- Email: [email protected]
Artificially produced [7-formyl]-chlorophyll d functions as an antenna pigment in the photosystem II isolated from the chlorophyllide a oxygenase-expressing Acaryochloris marina
Biochimica et Biophysica Acta 1817 (2012) 1285–1291
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbabio
Artificially produced [7-formyl]-chlorophyll d functions as an antenna pigment in the photosystem II isolated from the chlorophyllide a oxygenase-expressing Acaryochloris marina☆ Tohru Tsuchiya a,⁎, Seiji Akimoto b, Tadashi Mizoguchi c, Kazuyuki Watabe a, Hayato Kindo d, Tatsuya Tomo d, e, Hitoshi Tamiaki c, Mamoru Mimuro a a
Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606–8501, Japan Molecular Photoscience Research Center, Kobe University, Kobe 657–8501, Japan c Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, Kusatsu, Shiga 525–8577, Japan d Department of Biology, Faculty of Science, Tokyo University of Sciences, Kagurazaka, Shinjuku-ku, Tokyo 162–8601, Japan e Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332–0012, Japan b
a r t i c l e
i n f o
Article history: Received 17 November 2011 Received in revised form 17 February 2012 Accepted 20 February 2012 Available online 28 February 2012 Keywords: Chlorophyll d Cyanobacteria Photosystem II Acaryochloris marina
a b s t r a c t Acaryochloris marina, a chlorophyll (Chl) d-dominated cyanobacterium, is a model organism for studying photosynthesis driven by far-red light using Chl d. Furthermore, studies on A. marina may provide insights into understanding how the oxygenic photosynthetic organisms adapt after the acquisition of new Chl. To solve the reaction mechanism of its unique photosynthesis, photosystem (PS) II complexes were isolated from A. marina and analyzed. However, the lack of a molecular genetic method for A. marina prevented us from conducting further studies. We recently developed a transformation system for A. marina and we introduced a chlorophyllide a oxygenase gene into A. marina. The resultant transformant accumulated [7-formyl]-Chl d, which has never been found in nature. In the current study, we isolated PS II complexes that contained [7-formyl]-Chl d. The pigment composition of the [7-formyl]-Chl d-containing PS II complexes was 1.96 ± 0.04 Chl a, 53.21 ± 1.00 Chl d, and 5.48 ± 0.33 [7-formyl]-Chl d per two pheophytin a molecules. In contrast, the composition of the control PS II complexes was 2.01± 0.06 Chl a and 62.96 ± 2.49 Chl d. The steady-state fluorescence and excitation spectra of the PS II complexes revealed that energy transfer occurred from [7-formyl]-Chl d to the major Chl d species; however, the electron transfer was not affected by the presence of [7-formyl]-Chl d. These findings demonstrate that artificially produced [7-formyl]-Chl d molecules that are incorporated into PS II replace part of the Chl d molecules and function as the antenna. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Oxygenic photosynthetic organisms use chlorophylls (Chls) for photosynthesis, and the distribution of Chls varies among taxonomic groups. Certain Chls serve both as electron transfer components and as antenna, whereas others function solely as antenna [1]. The modification of the side chain(s) on the macrocyclic ring of a Chl molecule is critical for its functions because the change affects its chemical properties, such as its redox potential and chemical reactivity. Therefore, the properties of photosystems (PSs) and antenna proteins
Abbreviations: CAO, chlorophyllide a oxygenase; Chl, chlorophyll; CP43, 43-kDa chlorophyll protein; CP47, 47-kDa chlorophyll protein; Phe, pheophytin; PS, photosystem; TRFS, time-resolved fluorescence spectra ☆ This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. ⁎ Corresponding author. Tel.: + 81 75 753 6575; fax: + 81 75 753 7909. E-mail address: [email protected] (T. Tsuchiya). 0005-2728/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2012.02.021
primarily depend on the Chl species used in the organism. All oxygenic photosynthetic organisms produce Chl a (or [8-vinyl]-Chl a, in the case of Prochlorococcus spp.), other Chls are thought to have been acquired during the evolution of the organisms. Once a new Chl was acquired, the photosynthetic apparatus would adapt to facilitate the efficient use of the Chl [2,3]. In this sense, the acquisition of new Chl biosynthetic activities is a key driving force in the evolution of photosynthetic organisms. From the viewpoint of the acquisition of new Chl biosynthetic activity, Acaryochloris spp., marine unicellular cyanobacteria, are suitable models for the study of the evolution of photosynthesis. Acaryochloris spp. are the only known organisms that contain Chl d as a major Chl, and their content of Chl a is quite low (b 5%). Furthermore, Acaryochloris spp. can utilize far-red light for photosynthesis using Chl d [4]. Therefore, the photosynthetic reactions driven by Chl d have been intensively studied since the discovery of Acaryochloris marina [5,6]. It was shown that the special pair of PS I was a Chl d/Chl d’ heterodimer in A. marina [7,8]. Similarly, the special pair of PS II contains Chl d although there is
1286
T. Tsuchiya et al. / Biochimica et Biophysica Acta 1817 (2012) 1285–1291
an ongoing controversy as to whether the special pair is a Chl d/Chl d homodimer [9] or a Chl a/Chl d heterodimer [10]. In contrast, the primary electron acceptor of PS II was proven to be pheophytin (Phe) a [9–11]. The redox potential for water oxidation was estimated to be almost the same between a Chl a-dominated cyanobacterium Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis), and A. marina; this similarity indicates that the shortage of gain of light energy due to the utilization of Chl d by A. marina is compensated by a higher redox potential of Phe a in A. marina than in Synechocystis [12,13]. A similar result was shown in PS I: the special pairs of Synechocystis (P700) and A. marina (P740) yielded identical redox potentials [8,14]. These recent results revealed that the redox potentials of special pairs are determined by the oxidizing side. This finding is one of the prominent examples that support the consideration of the reaction principles in electron transfer system by comparing Chl a-based system with Chl d-based system. However, several unsolved problems still remain. One of these problems is the significance of the small amount of Chl a that is invariably found in PSs. To examine this problem, a perturbation that could affect the Chl composition in vivo was needed. Metabolic engineering of the Chl biosynthetic pathway is an effective way to replace Chls in PSs in vivo. For example, a gene encoding chlorophyllide a oxygenase (CAO), which is responsible for Chl b biosynthesis [15], was introduced into Synechocystis, and the resultant transformants produced Chl b [16,17]. The Chl b molecules in the transformants were incorporated into the PSs and functioned as antenna. These results indicate that the analyses of organisms produced by the strategy of artificial evolution are useful for the study of the evolution of oxygenic photosynthetic organisms. However, the genetic manipulation of A. marina has not yet been established. Therefore, we developed a transformation system for A. marina and introduced the CAO gene into this organism [18]. The CAO-expressing A. marina accumulated a novel Chl species instead of Chl b. The chemical structure of the novel Chl was determined to be [7-formyl]-Chl d, containing two formyl groups as its side chains (Fig. 1). [7-Formyl]-Chl d was hypothesized to be produced by the combined action of CAO and the enzyme(s) involved in Chl d biosynthesis. The molar ratio of [7formyl]-Chl d increased to 9.6% of the total Chl; however, the distribution of [7-formyl]-Chl d in the cells was not determined. We expected that the Chl a content might be reduced by the introduction of the CAO gene because CAO converts chlorophyllide a, a precursor of Chl a, into chlorophyllide b [19]. However, the content of Chl a in the CAO-expressing A. marina was similar to that of the control transformant [18]. This result indicates that the Chl a molecules in the PSs have a unique function that cannot be obtained from the Chl d molecules. In this study, we isolated the PS II complexes from CAO-expressing A. marina. The pigment composition and optical properties of the PS II complexes were analyzed. Based on our results, we discuss the function of Chl a and the newly synthesized [7-formyl]-Chl d in PS II and propose an asymmetric pigment configuration for A. marina PS II.
Fig. 1. The chemical structures of Chls related to this study.
2. Materials and methods 2.1. Culture of A. marina A. marina MBIC 11017 was cultured in an 8-liter culture bottle of IMK medium (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan) under photoautotrophic conditions; the light intensity was 10 μmol photons/(m 2 s) and the temperature was 298 K. For the transformants, spectinomycin was added to the medium (5 μg/ml). 2.2. Preparation and analysis of pigments The pigment compositions of PS II were analyzed following the approach of Zapata et al. [20]. The pigments were extracted with cold methanol from isolated PS II complexes, and the extracts were sonicated on ice. The supernatants of the centrifuged extracts were immediately analyzed by HPLC (Prominence series, Shimadzu, Kyoto, Japan) with a Symmetry C8 column (150 × 1.0 mm, 3.5-μm particle size; Waters, MA, USA). The elution profile was monitored with a photodiode array detector (SPD-M20A, Shimadzu). The compositions of the mobile phases were methanol:acetonitrile:0.25 M pyridine solution (50:25:25, v:v:v) and methanol:acetonitrile:acetone (20:60:20, v:v:v). The gradient profile was optimized for semimicro analysis. The calibration data were obtained using purified Chls as authentic samples. The Chls were quantified by the peak area of each Chl. Because the extinction coefficient for [7-formyl]Chl d had not been determined, the millimolar extinction coefficient for other Chl was applied. The formylation of the C-7 position of Chl a causes an approximately 45% decrease of the millimolar extinction coefficient [21], whereas little difference is observed from the formylation of the C-3 position of Chl a. Therefore, we applied the millimolar extinction coefficient for Chl b (38.55 mM - 1 cm - 1 for methanol) [21]. 2.3. Preparation of the PS II complexes PS II complexes in the thylakoid membranes of A. marina were solubilized in dodecyl-β-D-maltoside and then purified using a combination of sucrose density gradient centrifugation and anionexchange chromatography (UNO Q, Bio-Rad, CA, USA), as described previously [12]. The PS II core complexes of Synechocystis were prepared as reported previously [22]. The polypeptide composition of the purified complexes was analyzed by SDS-PAGE, using a 16–22% separating gel with a 6% stacking gel that contained 7.5 M urea [23]. After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250. 2.4. Spectroscopy The absorption and fluorescence spectra were measured as reported previously [24]. The fluorescence spectra were corrected for the spectral sensitivity of the detector. The Chl d concentration was adjusted to 2 μg/ml. For the fluorescence emission and excitation spectra, the respective bandwidths for excitation and fluorescence were 2 and 3 nm. The time-resolved fluorescence spectra (TRFS) and fluorescence decay curves were measured by the timecorrelated single-photon counting method at 77 K [25–27]. To examine differences in energy transfer process, TRFS were measured with a time interval of 2.4 ps/channel, whereas, to examine charge recombination process, fluorescence decay curves were measured at 685 nm with 24.4 ps/channel. A. marina cells and its PS II exhibited the delayed fluorescence around 685 nm, which was originated from charge recombination [9,25,31]. Although A. marina PS II exhibited fluorescences around 700 nm and 728 nm in later time region after laser excitation, any long-lived components, which could be assigned due to the delayed fluorescence, were not resolved in the fluorescence decay observed at
T. Tsuchiya et al. / Biochimica et Biophysica Acta 1817 (2012) 1285–1291
1287
these wavelengths [9]. Therefore, we selected 685 nm as the observed wavelength. The light source was a Ti:sapphire laser and the excitation wavelength was 400 nm, which brought about excitation of all Chls. 3. Results 3.1. Isolation and polypeptide composition of PS II complexes from the two transformants To investigate the function(s) of [7-formyl]-Chl d, we isolated PS II complexes from the CAO-expressing A. marina ([7-formyl]-Chl d-containing PS II complexes) and the control transformant (control PS II complexes). The polypeptide compositions of the complexes were examined by SDS-PAGE (Fig. 2). The peptide pattern of the [7-formyl]-Chl d-containing PS II complexes was very similar to that of the control PS II complexes; the principal bands were clearly detected in both preparations with almost identical stoichiometry. These PS II complexes consisted of a 47-kDa Chl protein (CP47), a 43-kDa Chl protein (CP43), D2, D1, the cytochrome b559 α-subunit, and low-molecular-weight proteins. 3.2. Pigment compositions of PS II complexes We analyzed the pigment compositions of the [7-formyl]-Chl dcontaining PS II complexes and the control PS II complexes by reversephase HPLC (Fig. 3). The pigment composition per two Phe a molecules was 2.01 ± 0.06 molecules of Chl a and 62.96 ± 2.49 molecules of Chl d in the control PS II complexes (Table 1). In contrast, the composition of the [7-formyl]-Chl d-containing PS II complexes was 1.96 ± 0.04 molecules of Chl a, 53.21 ± 1.00 molecules of Chl d, and 5.48±0.33 molecules of [7-formyl]-Chl d (Table 1). These results demonstrate that 9.0% of the total Chl molecules was [7-formyl]-Chl d in the [7-formyl]-Chl dcontaining PS II complexes, and that this value is partially correlated with a decrease in Chl d (15.5%). Moreover, we did not observe a difference in the Chl a content per Phe a between the two PS II complexes. Furthermore, we could not detect Phe d or any other pigment that was derived from Chl in the [7-formyl]-Chl d-containing PS II complexes (Fig. 3). Because the estimated Chl d content per two Phe a molecules
Fig. 3. Analyses of Chl compositions by HPLC. HPLC chromatograms of pigment extracts from the PS II complexes of control A. marina (A), [7-formyl]-Chl d-containing A. marina (B), and Synechocystis (C) monitored at 663 nm. 1, [7-formyl]-Chl d; 2, Chl d; 3, Chl a; 4, Phe a.
was significantly higher than the Chl a content per two Phe a molecules of Chl a-type PS II [28], we analyzed the pigment compositions of the PS II core complexes of Synechocystis as a control (Fig. 3C). The pigment composition per two Phe a molecules was 37.70±0.51 molecules of Chl a in the complexes (Table 1). Regarding the content of carotenoids, approximately eight and seven carotenoids per one Phe a molecule were detected in the control PS II complexes and the [7-formyl]-Chl d-containing PS II complexes, respectively. In contrast, approximately nine carotenoids per two Phe a molecules were found in the PS II core complexes of Synechocystis.
3.3. Low-temperature absorption spectra We measured the low-temperature absorption spectra of the PS II complexes. The results revealed clear differences in the Soret and Qy bands of [7-formyl]-Chl d at 80 K after normalization at the Soret band of Chl d (Fig. 4A). The difference in the absorption spectra between the two PS II complexes showed the presence of positive peaks at 482 and 675 nm and a few negative peaks at approximately 710 nm and at the Soret bands of Chl d, indicating several distinct forms of Chl d (Fig. 4B). These observations confirmed that part of the Chl d molecules were replaced by [7-formyl]-Chl d in the PS II complexes. This finding means that artificially produced [7-formyl]-
Table 1 The pigment compositions of PS II isolated from A. marina transformants and Synechocystis. Samples
Chl a
Chl d
[7-formyl]-Chl d1
carotenoids
A. marina
2.01 ± 0.06 (1.00 ± 0.03) 1.96 ± 0.04 (0.98 ± 0.02)
62.96 ± 2.49 (31.48 ± 1.25) 53.21 ± 1.00 (26.61 ± 0.50)
n.d. n.d. 5.48 ± 0.33 (2.74 ± 0.16)
15.17 ± 0.16 (7.59 ± 0.08) 13.31 ± 0.26 (6.65 ± 0.13)
37.70 ± 0.51
n.d.
n.d.
[7-formyl]-Chl d-containing A. marina Synechocystis Fig. 2. SDS-PAGE of PS II complexes isolated from A. marina transformants. Lane 1, control PS II complexes; lane 2, [7-formyl]-Chl d-containing PS II complexes. The individual symbols indicate CP47 (filled circle), CP43 (open circle), D2 (filled square), D1 (open square), the cytochrome b559 α-subunit (filled triangle), and low molecular-weight proteins (filled star).
9.12 ± 0.11
The values with standard deviations are normalized against two molecules of Phe a (n = 3). The values in parentheses are normalized against one molecule of Phe a. n.d., not detected. 1 The content of [7-formyl]-Chl d was estimated using the millimolar extinction coefficient for Chl b.
1288
T. Tsuchiya et al. / Biochimica et Biophysica Acta 1817 (2012) 1285–1291
Chl d molecules are incorporated into the pre-existing apoproteins without any amino-acid substitution.
3.4. Low-temperature excitation and emission spectra Clearly, [7-formyl]-Chl d was fully functional as an antenna pigment (Fig. 5). First, the emission spectra of the control PS II complexes were measured (Fig. 5A). When [7-formyl]-Chl d was excited at 482 nm in the [7-formyl]-Chl d-containing PS II complexes, the main emission was observed at 728 nm, with a small band at 678 nm (solid line, Fig. 5B). The former emission originated from PS II Chl d, as evidenced by the energy transfer from [7-formyl]-Chl d, and the latter, from free [7-formyl]-Chl d. Because of the very high fluorescence yield of free Chl, the amount of free [7-formyl]-Chl d was estimated to be very small, even though the fluorescence intensity was clear. In the control PS II complexes, fluorescence from free [7-formyl]-Chl d was not detected (Fig. 5A), confirming the absence of [7-formyl]-Chl d in the complexes. The energy transfer from [7-formyl]-Chl d to Chl d was evident from the excitation spectra (Fig. 5C). Compared with the spectrum of the control PS II complexes, the contribution of [7-formyl]-Chl d was evident at approximately 480 nm. Although a small amount of free [7formyl]-Chl d was present, the major energy flow was toward Chl d. These results demonstrated that [7-formyl]-Chl d was incorporated into the PS II complexes and that it functioned as a photosensitizer.
Fig. 5. The fluorescence and excitation spectra of the PS II complexes at 77 K. The fluorescence spectra of the control (A) and [7-formyl]-Chl d-containing PS II complexes (B). The excitation wavelengths were 457 (dotted lines) and 482 nm (solid lines). (C) The excitation spectra of the control (gray lines) and [7-formyl]-Chl d-containing PS II complexes (black lines). The emission wavelengths were 678 (solid lines) and 728 nm (dotted lines).
3.5. Time-resolved fluorescence spectra and delayed fluorescence
Fig. 4. The absorption and difference absorption spectra of the PS II complexes at 80 K. (A) The absorption spectra of the control (gray line) and [7-formyl]-Chl d-containing (black line) PS II complexes. (B) The difference absorption spectrum of the above PS II complexes. These spectra are normalized at the maximum of the Soret band.
We further measured TRFS (Fig. 6A) and delayed fluorescence in the nanosecond time region by the decay curves at 77 K (Fig. 6B) for the two PS II complexes. There were no significant differences in the spectra and decay, indicating that the incorporation of [7-formyl]-Chl d neither affected the energy transfer among the Chl d
T. Tsuchiya et al. / Biochimica et Biophysica Acta 1817 (2012) 1285–1291
molecules nor the primary electron transfer process. Accordingly, we concluded that the Chl d molecules were replaced by [7-formyl]-Chl d molecules at limited binding sites in the PS II complexes and that the [7-formyl]-Chl d functioned as an antenna. 4. Discussion 4.1. Re-examination of the pigment composition of the PS II complexes in A. marina From the wild type A. marina, we isolated the PS II complexes that contained an antenna protein, CP43′, instead of CP43 in the previous report [9]. The pigment composition of the PS II complexes was 3.0 ± 0.4 molecules of Chl a and 55 ± 7 molecules of Chl d per two Phe a molecules. In this study, we isolated the PS II complexes that consisted of CP47, CP43, D2, D1, the cytochrome b559 α-subunit, and low-molecular-weight proteins (Fig. 2). We re-examined the pigment composition of the PS II complexes using the control PS II complexes. We applied reverse-phase HPLC, as described by Zapata et al. [20], because pigments extracted with methanol can be directly analyzed using this technique. This method was highly reproducible, and the triplicate analyses were enough to obtain statistically significant results (Table 1). We detected 2.01 ± 0.06 molecules of Chl a, and 62.96 ± 2.49 molecules of Chl d per two Phe a molecules in the control PS II complexes. The Chl d content per two Phe a molecules was almost twice as high as the Chl a content per two Phe a molecules in PS II complexes that were isolated from Thermosynechococcus vulcanus (35 Chl a molecules/PS II monomer) [28]. There was one possibility that free CP43 or CP47 was contaminated in the purified samples. However, we excluded the possibility, because we applied sucrose density gradient centrifugation as a last step during the PS II isolation. Therefore, we hypothesize that there were not actually two Phe a molecules in the PS II monomer of A. marina. The pigment composition per one Phe a molecule was 1.00 ± 0.03 molecules of Chl a and 31.48 ± 1.25 molecules of Chl d; the total Chl content per one
1289
Phe a was 32.5. This value appears to be reasonable, although the Chl d content is slightly smaller than the Chl a content of T. vulcanus PS II. When we consider that the number of Chl-binding sites in PSII is conserved among different species, our hypothesis might not be unreasonable. Similarly, the carotenoid content supported this hypothesis. However, several errors might be caused by the extinction coefficient used for calibration. The difference in the content of Chl in PS II should be studied further. Our results demonstrated that equimolar amounts of Chl a and Phe a were present in A. marina PS II. Therefore, one Chl a molecule is present per one Phe a molecule in our A. marina PS II. In contrast to the present results, a value of 3.0 ± 0.4 Chl a molecules per two Phe a molecules was previously reported [9]. Because CP43′ contains a small amount of Chl a in addition to Chl d [29], approximately one Chl a per two Phe a molecules may be attributed to the presence of CP43′ in the complexes. As a control, we also analyzed the pigment composition of the PS II core complexes isolated from Synechocystis (Table 1). The result (37.70 ± 0.51 Chl a molecules per two Phe a molecules) was similar to the Chl a content of T. vulcanus PS II ([28]). This observation confirms that our biochemical analyses are precise enough to quantify the pigment compositions of the PS II complexes. 4.2. Limited function of [7-formyl]-Chl d in the PS II complexes Although we previously reported the production of [7-formyl]-Chl d in A. marina following the introduction of the CAO gene [18], it was unclear whether the [7-formyl]-Chl d molecules were incorporated into apoproteins. Therefore, the PS II complexes were isolated from the CAO-expressing A. marina, and the pigment composition of the complexes was analyzed by HPLC (Fig. 3B). The PS II complexes contained [7-formyl]-Chl d, and the pigment composition was 0.98 ± 0.02 molecules of Chl a, 26.61 ± 0.50 molecules of Chl d, and 2.74 ± 0.16 molecules of [7-formyl]-Chl d per one Phe a molecule (Table 1). Comparing the pigment compositions between the [7-formyl]-Chl d-containing PS II complexes and the control PS II complexes,
Fig. 6. The kinetic fluorescence properties of the PS II complexes. (A) The normalized time-resolved fluorescence spectra of the PS II complexes at 77 K. (B) The fluorescence decay curves at 685 nm of the PS II complexes at 77 K. The spectra of the control PS II complexes are indicated by black lines and those of the [7-formyl]-Chl d-containing PS II complexes are indicated by gray lines.
1290
T. Tsuchiya et al. / Biochimica et Biophysica Acta 1817 (2012) 1285–1291
approximately five molecules of Chl d were lost and, concomitantly, three molecules of [7-formyl]-Chl d were added in the [7-formyl]-Chl d-containing PS II complexes. In contrast, the content of Chl a was not affected by the acquisition of the [7-formyl]-Chl d molecules. It appears that after the replacement of three Chl d with [7-formyl]-Chl d molecules, two additional Chl d molecules were lost; however, these values may be within the margin of error of the estimation. Further investigations are needed to understand the effect of the incorporation of [7-formyl]-Chl d into PS II on the pigment composition in A. marina. We clearly demonstrated that a part of the Chl d molecules were replaced by artificially produced [7-formyl]-Chl d molecules in the [7-formyl]-Chl d-containing PS II complexes. However, the function of photosynthetic pigments is determined not only by their photophysical and photochemical properties but also by their interactions with amino acid residues in apoproteins. Accordingly, we analyzed the spectroscopic properties of the PS II complexes. Although the polypeptide compositions were almost identical between the control and [7-formyl]-Chl d-containing PS II complexes (Fig. 2), the lowtemperature absorption and steady state excitation and fluorescence spectra showed clear differences depending on the presence of [7formyl]-Chl d (Figs. 4 and 5). These findings indicated that the [7-formyl]-Chl d functioned as the antenna even if only 9% of the Chl d was replaced by [7-formyl]-Chl d. In contrast, TRFS and delayed fluorescence in the nanosecond time region showed no significant difference between the two PS II complexes (Fig. 6). We display Fig. 6A to examine the energy transfer dynamics, which can be obtained as changes in the fluorescence spectra in the early time region. However, it will be necessary for a discussion of the delayed fluorescence to observe fluorescence kinetics in a later time region. Due to the strong fluorescence from Chl d, the relative fluorescence intensity around 685 nm was small. However, by accumulating fluorescence counts, we obtained fluorescence decay curves at 685 nm (Fig. 6B). Delayed fluorescence from A. marina was found around 685 nm, which is identical to Synechocystis sp. PCC 6803 [9,25,31]. Chl a contributed to the fluorescence around 685 nm; therefore, Chl d fluorescence might mainly contribute to the 700-nm band. We have already reported that a difference in the delayed fluorescence was observed when Chl a molecules were replaced by [8-vinyl]-Chl a molecules [30]. Taking these results together, we concluded that the electron transfer components of PS II are not replaced and are not affected by [7-formyl]-Chl d. We proved that PS II is flexible enough to accept artificial novel Chl species as the antenna. 4.3. The presence of an indispensable Chl a molecule in the [7-formyl]-Chl d-containing PS II complexes A small amount of Chl a was always present in A. marina [31], and a part of the Chl a is considered to contribute to electron transfer. In the purified PS II complexes, one Chl a per one Phe a was estimated in this study (Table 1). Delayed fluorescence in the nanosecond time range at 77 K was observed only in the Chl a region for both cells and purified PS II complexes [9,25,31]. Therefore, Chl a appears to be involved in the electron transfer pathway of PS II. In this way, the recent biochemical and spectroscopic analyses of PS II emphasized the significance of Chl a in A. marina. However, molecular genetic studies were needed to elucidate the necessity of Chl a in vivo. Therefore, we developed a transformation system for A. marina to alter the Chl composition. We attempted to reduce the Chl a content in A. marina through the introduction of the CAO gene. However, the relative amount of Chl a in the cells was not changed, whereas the content of [7-formyl]-Chl d was approximately 10% of the total Chl [18]. In the case of PS II, the Chl a/Phe a ratio was almost the same value in the [7-formyl]-Chl d-containing PS II complexes and the control PS II complexes (Table 1). These results strongly suggested that the presence of a Chl a molecule is indispensable for the
photochemical reaction of A. marina PS II. Because we proposed that the special pair is a Chl d/Chl d homodimer, we assume that Chl a is located at the position of an accessory Chl in the active branch. However, the constitution of the special pair is still controversial; therefore, further studies are needed to elucidate the exact function and position of Chl a in PS II of A. marina. Regarding the function of Phe a, Phe a was reported to be the primary electron acceptor in A. marina PS II [9–11]. As described above, we have suggested the presence of one Phe a molecule per PS II monomer in A. marina. If this hypothesis is correct, Phe a must exist only in the active branch. Because we could not find any other Phe molecules, including Phe d, in the [7-formyl]-Chl d-containing PS II complexes (Fig. 3), we assume that the second Phe a molecule was replaced by a Chl d molecule. When we consider the formation of a Phe molecule, it is easier for a magnesium atom to be removed from Chl a than from the other Chls. Therefore, we propose that the second Phe a molecule, which locates in the inactive branch, can be replaced by a Chl molecule other than Chl a. In this case, a ligand to the central Mg of Chl d is needed. We tried to find the candidate amino acid substitution in the deduced amino acid sequences of both psbA and psbD from A. marina. However, no evident amino acid substitution in the region around Phe a was found. Therefore, we assume that a water molecule is a probable candidate of the ligand. However, this suggestion is only a possibility that remains to be verified. Our findings suggested that after the acquisition of Chl d biosynthetic activity by the ancestor of A. marina, only the functionally indispensable components (Chl a and Phe a) remained during the replacement of Chl a with Chl d. The current study clearly illustrates the flexibility of PS II in utilizing the newly acquired Chl. A re-examination of the pigment composition of the PS II complexes in A. marina may provide insight into the mechanism of PS II driven by far-red light. Acknowledgements This study was supported by a Grant-in-Aid for Creative Scientific Research (17GS0314) to M.M. from the Japan Society for the Promotion of Science (JSPS), by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) to T. Tomo (21570038), T.M. (22750017), H.T. (22245030), and T. Tsuchiya, S.A., T. Tomo, and M.M (22370017), by the Special Coordination Funds for Promoting Science and Technology, by a Grant-in-Aid to S.A. from the Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe) programs of MEXT, and by the Precursory Research for Embryonic Science and Technology program of the Japan Science and Technology Agency (to T. Tomo). References [1] H. Tamiaki, R. Shibata, T. Mizoguchi, The 17-propionate function of (bacterio) chlorophylls: biological implication of their long esterifying chains in photosynthetic systems, Photochem. Photobiol. 83 (2007) 152–162. [2] M. Mimuro, A. Murakami, T. Tomo, T. Tsuchiya, K. Watabe, M. Yokono, S. Akimoto, Molecular environments of divinyl chlorophylls in Prochlorococcus and Synechocystis: Differences in fluorescence properties with chlorophyll replacement, Biochim. Biophys. Acta 1807 (2011) 471–481. [3] H. Ito, A. Tanaka, Evolution of a divinyl chlorophyll-based photosystem in Prochlorococcus, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 18014–18019. [4] H. Miyashita, H. Ikemoto, N. Kurano, S. Miyachi, M. Chihara, Acaryochloris marina gen. et sp. nov. (cyanobacteria), an oxygenic photosynthetic prokaryote containing Chl d as a major pigment, J. Phycol. 39 (2003) 1247–1253. [5] H. Miyashita, K. Adachi, N. Kurano, H. Ikemoto, M. Chihara, S. Miyachi, Chlorophyll d as a major pigment, Nature 338 (1996) 402. [6] H. Miyashita, K. Adachi, N. Kurano, H. Ikemoto, M. Chihara, S. Miyachi, Pigment composition of a novel oxygenic photosynthetic prokaryote containing chlorophyll d as the major chlorophyll, Plant Cell Physiol. 38 (1997) 274–281. [7] Q. Hu, H. Miyashita, I. Iwasaki, N. Kurano, S. Miyachi, M. Iwaki, S. Itoh, A photosystem I reaction center driven by chlorophyll d in oxygenic photosynthesis, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 13319–13323. [8] T. Tomo, Y. Kato, T. Suzuki, S. Akimoto, T. Okubo, T. Noguchi, K. Hasegawa, T. Tsuchiya, K. Tanaka, M. Fukuya, N. Dohmae, T. Watanabe, M. Mimuro,
T. Tsuchiya et al. / Biochimica et Biophysica Acta 1817 (2012) 1285–1291
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
Characterization of highly purified photosystem I complexes from the chlorophyll d-dominated cyanobacterium Acaryochloris marina MBIC 11017, J. Biol. Chem. 283 (2008) 18198–18209. T. Tomo, T. Okubo, S. Akimoto, M. Yokono, H. Miyashita, T. Tsuchiya, T. Noguchi, M. Mimuro, Identification of the special pair of photosystem II in a chlorophyll d-dominated cyanobacterium, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 7283–7288. E. Schlodder, M. Çetin, H.-J. Eckert, F.-J. Schmitt, J. Barber, A. Telfer, Both chlorophylls a and d are essential for the photochemistry in photosystem II of the cyanobacteria, Acaryochloris marina, Biochim. Biophys. Acta 1767 (2007) 589–595. M.R. Razeghifard, M. Chen, J.L. Hughes, J. Freeman, E. Krausz, T. Wydrzynski, Spectroscopic studies of photosystem II in chlorophyll d-containing Acaryochloris marina, Biochemistry 44 (2005) 11178–11187. S.I. Allakhverdiev, T. Tomo, Y. Shimada, H. Kindo, R. Nagao, V.V. Klimov, M. Mimuro, Redox potential of pheophytin a in photosystem II of two cyanobacteria having the different special pair chlorophylls, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 3924–3929. S.I. Allakhverdiev, T. Tsuchiya, K. Watabe, A. Kojima, D.A. Los, T. Tomo, V.V. Klimov, M. Mimuro, Redox potentials of primary electron acceptor quinone molecule (QA)- and conserved energetics of photosystem II in cyanobacteria with chlorophyll a and chlorophyll d, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 8054–8058. M. Schenderlein, M. Çetin, J. Barber, A. Telfer, E. Schlodder, Spectroscopic studies of the chlorophyll d containing photosystem I from the cyanobacterium, Acaryochloris marina, Biochim. Biophys. Acta 1777 (2008) 1400–1408. A. Tanaka, H. Ito, R. Tanaka, N.K. Tanaka, K. Yoshida, K. Okada, Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from chlorophyll a, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 12719–12723. S. Satoh, M. Ikeuchi, M. Mimuro, A. Tanaka, Chlorophyll b expressed in cyanobacteria functions as a light-harvesting antenna in photosystem I through flexibility of the proteins, J. Biol. Chem. 276 (2001) 4293–4297. H. Xu, D. Vavilin, W. Vermaas, Chlorophyll b can serve as the major pigment in functional photosystem II complexes of cyanobacteria, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 14168–14173. T. Tsuchiya, T. Mizoguchi, S. Akimoto, T. Tomo, H. Tamiaki, M. Mimuro, Metabolic engineering of the chlorophyll d-dominated cyanobacterium Acaryochloris marina: Production of a novel chlorophyll species by the introduction of the chlorophyllide a oxygenase gene, Plant Cell Physiol (2012) doi:10.1093/pcp/pcs007. U. Oster, R. Tanaka, A. Tanaka, W. Rüdiger, Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana, Plant J. 21 (2000) 305–310.
1291
[20] M. Zapata, F. Rodríguez, J.L. Garrid, Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C8 column and pyridine-containing mobile phases, Mar. Ecol. Prog. Ser. 195 (2000) 29–45. [21] R.J. Porra, W.A. Thompson, P.E. Kriedemann, Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy, Biochim. Biophys. Acta 975 (1989) 384–394. [22] M. Mimuro, S. Akimoto, T. Tomo, M. Yokono, H. Miyashita, T. Tsuchiya, Delayed fluorescence observed in the nanosecond time region at 77 K originates directly from the photosystem II reaction center, Biochim. Biophys. Acta 1767 (2007) 327–334. [23] M. Ikeuchi, Y. Inoue, A new 4.8-kDa polypeptide intrinsic to the PS II reaction center, as revealed by modified SDS-PAGE with improved resolution of lowmolecular-weight proteins, Plant Cell Physiol. 29 (1988) 1233–1239. [24] T. Tomo, S. Akimoto, T. Tsuchiya, M. Fukuya, K. Tanaka, M. Mimuro, Isolation and spectral characterization of Photosystem II reaction center from Synechocystis sp. PCC 6803, Photosynth. Res. 98 (2008) 293–302. [25] M. Mimuro, S. Akimoto, I. Yamazaki, H. Miyashita, S. Miyachi, Fluorescence properties of chlorophyll d-dominating prokaryotic alga, Acaryochloris marina: studies using time-resolved fluorescence spectroscopy on intact cells, Biochim. Biophys. Acta 1412 (1999) 37–46. [26] M. Yokono, S. Akimoto, A. Tanaka, Seasonal changes of excitation energy transfer and thylakoid stacking in the evergreen tree Taxus cuspidata: How does it divert excess energy from photosynthetic reaction center? Biochim. Biophys. Acta 1777 (2008) 379–387. [27] M. Yokono, A. Murakami, S. Akimoto, Excitation energy transfer between photosystem II and photosystem I in red algae: Larger amounts of phycobilisome enhance spillover, Biochim. Biophys. Acta 1807 (2011) 847–853. [28] Y. Umena, K. Kawakami, J.-R. Shen, N. Kamiya, Crystal structure of oxygenevolving photosystem II at a resolution of 1.9 Å, Nature 473 (2011) 55–60. [29] M. Chen, R.G. Quinnell, A.W.D. Larkum, The major light-harvesting pigment protein of Acaryochloris marina, FEBS Lett. 514 (2002) 149–152. [30] T. Tomo, S. Akimoto, H. Ito, T. Tsuchiya, M. Fukuya, A. Tanaka, M. Mimuro, Replacement of chlorophyll with di-vinyl chlorophyll in the antenna and reaction center complexes of the cyanobacterium Synechocystis sp. PCC 6803: Characterization of spectral and photochemical properties, Biochim. Biophys. Acta 1787 (2009) 191–200. [31] M. Mimuro, S. Akimoto, T. Gotoh, M. Yokono, M. Akiyama, T. Tsuchiya, H. Miyashita, M. Kobayashi, I. Yamazaki, Identification of the primary electron donor in PS II of the Chl d-dominated cyanobacterium Acaryochloris marina, FEBS Lett. 556 (2004) 95–98.
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbabio
Artificially produced [7-formyl]-chlorophyll d functions as an antenna pigment in the photosystem II isolated from the chlorophyllide a oxygenase-expressing Acaryochloris marina☆ Tohru Tsuchiya a,⁎, Seiji Akimoto b, Tadashi Mizoguchi c, Kazuyuki Watabe a, Hayato Kindo d, Tatsuya Tomo d, e, Hitoshi Tamiaki c, Mamoru Mimuro a a
Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606–8501, Japan Molecular Photoscience Research Center, Kobe University, Kobe 657–8501, Japan c Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, Kusatsu, Shiga 525–8577, Japan d Department of Biology, Faculty of Science, Tokyo University of Sciences, Kagurazaka, Shinjuku-ku, Tokyo 162–8601, Japan e Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332–0012, Japan b
a r t i c l e
i n f o
Article history: Received 17 November 2011 Received in revised form 17 February 2012 Accepted 20 February 2012 Available online 28 February 2012 Keywords: Chlorophyll d Cyanobacteria Photosystem II Acaryochloris marina
a b s t r a c t Acaryochloris marina, a chlorophyll (Chl) d-dominated cyanobacterium, is a model organism for studying photosynthesis driven by far-red light using Chl d. Furthermore, studies on A. marina may provide insights into understanding how the oxygenic photosynthetic organisms adapt after the acquisition of new Chl. To solve the reaction mechanism of its unique photosynthesis, photosystem (PS) II complexes were isolated from A. marina and analyzed. However, the lack of a molecular genetic method for A. marina prevented us from conducting further studies. We recently developed a transformation system for A. marina and we introduced a chlorophyllide a oxygenase gene into A. marina. The resultant transformant accumulated [7-formyl]-Chl d, which has never been found in nature. In the current study, we isolated PS II complexes that contained [7-formyl]-Chl d. The pigment composition of the [7-formyl]-Chl d-containing PS II complexes was 1.96 ± 0.04 Chl a, 53.21 ± 1.00 Chl d, and 5.48 ± 0.33 [7-formyl]-Chl d per two pheophytin a molecules. In contrast, the composition of the control PS II complexes was 2.01± 0.06 Chl a and 62.96 ± 2.49 Chl d. The steady-state fluorescence and excitation spectra of the PS II complexes revealed that energy transfer occurred from [7-formyl]-Chl d to the major Chl d species; however, the electron transfer was not affected by the presence of [7-formyl]-Chl d. These findings demonstrate that artificially produced [7-formyl]-Chl d molecules that are incorporated into PS II replace part of the Chl d molecules and function as the antenna. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Oxygenic photosynthetic organisms use chlorophylls (Chls) for photosynthesis, and the distribution of Chls varies among taxonomic groups. Certain Chls serve both as electron transfer components and as antenna, whereas others function solely as antenna [1]. The modification of the side chain(s) on the macrocyclic ring of a Chl molecule is critical for its functions because the change affects its chemical properties, such as its redox potential and chemical reactivity. Therefore, the properties of photosystems (PSs) and antenna proteins
Abbreviations: CAO, chlorophyllide a oxygenase; Chl, chlorophyll; CP43, 43-kDa chlorophyll protein; CP47, 47-kDa chlorophyll protein; Phe, pheophytin; PS, photosystem; TRFS, time-resolved fluorescence spectra ☆ This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. ⁎ Corresponding author. Tel.: + 81 75 753 6575; fax: + 81 75 753 7909. E-mail address: [email protected] (T. Tsuchiya). 0005-2728/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2012.02.021
primarily depend on the Chl species used in the organism. All oxygenic photosynthetic organisms produce Chl a (or [8-vinyl]-Chl a, in the case of Prochlorococcus spp.), other Chls are thought to have been acquired during the evolution of the organisms. Once a new Chl was acquired, the photosynthetic apparatus would adapt to facilitate the efficient use of the Chl [2,3]. In this sense, the acquisition of new Chl biosynthetic activities is a key driving force in the evolution of photosynthetic organisms. From the viewpoint of the acquisition of new Chl biosynthetic activity, Acaryochloris spp., marine unicellular cyanobacteria, are suitable models for the study of the evolution of photosynthesis. Acaryochloris spp. are the only known organisms that contain Chl d as a major Chl, and their content of Chl a is quite low (b 5%). Furthermore, Acaryochloris spp. can utilize far-red light for photosynthesis using Chl d [4]. Therefore, the photosynthetic reactions driven by Chl d have been intensively studied since the discovery of Acaryochloris marina [5,6]. It was shown that the special pair of PS I was a Chl d/Chl d’ heterodimer in A. marina [7,8]. Similarly, the special pair of PS II contains Chl d although there is
1286
T. Tsuchiya et al. / Biochimica et Biophysica Acta 1817 (2012) 1285–1291
an ongoing controversy as to whether the special pair is a Chl d/Chl d homodimer [9] or a Chl a/Chl d heterodimer [10]. In contrast, the primary electron acceptor of PS II was proven to be pheophytin (Phe) a [9–11]. The redox potential for water oxidation was estimated to be almost the same between a Chl a-dominated cyanobacterium Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis), and A. marina; this similarity indicates that the shortage of gain of light energy due to the utilization of Chl d by A. marina is compensated by a higher redox potential of Phe a in A. marina than in Synechocystis [12,13]. A similar result was shown in PS I: the special pairs of Synechocystis (P700) and A. marina (P740) yielded identical redox potentials [8,14]. These recent results revealed that the redox potentials of special pairs are determined by the oxidizing side. This finding is one of the prominent examples that support the consideration of the reaction principles in electron transfer system by comparing Chl a-based system with Chl d-based system. However, several unsolved problems still remain. One of these problems is the significance of the small amount of Chl a that is invariably found in PSs. To examine this problem, a perturbation that could affect the Chl composition in vivo was needed. Metabolic engineering of the Chl biosynthetic pathway is an effective way to replace Chls in PSs in vivo. For example, a gene encoding chlorophyllide a oxygenase (CAO), which is responsible for Chl b biosynthesis [15], was introduced into Synechocystis, and the resultant transformants produced Chl b [16,17]. The Chl b molecules in the transformants were incorporated into the PSs and functioned as antenna. These results indicate that the analyses of organisms produced by the strategy of artificial evolution are useful for the study of the evolution of oxygenic photosynthetic organisms. However, the genetic manipulation of A. marina has not yet been established. Therefore, we developed a transformation system for A. marina and introduced the CAO gene into this organism [18]. The CAO-expressing A. marina accumulated a novel Chl species instead of Chl b. The chemical structure of the novel Chl was determined to be [7-formyl]-Chl d, containing two formyl groups as its side chains (Fig. 1). [7-Formyl]-Chl d was hypothesized to be produced by the combined action of CAO and the enzyme(s) involved in Chl d biosynthesis. The molar ratio of [7formyl]-Chl d increased to 9.6% of the total Chl; however, the distribution of [7-formyl]-Chl d in the cells was not determined. We expected that the Chl a content might be reduced by the introduction of the CAO gene because CAO converts chlorophyllide a, a precursor of Chl a, into chlorophyllide b [19]. However, the content of Chl a in the CAO-expressing A. marina was similar to that of the control transformant [18]. This result indicates that the Chl a molecules in the PSs have a unique function that cannot be obtained from the Chl d molecules. In this study, we isolated the PS II complexes from CAO-expressing A. marina. The pigment composition and optical properties of the PS II complexes were analyzed. Based on our results, we discuss the function of Chl a and the newly synthesized [7-formyl]-Chl d in PS II and propose an asymmetric pigment configuration for A. marina PS II.
Fig. 1. The chemical structures of Chls related to this study.
2. Materials and methods 2.1. Culture of A. marina A. marina MBIC 11017 was cultured in an 8-liter culture bottle of IMK medium (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan) under photoautotrophic conditions; the light intensity was 10 μmol photons/(m 2 s) and the temperature was 298 K. For the transformants, spectinomycin was added to the medium (5 μg/ml). 2.2. Preparation and analysis of pigments The pigment compositions of PS II were analyzed following the approach of Zapata et al. [20]. The pigments were extracted with cold methanol from isolated PS II complexes, and the extracts were sonicated on ice. The supernatants of the centrifuged extracts were immediately analyzed by HPLC (Prominence series, Shimadzu, Kyoto, Japan) with a Symmetry C8 column (150 × 1.0 mm, 3.5-μm particle size; Waters, MA, USA). The elution profile was monitored with a photodiode array detector (SPD-M20A, Shimadzu). The compositions of the mobile phases were methanol:acetonitrile:0.25 M pyridine solution (50:25:25, v:v:v) and methanol:acetonitrile:acetone (20:60:20, v:v:v). The gradient profile was optimized for semimicro analysis. The calibration data were obtained using purified Chls as authentic samples. The Chls were quantified by the peak area of each Chl. Because the extinction coefficient for [7-formyl]Chl d had not been determined, the millimolar extinction coefficient for other Chl was applied. The formylation of the C-7 position of Chl a causes an approximately 45% decrease of the millimolar extinction coefficient [21], whereas little difference is observed from the formylation of the C-3 position of Chl a. Therefore, we applied the millimolar extinction coefficient for Chl b (38.55 mM - 1 cm - 1 for methanol) [21]. 2.3. Preparation of the PS II complexes PS II complexes in the thylakoid membranes of A. marina were solubilized in dodecyl-β-D-maltoside and then purified using a combination of sucrose density gradient centrifugation and anionexchange chromatography (UNO Q, Bio-Rad, CA, USA), as described previously [12]. The PS II core complexes of Synechocystis were prepared as reported previously [22]. The polypeptide composition of the purified complexes was analyzed by SDS-PAGE, using a 16–22% separating gel with a 6% stacking gel that contained 7.5 M urea [23]. After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250. 2.4. Spectroscopy The absorption and fluorescence spectra were measured as reported previously [24]. The fluorescence spectra were corrected for the spectral sensitivity of the detector. The Chl d concentration was adjusted to 2 μg/ml. For the fluorescence emission and excitation spectra, the respective bandwidths for excitation and fluorescence were 2 and 3 nm. The time-resolved fluorescence spectra (TRFS) and fluorescence decay curves were measured by the timecorrelated single-photon counting method at 77 K [25–27]. To examine differences in energy transfer process, TRFS were measured with a time interval of 2.4 ps/channel, whereas, to examine charge recombination process, fluorescence decay curves were measured at 685 nm with 24.4 ps/channel. A. marina cells and its PS II exhibited the delayed fluorescence around 685 nm, which was originated from charge recombination [9,25,31]. Although A. marina PS II exhibited fluorescences around 700 nm and 728 nm in later time region after laser excitation, any long-lived components, which could be assigned due to the delayed fluorescence, were not resolved in the fluorescence decay observed at
T. Tsuchiya et al. / Biochimica et Biophysica Acta 1817 (2012) 1285–1291
1287
these wavelengths [9]. Therefore, we selected 685 nm as the observed wavelength. The light source was a Ti:sapphire laser and the excitation wavelength was 400 nm, which brought about excitation of all Chls. 3. Results 3.1. Isolation and polypeptide composition of PS II complexes from the two transformants To investigate the function(s) of [7-formyl]-Chl d, we isolated PS II complexes from the CAO-expressing A. marina ([7-formyl]-Chl d-containing PS II complexes) and the control transformant (control PS II complexes). The polypeptide compositions of the complexes were examined by SDS-PAGE (Fig. 2). The peptide pattern of the [7-formyl]-Chl d-containing PS II complexes was very similar to that of the control PS II complexes; the principal bands were clearly detected in both preparations with almost identical stoichiometry. These PS II complexes consisted of a 47-kDa Chl protein (CP47), a 43-kDa Chl protein (CP43), D2, D1, the cytochrome b559 α-subunit, and low-molecular-weight proteins. 3.2. Pigment compositions of PS II complexes We analyzed the pigment compositions of the [7-formyl]-Chl dcontaining PS II complexes and the control PS II complexes by reversephase HPLC (Fig. 3). The pigment composition per two Phe a molecules was 2.01 ± 0.06 molecules of Chl a and 62.96 ± 2.49 molecules of Chl d in the control PS II complexes (Table 1). In contrast, the composition of the [7-formyl]-Chl d-containing PS II complexes was 1.96 ± 0.04 molecules of Chl a, 53.21 ± 1.00 molecules of Chl d, and 5.48±0.33 molecules of [7-formyl]-Chl d (Table 1). These results demonstrate that 9.0% of the total Chl molecules was [7-formyl]-Chl d in the [7-formyl]-Chl dcontaining PS II complexes, and that this value is partially correlated with a decrease in Chl d (15.5%). Moreover, we did not observe a difference in the Chl a content per Phe a between the two PS II complexes. Furthermore, we could not detect Phe d or any other pigment that was derived from Chl in the [7-formyl]-Chl d-containing PS II complexes (Fig. 3). Because the estimated Chl d content per two Phe a molecules
Fig. 3. Analyses of Chl compositions by HPLC. HPLC chromatograms of pigment extracts from the PS II complexes of control A. marina (A), [7-formyl]-Chl d-containing A. marina (B), and Synechocystis (C) monitored at 663 nm. 1, [7-formyl]-Chl d; 2, Chl d; 3, Chl a; 4, Phe a.
was significantly higher than the Chl a content per two Phe a molecules of Chl a-type PS II [28], we analyzed the pigment compositions of the PS II core complexes of Synechocystis as a control (Fig. 3C). The pigment composition per two Phe a molecules was 37.70±0.51 molecules of Chl a in the complexes (Table 1). Regarding the content of carotenoids, approximately eight and seven carotenoids per one Phe a molecule were detected in the control PS II complexes and the [7-formyl]-Chl d-containing PS II complexes, respectively. In contrast, approximately nine carotenoids per two Phe a molecules were found in the PS II core complexes of Synechocystis.
3.3. Low-temperature absorption spectra We measured the low-temperature absorption spectra of the PS II complexes. The results revealed clear differences in the Soret and Qy bands of [7-formyl]-Chl d at 80 K after normalization at the Soret band of Chl d (Fig. 4A). The difference in the absorption spectra between the two PS II complexes showed the presence of positive peaks at 482 and 675 nm and a few negative peaks at approximately 710 nm and at the Soret bands of Chl d, indicating several distinct forms of Chl d (Fig. 4B). These observations confirmed that part of the Chl d molecules were replaced by [7-formyl]-Chl d in the PS II complexes. This finding means that artificially produced [7-formyl]-
Table 1 The pigment compositions of PS II isolated from A. marina transformants and Synechocystis. Samples
Chl a
Chl d
[7-formyl]-Chl d1
carotenoids
A. marina
2.01 ± 0.06 (1.00 ± 0.03) 1.96 ± 0.04 (0.98 ± 0.02)
62.96 ± 2.49 (31.48 ± 1.25) 53.21 ± 1.00 (26.61 ± 0.50)
n.d. n.d. 5.48 ± 0.33 (2.74 ± 0.16)
15.17 ± 0.16 (7.59 ± 0.08) 13.31 ± 0.26 (6.65 ± 0.13)
37.70 ± 0.51
n.d.
n.d.
[7-formyl]-Chl d-containing A. marina Synechocystis Fig. 2. SDS-PAGE of PS II complexes isolated from A. marina transformants. Lane 1, control PS II complexes; lane 2, [7-formyl]-Chl d-containing PS II complexes. The individual symbols indicate CP47 (filled circle), CP43 (open circle), D2 (filled square), D1 (open square), the cytochrome b559 α-subunit (filled triangle), and low molecular-weight proteins (filled star).
9.12 ± 0.11
The values with standard deviations are normalized against two molecules of Phe a (n = 3). The values in parentheses are normalized against one molecule of Phe a. n.d., not detected. 1 The content of [7-formyl]-Chl d was estimated using the millimolar extinction coefficient for Chl b.
1288
T. Tsuchiya et al. / Biochimica et Biophysica Acta 1817 (2012) 1285–1291
Chl d molecules are incorporated into the pre-existing apoproteins without any amino-acid substitution.
3.4. Low-temperature excitation and emission spectra Clearly, [7-formyl]-Chl d was fully functional as an antenna pigment (Fig. 5). First, the emission spectra of the control PS II complexes were measured (Fig. 5A). When [7-formyl]-Chl d was excited at 482 nm in the [7-formyl]-Chl d-containing PS II complexes, the main emission was observed at 728 nm, with a small band at 678 nm (solid line, Fig. 5B). The former emission originated from PS II Chl d, as evidenced by the energy transfer from [7-formyl]-Chl d, and the latter, from free [7-formyl]-Chl d. Because of the very high fluorescence yield of free Chl, the amount of free [7-formyl]-Chl d was estimated to be very small, even though the fluorescence intensity was clear. In the control PS II complexes, fluorescence from free [7-formyl]-Chl d was not detected (Fig. 5A), confirming the absence of [7-formyl]-Chl d in the complexes. The energy transfer from [7-formyl]-Chl d to Chl d was evident from the excitation spectra (Fig. 5C). Compared with the spectrum of the control PS II complexes, the contribution of [7-formyl]-Chl d was evident at approximately 480 nm. Although a small amount of free [7formyl]-Chl d was present, the major energy flow was toward Chl d. These results demonstrated that [7-formyl]-Chl d was incorporated into the PS II complexes and that it functioned as a photosensitizer.
Fig. 5. The fluorescence and excitation spectra of the PS II complexes at 77 K. The fluorescence spectra of the control (A) and [7-formyl]-Chl d-containing PS II complexes (B). The excitation wavelengths were 457 (dotted lines) and 482 nm (solid lines). (C) The excitation spectra of the control (gray lines) and [7-formyl]-Chl d-containing PS II complexes (black lines). The emission wavelengths were 678 (solid lines) and 728 nm (dotted lines).
3.5. Time-resolved fluorescence spectra and delayed fluorescence
Fig. 4. The absorption and difference absorption spectra of the PS II complexes at 80 K. (A) The absorption spectra of the control (gray line) and [7-formyl]-Chl d-containing (black line) PS II complexes. (B) The difference absorption spectrum of the above PS II complexes. These spectra are normalized at the maximum of the Soret band.
We further measured TRFS (Fig. 6A) and delayed fluorescence in the nanosecond time region by the decay curves at 77 K (Fig. 6B) for the two PS II complexes. There were no significant differences in the spectra and decay, indicating that the incorporation of [7-formyl]-Chl d neither affected the energy transfer among the Chl d
T. Tsuchiya et al. / Biochimica et Biophysica Acta 1817 (2012) 1285–1291
molecules nor the primary electron transfer process. Accordingly, we concluded that the Chl d molecules were replaced by [7-formyl]-Chl d molecules at limited binding sites in the PS II complexes and that the [7-formyl]-Chl d functioned as an antenna. 4. Discussion 4.1. Re-examination of the pigment composition of the PS II complexes in A. marina From the wild type A. marina, we isolated the PS II complexes that contained an antenna protein, CP43′, instead of CP43 in the previous report [9]. The pigment composition of the PS II complexes was 3.0 ± 0.4 molecules of Chl a and 55 ± 7 molecules of Chl d per two Phe a molecules. In this study, we isolated the PS II complexes that consisted of CP47, CP43, D2, D1, the cytochrome b559 α-subunit, and low-molecular-weight proteins (Fig. 2). We re-examined the pigment composition of the PS II complexes using the control PS II complexes. We applied reverse-phase HPLC, as described by Zapata et al. [20], because pigments extracted with methanol can be directly analyzed using this technique. This method was highly reproducible, and the triplicate analyses were enough to obtain statistically significant results (Table 1). We detected 2.01 ± 0.06 molecules of Chl a, and 62.96 ± 2.49 molecules of Chl d per two Phe a molecules in the control PS II complexes. The Chl d content per two Phe a molecules was almost twice as high as the Chl a content per two Phe a molecules in PS II complexes that were isolated from Thermosynechococcus vulcanus (35 Chl a molecules/PS II monomer) [28]. There was one possibility that free CP43 or CP47 was contaminated in the purified samples. However, we excluded the possibility, because we applied sucrose density gradient centrifugation as a last step during the PS II isolation. Therefore, we hypothesize that there were not actually two Phe a molecules in the PS II monomer of A. marina. The pigment composition per one Phe a molecule was 1.00 ± 0.03 molecules of Chl a and 31.48 ± 1.25 molecules of Chl d; the total Chl content per one
1289
Phe a was 32.5. This value appears to be reasonable, although the Chl d content is slightly smaller than the Chl a content of T. vulcanus PS II. When we consider that the number of Chl-binding sites in PSII is conserved among different species, our hypothesis might not be unreasonable. Similarly, the carotenoid content supported this hypothesis. However, several errors might be caused by the extinction coefficient used for calibration. The difference in the content of Chl in PS II should be studied further. Our results demonstrated that equimolar amounts of Chl a and Phe a were present in A. marina PS II. Therefore, one Chl a molecule is present per one Phe a molecule in our A. marina PS II. In contrast to the present results, a value of 3.0 ± 0.4 Chl a molecules per two Phe a molecules was previously reported [9]. Because CP43′ contains a small amount of Chl a in addition to Chl d [29], approximately one Chl a per two Phe a molecules may be attributed to the presence of CP43′ in the complexes. As a control, we also analyzed the pigment composition of the PS II core complexes isolated from Synechocystis (Table 1). The result (37.70 ± 0.51 Chl a molecules per two Phe a molecules) was similar to the Chl a content of T. vulcanus PS II ([28]). This observation confirms that our biochemical analyses are precise enough to quantify the pigment compositions of the PS II complexes. 4.2. Limited function of [7-formyl]-Chl d in the PS II complexes Although we previously reported the production of [7-formyl]-Chl d in A. marina following the introduction of the CAO gene [18], it was unclear whether the [7-formyl]-Chl d molecules were incorporated into apoproteins. Therefore, the PS II complexes were isolated from the CAO-expressing A. marina, and the pigment composition of the complexes was analyzed by HPLC (Fig. 3B). The PS II complexes contained [7-formyl]-Chl d, and the pigment composition was 0.98 ± 0.02 molecules of Chl a, 26.61 ± 0.50 molecules of Chl d, and 2.74 ± 0.16 molecules of [7-formyl]-Chl d per one Phe a molecule (Table 1). Comparing the pigment compositions between the [7-formyl]-Chl d-containing PS II complexes and the control PS II complexes,
Fig. 6. The kinetic fluorescence properties of the PS II complexes. (A) The normalized time-resolved fluorescence spectra of the PS II complexes at 77 K. (B) The fluorescence decay curves at 685 nm of the PS II complexes at 77 K. The spectra of the control PS II complexes are indicated by black lines and those of the [7-formyl]-Chl d-containing PS II complexes are indicated by gray lines.
1290
T. Tsuchiya et al. / Biochimica et Biophysica Acta 1817 (2012) 1285–1291
approximately five molecules of Chl d were lost and, concomitantly, three molecules of [7-formyl]-Chl d were added in the [7-formyl]-Chl d-containing PS II complexes. In contrast, the content of Chl a was not affected by the acquisition of the [7-formyl]-Chl d molecules. It appears that after the replacement of three Chl d with [7-formyl]-Chl d molecules, two additional Chl d molecules were lost; however, these values may be within the margin of error of the estimation. Further investigations are needed to understand the effect of the incorporation of [7-formyl]-Chl d into PS II on the pigment composition in A. marina. We clearly demonstrated that a part of the Chl d molecules were replaced by artificially produced [7-formyl]-Chl d molecules in the [7-formyl]-Chl d-containing PS II complexes. However, the function of photosynthetic pigments is determined not only by their photophysical and photochemical properties but also by their interactions with amino acid residues in apoproteins. Accordingly, we analyzed the spectroscopic properties of the PS II complexes. Although the polypeptide compositions were almost identical between the control and [7-formyl]-Chl d-containing PS II complexes (Fig. 2), the lowtemperature absorption and steady state excitation and fluorescence spectra showed clear differences depending on the presence of [7formyl]-Chl d (Figs. 4 and 5). These findings indicated that the [7-formyl]-Chl d functioned as the antenna even if only 9% of the Chl d was replaced by [7-formyl]-Chl d. In contrast, TRFS and delayed fluorescence in the nanosecond time region showed no significant difference between the two PS II complexes (Fig. 6). We display Fig. 6A to examine the energy transfer dynamics, which can be obtained as changes in the fluorescence spectra in the early time region. However, it will be necessary for a discussion of the delayed fluorescence to observe fluorescence kinetics in a later time region. Due to the strong fluorescence from Chl d, the relative fluorescence intensity around 685 nm was small. However, by accumulating fluorescence counts, we obtained fluorescence decay curves at 685 nm (Fig. 6B). Delayed fluorescence from A. marina was found around 685 nm, which is identical to Synechocystis sp. PCC 6803 [9,25,31]. Chl a contributed to the fluorescence around 685 nm; therefore, Chl d fluorescence might mainly contribute to the 700-nm band. We have already reported that a difference in the delayed fluorescence was observed when Chl a molecules were replaced by [8-vinyl]-Chl a molecules [30]. Taking these results together, we concluded that the electron transfer components of PS II are not replaced and are not affected by [7-formyl]-Chl d. We proved that PS II is flexible enough to accept artificial novel Chl species as the antenna. 4.3. The presence of an indispensable Chl a molecule in the [7-formyl]-Chl d-containing PS II complexes A small amount of Chl a was always present in A. marina [31], and a part of the Chl a is considered to contribute to electron transfer. In the purified PS II complexes, one Chl a per one Phe a was estimated in this study (Table 1). Delayed fluorescence in the nanosecond time range at 77 K was observed only in the Chl a region for both cells and purified PS II complexes [9,25,31]. Therefore, Chl a appears to be involved in the electron transfer pathway of PS II. In this way, the recent biochemical and spectroscopic analyses of PS II emphasized the significance of Chl a in A. marina. However, molecular genetic studies were needed to elucidate the necessity of Chl a in vivo. Therefore, we developed a transformation system for A. marina to alter the Chl composition. We attempted to reduce the Chl a content in A. marina through the introduction of the CAO gene. However, the relative amount of Chl a in the cells was not changed, whereas the content of [7-formyl]-Chl d was approximately 10% of the total Chl [18]. In the case of PS II, the Chl a/Phe a ratio was almost the same value in the [7-formyl]-Chl d-containing PS II complexes and the control PS II complexes (Table 1). These results strongly suggested that the presence of a Chl a molecule is indispensable for the
photochemical reaction of A. marina PS II. Because we proposed that the special pair is a Chl d/Chl d homodimer, we assume that Chl a is located at the position of an accessory Chl in the active branch. However, the constitution of the special pair is still controversial; therefore, further studies are needed to elucidate the exact function and position of Chl a in PS II of A. marina. Regarding the function of Phe a, Phe a was reported to be the primary electron acceptor in A. marina PS II [9–11]. As described above, we have suggested the presence of one Phe a molecule per PS II monomer in A. marina. If this hypothesis is correct, Phe a must exist only in the active branch. Because we could not find any other Phe molecules, including Phe d, in the [7-formyl]-Chl d-containing PS II complexes (Fig. 3), we assume that the second Phe a molecule was replaced by a Chl d molecule. When we consider the formation of a Phe molecule, it is easier for a magnesium atom to be removed from Chl a than from the other Chls. Therefore, we propose that the second Phe a molecule, which locates in the inactive branch, can be replaced by a Chl molecule other than Chl a. In this case, a ligand to the central Mg of Chl d is needed. We tried to find the candidate amino acid substitution in the deduced amino acid sequences of both psbA and psbD from A. marina. However, no evident amino acid substitution in the region around Phe a was found. Therefore, we assume that a water molecule is a probable candidate of the ligand. However, this suggestion is only a possibility that remains to be verified. Our findings suggested that after the acquisition of Chl d biosynthetic activity by the ancestor of A. marina, only the functionally indispensable components (Chl a and Phe a) remained during the replacement of Chl a with Chl d. The current study clearly illustrates the flexibility of PS II in utilizing the newly acquired Chl. A re-examination of the pigment composition of the PS II complexes in A. marina may provide insight into the mechanism of PS II driven by far-red light. Acknowledgements This study was supported by a Grant-in-Aid for Creative Scientific Research (17GS0314) to M.M. from the Japan Society for the Promotion of Science (JSPS), by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) to T. Tomo (21570038), T.M. (22750017), H.T. (22245030), and T. Tsuchiya, S.A., T. Tomo, and M.M (22370017), by the Special Coordination Funds for Promoting Science and Technology, by a Grant-in-Aid to S.A. from the Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe) programs of MEXT, and by the Precursory Research for Embryonic Science and Technology program of the Japan Science and Technology Agency (to T. Tomo). References [1] H. Tamiaki, R. Shibata, T. Mizoguchi, The 17-propionate function of (bacterio) chlorophylls: biological implication of their long esterifying chains in photosynthetic systems, Photochem. Photobiol. 83 (2007) 152–162. [2] M. Mimuro, A. Murakami, T. Tomo, T. Tsuchiya, K. Watabe, M. Yokono, S. Akimoto, Molecular environments of divinyl chlorophylls in Prochlorococcus and Synechocystis: Differences in fluorescence properties with chlorophyll replacement, Biochim. Biophys. Acta 1807 (2011) 471–481. [3] H. Ito, A. Tanaka, Evolution of a divinyl chlorophyll-based photosystem in Prochlorococcus, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 18014–18019. [4] H. Miyashita, H. Ikemoto, N. Kurano, S. Miyachi, M. Chihara, Acaryochloris marina gen. et sp. nov. (cyanobacteria), an oxygenic photosynthetic prokaryote containing Chl d as a major pigment, J. Phycol. 39 (2003) 1247–1253. [5] H. Miyashita, K. Adachi, N. Kurano, H. Ikemoto, M. Chihara, S. Miyachi, Chlorophyll d as a major pigment, Nature 338 (1996) 402. [6] H. Miyashita, K. Adachi, N. Kurano, H. Ikemoto, M. Chihara, S. Miyachi, Pigment composition of a novel oxygenic photosynthetic prokaryote containing chlorophyll d as the major chlorophyll, Plant Cell Physiol. 38 (1997) 274–281. [7] Q. Hu, H. Miyashita, I. Iwasaki, N. Kurano, S. Miyachi, M. Iwaki, S. Itoh, A photosystem I reaction center driven by chlorophyll d in oxygenic photosynthesis, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 13319–13323. [8] T. Tomo, Y. Kato, T. Suzuki, S. Akimoto, T. Okubo, T. Noguchi, K. Hasegawa, T. Tsuchiya, K. Tanaka, M. Fukuya, N. Dohmae, T. Watanabe, M. Mimuro,
T. Tsuchiya et al. / Biochimica et Biophysica Acta 1817 (2012) 1285–1291
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
Characterization of highly purified photosystem I complexes from the chlorophyll d-dominated cyanobacterium Acaryochloris marina MBIC 11017, J. Biol. Chem. 283 (2008) 18198–18209. T. Tomo, T. Okubo, S. Akimoto, M. Yokono, H. Miyashita, T. Tsuchiya, T. Noguchi, M. Mimuro, Identification of the special pair of photosystem II in a chlorophyll d-dominated cyanobacterium, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 7283–7288. E. Schlodder, M. Çetin, H.-J. Eckert, F.-J. Schmitt, J. Barber, A. Telfer, Both chlorophylls a and d are essential for the photochemistry in photosystem II of the cyanobacteria, Acaryochloris marina, Biochim. Biophys. Acta 1767 (2007) 589–595. M.R. Razeghifard, M. Chen, J.L. Hughes, J. Freeman, E. Krausz, T. Wydrzynski, Spectroscopic studies of photosystem II in chlorophyll d-containing Acaryochloris marina, Biochemistry 44 (2005) 11178–11187. S.I. Allakhverdiev, T. Tomo, Y. Shimada, H. Kindo, R. Nagao, V.V. Klimov, M. Mimuro, Redox potential of pheophytin a in photosystem II of two cyanobacteria having the different special pair chlorophylls, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 3924–3929. S.I. Allakhverdiev, T. Tsuchiya, K. Watabe, A. Kojima, D.A. Los, T. Tomo, V.V. Klimov, M. Mimuro, Redox potentials of primary electron acceptor quinone molecule (QA)- and conserved energetics of photosystem II in cyanobacteria with chlorophyll a and chlorophyll d, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 8054–8058. M. Schenderlein, M. Çetin, J. Barber, A. Telfer, E. Schlodder, Spectroscopic studies of the chlorophyll d containing photosystem I from the cyanobacterium, Acaryochloris marina, Biochim. Biophys. Acta 1777 (2008) 1400–1408. A. Tanaka, H. Ito, R. Tanaka, N.K. Tanaka, K. Yoshida, K. Okada, Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from chlorophyll a, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 12719–12723. S. Satoh, M. Ikeuchi, M. Mimuro, A. Tanaka, Chlorophyll b expressed in cyanobacteria functions as a light-harvesting antenna in photosystem I through flexibility of the proteins, J. Biol. Chem. 276 (2001) 4293–4297. H. Xu, D. Vavilin, W. Vermaas, Chlorophyll b can serve as the major pigment in functional photosystem II complexes of cyanobacteria, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 14168–14173. T. Tsuchiya, T. Mizoguchi, S. Akimoto, T. Tomo, H. Tamiaki, M. Mimuro, Metabolic engineering of the chlorophyll d-dominated cyanobacterium Acaryochloris marina: Production of a novel chlorophyll species by the introduction of the chlorophyllide a oxygenase gene, Plant Cell Physiol (2012) doi:10.1093/pcp/pcs007. U. Oster, R. Tanaka, A. Tanaka, W. Rüdiger, Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana, Plant J. 21 (2000) 305–310.
1291
[20] M. Zapata, F. Rodríguez, J.L. Garrid, Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C8 column and pyridine-containing mobile phases, Mar. Ecol. Prog. Ser. 195 (2000) 29–45. [21] R.J. Porra, W.A. Thompson, P.E. Kriedemann, Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy, Biochim. Biophys. Acta 975 (1989) 384–394. [22] M. Mimuro, S. Akimoto, T. Tomo, M. Yokono, H. Miyashita, T. Tsuchiya, Delayed fluorescence observed in the nanosecond time region at 77 K originates directly from the photosystem II reaction center, Biochim. Biophys. Acta 1767 (2007) 327–334. [23] M. Ikeuchi, Y. Inoue, A new 4.8-kDa polypeptide intrinsic to the PS II reaction center, as revealed by modified SDS-PAGE with improved resolution of lowmolecular-weight proteins, Plant Cell Physiol. 29 (1988) 1233–1239. [24] T. Tomo, S. Akimoto, T. Tsuchiya, M. Fukuya, K. Tanaka, M. Mimuro, Isolation and spectral characterization of Photosystem II reaction center from Synechocystis sp. PCC 6803, Photosynth. Res. 98 (2008) 293–302. [25] M. Mimuro, S. Akimoto, I. Yamazaki, H. Miyashita, S. Miyachi, Fluorescence properties of chlorophyll d-dominating prokaryotic alga, Acaryochloris marina: studies using time-resolved fluorescence spectroscopy on intact cells, Biochim. Biophys. Acta 1412 (1999) 37–46. [26] M. Yokono, S. Akimoto, A. Tanaka, Seasonal changes of excitation energy transfer and thylakoid stacking in the evergreen tree Taxus cuspidata: How does it divert excess energy from photosynthetic reaction center? Biochim. Biophys. Acta 1777 (2008) 379–387. [27] M. Yokono, A. Murakami, S. Akimoto, Excitation energy transfer between photosystem II and photosystem I in red algae: Larger amounts of phycobilisome enhance spillover, Biochim. Biophys. Acta 1807 (2011) 847–853. [28] Y. Umena, K. Kawakami, J.-R. Shen, N. Kamiya, Crystal structure of oxygenevolving photosystem II at a resolution of 1.9 Å, Nature 473 (2011) 55–60. [29] M. Chen, R.G. Quinnell, A.W.D. Larkum, The major light-harvesting pigment protein of Acaryochloris marina, FEBS Lett. 514 (2002) 149–152. [30] T. Tomo, S. Akimoto, H. Ito, T. Tsuchiya, M. Fukuya, A. Tanaka, M. Mimuro, Replacement of chlorophyll with di-vinyl chlorophyll in the antenna and reaction center complexes of the cyanobacterium Synechocystis sp. PCC 6803: Characterization of spectral and photochemical properties, Biochim. Biophys. Acta 1787 (2009) 191–200. [31] M. Mimuro, S. Akimoto, T. Gotoh, M. Yokono, M. Akiyama, T. Tsuchiya, H. Miyashita, M. Kobayashi, I. Yamazaki, Identification of the primary electron donor in PS II of the Chl d-dominated cyanobacterium Acaryochloris marina, FEBS Lett. 556 (2004) 95–98.