Energy Transfer in Cyanobacteria and Red Algae: Confirmation of

Aug 26, 2016 - †Graduate School of Science, ‡Graduate School of Engineering, and §Molecular Photoscience Research Center, Kobe University, Kobe ...
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Energy Transfer in Cyanobacteria and Red Algae: Confirmation of Spillover in Intact Megacomplexes of Phycobilisome and Both Photosystems Yoshifumi Ueno,† Shimpei Aikawa,‡ Akihiko Kondo,‡ and Seiji Akimoto*,†,§ †

Graduate School of Science, ‡Graduate School of Engineering, and §Molecular Photoscience Research Center, Kobe University, Kobe 657-8501, Japan S Supporting Information *

ABSTRACT: Cyanobacteria and red algae control the energy distributions of two photosystems (PSI and PSII) by changing the energy transfer among phycobilisome (PBS), PSI, and PSII. However, whether PSII → PSI energy transfer (spillover) occurs in the intact megacomplexes composed of PBS, PSI, and PSII (PBS−PSII−PSI megacomplexes) in vivo remains controversial. In this study, we measured the delayed fluorescence spectra of PBS-selective excitation in cyanobacterial and red algal cells. In the absence of spillover, 7% of the PBS (at most) would combine with PSII, inconsistent with the PBSs’ function as the antenna pigment−protein complexes of PSII. Therefore, we conclude that spillover occurs in vivo in PBS−PSII−PSI megacomplexes of both cyanobacteria and red algae.

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reported spillover was unlikely, although PBS was found to supply energy to both PSs in the isolated megacomplexes.14 Therefore, although the importance of spillover is understood in vivo, its occurrence in intact PBS−PSII−PSI megacomplexes remains controversial. In the present study, we examined whether spillover occurs in the intact PBS−PSII−PSI megacomplexes of two cyanobacterial species and a red alga in vivo. Figure 1 shows the absorption spectra of Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis), Synechococcus sp. PCC 7002 (Synechococcus), and Cyanidioschyzon merolae at 77 K. Each spectrum is normalized by the Chl Qy (0,0) band. Chl exhibits two absorption bands around 440 and 680 nm, corresponding to the Soret band and the Qy (0,0) band, respectively. PBS shows two absorption peaks around 630 and 650 nm, corresponding to phycocyanin (PC) and allophycocyanin (APC), respectively. The absorption spectra of the red alga C. merolae exhibits no APC peak, but a transition energy appears between the Chl Qy (0,0) band and the PC band. The absorption peaks in the 450−550 nm region were assigned to Car. We should note that the Qy (1,0) band of Chl appears around 630 nm. Therefore, laser excitation at 633 nm directly excites Chl as well as PBS. In PSs isolated from a cyanobacterium Synechocystis, the relative absorbance of the Qy (1,0) band to the Chl Qy (0,0) band at 633 nm was estimated as 0.20−0.24.15 To obtain the contribution of PBS to the relative absorbance, we subtracted 0.22 from the observed

xygenic photosynthetic organisms possess two photosystems, photosystem I (PSI) and photosystem II (PSII), each containing chlorophyll (Chl) and carotenoid (Car) as the major light-harvesting pigments. Efficient photosynthesis relies on a balanced excitation between PSI and PSII. As recently revealed, higher plants adapt to changing light conditions by controlling the energy transfer between both PSs (the PSII → PSI energy transfer is known as spillover).1 Cyanobacteria and red algae contain specific antenna pigment−protein complexes called phycobilisomes (PBSs), which extend their lightharvesting ability.2,3 The efficiency of the light energy transfer from PBS (which absorbs the light) to the Chl in PSs approaches 100%.4 PBSs are generally considered as the antenna pigment−protein complexes for PSII, but under intense light or PSII-exciting light, they can transfer light energy to PSI.5 Energy transfer from PBS to PSI is thought to occur by one of two pathways; direct PBS to PSI energy transfer (PBS → PSI transfer)6,7 or energy transfer from PBS to PSI via PSII (spillover after PBS → PSII transfer, i.e. PBS → PSII → PSI transfer).8−10 PBS → PSII → PSI transfer was first proposed by Murata,8 and has since been accepted by many researchers.9,10 In the later studies, PBS → PSI transfer was proposed.6,7 Recently, spillover has been reported by several research groups. Yokono et al. demonstrated the existence of spillover in red algae by measuring their delayed fluorescence spectra,11 which derives from charge recombination at the PSII reaction center.12 Kowalczyk et al. similarly confirmed PBS → PSII → PSI transfer in intact red macroalga Chondrus crispus.13 More recently, Liu et al. isolated PBS−PSII−PSI megacomplexes from the cyanobacterium Synechocystis sp. PCC 6803, and © 2016 American Chemical Society

Received: July 21, 2016 Accepted: August 26, 2016 Published: August 26, 2016 3567

DOI: 10.1021/acs.jpclett.6b01609 J. Phys. Chem. Lett. 2016, 7, 3567−3571

Letter

The Journal of Physical Chemistry Letters

the observed PSI fluorescence in the delayed fluorescence is attributed to spillover.11 To examine the energy transfer processes among PBS, PSI, and PSII, we classified the pigment−protein complexes into six categories, as depicted in Figure S1 (A: PSII complex; B: PSI complex; C: PSII−PSI supercomplex; D: PBS−PSII supercomplex; E: PBS−PSI supercomplex; F: PBS−PSII−PSI megacomplex). Only the PSII complexes emitted delayed fluorescence (Figure S1, A and D). Among these, the complexes containing both PSII and PSI exhibited delayed fluorescence in the PSI fluorescence region (Figure S1, C and F) with a single peak above 700 nm. In contrast, the PSII fluorescence region showed two fluorescence bands assignable to CP43 (686 nm) and CP47 (694 nm). Therefore, the PSII fluorescence intensity in the delayed fluorescence (DFPSII) was determined by averaging the fluorescence intensities of the CP43 and CP47 bands. The vibrational bands of the PSII and PSI fluorescences overlap. In isolated PSII, the intensity of the PSII vibrational band was ∼20% that of the main PSII fluorescence peak.1 Therefore, the PSI fluorescence intensity in the delayed fluorescence (DFPSI) was determined by subtracting 0.2 × DFPSII from the observed fluorescence intensity at the PSI peak.1 The corrected fluorescence intensity ratios of PSII to PSI in the delayed fluorescence (DFPSII/DFPSI) are summarized in Table 2. To discuss the energy transfer pathways in vivo, we assumed no spillover in the PBS−PSII−PSI megacomplexes. Under this assumption, F would become a combination of D and E, and the delayed fluorescence in the PSI fluorescence region would originate from C alone. Here we calculate the ratios of PBS− PSII in the PBS-associated complexes, assuming the existences of A−E in Figure S1. Given the relative absorbances of PSs and PBS (APSs and APBS, respectively), the probability of PSs excitation at 633 nm is calculated by eq 1.

Figure 1. Absorption spectra of Synechocystis sp. PCC 6803 (a), Synechococcus sp. PCC 7002 (b), and C. merolae (c) at 77 K. Each spectrum is normalized by the Chl Qy (0,0) band.

relative absorbance at 633 nm (see Table 1). Accordingly, we obtained the relative absorbance ratios of the PSs to PBS at 633 nm (APSs/APBS) in each sample (Table 1).

APSs APSs + APBS

PSI and PSII contain ∼100 Chl molecules per monomer18 and ∼35 Chl molecules per monomer,19 respectively. Therefore, the Chl content is approximately three times higher in PSI than in PSII. The probability of PSII excitation at 633 nm is then given by

Table 1. Relative Absorbance Ratios of Photosystems (PSs) to Phycobilisome (PBS) at 633 nm in the Absorption Spectra of Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7002, and C. merolae

APSs/APBS

Synechocystis sp. PCC 6803

Synechococcus sp. PCC 7002

C. merolae

0.28

0.35

0.23

(1)

APSs 1 × APSs + APBS 3α + 1

(2)

where α is the reaction center ratio of PSI to PSII. From eq 2, the DFPSII and DFPSI after direct PS-excitation are given by eqs 3 and 4, respectively.

Figure 2 shows the time-resolved fluorescence spectra (TRFS) of Synechocystis, Synechococcus, and C. merolae under 633 nm excitation at 77 K. Fluorescences in the 675−700 nm and 700−750 nm regions originate from PSII and PSI, respectively. Up to 4.0 ns postexcitation, the relative intensities and peak wavelengths of the fluorescence bands are dynamic, reflecting the energy transfer within or among PBS, PSI, and PSII. At 47−76 ns and 77 K, the TRFS exhibits delayed fluorescence with a lifetime of 15−25 ns (bottom panels in Figure 2).12 It was reported that, when dithionite was added and strong light was irradiated, PSI isolated from a higher plant shows delayed fluorescence in later time region at 77 K.16 However, we found that PSI isolated from a cyanobacterium Synechocystis and PSI−LHCI isolated from a higher plant Arabidopsis thaliana does not exhibit delayed fluorescence in the time region examined in the present study.1,17 Therefore,

APSs 1 a+d × × APSs + APBS 3α + 1 a+c+d

(3)

APSs 1 c × × APSs + APBS 3α + 1 a+c+d

(4)

where a, c, and d denote the abundance ratios of A, C, and D, respectively. The DFPSII after PBS → PSII transfer is computed by eq 5.

APBS d × APSs + APBS d+e

(5)

where e is the abundance ratio of E. DFPSII/DFPSI is then computed by eq 6. 3568

DOI: 10.1021/acs.jpclett.6b01609 J. Phys. Chem. Lett. 2016, 7, 3567−3571

Letter

The Journal of Physical Chemistry Letters

Figure 2. Time-resolved fluorescence spectra of Synechocystis sp. PCC 6803 (a), Synechococcus sp. PCC 7002 (b), and C. merolae (c) at 77 K. The excitation wavelength was 633 nm. Zero time corresponds to the time of maximum intensity of the excitation laser pulse. Each spectrum is normalized by its maximum intensity, and the numbers shown on each panel indicate magnification factors relative to the most intense spectrum.

ratios in Synechocystis, Synechococcus, and C. merolae become