FEBS Letters 589 (2015) 1761–1765

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Characterization of the quinones in purple sulfur bacterium Thermochromatium tepidum Yuuka Kimura a,1, Tomoaki Kawakami a,1, Long-Jiang Yu a,2, Miku Yoshimura a, Masayuki Kobayashi b, Zheng-Yu Wang-Otomo a,⇑ a b

Faculty of Science, Ibaraki University, Mito 310-8512, Japan Institute of National Colleges of Technology, Ariake College, Omuta, Fukuoka 836-8585, Japan

a r t i c l e

i n f o

Article history: Received 20 April 2015 Revised 21 May 2015 Accepted 22 May 2015 Available online 3 June 2015 Edited by Richard Cogdell Keywords: Photosynthesis Reaction center Electron transport Chromatophore Light-harvesting

a b s t r a c t Quinone distributions in the thermophilic purple sulfur bacterium Thermochromatium tepidum have been investigated at different levels of the photosynthetic apparatus. Here we show that, on average, the intracytoplasmic membrane contains 18 ubiquinones (UQ) and 4 menaquinones (MQ) per reaction center (RC). About one-third of the quinones are retained in the light-harvest ing–reaction center core complex (LH1–RC) with a similar ratio of UQ to MQ. The numbers of quinones essentially remains unchanged during crystallization of the LH1–RC. There are 1–2 UQ and 1 MQ associated with the RC-only complex in the purified solution sample. Our results suggest that a large proportion of the quinones are confined to the core complex and at least five UQs remain invisible in the current LH1–RC crystal structure. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Quinones are membrane-soluble redox molecules found in nearly all living organisms [1]. They mainly exist in photosynthetic and respiratory electron transport chains and function as electron and proton carriers to produce transmembrane proton gradients. Four types of quinone have been reported in anoxygenic photosynthetic bacteria [2]: ubiquinone (UQ), menaquinone (MQ), rhodoquinone (RQ) and chlorobiumquinone (CQ). Both UQ and RQ belong to the benzoquinones that are considered to be evolutionarily younger than the naphthoquinones to which the MQ and

Abbreviations: CQ, chlorobiumquinone; DDM, n-dodecyl b-D-maltopyranoside; LDAO, lauryl dimethylamine N-oxide; LH, light-harvesting; MALDI TOF, matrix-assisted laser desorption/ionization time-of-flight; MQ, menaquinone; RC, reaction center; RQ, rhodoquinone; THAP, 2,4,6-trihydroxy acetophenone; TFA, trifluoroacetic acid; UQ, ubiquinone Author contributions: ZYWO designed and supervised the study. YK and TK prepared samples and performed experiments. LJY, MY and MK contributed to sample preparation and data analysis. ZYWO wrote the manuscript. ⇑ Corresponding author. E-mail address: [email protected] (Z.-Y. Wang-Otomo). 1 These authors contributed equally to this work. 2 Present address: Department of Biology, Faculty of Science, Okayama University, Okayama, Japan.

CQ belong. UQs are present in all purple phototropic bacteria [3] and have been shown to play a crucial role in the photochemical reactions of bacterial photosynthesis [4–6]. MQs are found in some purple bacteria and are the major quinone compounds in green bacteria and heliobacteria. RQs are present in some purple non-sulfur bacteria, and CQs are only found in the green sulfur bacteria. Although these quinones have been identified at the levels of cell or photosynthetic membrane, we have only limited and fragmentary knowledge on their distribution and composition in the specific apparatus or protein complexes to which they are associated. In the well studied Rhodobacter (Rba.) sphaeroides that contains solely UQ10 (subscript number specifies the number of isoprenoid units in the side chain), about 20–30 UQ10 per reaction center (RC) were estimated for the so-called quinone pool in chromatophores based on both biochemical and spectroscopic analyses [7–10]. The stoichiometric ratio decreased to about 10–15 in the light-harvesting–reaction center core complex (LH1–RC, per monomer) [9–11] and further to 1–2 in the RC [6,9]. Two UQ10 molecules were confirmed in the crystal structure of the RC [12]. A similar number of 25 quinones/RC was reported for chromatophores from Phaeospirillum (Pha.) molischianum that contains both UQ9 and MQ9 [3,13]. However, the diffusion rate of quinone/quinol exchange was found to be 30 times slower than that in

http://dx.doi.org/10.1016/j.febslet.2015.05.043 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Y. Kimura et al. / FEBS Letters 589 (2015) 1761–1765

2. Materials and methods 2.1. Preparations of chromatophores, LH1–RC and RC complexes Tch. tepidum cells were cultured for seven days. Chromatophores were prepared as previously reported [24]. The chromatophores were suspended in 0.5 mL of 20 mM Tris–HCl (pH 7.5) at concentration of A850 = 120, and were then lyophilized. LH1–RC complex was isolated from the chromatophores and purified following the same procedure described before [25]. Crystals of the LH1–RC were the same as those used in the structure determination [22]. About 200 crystals were collected and dissolved in 20 mM Tris–HCl (pH 7.5) buffer containing 0.05% w/v n-dodecyl b-D-maltopyranoside (DDM). RC complex was prepared by the same procedure as reported [26]. After recorded the absorption spectra, all samples in the solutions (0.5 mL) were precipitated by adding the same volume of 50% w/v PEG3000, followed by washing the precipitates with 17% w/v PEG3000. The pellets were dried and stored at 80 °C. Absorption spectra of the samples used in this study are shown in Fig. 1. 2.2. Extraction and separation of the quinones All chemicals used were purchased from Wako Pure Chemical Industries, Ltd. (Japan) and Sigma Chemical Co. (U. S. A), unless otherwise noted. Three methods of quinone extraction were tested: (1) the quinones were extracted with a mixture of 1:1 acetone/methanol; (2) the quinones were first extracted by 1:1 acetone/methanol, followed by petroleum ether (bp. 30–70 °C) extraction [27]; and (3) the quinones were extracted with a mixture of 2:1 chloroform/methanol [21,28]. All extractions were repeated twice and the combined extracts were dried under argon stream. The dried extracts were dissolved in ethanol (0.2 mL) and injected onto a reverse-phase HPLC column (TOSOH, TSKgel ODS-80Ts, 4.6  250 mm). The quinones and pigments were eluted isocratically at 25 °C by 7:3 methanol/isopropanol at a flow rate of 0.7 mL/min and were monitored by a multi-wavelength UV detector (ÄKTA Purifier, UV-900) at 270 nm, 370 nm and 500 nm.

1.5

Absorbance

Chromatophore

measured

LH1

1.0 700

800

900

1000

Wavelength (nm)

0.5

0.0 1.0 Absorbance

Rba. sphaeroides. In the purified RC solution of Blastochloris (Blc., formerly Rhodopseudomonas) viridis, one UQ9 and one MQ9 per RC were measured, but nearly half of the UQ9 were lost during crystallization with a final ratio of UQ9/MQ9 = 0.6 in the RC crystals [14]. The UQ9 at QB site in the crystal structures is characterized by low occupancy and heterogeneity in position [15–18]. In Allochromatium (Alc.) vinosum that contains both UQ8 and MQ8 [3,19,20], a smaller number of 5–10 quinones/RC was estimated in the chromatophores [5]. In Thermochromatium (Tch.) tepidum, which is a close relative to the Alc. vinosum, a ratio of UQ/MQ = 4.3 was reported for whole cell extracts [21]. The MQs consisted of a mixture of isoprenoid chains of lengths 6, 7 and 8 units in a ratio of 11:4:85, respectively, and only MQ8 was detected in the isolated RC solution [21]. In the recently published crystal structure of the Tch. tepidum LH1–RC core complex at 3.0 Å resolution, we have identified one UQ8 and one MQ8 [22]. However, the UQ8 was not found in a RC-only structure at much higher resolution [23]. Since the LH1–RC core complex is a more natural form than the RC-only complex, the above results prompt us to ask how many quinones could be expected in the Tch. tepidum LH1– RC and whether there is a loss during crystallization of the LH1– RC. To answer these questions, we have conducted a quantitative study to investigate the distribution of the quinones in Tch. tepidum and to compare their compositions among chromatophore, LH1–RC solution and crystal samples, and the RC-only complex.

LH1-RC (solution) LH1-RC (crystal)

0.5

0.0 1.5 Absorbance

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RC (solution) 1.0

0.5

0.0

400

600

800

1000

Wavelength (nm) Fig. 1. Absorption spectra of the samples used in this study. Top: chromatophores (inset: deconvoluted region, dotted lines show the components). Middle: LH1–RC solution and crystal samples. Bottom: purified RC.

2.3. Determination of quinone content The quinone identities were determined by comparing their absorption and mass spectra with those of authentic UQ10 and MQ4 purchased from Sigma–Aldrich (U. S. A). The absorption spectra were recorded on a Agilent 8453 UV–Vis spectrophotometer. The mass spectra were measured on a 4800 Plus MALDI TOF/TOF Analyzer (Applied Biosystems, MDS SCIEX). 2,4,6-Trihydroxy acetophenone (THAP) was used as matrix and was dissolved in 50% acetonitrile solution containing 0.3% TFA with the final concentration of 20 mg/mL. The quinones dissolved in chloroform were mixed with the THAP solution in a ratio of 1:1 (v/v) and then loaded onto the sample stage for co-crystallization. Measurement was performed in positive and reflector modes. The spectra obtained were calibrated externally using the authentic UQ10, MQ4 and the [M+H+] ions from five standards: angiotensin I (m/z 1297.51), ACTH (clip 1–17) (m/z 2094.46), ACTH (clip 18–36) (m/z 2466.72), ACTH (clip 7–38) (m/z 3660.19) and insulin (bovine) (m/z 2867.80, z = 2). Quantification of the quinone contents was conducted by integrating the quinone peaks in chromatogram based on the calibration using authentic UQ10 and MQ4 as the standards. Following extinction coefficients were used to calculate the number of quinones per RC: 14.7 mM 1 cm 1 at 275 nm for the UQ in ethanol [11,14], 17.3 mM 1 cm 1 at 270 nm for the MQ in ethanol [29], 4320 mM 1 cm 1 at 915 nm for the Tch. tepidum LH1–RC

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3. Results Using a multi-wavelength UV detector, the quinones eluted from HPLC can be easily distinguished from other pigments as shown in Fig. 2a. The absorption spectra in Fig. 2b confirm that the fractions eluted at 23.5 min and 38.6 min are UQ and MQ, respectively. Both the UQ and MQ extracted are in oxidized form as confirmed by treatment with 2 mM FeCl3. Mass spectra yielded m/z values of 751.5 and 739.5 for the UQ and MQ, respectively, indicating that both of them were detected as Na+-associated ion species and correspond to the quinones with eight isoprenoid units. No other quinones with different chain lengths were detected from all samples.

200 mAU

MQ8 Chromatophore

LH1-RC (solution)

LH1-RC (crystal)

RC (solution)

20

25

30

35

40

45

50

Elution time (min) Fig. 3. Chromatograms of the quinone extracts from Tch. tepidum chromatophores, LH1–RC complexes in solution and crystals, and RC-only complex. The chromatograms are normalized to the peak height of MQ8 for comparison.

BChl

Table 1 Numbers of the quinones per RC in chromatophores, purified LH1–RC, LH1–RC crystals and RC-only complex from Tch. tepidum.

270 nm 370 nm 500 nm

Absorbance

(a)

UQ8

Absorbance at 270 nm

[30] and 288 mM 1 cm 1 at 802 nm for the RC complex [31]. The number of LH1–RC complex in chromatophore was evaluated by deconvolution of the absorption spectrum of the chromatophores from 630 to 1000 nm in order to extract the contribution of LH1 at 915 nm using the same procedure described previously [24] (see Fig. 1). This is also the number of RC assuming that each LH1 ring surrounds a RC.

Spir

BPhe

UQ8

Sample

UQ8

MQ8

UQ8/MQ8

Chromatophores LH1–RC (solution) LH1–RC (crystal) Purified RC

18 ± 2 6.1 ± 0.8 6.7 ± 0.2 1.6 ± 0.1

3.7 ± 1.1 1.3 ± 0.1 1.4 ± 0.1 0.6 ± 0.1

5.0 ± 0.8 4.6 ± 0.5 4.7 ± 0.1 2.8 ± 0.2

MQ8

10

20

30

40

50

Elution time (min)

(b)

Fig. 2. (a) A representative HPLC chromatogram of the pigments extracted from Tch. tepidum LH1–RC complex with 2:1 chloroform/methanol. The absorbance was monitored at three wavelengths as indicated. BChl: bacteriochlorophyll a, BPhe: bacteriopheophytin a, Spir: spirilloxanthin. (b) Absorption spectra of the quinone fractions eluted at 23.5 min (upper panel, solid line with the scale on left axis) and 38.6 min (lower panel, solid line with the scale on left axis) from the HPLC of chromatophore extracts. Comparison of the spectra with those of the authentic UQ10 (upper panel, dotted line with the scale on right axis) and MQ4 (lower panel, dotted line with the scale on right axis) confirms that the fractions at 23.5 min and 38.6 min are the UQ and MQ, respectively.

Of the three methods of extraction we have tested, the mixture of 2:1 chloroform/methanol was found to be most efficient by giving the highest yield. Thereafter, this solvent mixture was used throughout all experiments. Fig. 3 shows chromatograms of the quinones extracted from chromatophores, LH1–RC in purified solution, LH1–RC crystals and RC-only solution sample. Apparently, the chromatophore and both LH1–RC samples exhibited a similar ratio of UQ8/MQ8, whereas the RC showed a much reduced value. A quantitative analysis on the number of the quinones per RC is shown in Table 1. There are about 18 UQ8 and 4 MQ8 per RC in the Tch. tepidum chromatophore. The total number of the quinones/RC is compatible with those reported for Rba. sphaeroides [7–10] and Pha. molischianum [13], but is greater than that estimated for Alc. vinosum [5]. The ratio of UQ8 to MQ8 is consistent with that previously reported for the whole cell extracts from Tch. tepidum [21]. The numbers of quinone per RC decreased to about 6 for UQ8 and about 1.3 for MQ8 in the LH1–RCs in both solution and crystal samples with the UQ8/MQ8 ratios similar to that in chromatophore. The result indicates that about one-third of both UQ8 and MQ8 in the photosynthetic membrane are located within the LH1–RC core complex and there is no essential loss of these quinones during the crystallization. However, the UQ8 and MQ8 in the purified RC-only complex are much fewer than those in LH1–RC complex, only a quarter of the UQ8 and half of the MQ8 are retained in the RC with a reduced ratio of UQ8/MQ8 = 2.8. The numbers of quinones in the purified RC are in agreement with those measured for Rba. sphaeroides [6,9] and Blc. viridis [14]. A major source of errors in estimating the quinone contents in chromatophore and core complex may arise from the uncertainty in the extinction coefficient of LH1–RC complex. To investigate this, we extracted the carotenoids from the purified LH1–RC with

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methanol and benzene. Spirilloxanthin constitutes more than 90% of the carotenoids in Tch. tepidum LH1–RC [25]. Using the extinction coefficient of spirilloxanthin in benzene (147 mM 1 cm 1 at 510 nm), a number of 15.3 carotenoids per LH1–RC was calculated. This is very close to the number identified from the crystal structure [22], confirming the correctness of the extinction coefficient of LH1–RC. 4. Discussion In this study, we have determined the quinone contents in various portions of the thermophilic purple sulfur bacterium Tch. tepidum. The results provide a comprehensive and quantitative analysis of the quinone distribution at different levels of the photosynthetic apparatus. Size of the quinone pool (22 quinones/RC) in the intracytoplasmic membrane is about the same as those reported for other photosynthetic bacteria. However, the distribution appears to be inhomogeneous. About one-third of the quinones are confined to the LH1–RC core complex with the UQ8/MQ8 ratio similar to that in chromatophores. About 12 UQ8 and 2.4 MQ8 per RC remain outside the core complex possibly as mobile quinones in the membrane. Taking into account that only 1–2 quinones are present in LH2 complex [11], the result suggests a specific affinity of the quinones for the core complex, which is consistent with that reported for Rba. sphaeroides where about 25–40% of the endogenous UQ10 in quinone pool are associated with the core complex [9–11]. Although we have relatively detailed information on the UQ so far, little is known about the behavior of MQ. The finding of this work reveals that MQs are also present in the quinone pool outside the core complex with a stoichiometric ratio of MQ/UQ = 0.2. The MQ is tightly bound at QA site in the RCs of Tch. tepidum and Blc. viridis, but the quinone in this position has been shown by numerous studies (in most cases the UQ10 in Rba. sphaeroides) to be replaceable by a large variety of quinone molecules [4,6,32]. Our result implies a possibility of the MQ exchange between the QA site and quinone pool but at a much reduced rate with respect to the UQ at QB site, because the binding affinity of the quinone at QA site is much greater than that of the quinone at QB site ( 21 vs. 11 kJ/mol for DG°) [33]. This might also be reflected in the small ratio of MQ to UQ. Different from the situation of RC-only complex, the numbers of UQ8 and MQ8 in Tch. tepidum LH1–RC complex remained essentially unchanged during the crystallization. However, only one UQ8 and one MQ8 were confirmed in the LH1–RC crystal structure [22], occupying the QB (proximal) and QA sites, respectively. This means that about five UQ8 were still invisible. Inspecting the published LH1–RC structure at 3.0 Å resolution, there are sufficient cavities between the LH1 ring and RC to accommodate several quinone molecules. In fact, from the electron density map we can see a number of areas with residual densities, which might correspond to the fragments of quinones and/or lipids. The invisible quinones may be scattered over the interior of the LH1–RC and could exist in a transient state between the quinone and quinol exchange as the structure shows that the LH1 ring has multiple channels potentially available for the quinone passage. These mobile quinones should be structurally disordered, making them difficult to be detected in the crystal structure. There is also a possibility that additional quinone-binding sites are in the LH1–RC complex as in the case of photosystem II, in which a third quinone-binding site (QC) was identified [34–36]. Such quinones would be difficult to identify in the RC-only complex but may become visible from the higher resolution structures of the LH1–RC core complex through improvement of the crystal quality. It is noted that electron density tentatively assigned to the extra quinone is recently reported from the high resolution structures of RCs [18].

In the crystal structure of the Tch. tepidum RC-only complex, a MQ8 is bound to QA site but no quinone is found in the QB position [23]. Usually, the QB site in Blc. viridis RC has poorly defined electron density due to the low occupancy [16–18]. Based on our result, it is reasonable to estimate that there are 1–2 UQ8 and one MQ8 associated with the RC in the purified sample prior to crystallization. These are much fewer than those in the LH1–RC core complex, indicating that the LH1 ring plays an important role as the protective fence in confinement of the quinone molecules. The UQ8 may be loosely bound to the RC in solution but a majority of them are lost during the crystallization as in the case of Blc. viridis RC [14]. The result is not surprising because the crystallization procedure of RC-only complex usually involves using lauryl dimethylamine N-oxide (LDAO) that is known as an effective detergent for the quinone extraction [4,6]. Moreover, the RC-only complex may have been partly modified or even damaged during the isolation and purification processes as evidenced in the LH1–RC structure. The conformation of a portion in the C-subunit is markedly different from that in the RC-only structure and a fragment of the H-subunit, which was invisible in the RC-only structure, reveals clear electron density in the LH1–RC structure [22]. Combining other findings that show crucial roles of the surrounding LH1 in stabilizing the charge separation process of RC [9,11], our results support the conclusion that it is more desirable for investigation of the structure and function of the intact RC by using the LH1–RC core complex as a starting material. Acknowledgments We thank Y. Takasu and S. Igarashi for experimental assistance. This work was supported in part by Grant for Basic Scientific Research Projects from The Sumitomo Foundation and The Kurata Memorial Hitachi Science and Technology Foundation. We thank Kao Corporation for kindly providing the LDAO. References [1] Nowicka, B. and Kruk, J. (2010) Occurrence, biosynthesis and function of isoprenoid quinones. Biochim. Biophys. Acta 1797, 1587–1605. [2] Imhoff, J.F. and Bias-Imhoff, U. (1995) Lipids, quinones and fatty acids of anoxygenic phototrophic bacteria in: Anoxygenic Photosynthetic Bacteria (Blankenship, R.E., Madigan, M.T. and Bauer, C.D., Eds.), pp. 179–205, Kluwer Academic Publishers, Dordrecht, The Netherlands. [3] Imhoff, J.F. (1984) Quinones of phototrophic purple bacteria. FEMS Microbiol. Lett. 25, 85–89. [4] Cogdell, R.J., Brune, D.C. and Clayton, R.K. (1974) Effects of extraction and replacement of ubiquinone upon the photochemical activity of reaction centers and chromatophores from Rhodopseudomonas spheriodes. FEBS Lett. 45, 344–347. [5] Halsey, Y.D. and Parson, W.W. (1974) Identification of ubiquinone as the secondary electron acceptor in the photosynthetic apparatus of Chromatium vinosum. Biochim. Biophys. Acta 347, 404–416. [6] Okamura, M.Y., Isaacson, R.A. and Feher, G. (1975) Primary acceptor in bacterial photosynthesis: obligatory role of ubiquinone in photoactive reaction centers of Rhodopseudomonas spheroides. Proc. Natl. Acad. Sci. U.S.A. 72, 3491–3495. [7] Takamiya, K. and Dutton, P.L. (1979) Ubiquinone in Rhodopseudomonas sphaeroides. Some thermodynamic properties. Biochim. Biophys. Acta 546, 1–16. [8] Mezzetti, A., Leibl, W., Breton, J. and Nabedryk, E. (2003) Photoreduction of the quinone pool in the bacterial photosynthetic membrane: identification of infrared marker bands for quinol formation. FEBS Lett. 537, 161–165. [9] Francia, F., Dezi, M., Rebecchi, A., Mallardi, A., Palazzo, G., Melandri, B.A. and Venturoli, G. (2004) Light-harvesting complex I stabilizes P+QB charge separation in reaction centers of Rhodobacter sphaeroides. Biochemistry 43, 14199–14210. [10] Comayras, F., Jungas, C. and Lavergne, J. (2005) Functional consequences of the organization of the photosynthetic apparatus in Rhodobacter sphaeroides. I. Quinone domains and excitation transfer in chromatophores and reaction center-antenna complexes. J. Biol. Chem. 280, 11203–11213. [11] Dezi, M., Francia, F., Mallardi, A., Colafemmina, G., Palazzo, G. and Venturoli, G. (2007) Stabilization of charge separation and cardiolipin confinement in antenna-reaction center complexes purified from Rhodobacter sphaeroides. Biochim. Biophys. Acta 1767, 1041–1056.

Y. Kimura et al. / FEBS Letters 589 (2015) 1761–1765 [12] Allen, J.P., Feher, G., Yeates, T.O., Komiya, H. and Rees, D.C. (1987) Structure of the reaction center from Rhodobacter sphaeroides R-26: the cofactors. Proc. Natl. Acad. Sci. U.S.A. 84, 5730–5734. [13] Mascle-Allemand, C., Lavergne, J., Bernadac, A. and Sturgis, J.N. (2008) Organisation and function of the Phaeospirillum molischianum photosynthetic apparatus. Biochim. Biophys. Acta 1777, 1552–1559. [14] Gast, P., Michalski, T.J., Hunt, J.E. and Norris, J.R. (1985) Determination of the amount and the type of quinones present in single crystals from reaction center protein from the photosynthetic bacterium Rhodopseudomonas viridis. FEBS Lett. 179, 325–328. [15] Deisenhofer, J., Epp, O., Sinning, I. and Michel, H. (1995) Crystallographic refinement as 2.3 Å resolution and refined model of the photosynthetic reaction centre from Rhodopseudomonas viridis. J. Mol. Biol. 246, 429–457. [16] Lancaster, C.R. and Michel, H. (1997) The coupling of light-induced electron transfer and proton uptake as derived from crystal structures of reaction centres from Rhodopseudomonas viridis modified at the binding site of the secondary quinone, QB. Structure 5, 1339–1359. [17] Baxter, R.H.G., Seagle, B.-L., Ponomarenko, N. and Norris, J.R. (2005) Cryogenic structure of the photosynthetic reaction center of Blastochloris viridis in the light and dark. Acta Crystallogr. D 61, 605–612. [18] Roszak, A.W., Moulisová, V., Reksodipuro, A.D.P., Gardiner, A.T., Fujii, R., Hashimoto, H., Isaac, N.W. and Cogdell, R.J. (2012) New insights into the structure of the reaction centre from Blastochloris viridis: evolution in the laboratory. Biochem. J. 442, 27–37. [19] Takamiya, K., Nishimura, M. and Takamiya, A. (1967) Distribution of quinones in some photosynthetic bacteria and algae. Plant Cell Physiol. 8, 79–86. [20] Hale, M.B., Blankenship, R.E. and Fuller, R.C. (1983) Menaquinone is the sole quinone in the facultatively aerobic green photosynthetic bacterium Chloroflexus aurantiacus. Biochim. Biophys. Acta 723, 376–382. [21] Nozawa, T., Trost, J.T., Fukuda, T., Hatano, M., McManus, J.D. and Blankenship, R.E. (1987) Properties of the reaction center of the thermophilic purple photosynthetic bacterium Chromatium tepidum. Biochim. Biophys. Acta 894, 468–476. [22] Niwa, S., Yu, L.-J., Takeda, K., Hirano, Y., Kawakami, T., Wang-Otomo, Z.-Y. and Miki, K. (2014) Structure of the LH1-RC complex from Thermochromatium tepidum at 3.0 Å. Nature 508, 228–232. [23] Nogi, T., Fathir, I., Kobayashi, M., Nozawa, T. and Miki, K. (2000) Crystal structures of photosynthetic reaction center and high-potential iron-sulfur protein from Thermochromatium tepidum: thermostability and electron transfer. Proc. Natl. Acad. Sci. U.S.A. 97, 13561–13566.

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[24] Yu, L.-J., Kato, S. and Wang, Z.-Y. (2010) Examination of the putative Ca2+binding site in the light-harvesting complex 1 of thermophilic purple sulfur bacterium Thermochromatium tepidum. Photosynth. Res. 106, 215–220. [25] Suzuki, H., Hirano, Y., Kimura, Y., Takaichi, S., Kobayashi, M., Miki, K. and Wang, Z.-Y. (2007) Purification, characterization and crystallization of the core complex from thermophilic purple sulfur bacterium Thermochromatium tepidum. Biochim. Biophys. Acta 1767, 1057–1063. [26] Kobayashi, M. and Nozawa, T. (1993) Purification and crystallization of the reaction center from the thermophilic purple sulfur bacterium Chromatium tepidum. Bull. Chem. Soc. Jpn. 66, 3834–3836. [27] Trost, J.T. and Blankenship, R.E. (1989) Isolation of a photoactive photosynthetic reaction center-core antenna complex from Heliobacillus mobilis. Biochemistry 28, 9898–9904. [28] Collins, M.D. (1985) Analysis of isoprenoid quinones. Methods Microbiol. 18, 329–366. [29] Dadák, V. and Krivánková, L. (1980) Spectroscopic determination of vitamin K after reduction. Methods Enzymol. 67, 128–134. [30] Kimura, Y., Hirano, Y., Yu, L.-J., Suzuki, H., Kobayashi, M. and Wang, Z.-Y. (2008) Calcium ions are involved in the unusual red shift of the lightharvesting 1 Qy transition of the core complex in thermophilic purple sulfur bacterium Thermochromatium tepidum. J. Biol. Chem. 283, 13867–13873. [31] Straley, S.C., Parson, W.W., Mauzerall, D.C. and Clayton, R.K. (1973) Pigment content and molar extinction coefficients of photochemical reaction centers from Rhodopseudomonas spheroides. Biochim. Biophys. Acta 305, 597–609. [32] Jones, M.R. (2009) Structural plasticity of reaction centers from purple bacteria in: The Purple Phototrophic Bacteria (Hunter, C.N., Daldal, F. and Beatty, J.T., Eds.), pp. 295–321, Springer. [33] Mallardi, A., Giustini, M. and Palazzo, G. (1998) Binding of ubiquinone to photosynthetic reaction centers. 2. Determination of enthalpy and entropy changes for the binding to the QA site in reverse micelles. J. Phys. Chem. B 102, 9168–9173. [34] Krivanek, R., Kern, J., Zouni, A., Dau, H. and Haumann, M. (2007) Spare quinones in the QB cavity of crystallized photosystem II from Thermosynechococcus elongatus. Biochim. Biophys. Acta 1767, 520–527. [35] Kaminskaya, O., Shuvalov, V.A. and Renger, G. (2007) Evidence for a novel quinone-binding site in the photosystem II (PS II) complex that regulates the redox potential of cytochrome b559. Biochemistry 46, 1091–1105. [36] Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A. and Saenger, W. (2009) Cyanobacterial photosystem II at 2.9-Å resolution and the role of quinones, lipids, channels and chloride. Nat. Struct. Mol. Biol. 16, 334–342.