Control Mechanism of Excitation Energy Transfer in a Complex

Aug 15, 2014 - Daisuke Kosumi , Tomoko Horibe , Mitsuru Sugisaki , Richard J. Cogdell , and Hideki Hashimoto. The Journal of Physical Chemistry B 2016...
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Letter pubs.acs.org/JPCL

Control Mechanism of Excitation Energy Transfer in a Complex Consisting of Photosystem II and Fucoxanthin Chlorophyll a/ c‑Binding Protein Ryo Nagao,*,†,¶ Makio Yokono,‡,¶ Tatsuya Tomo,§,∥ and Seiji Akimoto‡,⊥ †

Division of Material Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan Molecular Photoscience Research Center, Kobe University, Kobe 657-8501, Japan § Department of Biology, Faculty of Science, Tokyo University of Science, Kagurazaka 1-3, Shinjuku-ku, Tokyo 162-8601, Japan ∥ PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan ⊥ JST, CREST, Kobe 657-8501, Japan ‡

S Supporting Information *

ABSTRACT: Fucoxanthin chlorophyll (Chl) a/c-binding protein (FCP) is a unique light-harvesting antenna in diatoms, which are photosynthesizing algae ubiquitous in aquatic environments. However, it is unknown how excitation energy is trapped and quenched in a complex consisting of photosystem II and FCP (PSII−FCPII complex). Here, we report the control mechanism of excitation energy transfer in the PSII−FCPII complexes isolated from a diatom, Chaetoceros gracilis, as revealed by picosecond timeresolved fluorescence spectroscopy. The results showed that Chl-excitation energy is harvested in low-energy Chls near/within FCPII under the 77 K conditions, whereas most of the energy is trapped in reaction center Chls in PSII under the 283 K conditions. Surprisingly, excitation energy quenching was observed in a part of PSII−FCPII complexes with the time constants of hundreds of picosecond, thus indicating the large contribution of FCPII to energy trapping and quenching. On the basis of these results, we discuss the light-harvesting strategy of diatoms. SECTION: Energy Conversion and Storage; Energy and Charge Transport

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hence, elucidation of light-harvesting strategies around PSII will provide an answer to the long-standing question of how diatoms survive in a wide range of aquatic environments such as freshwater, marine, and terrestrial habitats. In this study, we investigate the control mechanism of excitation energy transfer in two types of PSII preparations isolated from the diatom Chaetoceros gracilis. Oxygen-evolving PSII particles containing FCPII that are specifically associated with PSII12 and the oxygen-evolving PSII complexes13 (hereafter referred to as PSII−FCPII and PSII complexes, respectively) were used for picosecond time-resolved fluorescence measurements. Figure 1 shows TRFS of the PSII−FCPII and PSII complexes at 77 K. During the time regions from 0 ps to 2.0 ns, the fluorescence maximum of the PSII−FCPII complex was shifted from 686 to 691 nm, whereas the fluorescence maximum of the PSII complex was shifted from 688 to 695 nm. In the PSII complex, the fluorescence intensity decreased to approximately 40% within 200 ps and further decreased to approximately 8% in 2.0 ns. However, the fluorescence intensity

hotosynthetic organisms have developed their lightharvesting antenna to overcome the inescapable fate of both efficient collection of sunlight and dissipation of excess sunlight under changing light intensities throughout the day. This has resulted in an optimum supply of light energy to two photosystems (PSI and PSII) followed by charge-separation processes.1−5 It is generally known that antenna, which efficiently collect sunlight, form a light-harvesting state, whereas antenna dissipating excess sunlight form a quenching state. These two states contribute to the control of charge-separation reactions of PSII, as revealed with both light-harvesting chlorophyll (Chl) a/b-binding protein of PSII (LHCII) and PSII−LHCII complexes in higher plants and green algae,1−5 although such control mechanisms are still unclear in other oxyphototrophs. Diatoms are believed to be the most successful phytoplankton in aquatic ecosystems and to be responsible for approximately 20% of primary productivity.6,7 Their lightharvesting apparatus is a fucoxanthin Chl a/c-binding protein (FCP), whose pigment (Chl c, fucoxanthin, diadinoxanthin, and diatoxanthin) and polypeptide (Lhcf and Lhcr) compositions are notably different from those of LHC (Chl b, lutein, 9′-cis neoxanthin, violaxanthin, and zeaxanthin).8−11 The detailed mechanism of excitation energy transfer and quenching in a complex consisting of PSII and FCP remains largely unknown; © 2014 American Chemical Society

Received: July 17, 2014 Accepted: August 15, 2014 Published: August 15, 2014 2983

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Figure 1. TRFS of the PSII−FCPII (red lines) and PSII (black lines) complexes at 77 K. Each spectrum was normalized with the 0 ps fluorescence peak intensity of the PSII complex. The right and left vertical axes represent fluorescence intensities of the PSII−FCPII and PSII complexes, respectively.

Figure 2. FDAS of the PSII−FCPII (red lines) and PSII (black lines) complexes at 77 K. Each spectrum was normalized with the peak intensity of the 25 ps fluorescence decay of the PSII complex. The right and left vertical axes represent fluorescence amplitudes of the PSII−FCPII and PSII complexes, respectively.

of the PSII−FCPII complex remained almost unchanged within 200 ps and was maintained at approximately 41% even for 2.0 ns. Moreover, the spectral widths in the PSII−FCPII complex were considerably broader than those in the PSII complex at each time constant. These differences between the PSII−FCPII and PSII complexes are caused by the binding of FCPII, whose TRFS showed that its fluorescence maximum was shifted from 677 to 686 nm in 6.0 ns,14 thereby reflecting the functional binding of FCPII to PSII in the isolated PSII−FCPII fraction. These findings suggest that the PSII−FCPII complex contains Chls with a wide variety of excitation levels, some of which originate from low-energy Chls generated by Chl−Chl exitonic interactions at the interface between PSII and FCPII and/or within FCPII. After 30 ns, the fluorescence spectra exhibited a 693 nm peak in both complexes, with a similar intensity and width. The 30 ns fluorescence spectra can be assigned as delayed fluorescence, which is generated by charge recombination between the special pair Chl (P) and the primary electron acceptor pheophytin in PSII.15 Figure 2 shows FDAS of the PSII−FCPII and PSII complexes. An expanded view from 620 to 660 nm in FDAS is presented in Supporting Information Figure S1. Five lifetime components (tens of ps, hundreds of ps, and approximately 2.4, 5.6, and 33 ns) were necessary to fit the fluorescence rise and decay profiles. Fluorescence decay and rise components are represented as positive and negative peaks, respectively. The sets of positive and negative peaks reflect excitation energy transfer. In the PSII−FCPII complex, the 20 ps component represented a set of positive peaks around 643, 674, and 684 nm and a negative peak around 695 nm. The 480 ps component represented positive peaks around 678 and 690 nm in addition to small peaks around 711 and 733 nm, which reflect Chl vibration bands. The 2.4 ns component represented a broad positive peak around 686 nm, including a shoulder at 690−705 nm. The 5.6 ns component represented a broad positive peak around 691 nm, including a shoulder at 695−705

nm. The 33 ns component represented only a positive peak around 693 nm. In the PSII complex, on the other hand, the 25 ps component represented a set of positive peak around 686 nm and a negative peak around 695 nm. The 280 ps component represented only a positive peak around 690 nm. The 1.4 ns component represented only a positive peak around 695 nm. The 5.2 ns component represented a broad positive peak around 695 nm, including a shoulder at 680−692 nm. The 31 ns component represented only a positive peak around 693 nm. Note that the 643 nm peak originated from Chl c, and the other peaks originated from Chl a. In the tens of ps, the 643 and 674 nm decays were observed only from the PSII−FCPII complex, originating from FCPII. The other fluorescence components probably originate from PSII core antennae. In the PSII complex, the amplitude of the 686 nm decay was significantly higher than that of the 695 nm rise. The different amplitudes between the decay and rise are due to the charge-separation reactions of PSII. When excitation energy is trapped in reaction center (RC) Chls in PSII, the energy is utilized for charge separation after tens of ps, resulting in no observation of fluorescence rise from RC Chls.16 Note that RC Chls contain PD1/PD2 and the accessary Chls (ChlD1/ ChlD2). The Chl component around 686 nm acts as an energy donor to RC Chls. This is in agreement with a previous study, which shows that the fluorescence rise and decay at tens of ps are assigned as excitation energy trapping components in the cyanobacterial PSII complexes.17 Chl686 also acts as an energy donor to the Chl component around 695 nm with a much lower contribution. Because Chl695 is identical to the lowenergy Chl694 in the core antenna protein CP47 of PSII,18 the results indicates that a slight amount of excitation energy is transferred to Chl695 in CP47. In the PSII−FCPII complex, in contrast, the amplitude of the 695 nm rise was higher than that of the 684 nm decay. This reflects energy transfer from FCPII 2984

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Table 1. 77 K Fluorescence Lifetimes and Their Amplitudes for the PSII, PSII−FCPII, and FCPII Complexes Observed at 683 nm Normalized amplitudea (lifetime)b PSII PSII−FCPII FCPII

62% (20 ps) −100% (14 ps) −18% (52 ps)

25% (120 ps) 17% (280 ps) −20% (100 ps)

8% (460 ps) 16% (810 ps) −31% (330 ps)

4% (1.5 ns) 28% (2.3 ns) 100% (4.0 ns)

1% (6.6 ns) 39% (4.9 ns)

a

Decay and rise (positive and negative values, respectively) are normalized to the overall decay. bThe time window of measurements was 10 ns (Supporting Information Figure S2).

to CP47. The high amplitude of fluorescence rise at tens of ps has been observed in FDAS of spinach PSII membrane.19 In the hundreds of ps, the 690 nm decay was observed from both the complexes, whereas no fluorescence rise was observed, reflecting that excitation energy was trapped in RC Chls.16 This FDAS feature has been observed in cyanobacterial PSII complexes, whose FDAS exhibit only the 688 nm fluorescence decay with the time constant of 200 ps,18 suggesting that the fluorescence decay around 690 nm is generally characteristic of the PSII complexes. However, the time constant of the FDAS of PSII−FCPII complex (480 ps) was approximately 1.7-fold longer than that of PSII complex (280 ps). This is likely due to the existence of low-energy Chls, which may construct different excitation energy transfer pathways by the binding of FCPII to PSII, thereby resulting in the longer time constants for excitation energy trapping and migration in the PSII−FCPII complex. The 678 nm decay was observed only from the PSII−FCPII complex, reflecting that the Chl component around 678 nm exists in FCPII. It appears that Chl678 is involved in either excitation energy transfer or quenching. The former is that Chl678 transfers excitation energy to RC Chls followed by the charge-separation reactions. In contrast, the latter is that Chl678 transfers excitation energy to carotenoids followed by quenching. This quenching model using carotenoids has been proposed in various light-harvesting apparatus4 including diatom FCP.20 The quenching reaction is supported by the prominent Chl vibration bands around 711 and 733 nm, because Miloslavina and co-workers identified such Chl vibration bands as the feature of quenching in diatom cells exposed to strong irradiance (600 μmol photons m−2 s−1).21 However, it is quite unlikely that one PSII−FCPII complex plays dual roles in excitation energy transfer and quenching, suggesting that a part of the isolated PSII−FCPII complex forms a quenching state, although the remainder forms a lightharvesting state. In a few ns (approximately 2.4 ns), the 695 nm decay was observed in the PSII complex, indicating migration of the excitation energy to Chl695 in CP47. The 686 nm decay was mainly observed in the PSII−FCPII complex, reflecting that the Chl component around 686 nm represents low-energy Chl near/within FCPII. Chl686 likely contributes to the relatively long time constant in FDAS of the PSII−FCPII complex (2.4 ns). Furthermore, the fluorescence amplitude in 2.4 ns FDAS of the PSII−FCPII complex was significantly higher than that in 1.4 ns FDAS of the PSII complex. These findings suggest that low-energy Chls near/within FCPII, such as Chl686, maintain a large amount of excitation energy without transferring it to PSII. However, it should be noted that slow energy quenching, including nonradiative processes, are present in this time region;20 hence, Chl686 may be involved in energy quenching. In approximately 5.6 ns, the 695 nm decay was observed in the PSII complex, whereas the 691 nm decay was observed in

the PSII−FCPII complex. Because this time constant is the fluorescence lifetime of Chl a, the Chl components around 691 and 695 nm in the PSII−FCPII and PSII complexes, respectively, are the termination of excitation energy. Chl695 probably originates from CP47, whereas Chl691 is low-energy Chl near/within FCPII. Moreover, the fluorescence amplitude in 5.6 ns FDAS of the PSII−FCPII complex was significantly higher than that in 5.2 ns FDAS of the PSII complex, indicating that the binding of FCPII to PSII induces the excitation energy transfer to low-energy Chls near/within FCPII, without utilizing the charge-separation reaction in PSII. In approximately 33 ns, the 693 nm fluorescence decay was observed as delayed fluorescence in both the complexes. To examine the contribution of FCPII as energy donor, the 77 K fluorescence decay curves at 683 nm were compared among the PSII, PSII−FCPII, and isolated FCPII complexes (Supporting Information Figure S2). The curve representing the isolated FCPII complex was analyzed using previous data.14 The fluorescence amplitudes and lifetimes were determined by fitting the decay curves (Table 1). Fluorescence decays within hundreds of picoseconds are involved in excitation energy trapping in RC Chls in PSII complexes;18 therefore, the proportions of total trapping in the PSII and PSII-FCPII complexes can be estimated to 95% and 33%, respectively. Because almost all of the excitation energy originating from PSII core antenna in the PSII−FCPII complex is most probably trapped in RC Chls, it is indicated that approximately 67% of the excitation energy originating from FCPII is not utilized for the charge-separation reaction. In contrast, under the 283 K conditions (Table 2), the longest-lived component (approxTable 2. 283 K Fluorescence Lifetimes and Their Amplitudes for the PSII and PSII−FCPII Complexes Observed at 683 nm Normalized amplitude (lifetime)a PSII PSII−FCPII a

57% (220 ps) 51% (310 ps)

37% (1.0 ns) 31% (1.4 ns)

6% (2.0 ns) 18% (2.4 ns)

The time window of measurements was 10 ns.

imately 5 ns) was lost in the PSII−FCPII complex; instead, the 2.4 ns component appeared although its contribution was low (18%). This is due to the uphill energy transfer from the lowenergy Chls to RC Chls. Furthermore, the amplitudes and lifetimes of the PSII−FCPII complex were roughly comparable to those of the PSII complex (Table 2), although quenching component is likely included in the fluorescence decays within 1.4 ns in the PSII−FCPII complex. These results strongly indicate that excitation energy harvested in low-energy Chls near/within FCPII is effectively utilized for the chargeseparation reaction in the PSII−FCPII complex under physiological temperature. 2985

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Letter

EXPERIMENTAL METHODS The PSII−FCPII and PSII complexes were isolated according to the procedure described by Nagao et al.12,13 and were suspended in 50 mM MES-NaOH buffer (pH 6.5) containing 1 M betaine. Time-resolved fluorescence spectra (TRFS) of the PSII−FCPII and PSII complexes were measured using a timecorrelated single-photon counting system.22 Fluorescence decay-associated spectra (FDAS) were constructed from a global fitting analysis of the fluorescence kinetics.20

In the case of the isolated FCPII complex (Table 1), almost all the fluorescence decays were observed with a lifetime of 4.0 ns, whereas the fluorescence rises were observed with lifetimes of 52, 100, and 330 ps and were interpreted as the intercomplex energy transfer between FCP-A oligomer and FCP-B/C trimer in FCPII.14 The fluorescence rises with the hundreds of ps were not observed in the PSII−FCPII complex, reflecting that the PSII−FCPII complex creates novel excitation energy transfer pathways from FCPII to PSII when FCPII binds to PSII. The novel energy pathways likely contribute to the significant high fluorescence rise at 14 ps and decays at 2.3 and 4.9 ns in the PSII−FCPII complex. The characteristic fluorescence components in the PSII−FCPII complex were not present in the PSII and isolated FCPII complexes, reinforcing the view that FCPII is functionally associated with PSII. Moreover, the existence of free Chls a/c can be completely excluded in both PSII complexes because there were no fluorescence decays at 643 and 670−673 nm in approximately 5 ns FDAS, thus providing evidence that the PSII−FCPII and PSII complexes presented here are functional complexes. In conclusion, for the first time, we have demonstrated the spectral features of excitation energy transfer and quenching in the PSII−FCPII complex, whose FDAS exhibited the high amplitudes of fluorescence rise and decay in spite of the functional binding of FCPII to PSII. The high amplitudes and relatively long time constants in the PSII−FCPII complex are attributed to the complicated energy transfer pathways before trapping (Figure 3). Most of Chls in FCPII exists as a bulk Chl,

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ASSOCIATED CONTENT

S Supporting Information *

Details of experimental procedures. Possibility of low-light induced quenching. An expanded view of FDAS (Figure S1). Fluorescence decay curves at 77 K (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ¶

R.N. and M.Y. are the first coauthors.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a Grant-in-Aids for Scientific Research from the Ministry of Education of Japan (21570038, 24370025, and 26220801 to T.T. and 22370017 to T.T. and S.A.), a Grant-in-Aid for JSPS Fellows No. 21-2944 (M.Y.), a grant from JST PRESTO (to T.T.), and a grant from Australian Research Council’s Discovery Projects funding scheme (project number DP12101360 to T.T.).

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Figure 3. Schematic representation of excitation energy transfer pathways in the PSII−FCPII complexes. Gray dashed circles represent FCPII and PSII. Blue and red arrows represent downhill and uphill energy transfer, respectively. Bulk Chls (Chl643, Chl674, and Chl678) and low-energy Chls (Chl686 and Chl691) represent closed gray and black circles, respectively.

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which transfers excitation energy to RC Chls in PSII or lowenergy Chls near/within FCPII via downhill energy transfer under the 77-K conditions. The excitation energy harvested in the low-energy Chls is transferred to RC Chls via uphill energy transfer under the 283-K conditions. These findings provide strong evidence that uphill energy transfer from low-energy Chls is of significant importance for excitation energy trapping in RC Chls in the PSII−FCPII complex. Intriguingly, the quenching mode of PSII−FCPII complex was detected in 480 ps FDAS, although the PSII−FCPII and PSII complexes were isolated from C. gracilis grown at 30 μmol photons m−2 s−1 (the possibility of low-light induced quenching is discussed in Supporting Information). It is therefore likely that the isolated PSII-FCPII complexes form either light-harvesting state or quenching state even under the low-light conditions, implying that C. gracilis precisely controls excitation energy by properly utilizing the different states of the PSII−FCPII complexes. The unique control system would contribute to the optimum supply of light energy to PSII under the fluctuating light conditions in aquatic environments, leading to the successful prosperity of diatom species on the Earth. 2986

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