. (4) Linear Polyene Electronic Structure and Potential Surfaces; Hudson, B. S.; Kohler, B. E.; Schulten, K., Eds.; Academic Press: New York, 1982; Vol. 6. (5) Mimuro, M.; Nagashima, U.; Takaichi, S.; Nishimura, Y.; Yamazaki, I.; Katoh, T. Molecular Structure and Optical Properties of Carotenoids for the in Vivo Energy Transfer Function in the Algal Photosynthetic Pigment System. Biochim. Biophys. Acta 1992, 1098, 271−274. (6) Bautista, J. A.; Connors, R. E.; Raju, B. B.; Hiller, R. G.; Sharples, F. P.; Gosztola, D.; Wasielewski, M. R.; Frank, H. A. Excited State Properties of Peridinin: Observation of a Solvent Dependence of the Lowest Excited Singlet State Lifetime and Spectral Behavior Unique among Carotenoids. J. Phys. Chem. B 1999, 103, 8751−8758. (7) Zigmantas, D.; Polívka, T.; Hiller, R. G.; Yartsev, A.; Sundström, V. Spectroscopic and Dynamic Properties of the Peridinin Lowest Singlet Excited States. J. Phys. Chem. A 2001, 105, 10296−10306. (8) Zigmantas, D.; Hiller, R. G.; Sharples, F. P.; Frank, H. A.; Sundström, V.; Polívka, T. Effect of a Conjugated Carbonyl Group on the Photophysical Properties of Carotenoids. Phys. Chem. Chem. Phys. 2004, 6, 3009−3016. (9) Frank, H. A.; Bautista, J. A.; Josue, J.; Pendon, Z.; Hiller, R. G.; Sharples, F. P.; Gosztola, D.; Wasielewski, M. R. Effect of the Solvent Environment on the Spectroscopic Properties and Dynamics of the Lowest Excited States of Carotenoids. J. Phys. Chem. B 2000, 104, 4569−4577. (10) Niedzwiedzki, D. M.; Chatterjee, N.; Enriquez, M. M.; Kajikawa, T.; Hasegawa, S.; Katsumura, S.; Frank, H. A. Spectroscopic Investigation of Peridinin Analogues Having Different π-Electron Conjugated Chain Lengths: Exploring the Nature of the Intramolecular Charge Transfer State. J. Phys. Chem. B 2009, 113, 13604− 13612. (11) Chatterjee, N.; Niedzwiedzki, D. M.; Aoki, K.; Kajikawa, T.; Katsumura, S.; Hashimoto, H.; Frank, H. A. Effect of Structural Modifications on the Spectroscopic Properties and Dynamics of the Excited States of Peridinin. Arch. Biochem. Biophys. 2009, 483, 146− 155. (12) Oum, K.; Lohse, P. W.; Ehlers, F.; Scholz, M.; Kopczynski, M.; Lenzer, T. 12′-Apo-β-caroten-12′-al: An Ultrafast “Spy” Molecule for Probing Local Interactions in Ionic Liquids. Angew. Chem., Int. Ed. 2010, 49, 2230−2232. (13) Zigmantas, D.; Hiller, R. G.; Sundström, V.; Polívka, T. Carotenoid to Chlorophyll Energy Transfer in the PeridininChlorophyll-Protein Complex Involves an Intramolecular Charge Transfer State. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16760−16765. (14) Schulte, T.; Niedzwiedzki, D. M.; Birge, R. R.; Hiller, R. G.; Polívka, T.; Hofmann, E.; Frank, H. A. Identification of a Single

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EXPERIMENTAL METHODS Femtosecond pump−probe spectroscopic measurements based on a mode-locked Ti:Sapphire regenerative amplifier are described in detail elsewhere.19,39 Visible excitation pulses were generated using an optical parametric amplifier. A whitelight continuum probe pulse was generated by a 5.0 mm thick sapphire plate. Relative polarization between the excitation and probe pulses was set to the magic angle. The instrumental response function of the system was determined by examining the cross correlation between the excitation and probe pulses and was found to be better than 100 fs. The cross correlation function was used to determine the precise zero time delay for each probe energy. After chirp compensation, the uncertainty in the zero time delay was better than 10 fs. FCP complexes were extracted and purified from the brown algae Cladosiphon okamuranus TOKIDA. The pigment stoichiometry of the purified complexes was determined to be Chl-a/Chl-c1/Chl-c2/Fx = 4.6:1.1:1.0:5.5.30 For optical measurements, the samples were dispersed in 25 mM Tris-HCl buffer (pH 7.5 with 2 mM KCl) containing 0.03% DDM and circulated in a 1.0 mm optical path length flow cell. The absorbance of all samples was adjusted to 0.5 at the S2 absorption band of Fx. All optical measurements were performed at room temperature.

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ASSOCIATED CONTENT

* Supporting Information S

Complete sets of the photoinduced absorption spectra, kinetic traces, and EADS obtained using different excitation energies. This material is available free of charge via the Internet at http://pubs.acs.org. 2663



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