Article pubs.acs.org/JPCB
High Excitation Energy Quenching in Fucoxanthin Chlorophyll a/c‑ Binding Protein Complexes from the Diatom Chaetoceros gracilis Ryo Nagao,*,†,∥ Makio Yokono,‡ Seiji Akimoto,‡,§ and Tatsuya Tomo¶,⊥ †
Department of Integrated Sciences in Physics and Biology, College of Humanities and Sciences, Nihon University, 3-25-40 Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan ‡ Molecular Photoscience Research Center, Kobe University, Kobe 657-8501, Japan § CREST, Japan Science and Technology Agency (JST), 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 S Supporting Information *
ABSTRACT: The fucoxanthin chlorophyll (Chl) a/c-binding protein (FCP) is responsible for excellent light-harvesting strategies that enable survival in fluctuating light conditions. Here, we report the light-harvesting and quenching states of two FCP complexes, FCP-A and FCP-B/C, isolated from the diatom Chaetoceros gracilis. Pigment analysis revealed that FCP-A is enriched in Chl c, whereas FCP-B/C is enriched in diadinoxanthin, reflecting differences in low-temperature steady-state absorption and fluorescence spectra of each FCP complex. Time-resolved fluorescence spectra were measured at 77 K, and the characteristic lifetimes were determined using global fitting analysis of the spectra. Tens of picosecond (ps) components revealed energy transfer to low-energy Chl a from Chls a and c, whereas the other components showed only fluorescence decay components with no concomitant rise components. The normalized amplitudes of hundreds of picosecond components were relatively 30% in the total fluorescence, whereas those of longest-lived components were 60%. The hundreds of picosecond components were assigned as excitation energy quenching, whereas the longest-lived components were assigned as fluorescence from the final energy traps. These results suggest that 30% of FCP complex forming quenching state and the other 60% of FCP complex forming light-harvesting state exist heterogeneously in each FCP fraction under continuous low-light condition. fluorescence decay-associated spectra (FDAS), which is obtained through the global fitting of TRFS. Chls having the decay component are frequently recognized as low-energy Chls. It has been proposed that low-energy Chls are generated by excitonic interactions within a trimer upon oligomerization.7 The excitation energy harvested in low-energy Chls appears to be quenched through xanthophyll carotenoids that have a sufficiently lower energy level (S1 excited state).8,9 The deepoxidized form of xanthophylls, zeaxanthin (Zx), is mainly involved in NPQ and naturally functions in LHCII in response to a sudden increase in irradiance.1−3 However, very little is known about the excitation energy quenching state in other light-harvesting apparatus. Diatoms are a major group of microalgae ubiquitous in the world’s oceans and freshwater environments.10 They are currently the most successful phytoplankton in terms of their primary production and the number of species.11 Their successful prosperity is partly a result of their light-harvesting
1. INTRODUCTION Photosynthetic organisms have developed numerous strategies to optimize the realized quantum yield of photosynthesis in naturally variable light environments. To achieve effective photosynthesis, light-harvesting antenna complexes are utilized under low-light conditions. On the other hand, the antennae are involved in the non-photochemical quenching (NPQ) of excess excitation energy under high-light conditions. The quenching reaction results in a decrease in the total fluorescence yield by harmlessly dissipating excess energy as heat, which protects photosystem II (PSII) from light-induced damage.1−3 Thus, light-harvesting antenna complexes alter their conditions from light-harvesting state to quenching state in response to various light irradiances, and vice versa. The detailed light-harvesting and quenching states have been examined by time-resolved fluorescence spectra (TRFS) using a major antenna from plants, the light-harvesting chlorophyll a/bbinding protein complex of PSII (LHCII). The lifetime in the 100 ps region is resolved as the characteristic time constant for NPQ in oligomeric LHCII, which is an aggregation form of trimeric LHCII.4−7 With the lifetime in this time region, a dominant fluorescence decay component is represented as © XXXX American Chemical Society
Received: February 8, 2013 Revised: May 13, 2013
A
dx.doi.org/10.1021/jp403923q | J. Phys. Chem. B XXXX, XXX, XXX−XXX
The Journal of Physical Chemistry B
Article
Table 1. Pigment Compositions of FCP Complexes by Reversed-Phase HPLCa FCP-A FCP-B/C a
Chl c1
Chl c2
Fx
Ddx
Chl a
0.456 ± 0.029 0.293 ± 0.013
0.388 ± 0.029 0.313 ± 0.011
1.644 ± 0.058 1.880 ± 0.014
0.033 ± 0.001 0.101 ± 0.003
1.000 1.000
Values represent mole of pigment/mole of Chl a. Ratios are given as the mean ±standard deviation of four measurements.
2.2. Reversed-Phase HPLC. An Inertsil C8 column (150 × 4.6 mm, 5 μm particle size; GL Sciences, Japan) was equipped with a photodiode array detector (MD-2018 Plus; JASCO, Japan). Samples were extracted with 90% acetone. After centrifugation at 40 000g for 10 min, the supernatant was diluted to 75% acetone with distilled water, and the sample was injected into the HPLC system. Pigments were eluted according to Zapata et al.;28 eluent A was a mixture of methanol:acetonitrile:0.25 M pyridine (50:25:25 v:v:v), whereas eluent B was a mixture of methanol:acetonitrile:acetone (20:60:20 v:v:v). The flow rate was set to 0.9 mL min−1. For calibration standards, Chl a was quantified in 100% acetone at 661.6 nm using an extinction coefficient of 8.13 × 104 M−1 cm−1.29 Chls c1 and c2 were quantified in 100% acetone (+1% pyridine) at 446 and 445 nm using extinction coefficients of 2.12 × 105 and 1.96 × 105 M−1 cm−1, respectively.30 Fx and Ddx were quantified in 100% acetone at 448 nm using extinction coefficients of 1.09 × 105 and 1.29 × 105 M−1 cm−1, respectively.31 2.3. Low-Temperature Absorption Spectra. Absorption spectra were measured using a V-660 spectrophotometer (JASCO) at 80 K. For low-temperature spectra, a cryostat (OptistatDN; Oxford, UK) was used in conjunction with a controller (Oxford ITC-601PT). 2.4. Steady-State Fluorescence Spectra. Steady-state fluorescence spectra were recorded using a spectrofluorometer (FP 6600, JASCO) at 77 K. The excitation wavelengths were varied from 420 to 690 nm using at 1 nm intervals, and fluorescence spectra were recorded at 1 nm intervals. The fluorescence emission and excitation spectra were corrected. 2.5. Time-Resolved Fluorescence Spectra. TRFS was measured using the time-correlated single-photon-counting method at 77 K.32 Excitation laser intensity was set to give fluorescence signals of less than 10 000 counts/s around fluorescence peak wavelengths, and in this condition samples did not suffer damage from the laser excitation with a repetition rate of 2.9 MHz. Fluorescence decay kinetics showed little change (
