Ultrafast Excitation-Energy Dynamics in Diatom Photosystem I

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Ultrafast Excitation-Energy Dynamics in Diatom Photosystem IAntenna Complex: A Femtosecond Fluorescence Upconversion Study Ryo Nagao, Kohei Kagatani, Yoshifumi Ueno, Jian-Ren Shen, and Seiji Akimoto J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b12086 • Publication Date (Web): 26 Feb 2019 Downloaded from http://pubs.acs.org on March 5, 2019

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The Journal of Physical Chemistry

Ultrafast Excitation-Energy Dynamics in Diatom Photosystem I-Antenna Complex: A Femtosecond Fluorescence Upconversion Study

Ryo Nagao,¶,* Kohei Kagatani,┴ Yoshifumi Ueno,┴ Jian-Ren Shen,¶ and Seiji Akimoto┴,*

¶Research

Institute for Interdisciplinary Science and Graduate School of Natural

Science and Technology, Okayama University, Okayama 700-8530, Japan ┴Graduate

School of Science, Kobe University, Kobe 657-8501, Japan

*Corresponding Authors: Ryo Nagao, TEL/FAX: +81-86-251-8630, E-mail: [email protected] Seiji Akimoto, TEL/FAX: +81-78-803-5705, E-mail: [email protected]

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ABSTRACT Fucoxanthin chlorophyll (Chl) a/c-binding protein (FCP) is unique light-harvesting antennas in diatoms. Recent time-resolved fluorescence analysis of photosystem I with FCP associated (PSI-FCPI) has mainly shown excitation-energy transfer among Chls a from FCPI to PSI in tens of ps. However, it remains unclear how each pigment, especially carotenoids and Chl c, in the FCPI is functionally related to the energy transfer in a femtosecond-time range. Here we reveal ultrafast excitation-energy-transfer mechanism in the PSI-FCPI preparations isolated from a diatom, Chaetoceros gracilis, by means of femtosecond time-resolved fluorescence spectroscopy with an upconversion system. Compared with the fluorescence-lifetime components of PSI-core like complexes, the energy transfer of Chl cChl a in the FCPI was observed within hudreds of fs, and the energy in the FCPI was transferred to PSI in ~2 ps. The comparative fluorescence analyses provide physical insights into the energy-transfer machinery

within

FCPI

and

from


FCPI

to

PSI.

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INTRODUCTION For photosynthetic organisms, management of the solar energy is an important task to achieve efficient photochemical reactions in two photosystems (PSI and PSII).1 The energy is precisely handled by the photosystems and their light-harvesting antennas, among which, the latter bind to the outer part of the photosystem cores and play a pivotal role in the energy transfer to the cores. It is known that there are a wide variety of antennas among the organisms, with different pigment and subunit compositions and structural organizations.2 This is a result of long time evolution, during which the light-harvesting system has diversified remarkably in response to either the light quality or quantity that every photosynthetic organism experiences, which contributed to better survivals of the organisms in their respective living environments. Charge-separation reactions in PSI and PSII take place in a few ps-time range1,3-5; therefore, excitation energy from the light-harvesting antennas should be transferred to the photosystem cores in a shorter time range prior to the charge separation. Time-resolved fluorescence (TRF) spectroscopy is a powerful method for investigating the excitation-energy dynamics from the antennas to the cores. Femtosecond TRF measurements with an upconversion system reveal excitation relaxation by carotenoid and chlorophyll (Chl) under room-temperature conditions,6,7 e.g., the excitation-energy transfer of luteinlutein, luteinChl a, and Chl bChl a in plant

light-harvesting

Chl

a/b-binding

protein

complex

(LHC)8

and

of

fucoxanthinChl a and Chl cChl a in diatom fucoxanthin Chl a/c-binding protein (FCP)9. In addition to these antennas, β-caroteneChl a energy transfer has been observed in cyanobacterial PSI trimers and plant PSI-LHCI.6,10 In contrast, picosecond 3 ACS Paragon Plus Environment


TRF measurements with a time-correlated single photon counting system mainly focus on Chl a-excitation dynamics including the charge-separation reactions in the cores in addition to slower excitation-energy transfer in the antennas under liquid nitrogen conditions.11 The optical advantage of the fluorescence measurements is an employment of extremely low laser power, which is sufficient for the excitation of each pigment in the pigment-protein complexes, making artificial excitation reactions negligibly low. This is entirely different from the time-resolved absorption measurements where significantly higher laser power is employed. Thus, TRF analyses are suited to evaluate contributions of the light-harvesting antennas to the excitation-energy dynamics. Among the photosynthetic organisms, diatoms are one of the successful phytoplanktons in marine and freshwater environments.12 One important factor that is considered to contribute to the success of diatoms is the presence of unique light-harvesting apparatus, FCP, whose pigment and subunit compositions are largely different from those of LHCs found in green lineage organisms.13 Diatom PSI and PSII possess their own FCPs, FCPI14-18 and FCPII16,19,20, respectively, and functional energy flows from the FCPs to the photosystem cores have been revealed by picosecond TRF spectroscopy using PSI-FCPI membranes21 and PSII-FCPII complexes22. The picosecond TRF measurements showed mainly energy dynamics among Chl a molecules, whereas little has been known regarding the energy transfer processes of Chl c and carotenoids. Yamagishi et al. have shown that excitation-energy transfer of Chl cChl a in PSI-FCPI preparations occurred in a time range of hundreds of femtoseconds under cryogenic temperatures.23 However, it is still unknown how carotenoids as well as Chl c in FCPI transfer their excitation energy to Chl a in PSI


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under physiological temperature conditions, under which the uphill energy transfer is not suppressed. In this study, we examined the femtosecond florescence dynamics of PSI-FCPI membranes from a diatom by the upconversion TRF spectroscopy under room-temperature conditions. Compared with the fluorescence properties of PSI-core preparations, functional energy flows from the FCPI to PSI were observed in the PSI-FCPI membranes prior to an energy equilibrium. Based on this finding together with our previous results of upconversion analysis of the FCPII,9 we discuss functional differences between FCPI and FCPII in diatoms.

EXPERIMENTAL METHODS PSI-FCPI membranes and PSI core complexes were isolated from the diatom Chaetoceros gracilis according to the procedure described previously.12 The PSI-FCPI membranes were suspended in a 50 mM Mes-NaOH buffer (pH 6.5) containing 0.2 M sucrose, 5 mM EDTA, and 10 mM NaCl, while the PSI complexes were suspended in a 50 mM Mes-NaOH buffer (pH 6.5) containing 0.2 M sucrose, 0.03% (w/v) n-dodecyl-β-D-maltoside, and 10 mM NaCl. Steady-state absorption spectra were measured with a JASCO V-660 spectrophotometer (JASCO, Tokyo, Japan) at 298 K. Fluorescence rise and decay curves were measured with a fluorescence upconversion method at 296 K. The excitation source was the second harmonic (425 nm) of a Ti:Sapphire laser (Tsunami, Spectra-Physics, Mountain View, CA, USA), which simultaneously excites all pigments.9 At 425 nm, contributions of PSI and FCPI to the total absorbance of PSI-FCPI are estimated to be 69% and 31%, respectively (Fig. S1). The pulse energy of the excitation was at most around 0.23×10–9 J pulse–1, and the spot 5 ACS Paragon Plus Environment


diameter was about 0.5 mm. This corresponds to a photon density of 6.2×1010 photons pulse–1 cm–2, which should be low enough to allow ignorance of the annihilation effect by one pulse, judging from the data reported previously.24 The sample was exchanged to a fresh one every 1 h during the data collections.

RESULTS AND DISCUSSION Room-temperature absorption spectra of the PSI-FCPI membranes (red line) and PSI core complexes (black line) are shown in Figure 1. The spectrum of the PSI complexes is virtually identical to that of PSI core complexes from cyanobacteria, as mentioned previously.17 The Qy peak of Chl a in the PSI-FCPI spectrum is slightly shifted to a longer wavelength at 681 nm than that in the PSI core spectrum. Moreover, the spectrum of PSI-FCPI exhibits relatively high intensity in the region of 450–650 nm. The enhancement of absorbance in the regions of 560–650 and 450–550 nm is mainly contributed by Chl c and fucoxanthin/diadinoxanthin, respectively. The Soret band of Chl c also contributes to the absorbance around 460 nm. Pigment compositions of the two PSI preparations have been reported previously; the PSI-FCPI membranes contain Chls a and c, fucoxanthin, diadinoxanthin, and β-carotene, whereas the PSI complexes contain Chl a and β-carotene.21


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Normalized absorbance

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400

500

600

700

Wavelength (nm) Figure 1. Room-temperature absorption spectra of the PSI-FCPI membranes (red line) and PSI complexes (black line). The spectra were normalized at the Qy absorption intensity of Chl a.



Fluorescence rise and decay curves of the PSI complexes and PSI-FCPI membranes are shown in Figures 2 and 3, respectively. The fluorescence curves were analyzed by multi-exponential functions with three lifetime components in the carotenoid and Chl fluorescence regions (560 nm and 640/655/677/690/710 nm, respectively). The lifetimes of the longest-lived component were assumed to be common in each sample, which were obtained to be 10.0 and 18.7 ps for PSI and PSI-FCPI, respectively. Table 1 summarizes the lifetime values and their amplitudes obtained. The 1st-lifetime component of the PSI complexes is represented by a 85-fs fluorescence decay at 560 nm. Since the S2S1 internal conversion of β-carotenes occurs in a time range of 100–200 fs,6,25 the shorter lifetime found in the PSI complexes seems to represent energy transfer from the S2 state of β-carotene to other pigments. The time constant of this energy transfer is estimated to be ~200 fs (=((85 fs)–1–(150 fs)–1)– 1),

when the S2S1 internal conversion of β-carotene is assumed to occur with a time

constant of 150 fs (an average value of 100–200 fs). Plausible candidates for the energy acceptor from β-carotene are the second and first singlet excited states of Chl a, so-called Qx and Qy, respectively, whose fluorescence rises appear at 677, 690, and 710 nm with time constants of 100, 155, and 435 fs, respectively. In addition, fluorescence decays at 640 and 655 nm exhibit lifetimes of 70 and 115 fs, respectively. These decay components appear to correspond to the transfer of excitation energy to pigments with fluorescence rises at 677/690/710-nm, which likely reflect the Qx to Qy internal conversion in Chl a.9 It is possible that the 640 and 655-nm decays may include a small part of the fluorescence decay from the S2 state of β-carotenes, and that the 710-nm rise includes a component of vibrational relaxation.


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Normalized fluorescence intensity

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1

560 nm

0 1

640 nm

0 1

655 nm

0 1

677 nm

0 1

690 nm

0 1

710 nm

0 0

2

4

6

8

Time (ps) Figure 2. Fluorescence rise and decay curves of the PSI complexes. Black and red lines show the observed fluorescence rise and decay curves and best-fit functions, respectively. Excitation wavelength was 425 nm and the fluorescence curves were monitored at 560, 640, 655, 677, 690, and 710 nm, respectively.



1

560 nm

0

Normalized fluorescence intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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640 nm

1

0

655 nm

1

0

677 nm

1

0

690 nm

1

0

710 nm

1

0

0

2

4

6

8

Time (ps) Figure 3. Fluorescence rise and decay curves of the PSI-FCPI membranes. Black and red lines show the observed fluorescence rise and decay curves and best-fit functions, respectively. Excitation wavelength was 425 nm and the fluorescence curves were monitored at 560, 640, 655, 677, 690, and 710 nm, respectively.


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In the 2nd-lifetime components of PSI, the decay components of fluorescence at 640, 655, 677, and 690 nm exhibit lifetimes of 950 fs, 690 fs, 1.9 ps, and 4.0 ps, respectively. The 640 and 655-nm decay components likely originate from fluorescence of vibrationally excited states of Qy; therefore, this process may be responsible for a relaxation of the excess vibrational energy in Chl a prior to thermal equilibrium. In the 3rd-lifetime components, the decay of fluorescence had a lifetime of 10 ps, providing evidence for the energy equilibrium among Chls a. The 1st-lifetime component of the PSI-FCPI membranes is represented by a 80-fs fluorescence decay at 560 nm (Table 1). Similar to the decay of β-carotenes in PSI (Table 1), the S2S1 internal conversion of fucoxanthin takes place in 80 fs,9 while a time constant for the internal conversion of diadinoxanthin is expected to be 140–200 fs taking into account its conjugation length.8,26 These observations suggest that the 80-fs lifetime component includes two events: an internal conversion (S2S1) in fucoxanthin and an energy transfer to Chl(s) from diadinoxanthin (S2) as well as β-carotene (S2). The time constant of the energy transfer from the diadinoxanthin (S2) is estimated to be ~150 fs (=((80 fs)–1–(170 fs)–1)–1), whereas the S2S1 internal conversion of diadinoxanthin is assumed to occur with a time constant of 170 fs (an average value of 140–200 fs). Energy acceptors from the carotenoids are likely relevant to three fluorescence-rise components at 677/690/710 nm with lifetimes of 105–170 fs. In addition, fluorescence decays at 640 and 655 nm exhibit lifetimes of 80 and 115 fs, respectively, and hence, may be associated with the rises at 677/690/710-nm. It should be noted that energy transfer from Chl c to Chl a, namely the set of 640-nm decay and 677/690/710-nm rises, does not work as a main pathway with 80 fs, because the excitation-energy transfer from Chl c to Chl a mainly occurs in hundreds of fs.9 These results indicate that the 11 ACS Paragon Plus Environment


677/690/710-nm rises represent the energy transfer from the carotenoids to Chl a and/or the Qx to Qy internal conversion in Chl a. In contrast, a highly efficient energy transfer from fucoxanthin (S1) to Chl has been observed in ~300 fs.9 A contribution of the S1 state of fucoxanthin to the energy dynamics in the PSI-FCPI can be explained by the higher magnitude at 677 nm with a slightly longer lifetime (170 fs) than that in the PSI, i.e., the energy transfer from fucoxanthin (S1) to the relatively high-energy Chl a. In the 2nd-lifetime components of PSI-FCPI, fluorescence decays at 640 and 655 nm are observed with lifetimes of 550 and 660 fs, respectively, whereas fluorescence rises at 690 and 710 nm are also observed with lifetimes of 1.1 and 1.9 ps, respectively. It is interesting to note that we cannot obtain any lifetime components from the 677-nm fluorescence by the fitting analysis. This is likely because the 677-nm fluorescence curve includes both fluorescence rise and decay with similar time constants, i.e., the 677-nm fluorescence rise and decay are seemingly canceled by both the energy acceptance from the pigments with 640/655-nm decays and the energy supply to the pigments with 690/710-nm rises. The energy-transfer events with the different time constants may occur in a pigment pool of Chls a and c toward the energy equilibrium in the PSI cores, suggesting that the pigment pool with the relatively high and low energy levels in the FCPI is responsible for the Chl cChl a energy transfer in the FCPI and the energy transfer from Chl a in the FCPI to Chl a in the PSI cores. The time constant of energy transfer from Chl c is virtually identical to that in the isolated FCPII complexes.9 On the other hand, a fluorescence curve at 560 nm represents a decay of 2.2 ps with a tiny amplitude of 4%; however, the origin of this component is not clear but required for the best fitting. In the 3rd-lifetime components, all of the


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Chl-fluorescence components exhibit a decay of 18.7 ps, reflecting the energy equilibrium among Chls a.



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Table 1. Fluorescence lifetimes (τi) and amplitudes (Ai) of fluorescence rise and decay curves for the PSI complexes and PSI–FCPI membranes obtained in this study

Wavelengtha

τ1 (A1b)

τ2 (A2b)

τ3 (A3 b)

560 nm

85 fs (1.00)

−

−

640 nm

70 fs (0.89)

950 fs (0.09)

10.0 ps (0.02)

655 nm

115 fs (0.53)

690 fs (0.31)

10.0 ps (0.16)

677 nm

100 fs (–0.26)

1.9 ps (0.18)

10.0 ps (0.82)

690 nm

155 fs (–0.43)

4.0 ps (0.10)

10.0 ps (0.90)

710 nm

435 fs (–0.20)

−

10.0 ps (1.00)

560 nm

80 fs (0.96)

2.2 ps (0.04)

−

640 nm

80 fs (0.69)

550 fs (0.15)

18.7 ps (0.16)

655 nm

115 fs (0.56)

660 fs (0.17)

18.7 ps (0.27)

677 nm

170 fs (–0.43)

−

18.7 ps (1.00)

690 nm

105 fs (–0.60)

1.1 ps (–0.10)

18.7 ps (1.00)

710 nm

135 fs (–0.20)

1.9 ps (–0.14)

18.7 ps (1.00)

PSI

PSI-FCPI

aWavelengths

for detection of the decay curves.

bAmplitudes

of decay and rise (positive and negative values, respectively) are

normalized by the amplitudes of the overall decay.


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Among the three lifetime components in the PSI-FCPI and PSI, there is a large difference in the 2nd-lifetime components. The characteristic set of the fluorescence decay and rise components in the PSI-FCPI membranes reflect the energy transfer from FCPI to PSI, whereas the only decays in the PSI complexes are indicative of the fluorescence relaxation from the vibrational states of Chl a toward the thermal equilibrium. By contrast, in the 1st-lifetime components, the lifetimes and amplitudes for the energy transfer of carotenoid→Chl and the internal conversion in Chl a are similar between the two samples, irrespective of the association of FCPI with PSI. Thus, we

successfully

observed

a

functional

energy

flow

of

FCPI→PSI

under

room-temperature conditions. From our recent picosecond TRF analyses with a time-correlated single photon counting system under room-temperature conditions, the PSI-FCPI membranes exhibited significant fluorescence rises of ~20 ps followed by fluorescence decays at ~40 ps, whereas the PSI complexes showed only fluorescence decays at ~30 ps.21 In this study, the characteristic rises at 1.1 and 1.9 ps in the 2nd-lifetime components of the PSI-FCPI, which were not observed in the PSI complexes (Table 1), may be related to the ~20-ps rise components observed in the picosecond TRF measurements. The lifetime components are interpreted as the excitation-energy transfers from the higher energy Chls a/c in the FCPI to the lower energy Chls a in the PSI cores, strongly indicating that the PSI cores accept excitation energy from the FCPI within 2.0 ps. The 560-nm fluorescence comes from carotenoids in the two preparations (Figures 2 and 3). The single fluorescence lifetimes from the S2 state are observed with around 80 fs irrespective of the association of FCPI with PSI. Since Ikeda et al. reported that a major carotenoid in FCPI is fucoxanthin with a lesser amount of diadinoxanthin 15 ACS Paragon Plus Environment


and a negligibly low level of β-carotene,15 the main component in the 80-fs decay of the PSI-FCPI membranes may be the S2 fluorescence of fucoxanthin rather than diadinoxanthin in the FCPI. The single lifetime component also suggests that fucoxanthin and diadinoxanthin in the FCPI have similar energy levels, which are largely different from the isolated FCPII complexes,9 whose fucoxanthins have two different energy levels. These observations imply that the binding properties of carotenoids differ significantly between the FCPI and FCPII. As for the Chl fluorescence regions, we suggested a single energy level of Chl c with the time constant of hundreds of fs in the PSI-FCPI membranes. In the FCPII,9 we have proposed a model of Chl c having two different energy levels, which is in agreement with a recent structural analysis of an FCP dimer showing that each FCP binds two Chls c.27 In our PSI-FCPI and PSII-FCPII preparations,16,17 the content of Chl c in the FCPI is considered to be smaller than that in the FCPII, based on the Chl a/c ratio of approximately 14.2 and 2.5 in the PSI-FCPI and PSII-FCPII, respectively. The variation of Chl contents and energy levels between the FCPI and FCPII suggests that the excitation-energy dynamics of FCPI is reasonably different with that of FCPII. Based on these observations, we propose a schematic model of excitation-energy-transfer processes in the PSI-FCPI membranes (Figure 4). In the PSI core, the time constant of β-carotene (S2)Chl a transfer is ~200 fs, whereas the internal conversion in Chl a occurs with a time constant of ~150 fs. By contrast, in the FCPI, the S2 state of diadinoxanthin may transfer excitation energy to the S2 state of Chl a with ~150 fs taking into account its conjugation length,8,26 whereas the S1 state of fucoxanthin seems to transfer energy to the S1 state of Chl a with ~300 fs as suggested by the fluorescence measurements of isolated FCPII.9 The energy transfer between the 16 ACS Paragon Plus Environment

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S1 states of Chl cChl a is likely to occur with ~600 fs, which is consistent with the observation in isolated FCPII.9 The time constant of internal conversion in Chl a is also ~150 fs in FCPI, and then the energy trapped by Chl a in FCPI is transferred to Chl a in the PSI core with ~2 ps.



Figure 4. A model for the excitation-energy transfer in PSI-FCPI and PSI core complexes. Fx and Ddx represent fucoxanthin and diadinoxanthin, respectively.


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CONCLUSIONS This study demonstrated the functional excitation-energy transfer from the FCPI to PSI under room-temperature conditions by the femtosecond TRF spectroscopy. The unique energy levels of carotenoids and Chls in FCPI enable the energy transfer between FCPI and PSI in the time order of 1.1–1.9 ps. The ultrafast spectroscopic findings, together with our previous TRF observations of the FCPII,9 provide functional and physical insights into the different energy-transfer dynamics between FCPI and FCPII. The handlings of solar energy with such different manners in FCPI and FCPII may provide an important means for diatoms to adapt to the ecological niches they live that are enriched in the blue-green spectral components under the water surface.

ASSOCIATED CONTENT Supporting Information Absorption spectra of the PSI complexes and PSI-FCPI membranes normalized by the amount of β-carotene (Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org.

Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT This work was supported by the Grants-in-Aid for Scientific Research from Japan 19 ACS Paragon Plus Environment


Society for the Promotion of Science JP17K07442 (to R. N.), JP17H06433 (to J.-R. S.), and JP16H06553 (to S. A.).

REFERENCES (1) Blankenship, R. E. Molecular mechanisms of photosynthesis, 2nd ed., Wiley-Blackwell Oxford, U.K., 2014. (2) Falkowski, P. G.; Katz, M. E.; Knoll, A. H.; Quigg, A.; Raven, J. A.; Schofield, O.; Taylor, F. J. R. The evolution of modern eukaryotic phytoplankton. Science 2004, 305, 354-360. (3) Brettel, K.; Leibl, W. Electron transfer in photosystem I. Biochim. Biophys. Acta 2001, 1507, 100-114. (4) Diner, B. A.; Rappaport, F. Structure, dynamics, and energetics of the primary photochemistry of photosystem II of oxygenic photosynthesis. Annu. Rev. Plant Biol. 2002, 53, 551-580. (5) Byrdin, M.; Rimke, I.; Schlodder, E.; Stehlik, D.; Roelofs, T. A. Decay kinetics and quantum yields of fluorescence in photosystem I from Synechococcus elongatus with P700 in the reduced and oxidized state: Are the kinetics of excited state decay trap-limited or transfer-limited? Biophys J. 2000, 79, 992-1007. (6) Holt, N. E.; Kennis, J. T. M.; Fleming, G. R. Femtosecond fluorescence upconversion studies of light harvesting by β -carotene in oxygenic photosynthetic core proteins. J. Phys. Chem. B 2004, 108, 19029-19035. (7) Akimoto, S.; Mimuro, M. Application of time-resolved polarization fluorescence spectroscopy in the femtosecond range to photosynthetic systems. Photochem. Photobiol. 2007, 83, 163-170. 20 ACS Paragon Plus Environment

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Ultrafast Excitation-Energy Dynamics in Diatom Photosystem I

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