Taiki Motomura

Michihiro Suga

Rainer Hienerwadel

Akiko Nakagawa

Thanh-Lan Lai

Wolfgang Nitschke

Takahiro Kuma

Miwa Sugiura

Alain Boussac

Jain-Ren Shen
email: shen@cc.okayama-u.ac.jp

Jian-Ren Shen

Jian-Ren Shen

Structure and properties of the heme-protein Tll0287 1 Crystal structure and redox properties of a novel cyanobacterial heme-protein with a His/Cys heme axial ligation and a per-arnt-sim (PAS)-like domain

Keywords: psbA gene, X-ray crystallography, His-Cys heme axial coordination, PAS-like domain

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Crystal structure and redox properties of a novel cyanobacterial heme-protein with a His/Cys heme axial ligation and a per-arnt-sim (PAS)-like domain

prokaryotic cyanobacteria to eukaryotic higher plants, and a typical PSII core from cyanobacteria consists of 17 trans-membrane subunits and 3 peripheral, membrane-extrinsic subunits, with an overall molecular mass of  350 kDa for a PSII monomer. PSII exists in a dimeric form, and the structure of cyanobacterial PSII dimer has been solved up to 1.9 Å resolution [START_REF] Umena | Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å[END_REF][START_REF] Suga | Native structure of photosystem II at 1.95 Å resolution viewed by femtosecond X-ray pulses[END_REF]. The core of PSII is formed by two subunits D1 and D2, which bind all the cofactors involved in the PSII electron transfer chain, and are surrounded by two large chlorophyll-protein subunits CP47 and CP43 [START_REF]Photosystem II, The Light-Driven Water: Plastoquinone Oxidoreductase[END_REF][START_REF] Shen | Structure of Photosystem II and the Mechanism of Water Oxidation in Photosynthesis[END_REF][START_REF] Kawakami | Structure of the catalytic, inorganic core of oxygen-evolving Photosystem II at 1.9 Å resolution[END_REF].

The D1 protein is encoded by the psbA gene which exists in a single copy in higher plants but in multiple forms in cyanobacteria [START_REF] Golden | Light-responsive gene expression in cyanobacteria[END_REF]. The isoforms of the D1 protein encoded by these different psbA genes in cyanobacteria have been reported to be differently expressed in response to various environmental conditions such as light intensity [START_REF] Schaefer | Light availability influences the ratio of two forms of D1 in cyanobacterial thylakoids[END_REF][START_REF] Schaefer | Differential expression of members of a cyanobacterial psbA gene family in response to light[END_REF][START_REF] Bustos | Different and rapid responses of four cyanobacterial psbA transcripts to changes in light intensity[END_REF], the presence of UV-B [START_REF] Campbell | The cyanobacterium Synechococcus resists UV-B by exchanging photosystem II reaction-center D1 proteins[END_REF][START_REF] Sicora | Cyanobacterial psbA families in Anabaena and Synechocystis encode trace, constitutive and UVB-induced D1 isoforms[END_REF], chilling stress [START_REF] Campbell | Electron transport regulates exchange of two forms of photosystem II D1 protein in the cyanobacterium Synechococcus[END_REF], reductive stress [START_REF] Sippola | Thiol redox state regulates expression of psbA genes in Synechococcus sp. PCC 7942[END_REF], and low O2 [START_REF] Summerfield | Low-oxygen induction of normally cryptic psbA genes in cyanobacteria[END_REF] or micro-aerobic conditions [START_REF] Sicora | Transcription of a "silent" cyanobacterial psbA gene is induced by microaerobic conditions[END_REF][START_REF] Mulo | Cyanobacterial psbA gene family: optimization of oxygenic photosynthesis[END_REF].

In the thermophilic cyanobacterium Thermosynechococcus elongatus 3 homologous genes, psbA1, psbA2 and psbA3, are present in the genome [START_REF] Nakamura | Complete genome structure of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1[END_REF]. The PsbA1, PsbA2 and PsbA3 D1 proteins encoded by the psbA1, psbA2 and psbA3 genes, are expressed under different environmental conditions [START_REF] Sicora | Transcription of a "silent" cyanobacterial psbA gene is induced by microaerobic conditions[END_REF][START_REF] Mulo | Cyanobacterial psbA gene family: optimization of oxygenic photosynthesis[END_REF][START_REF] Kós | Differential regulation of psbA and psbD gene expression, and the role of the different D1 protein copies in the cyanobacterium Thermosynechococcus elongatus BP-1[END_REF][START_REF] Sander | Role of the psbA gene family of PSII from the thermophilic cyanobacterium Thermosynechococcus elongatus[END_REF]. Out of the 344 amino acid residues, 31 differ between PsbA1 and PsbA2, 21 differ between PsbA1 and PsbA3, and 27 differ between PsbA2 and PsbA3 (see 20, 21 for reviews).

In T. elongatus, some of the properties of PSII were found to be different depending on the psbA gene product [START_REF] Sugiura | Some Photosystem II properties depending on the D1 protein variants in Thermosynechococcus elongatus[END_REF][START_REF] Sugiura | Photosystem II D1 protein variants in Thermosynechococcus elongatus[END_REF]. For example, while the O2-evolving activity was found to be similar in PsbA1-and PsbA2-containing PSII (PsbA1-PSII and PsbA2-PSII) [START_REF] Sugiura | Environment of TyrZ in Photosystem II from Thermosynechococcus elongatus in which PsbA2 is the D1 protein[END_REF], the PsbA3-containing PSII (PsbA3-PSII) had a higher O2 evolution activity [START_REF] Sugiura | Influence of histidine-198 of the D1 subunit on the properties of the primary electron donor, P 680, of photosystem II in Thermosynechococcus elongatus[END_REF][START_REF] Sugiura | The D1-173 amino acid is a structural determinant of the critical interaction between D1-Tyr161 (Tyr Z) and D1-His190 in Photosystem II[END_REF]. The redox potential of pheophytin, QA and QB, and the binding properties of some herbicides, were also reported to be different in PsbA1-PSII and PsbA3-PSII [START_REF] Sugiura | Energetics in Photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA1 or psbA3 gene[END_REF][START_REF] Boussac | Probing the quinone binding site of Photosystem II from Thermosynechococcus elongatus containing either PsbA1 or PsbA3 as the D1 protein through the binding characteristics of herbicides[END_REF]. On the other hand, the differences between PsbA2-PSII and PsbA1/A3-PSII are less documented. The structures of PsbA2-PSII and PsbA3-PSII have not yet been reported. However, a previous work showed that the geometry of the phenol group of TyrZ and its environment, likely the hydrogen bond between TyrZ and D1/His190, is modified in PsbA2-PSII in comparison with PsbA1/A3-PSII [START_REF] Sugiura | Environment of TyrZ in Photosystem II from Thermosynechococcus elongatus in which PsbA2 is the D1 protein[END_REF][START_REF] Sugiura | The D1-173 amino acid is a structural determinant of the critical interaction between D1-Tyr161 (Tyr Z) and D1-His190 in Photosystem II[END_REF].

Interestingly, a new electron paramagnetic resonance (EPR) signal was detected in whole cells expressing the psbA2 gene only [START_REF] Boussac | The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus[END_REF]. This EPR signal was identified to be originated from a hemoprotein which is the product of tll0287, a hypothetical gene encoding a ~19 kDa protein (consisting of 191 residues) with an unknown function [START_REF] Boussac | The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus[END_REF]. Homologous genes to tll0287 are also found in several cyanobacteria (see Discussion section). The amino acid sequence of the Tll0287 protein has a CXXCH motif characteristic for c-type cytochromes. Four different N-terminal sequences were detected for the mature Tll0287, suggesting multiple processing sites. In the present work, we purified the Tll0287 protein, solved its crystal structure, determined its midpoint potential, and investigated its cellular localization by immunoblot analysis. Possible functions of Tll0287 are discussed based on the sequence and structural homologies with other known proteins.

RESULTS

Expression and location of the Tll0287 protein-The previous study has detected the novel EPR signal ascribed to Tll0287 in both soluble and membrane fractions from the psbA2-expressing cells, but neither from the psbA1-nor psbA3-expressing cells [START_REF] Boussac | The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus[END_REF]. In order to confirm the different expression of Tll0287 in these three different strains, we used an anti-Tll0287 antibody. Fig. 1a shows a SDS-PAGE analysis of the purified Tll0287 protein used in this study, and Fig. 1c shows an immunoblot analysis with the anti-Tll0287 antibody in the whole cell and thylakoid fractions from WT (expressing mainly the psbA1 gene), WT*2 (expressing the psbA2 gene only) and WT*3 (expressing the psbA3 gene only) strains. The amount of proteins loaded in each lane of Fig. 1c are shown in Fig. 1b, where exactly the same amount of the samples were loaded in each lane as in Fig. 1c. The results in Fig. 1c clearly shows that Tll0287 is expressed in the WT*2 strain, but little or not at all in the WT and WT*3 strains. In the immunoblots of the purified Tll0287 protein and various subcellular fractions from the WT*2 strain, weak bands at twice and four times the molecular mass of Tll0287 were detected, which may correspond to non-native oligomeric form of Tll0287 that would be present even under SDS-PAGE conditions in a very low amounts. It should be noted that in lane 2 (labeled 'Tll' of Fig. 1b), the CBB-stained Tll0287 is barely visible, which is required to ensure that the immunoblot in Fig. 1c does not become too dense.

Tll0287 was also detected in a crude-PSII fraction (PSII purified with LDAO) from the WT*2 strain, but not in purified PSII after extensive purification procedures involving two different detergents (LDAO and β-DDM) (Fig. 1c). The previous result has suggested that Tll0287 is co-purified with a His-tagged PSII [START_REF] Boussac | The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus[END_REF]; however, the PSII used in the previous study was obtained with a simple step of purification employing a mild detergent-solubilization and nickel affinity column. The present results therefore suggest that Tll0287 is likely not bound to PSII specifically. However, it should be noted that cyt c-550, an extrinsic protein associated with PSII rather strongly, can also be dissociated from PsbA2-PSII, but not from PsbA1-PSII and PsbA3-PSII, during a size exclusion chromatography performed under high pressure (results not shown), implying that it is sometimes difficult to conclude whether the binding of non-membrane proteins is real or not based on the co-purification only.

Although previous studies [START_REF] Boussac | The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus[END_REF] have suggested the possible presence of Tll0287 at the lumenal side of the thylakoid membrane, its location remained to be confirmed. Therefore, the thylakoid fraction from the psbA2-expressing strain was treated with the protease thermolysin, together with the purified Tll0287 as a control, and the protease-treated samples were probed by immunoblot analysis (Fig. 2). The amount of the samples loaded in each lane for the immunoblot analysis was shown in Fig. 2a, where again the band of purified Tll0287 was barely visible by CBB-staining. While the purified Tll0287 is largely digested by thermolysin, it was poorly digested by the protease in the thylakoid fraction (Fig. 2b). An exactly same situation was found for the PsbO protein (Fig. 2c), which has a well-established lumenal location. The CBB-stained gel (Fig. 2b) showed that many bands were weakened in the thermolysin-treated thylakoid membrane fraction as well as the purified PSII, indicating the effective digestion by the protease. These results suggest that at least part of Tll0287 is located in the lumen of the thylakoid, or periplasm if we consider that there may be some contamination of cytoplasmic membranes in the thylakoid membrane fraction used in the present study. Determination of the midpoint potential of Tll0287-The redox titration of Tll0287 is shown in Fig. 3. Fig. 3a and3b show the absorption spectra recorded in the oxidative way and reductive way, respectively. The presence of well-defined isosbestic points in the two panels shows that the protein was fully stable in these experimental conditions. Fig. 3c shows a Nernst plot for an oxidation (blue points) and a reduction (red points) titration performed at pH 8.5. The continuous black line is a global fit with n = 1 and E1/2' = -255 ± 20 mV vs NHE. Finally, Fig. 3d shows the pH dependence of the E1/2' determined as above at different pH values (the data points are from electrochemical experiments performed on Tll0287 from 3 batches). The continuous line is the result of a fitting procedure with the following equation: E1/2' = E0' -57 log((Kox + [H + ]) / (Kred + [H + ])) where E0' is the midpoint redox potential of the fully protonated form and Kred and Kox are the ionization constants of the protonatable group when the iron is reduced and oxidized, respectively [START_REF] Dutton | Redox potentiometry: Determination of midpoint potentials of oxidation-reduction components of biological electron-transfer systems[END_REF]. The E0'value was found to be -255 ± 20 mV vs NHE at pH values above pKred = 7.2 and the pKox value was ≤ 5.5.

Structure determination-Tll0287 was purified from the pool of non-membrane proteins and crystals of the isolated protein were grown at 20°C for a few days. The crystals appeared as red needles with a maximum size of 0.4 mm x 0.02 mm x 0.02 mm. The crystals belong to the space group P42212 with unit cell dimensions a = b = 101.2 Å, c = 33.1 Å (Table 1). Phase information was obtained by the single-wavelength anomalous diffraction (SAD) method using anomalous diffraction from the Fe ion of the heme, and the model was refined to a resolution of 2.0 Å with an Rfactor and Rfree of 0.1765 and 0.2336, respectively.

The structure of Tll0287 is shown in Fig. 4a. It contains residues 26-186 (the full length of the protein is 191 residues), a heme-c, a tentatively assigned chloride ion, and 84 water molecules. The secondary structure of the domain was defined as follows: α-helices, α28-49, α2: 68-82, α3: 103-114, α4: 139-146, α5: 153-158; β-strands, β1: 85-89, β2: 120-125, β3: 128-136 and β4: 172-181. The helix α2 is antiparallel to the N-terminal end of helix α1. The β-strands are arranged in an antiparallel fashion and surrounded by three α-helices α1, α2 and α3.

Fig. 4b shows the surface charge distribution of Tll0287. The periphery of the heme ligand is relatively positive (Fig. 4c), and a concave pocket was found between the four β-strands bundle and α3 (Fig. 4b), which is also apparent in the overall structure of Tll0287. Fig. 4d shows that Tll0287 indeed exhibits a large hydrophobic surface as predicted from the properties observed during its purification [START_REF] Boussac | The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus[END_REF].

MALDI-TOF/MS analysis of the Tll0287 protein before crystallization detected peaks at m/z values of 18,633, 18,997, 19,162, 19,858 and 20,382 (Table 2). The peak at m/z 19,162 Da is equal to the molecular mass of the Tll0287 peptide with 24 residues truncated at the N-terminal region (Δ24) and with the heme bound [START_REF] Boussac | The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus[END_REF]. The other fragments may correspond to Tll0287 truncated at 11 or 12, 17 and 26. Because the previous study showed that the Δ24 fraction likely dominates the heme-bound form of Tll0287 [START_REF] Boussac | The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus[END_REF], our heme-containing structure likely corresponds to the Δ24-truncated protein. This is also in agreement with the sequence analysis by SignalP which suggested that this protein possesses a signal peptide that cleaves between 24 and 25 residues. The mass analysis, however, did not give information regarding the relative ratio of other fragments in the purified Tll0287 protein solution.

Heme in the Tll0287 protein-The 5 th and 6 th heme iron axial ligands are Cys68 and His145 (Fig. 4c); this is remarkably different from most of the c-type cytochromes in which His/Met or His/His axial ligands are found. It also appears that the Cys68 axial ligand is close to Arg45 and may be stabilized by it, whereas the His145 axial ligand is supported by the carbonyl group of Ala164 and is also close to Arg94 and Arg173.

Expression level of the Tll0287 protein in the dark-As mentioned in the Introduction section, the expression level of psbA2 was reported to increase under micro-aerobic conditions (14-16) and, as discussed below, in Synechocystis sp. PCC6803 it has been shown that sll5034 (analogous to tll0287) was transcribed at a higher level in the dark, an environment possibly comparable to micro-aerobic conditions [START_REF] Mitschke | An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp[END_REF]. We have therefore compared the level of expression of Tll0287 between the whole cells of WT, which mainly express psbA1, with those of WT*2 cells, under different light conditions. Fig. 5 shows the EPR spectra recorded from WT and WT*2 cells cultivated under either normal light conditions (i.e. 120 µE m -2 s -1 ) or after a further dark period of 3 to 4 hours. The magnetic field region shown allows us to compare the relative amplitude of 3 resonance signals, namely, the Fe 3+ superoxide dismutase signal labeled with an asterisk between 1300 and 2000 gauss [START_REF] Kerfeld | The 1.6 Å resolution structure of Fe-superoxide dismutase from the thermophilic cyanobacterium Thermosynechococcus elongatus[END_REF], the gz resonance of the soluble fraction of cyt c-550 labeled with an asterisk between 2200 and 2300 gauss [START_REF] Kerfeld | Structural and EPR Characterization of the soluble form of Cytochrome c-550 and of the psbV2 gene product from the cyanobacterium Thermosynechococcus elongatus[END_REF], and the gz resonance of Tll0287 labeled with an asterisk at around 2800 gauss [START_REF] Boussac | The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus[END_REF]. The large signal from Mn 2+ present in the cells precluded the detection of resonances other than cytochromes. Data in Fig. 5 clearly shows that, while the gz resonance signal of Tll0287 was virtually not detected in the WT cells grown either under normal light conditions (spectrum a) or after a further dark period (spectrum b), the level of expression of Tll0287 was approximately doubled upon a short dark period in the WT*2 cells when compared to other EPR detectable proteins (spectra c and d).

DISCUSSION

Location, structure and physical chemical properties of Tll0287-The present results confirmed that the Tll0287 protein is highly expressed in a strain expressing psbA2, but not in the WT (expressing predominantly psbA1), and WT*3 (expressing only psbA3) strains. Furthermore, it is shown that this protein is likely located in the thylakoid lumen and/or in the periplasm based on the protease digestion results. This location is in agreement with sequence analysis by SignalP which suggested that this protein possesses a cleavage site between residues 24 and 25 (not shown). The lumenal or periplasmic location is also consistent with the structural analysis showing that this protein has a globally similar structure to the PhoQ-DcuS-CitA (PDC) domain of some proteins involved in signal transduction with a periplasmic PAS fold instead of a cytoplasmic one (see below). However, we cannot exclude the possibility that another fraction of the Tll0287 protein may be located in the cytoplasm. This would be consistent with the results of MS analysis showing that the N-terminal of the purified protein had several N-termini [START_REF] Boussac | The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus[END_REF] consistent with different cleavage sites. Thus, it is possible that the protein has a dual location and possibly multiple functions. Interestingly, three other possible cleavage sites at the N-terminal region are also predicted by SignalP, but with a lower probability. These predicted sites correspond to the other N-terminal sequences found for Tll0287 although in these cases probably no heme is incorporated [START_REF] Boussac | The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus[END_REF]. Further studies are required to understand these observations.

The crystal structure of Tll0287 showed that the heme has a His/Cys ligation pattern, which is distinctly different from the typical His/Met or His/His ligand pairs for most of the c-type cytochromes. Such c-type heme with His/Cys axial ligation generally have a lower midpoint potential, typically below -400 mV, than other cytochromes with either a His/His or a His/Met axial coordination, e.g. [START_REF] Grein | DsrJ, an essential part of the DsrMKJOP transmembrane complex in the purple sulfur bacterium Allochromatium vinosum, is an unusual trihem cytochrome c[END_REF][START_REF] Ukita | Heterodimeric cytochrome c complex with a very low redox potential from an anaerobic ammonium-oxidizing enrichment culture[END_REF][START_REF] Kappler | The bacterial SoxAX cytochromes[END_REF][START_REF] Alric | Structural and functional characterization of the unusual triheme cytochrome bound to the reaction center of Rhodovulum sulfidophilum[END_REF][START_REF] Vandriessche | GHP, a new c-type green heme protein from Halobacterium salexigens and other proteobacteria[END_REF]. Here, the midpoint potential of Tll0287 was determined to be -255 ± 20 mV vs NHE at pH 8.5 and to be pH-dependent involving a protonatable group with a pK at 7.2 ± 0.3 in the reduced state. The nature of this group is under investigation by using vibrational spectroscopy.

The His/Cys ligation pattern for the heme in Tll0287 is the second one found in cyanobacteria. The first one is PsbV2 (37), a c-type heme protein similar to PsbV (i.e. the cyt c-550), but with unknown function, although it has been reported that this protein may partially support the photoautrophic growth of Synechocystis 6803 in place of the PsbV protein [START_REF] Katoh | Functional analysis of psbV and a novel c-type cytochrome gene psbV2 of the thermophilic cyanobacterium Thermosynechococcus elongatus strain BP-1[END_REF]. Fig. 6a shows the superimposition of the structure of Tll0287 with that of PsbV2. Surprisingly, although the heme structures can be superimposed with a rmsd (root-mean square deviation) of 0.8 Å, the overall structures of the two proteins are remarkably different. Because the structure of PsbV2 belongs to the general folding of c-type cytochromes and is rather similar to other c-type cytochromes [START_REF] Suga | Crystal structure at 1.5 Å resolution of the PsbV2 cytochrome from the cyanobacterium Thermosynechococcus elongatus[END_REF], the present results indicate that the structure of Tll0287 is different from those of typical c-type cytochromes.

The ligand pair of His/Cys is different from the His/Tyr ligand pair previously erroneously predicted from the EPR spectrum [START_REF] Boussac | The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus[END_REF]. Indeed, using the formalism developed in [START_REF] Peisach | Electron paramagnetic resonance studies of iron porphyrin and chlorin systems[END_REF] (see also 40 for a review), the crystal field parameters such as the rhombicity (ν/Δ) and the tetragonality (Δ/) can be calculated from the gz, gy and gx values of the EPR signal. These structural parameters put Tll0287 in a domain close to the His/TyrO -domain (see Fig. S1). From the EPR data available in the literature, the rhombicity and tetragonality parameters can now be calculated for at least 10 hemoproteins with a His/Cys axial ligation. With the exception of the b-type heme, the cystathionine -synthase (41) and ATP-dependent potassium channels [START_REF] Santolini | A heme-binding domain controls regulation of ATP-dependent potassium channels[END_REF], the c-type heme PsbV2 [START_REF] Suga | Crystal structure at 1.5 Å resolution of the PsbV2 cytochrome from the cyanobacterium Thermosynechococcus elongatus[END_REF], DsrJ [START_REF] Grein | DsrJ, an essential part of the DsrMKJOP transmembrane complex in the purple sulfur bacterium Allochromatium vinosum, is an unusual trihem cytochrome c[END_REF], NaxLS [START_REF] Ukita | Heterodimeric cytochrome c complex with a very low redox potential from an anaerobic ammonium-oxidizing enrichment culture[END_REF], triheme in R. sulfidophilum [START_REF] Alric | Structural and functional characterization of the unusual triheme cytochrome bound to the reaction center of Rhodovulum sulfidophilum[END_REF], SoxAX [START_REF] Kappler | The bacterial SoxAX cytochromes[END_REF], cyt c6 M58C (43), RcoM 2 [START_REF] Smith | Identification of Cys94 as the distal ligand to the Fe(III) heme in the transcriptional regulator RcoM-2 from Burkholderia xenovorans[END_REF] and cyt c T58C/L79G/M80X mutant [START_REF] Zhong | Redox-dependent stability, protonation, and reactivity of cysteine-bound heme proteins[END_REF] are very close to each other and at the interface of three other domains already identified (Fig. S1). In contrast, Tll0287 is slightly outside of this small domain. This at least shows that such a graph can only be indicative mainly when it concerns with hemoproteins having a structure largely different from the others as mentioned above. Construction of a cyt c-550 mutant with His/Cys axial ligation has been reported in T. elongatus [START_REF] Kirilovsky | Cytochrome c550 in the cyanobacterium Thermosynechococcus elongatus: study of redox mutants[END_REF]. However, cyt c-550 purified from this mutated strain had the same EPR signals and redox potential as that of the WT cyt c-550, which strongly suggests that the construction failed in obtaining a His/Cys cyt c-550 mutant.

Sequence and structure similarity -Because the structure of Tll0287 is unique in that it does not resemble those of the typical c-type cytochromes, we searched for possible similar structures with the DALI server [START_REF] Holm | DALI server: conservation mapping in 3D[END_REF]. The results (Table S1) showed that the structure of Tll0287 is similar to that of a sensor domain contained in some kinases or sensor proteins possibly involved in cellular signal transduction, such as the sporulation kinase d domain from Bacillus subtilis (KinD, PDBID: 4JGO) (48) (Fig. 6b) and a methyl-accepting chemotaxis protein (Mcp, PDBID: 3C8C) (Fig 6c), even though they do not contain a heme. These proteins have a PAS-like domain, especially a PhoQ-DcuS-CitA (PDC) domain as extracellular sensors [START_REF] Cheung | Sensor domains of two-component regulatory systems[END_REF], although the rmsd values between the overall structures of Tll0287 and these proteins were approximately 2.2 Å and 3.2 Å, respectively. Fig. 7 shows the results of multiple structural alignments by the DALI server [START_REF] Holm | DALI server: conservation mapping in 3D[END_REF]. It is clear that although the primary sequences of Tll0287 and other proteins are not similar, the secondary structures appear to exhibit significant similarities. On the basis of the comparisons of the secondary (Fig. 7) and three-dimensional structures (Fig. 6), the structure of Tll0287 can be divided into three domains: a PAS-like domain (Ala26-Ala49, Val67-Gln138 and Val169-Ala181), a heme binding site (Ala139-Arg168), and a loop domain (Ser50-Ala66) that separates the PAS-like and heme-binding domains.

PAS domains were discovered as the large regions of per, arnt and sim genes in Drosophila [START_REF] Hoffman | Cloning of a factor required for activity of the Ah (dioxin) receptor[END_REF][START_REF] Nambu | The Drosophila single-minded gene encodes a helix-loop-helix protein that acts as a master regulator of CNS midline development[END_REF] that are related to circadian rhythmicity [START_REF] Dunlap | Genetic and molecular analysis of circadian rhythms[END_REF], and are signaling modules that monitor changes in the redox potential, light, oxygen level, small ligands, or overall energy level of the cell [START_REF] Taylor | PAS domains: internal sensors of oxygen, redox potential, and light[END_REF]. So far, proteins with the PAS domain are found as transcription factors in clock genes [START_REF] Crosthwaite | Neurospora wc-1 and wc-2: transcription, photoresponses, and the origins of circadian rhythmicity[END_REF], ion channels such as HERG [START_REF] Cabral | Crystal structure and functional analysis of the HERG potassium channel N terminus: a eukaryotic PAS domain[END_REF] and sensor proteins such as kinases [START_REF] Taylor | PAS domains: internal sensors of oxygen, redox potential, and light[END_REF]. The PAS domains are composed of a single antiparallel, five-stranded β-sheet with the strand order of 2-(1)-5-4-3 (some proteins have only four-stranded β-sheet without the first strand, such as the sensor domain of histidine kinase DcuS (PDB ID: 3BY8)) [START_REF] Wu | Insight into the sporulation phosphorelay: crystal structure of the sensor domain of Bacillus subtilis histidine kinase, KinD[END_REF] and several α-helices [START_REF] Möglich | Structure and signaling mechanism of Per-ARNT-Sim domains[END_REF]. Although the PAS domains have a conserved three-dimensional fold, these domains share little primary sequence identity [START_REF] Henry | Ligand binding PAS domains in a genomic, cellular, and structural context[END_REF]. The homologous structure of Tll0287 revealed in this study is consistent with these features, and suggests that Tll0287 also contains the PAS-like domain.

A subclass of the PAS domain is the PDC domain, which is a general feature of periplasmic PAS domains [START_REF] Kneuper | Sensing ligands by periplasmic sensing histidine kinases with sensory PAS domains[END_REF] and may bind multiple ligands [START_REF] Glekas | The Bacillus subtilis chemoreceptor McpC senses multiple ligands using two discrete mechanisms[END_REF]. The periplasmic PAS domains typically contain three N-terminal helices, a central helical linker region and a C-terminal α-helix, which are separated by two and three β-strands respectively, forming a 3α-2β-1/2α-3β-1α arrangement. The secondary structure of the Δ24-truncated Tll0287 revealed by our present structural analysis was 2α-1β-1α-2β-2α-1β(Fig. 4a, Fig. 7).

Considering the lack of the N-terminal region and the possible insertion of the α-helix in the heme binding site, it seems that Tll0287 is similar to the structure of the PDC domain, namely, the periplasmic PAS domain, rather than the cytoplasmic PAS domain which has a 1α-2β-4α-3β folding pattern [START_REF] Kneuper | Sensing ligands by periplasmic sensing histidine kinases with sensory PAS domains[END_REF].

Previous studies have suggested that the sensor domain of KinD has an extra ligand between the α-helix and β-strands, and the ligand binding site may accommodate small molecules related to metabolism such as pyruvate or propionate [START_REF] Wu | Insight into the sporulation phosphorelay: crystal structure of the sensor domain of Bacillus subtilis histidine kinase, KinD[END_REF]. In the electron density map of Tll0287, we indeed found an extra density at the similar position between the α-helix and β-strands (Fig. 8), which is in the concave pocket shown in Fig. 4. It seems that the extra density is surrounded by the guanidine group of Arg87, the carboxyl group of Asp105 and Thr179.

In order to identify the possible ligand corresponding to the extra density, we used the Arp/wARP program [START_REF] Langer | Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7[END_REF] for prediction of the extra ligand, which suggested that the extra ligand may be 2-oxoglutaric acid, although its abundance may be low in the thylakoid lumen. On the other hand, MALDI-TOF/MS measurements of the Tll0287 sample after trypsin-digestion showed extra masses of 617 Da, 74 Da and 118 Da (data not shown). The former two correspond to the masses of heme c and chlorine, respectively, whereas the 118 Da mass has no corresponding molecules and may therefore be derived from the extra ligand. This mass is similar to those of homoserine, valeric acid such as norvaline, or betaine which was used as an additive in the purification buffer. Although our current results do not allow the identification of the extra ligand, they suggest that Tll0287 has a PDC-like domain with an extra ligand.

The PAS-like domain found in the Tll0287 structure suggests that this protein may have a function in signal transduction, or in response to some environmental factors. In relation to this, it is interesting to note that the expression level of psbA2 is reported to increase under micro-aerobic conditions (14-16), and the EPR data in Fig. 5 show that under conditions in which O2 was not produced the expression level of Tll0287 increased in the WT*2 strain, at least transiently.

A second approach searching for structural similarities was done by using the Phyre 2 server (61), which revealed a good structural similarity with GSU0935, a methyl-accepting chemotaxis protein from Geobacter sulfurreducens (PDB 3B42) (62) (Fig. 6d). This b-type heme protein also shows a PAS-type fold but exhibits axial ligations (His-water and His-Met) different from that of Tll0287 with a high-spin configuration in the oxidized state. The overexpressed non-membrane part of GSU0935 was crystallized as a dimer [START_REF] Pokkuluri | Structures and solution properties of two novel periplasmic sensor domains with c-type heme from chemotaxis proteins of Geobacter sulfurreducens: Implications for signal transduction[END_REF]. Interestingly, as shown in Fig. 6d, the heme part of one monomer of GSU0935 fits very well with the heme part of Tll0287, and the two α-helices of the opposite monomer fit very well with two of the α-helices, α1 and α2 of Tll0287 albeit that they do not have counterparts in the first monomer. Such similarities may suggest that, in vivo, Tll0287 is present as a dimer with a structure similar to the dimer of GSU0935. These structurally similar proteins have a trans-membrane region and active site in the C-terminus after the PAS domain [START_REF] Holm | DALI server: conservation mapping in 3D[END_REF][START_REF] Pokkuluri | Structures and solution properties of two novel periplasmic sensor domains with c-type heme from chemotaxis proteins of Geobacter sulfurreducens: Implications for signal transduction[END_REF], whereas Tll0287 has no region after PAS domain. Instead, Tll0287 has a heme c inserted. It is possible that the heme c may sense some environmental conditions such as the oxygen level or redox state of the cell, which may therefore induce structural changes of the heme. This structural change may be transmitted and/or enlarged through the PAS-like domain.

Tll0287-homologs, genomic context and phylogeny-Homology sequences of Tll0287 were searched with BLAST for cyanobacteria as well as non-cyanobacterial prokaryotes. Genes with substantial sequence homologies (BLAST search expectation values exceeding e-50), in particular with the C-terminal CXXCH motif, were detected in several cyanobacteria but also outside the cyanobacterial phylum (Fig. S2). While some of these non-cyanobacterial genes coded for larger, two-domain proteins that likely correspond to two-component sensors, others encoded monotopic cytochromes with sizes similar to that of Tll0287 (see phylogenetic tree in Fig. S3A). In order to glean insight into their functions we compared the genomic context of all these Tll0287-and Tll0287-like genes. The following observations emerge: (a) No common features are obvious between cyanobacterial and non-cyanobacterial cases. (b) In cyanobacteria, Tll0287-homologous genes occur almost exclusively in close genomic association with a gene coding for sulphide:quinone-oxidoreductase (SQR). The only exception to this rule is Arthrospira platensis in which the Tll0287-homologue and the SQR genes are distantly positioned in the genome. It is noteworthy that the majority of these SQR-genes are annotated as NDH-2 (type-2 NADH-dehydrogenase) but the phylogenetic analysis (Fig. S3B) clearly shows that they all cluster together with bona fide SQR from Oscillatoria limnetica [START_REF] Arieli | Purification and characterisation of sulfide-quinone reductase, a novel enzyme driving anoxygenic photosynthesis in Oscillatoria limnetica[END_REF] and not with NDH-2. Remarkably, the tree of SQR-sequences retrieved from the cyanobacterial clade falls into two distinct sub-clades, one contains species in which SQR but not Tll0287-homologous genes are present while in the second sub-clade, SQR-genes co-occur with Tll0287-homologs. Two distinct scenarios can be proposed based on this pattern of co-occurrence: (i) The sulphide dehydrogenase in the clade where "SQR" and Tll0287-homologs co-occur is actually a sulphide:cytochrome-oxidoreductase rather than a sulphide:quinone-oxidoreductase and the Tll0287-gene product would in fact correspond to the cytochrome subunit of an H2S-oxidising enzyme, reminiscent of so-called flavocytochrome c-sulphide dehydrogenase (FCSD) observed in a number of prokaryotes [START_REF] Hirano | Structure analysis and comparative characterization of the cytochrome c' and flavocytochrome c from thermophilic purple photosynthetic bacterium Thermochromatium tepidum[END_REF];

(ii) The association of Tll0287-homologous genes to sulphide metabolism is more indirect and potentially even related to some kind of sensory mechanism (it could be noted here that several of the hemo-proteins with His-Cys axial ligation are involved in the sulphide metabolism). We presently consider the first possibility as less likely since (a) no co-purification of Tll0287 and SQR has been observed so far and, (b) the sequence motif involved in forming the quinone-binding pocket in SQR (GQMTEE in T. elegans, deduced from the 3D structure of Aquifex aeolicus SQR, pdb-entry 3HYW) is highly conserved in both sub-clades arguing for true SQR-type functional characteristics of all enzymes in both clades. However, in Synechocystis sp. PCC6803 it has been shown that sll5034 (analogous to tll0287) and the SQR (coded by sll5036) belong to the same operon, and are indeed co-transcribed with a higher level of transcription in the dark, possibly equivalent to micro-aerobic conditions [START_REF] Mitschke | An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp[END_REF]. Such a higher level of expression of Tll0287 in the dark was also observed in the WT*2 strain (Fig. 5), suggesting a role of this protein in sensing the environmental conditions. The actual functional significance of Tll0287-homologous genes in cyanobacteria therefore remains enigmatic, but an involvement in H2S-oxidation as studied in Oscillatoria limnetica [START_REF] Arieli | Purification and characterisation of sulfide-quinone reductase, a novel enzyme driving anoxygenic photosynthesis in Oscillatoria limnetica[END_REF] represents an intriguing lead. In this context, it is noteworthy that, with the sole exception of Pleurocapsa minor, we only found Tll0287 homologs in members of the Oscillatoriaceae.

EXPERIMENTAL PROCEDURES

Thermosynechococcus elongatus strain, cultivation of cells and purification of the Tll0287 protein-The T. elongatus strain used in this work was the WT*2 strain in which a His-tag was attached in the C-terminus of CP43, and both psbA1 and psbA3 were deleted so that only psbA2 is expressed for the D1 subunit [START_REF] Sugiura | Environment of TyrZ in Photosystem II from Thermosynechococcus elongatus in which PsbA2 is the D1 protein[END_REF][START_REF] Boussac | The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus[END_REF]. The WT*3 strain that expresses the psbA3 gene only was made as described in [START_REF] Sugiura | Influence of histidine-198 of the D1 subunit on the properties of the primary electron donor, P 680, of photosystem II in Thermosynechococcus elongatus[END_REF]. The cells were grown in 1-L cultures of DTN medium with a CO2-enriched atmosphere at 45°C under continuous light (Grolux, Sylvania). The cells were collected by centrifugation (Beckman) with a JA10 rotor. and were suspended in a buffer containing 100 mM NaCl, 10 mM Tris pH 8.5 and 0.03% -dodecyl maltoside (-DDM), 1 mM benzamidine, 1 mM -aminocaproic acid and DNase I, and disrupted by a French press. The Tll0287 protein was purified from the soluble proteins upon the breakage of the cells, i.e. after the removal of the membrane fraction by centrifugation based on the procedures described previously [START_REF] Boussac | The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus[END_REF] with some modifications. The supernatant was concentrated and then loaded onto a gel filtration column (Sephadex 75, HiLoad 26/60). The buffer used for this column contained 100 mM NaCl, 10 mM Tris pH 8.5 and 0.03% -DDM, and the flow-rate was 1 ml/min. The fraction of eluate containing Tll0287 (which was judged based on the approximate molecular masses eluted from the column and the reddish color of the heme together with the measurement of the absorption spectra taken every 4 ml) was collected and further purified with a Mono Q column (GE-Healthcare) at pH 8.5 with a NaCl gradient in the presence of 0.03% -DDM and a flow rate of 0.5 ml/min. Finally, the fraction eluted from the Mono Q column was diluted 3-5 folds and purified by a Mono S column (GE-Healthcare) at pH 8.5 with a NaCl gradient in the presence of 0.03% -DDM and a flow rate of 0.5 ml/min. In all cases, the fraction containing Tll0287 was easily followed by the color of the heme. Under the conditions used, Tll0287 was eluted from the Mono Q and Mono S column with a gradient of 100-150 mM NaCl but other proteins which are either more tightly or more loosely bound to the resins eluted differently from the two columns which allowed us to separate them efficiently. It should be noted that: 1) the detergent was added in all of the purification steps to prevent almost irreversible binding of the protein to the resins and to hydrophobic surfaces due to the high hydrophobic character of Tll0287; and 2) hydrophobic resins like phenyl-sepharose cannot be used because the protein binds almost irreversibly to such resins. Tll0287 was then concentrated using a Millipore Amicon Ultra-15 centrifugal filter devices with a cutoff value of 10 kDa. By comparing the relative contents of Tll0287 and soluble Cyt c-550 (i.e. the fraction of Cyt c-550 not bound to PSII easily identifiable by its EPR signal different from the PSII bound fraction) with EPR in whole cells before breakage and the amounts of Tll0287 and cyt c-550 purified in parallel from the same batch of the cells, the protocol described above allows the purification of a very large proportion of Tll0287 present in the cells.

Crystallization and X-ray data collection-Initial crystallization conditions were screened with an automatic dispensing machine (Mosquito, TTP Labtech) with over 800 commercially available screening conditions at a concentration of 2 mg protein/ml. One of these conditions composed of Na2HPO4, K2HPO4, (NH4)2HPO4 and Tris (pH 8.5) produced small crystals with a red color of Tll0287. These small crystals were used as seeds to grow larger crystals under the same condition for 2 weeks at 20°C. The crystals obtained were needle-type, and they were cryo-protected using a solution containing 0.7 M Na2HPO4, 0.7 M K2HPO4, 0.1 M (NH4)2HPO4 and 0.1 M Tris (pH 8.6) in either the presence or absence of 25% (w/v) glycerol in a stepwise fashion, and flash-frozen under liquid nitrogen gas. X-ray diffraction data were collected at beamlines BL41XU and BL44XU of SPring-8 at a wavelength of 0.900 Å for a native data set, and at 1.730 Å to measure the Fe anomalous signal for single wavelength anomalous dispersion (SAD) phasing at 100 K.

Structure determination-Diffraction data were indexed, integrated and scaled by XDS and XSCALE [START_REF] Kabsch | XDS[END_REF]. The initial phases were obtained using SHELX C/D/E [START_REF] Sheldrick | Experimental phasing with SHELXC/D/E: combining chain tracing with density modification[END_REF]. The iron site identified was provided to the program SHARP/autoSHARP [START_REF] Bricogne | Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0[END_REF][START_REF] Vonrhein | Automated structure solution with autoSHARP[END_REF] for density modification and automatic model building, which results in a model having 155 residues. The rest of the model was manually built with the program COOT [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF][START_REF] Emsley | Features and development of Coot[END_REF] and structural refinement was performed with the program Phenix [START_REF] Adams | PHENIX: a comprehensive Python-based system for macromolecular structure solution[END_REF]. Ramachandran plot was calculated with the MolProbity [START_REF] Chen | MolProbity: all-atom structure validation for macromolecular crystallography[END_REF]. The statistics of data collection and structural refinement were summarized in Table 1. For prediction of the extra ligand in the Tll0287 protein, the software Arp/wARP (60) was used. The mFO-DFC map and 2mFO-DFC map without introducing any ligands were calculated using FFT in the CCP4 suite [START_REF] Winn | Overview of the CCP4 suite and current developments[END_REF].

Homology search-Search of sequences homologous to Tll0287 was carried out with BLAST (76) and search of the three-dimensional structures similar to Tll0287 was performed by the DALI Server [START_REF] Holm | DALI server: conservation mapping in 3D[END_REF]. Superposition and root mean square deviation (rmsd) calculation were performed by the LSQ Superpose function of Coot [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF][START_REF] Emsley | Features and development of Coot[END_REF] at the heme c ligand for comparison with that of PsbV2, and the SSM Superpose function [START_REF] Krissinel | Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions[END_REF] for comparison with Per-Arnt-Sim (PAS)-like domain proteins. Protein structure homology was also investigated with the Phyre 2 server [START_REF] Kelley | The Phyre 2 web portal for protein modeling, prediction and analysis[END_REF].

MALDI-TOF mass analysis-In order to determine the molecular size of Tll0287 the sample was analyzed before the crystallization with MALDI-TOF/MS in linear mode (Voyager-DE PRO MALDI-TOF mass spectrometer; Applied Biosystems) as described previously [START_REF] Boussac | The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus[END_REF]. For identification of possible extra mass, the Tll0287 protein was digested with trypsin (Promega), and the polypeptides produced were analyzed with MALDI-TOF/MS in reflector mode after reduction and alkylation [START_REF] Sugiura | Evidence for an unprecedented histidine hydroxyl modification on D2-His336 in Photosystem II of Thermosynechoccocus vulcanus and Thermosynechoccocus elongatus[END_REF].

Prediction of the signal peptide and transmembrane helices-Possible peptide signal cleavage sites were predicted by the SignalP 4.1 Server (79), with the organism group set as Gram-negative bacteria. For prediction of the transmembrane helices, the HMMTOP server was used (80, 81).

Immunoblot analysis-Polyclonal antibodies against Tll0287 were obtained after several injections of purified Tll0287 (see Fig. S5) into rabbits (Service de Pharmacologie et Immunoanalyse, iBiTec-S, CEA Saclay).

Amersham ELC Prime Western Blotting Detection Reagents were used for immunoblot analysis (GE Healthcare). For the experiment in Fig. 1, whole cell samples were prepared by breaking the cell walls with lysozyme-treatment as described previously (82, 83), and the resulted fraction was loaded onto SDS-polyacrylamide gel directly without separation of the soluble and membrane fractions. Thylakoid fractions and purified PsbA2-PSII core complexes were purified from the WT*2 strain as described previously (82, 84). For comparison, the Tll0287 protein purified from the WT*2 strain, whole cells and thylakoid fractions of wild type expressing the psbA1 gene predominately (WT), and those of a WT*3 [START_REF] Boussac | The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus[END_REF] strain expressing the psbA3 gene only were compared.

For determining the cellular location of Tll0287, thylakoid fraction of WT*2 were digested with a protease thermolysin (Sigma-Aldrich) at 5 μg protease/ml for 6 hours at 25 o C, followed by detection with immunoblotting to determine the Tll0287 location. For comparison, the purified Tll0287 and PSII cores purified from the WT*2 strain was digested in the same way, and the PsbO protein was detected by immunoblotting with an anbi-PsbO antibody from Agrisera (Sweden).

Electrochemistry-The redox potential of Tll0287 was determined with the same samples as that used for crystallization. Details of the thin-layer (6 µm) electrochemical cell used for the experiments are given in (85) except that the diamond windows were replaced by CaF2 windows of 1 mm thickness. The working gold grid purchased from Euromip (France) was surface-modified by dipping it for 10 min into a 5 mM pyrimide-3-carboxyaldehyde thiosemicarbazone (PATS-3, Lancaster) solution heated to 80-90°C, followed by careful washing of the gold grid with ultra-pure water. The following redox mediators (each at 40 μM final concentration) were used to accelerate the electrochemical reaction: methyl viologen (Em = -440 mV vs normal hydrogen electrode, NHE), benzyl viologen (Em = -360 mV vs NHE), antraquinone 2-sulfonate (Em = -225 mV vs NHE), 2 hydroxy 1-4 naphtoquinone (Em = -128 mV vs NHE), 2,5 dihydroxy-p-benzoquinone (Em = -62 mV vs NHE), duroquinone (-8 mV vs NHE), phenazine ethosulfate (55 mV vs NHE).

Equilibrium redox titrations were performed by applying potentials to the working gold grid from -100 mV to -500 mV vs NHE with 20 mV intervals using a potentiostat (PRGE, Tacussel Electronique). For each potential step the sample was allowed to equilibrate for 5 minutes. Spectra from 400 to 650 nm were recorded using a modified Cary 14 spectrophotometer equipped with a Xenon light source and a UV enhanced silicon detector. Data was analyzed by calculating the integrated Soret absorption (422 to 457 nm) obtained from the difference of the spectrum recorded at a given potential minus the spectrum of fully oxidized Tll0287 obtained at -100 mV vs NHE. The electrochemical cell was thermostated at 15°C with a water circulation system and the sample compartment was purged with dry air.

Buffers at different pH values contained 200 mM NaCl, 0.03 % -DDM and 50 mM of buffer MES (pH 5.5, 6.0, 6.5), Tricine (pH 7.5), Tris (pH 8.5) or CAPS (pH 10.5). An aliquot of 9 µl of the Tll0287 sample solution adjusted to the buffer solution described above was mixed with 1 µl of mediator-buffer solution and dropped on the working gold grid. The path-length of the closed electrochemical cell was adjusted to about 6 µm by adjusting the Amide I/H2O absorption of the sample to about 0.8 using an FTIR-spectrophotometer (Bruker Tensor 27). protein in the purified Tll0287 sample and thylakoid membrane fraction (A2-Thy) from the WT*2 strain with an anti-Tll0287 antibody, either before (labeled '-') or after digestion of the samples with 5 μg/ml thermolysin for 6 hours (labeled '+TH'). c. Immunoblot analysis of the PsbO protein in the purified PSII and thylakoid membrane fraction from the WT*2 strain with an anti-PsbO antibody, before (-) or after digestion of the samples with thermolysin (+TH) at the same conditions as for Tll0287. [START_REF] Suga | Crystal structure at 1.5 Å resolution of the PsbV2 cytochrome from the cyanobacterium Thermosynechococcus elongatus[END_REF]. The colors for the Tll0287 protein and its heme are green and red, respectively, and those for the PsbV2 protein are as follows: heme, yellow; others, orange. b. Superposition of Tll0287 and the sporulation kinase D sensor domain from Bacillus subtilis (blue, A chain of PDB 4JGO; pink, the ligand of this protein, pyruvate) [START_REF] Wu | Insight into the sporulation phosphorelay: crystal structure of the sensor domain of Bacillus subtilis histidine kinase, KinD[END_REF]. c. Superimposition of Tll0287 and a methyl-accepting chemotaxis protein (Mcp) (purple, A chain of PDB 3C8C; cyan, the ligand of this protein, alanine). d. Superimposition of Tll0287 and the dimer of the non-membrane part of GSU0935 (PDB 3B42) [START_REF] Pokkuluri | Structures and solution properties of two novel periplasmic sensor domains with c-type heme from chemotaxis proteins of Geobacter sulfurreducens: Implications for signal transduction[END_REF].

FIGURE 7. Structural alignment of the Tll0287 protein with 11 homologous proteins

Eleven representative proteins are selected from Table S1 that show structural similarities with Tll0287, excluding their similar molecules. Inserted segment relative to the Tll0287 protein structure are hidden. The upper part shows the alignment of the amino acid sequence, and the lower part shows the secondary structure assignments performed by DSSP [START_REF] Touw | A series of PDB-related databanks for everyday needs[END_REF][START_REF] Kabsch | Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features[END_REF]. H: helix; E: strand; L: coil.

FIGURE 8. A possible extra ligand in Tll0287

a. The residues Arg87, Asp105 and Thr179 surrounding the concave pocket (shown in Fig. 4b) are depicted in stick models. b. Electron density map of a possible ligand corresponding to the ligand binding site in the structure of the sporulation kinase D sensor domain [START_REF] Wu | Insight into the sporulation phosphorelay: crystal structure of the sensor domain of Bacillus subtilis histidine kinase, KinD[END_REF]. The blue and red mesh represents mFO-DFC omit map (positive and negative, respectively) contoured at 3.5 σ distributions, and the grey mesh represents 2mFO-DFC map contoured at 1.0 σ distributions.

Biochemistry 52 ,FIGURE 1 .

 521 FIGURE LEGENDS

FIGURE 2 .

 2 FIGURE 2. Digestion of the Tll0287 protein with a protease thermolysin a. CBB-stained gel of the samples used for immunoblot analysis shown in Panels b and c. The samples used are the purified Tll0287 protein, thylakoid membrane fraction from the WT*2 strain (A2-Thy, expressing psbA2 only), and purified PSII from WT*2 (A2-PSII). The amount of the samples loaded on each lane was the same as that in Fig. 1b. b. Immunoblot analysis of the Tll0287

FIGURE 3 .

 3 FIGURE 3. Electrochemistry on Tll0287 Panel a and Panel b show the absorption spectra recorded at the indicated potential during the oxidative and reductive processes, respectively. Panel c shows a Nernst plot for an oxidation (blue points) and a reduction (red points) performed at pH 8.5 and T = 15°C. The continuous black line is a global fit with n fixed to 1 and E1/2' = -255 mV vs NHE. Panel d shows the pH dependence of the E1/2' determined as above at different pH values. The continuous line is the result of a fitting procedure with following equation: E1/2' = E0' -57 log((Kox + [H + ]) / (Kred + [H + ])) where E0' is the midpoint redox potential of the fully protonated form and Kred and Kox are the ionization constants of the protonatable group when the iron is reduced and oxidized, respectively. The pKred was found to be 7.2.

FIGURE 4 .

 4 FIGURE 4. Crystal structure of the Tll0287 protein a. Overall structure of the Tll0287 protein. The PAS-like domain composed by 3 α-helices and 4 β-strands was depicted in green, the heme and its binding part were depicted in red and yellow respectively, and the loop region was depicted in orange. b. Surface charge distribution of the Tll0287 protein. Blue and red colors represent negative and positive areas, respectively. The area surrounding the heme is largely positive. The black arrow indicates a concave pocket. c. Ligand environment of the heme c in Tll0287. The 5 th and 6 th heme iron axial ligands, Cys68 and His145 and the residues around them are depicted in stick models. d. Hydrophobicity characters of the surface of Tll0287. The structure of Tll0287 was depicted in a ball-model in the same direction (left-side) as that in Fig.3aand 3b, and in a direction rotated 180 o (right-side). Blue represents positively charged residues (R, K, H), red represents negatively charged residues (D, E), white are hydrophilic residues, and orange are hydrophobic residues.

FIGURE 5 .

 5 FIGURE 5. Expression level of Tll0287 under dark conditions.Spectrum a, WT whole cells cultivated for 2 days under normal light conditions. Spectrum b, WT whole cells after a further dark period of 3 to 4 hours. Spectrum c, WT*2 whole cells cultivated for 2 days under normal light conditions. Spectrum d, WT*2 whole cells after a further dark period of 3 to 4 hours. For sake of comparison, the spectra were approximately scaled by using both the Fe 3+ SOD signals (labeled by '*' between 1300 and 2000 gausses) and the gz signal of cyt-c550 (labeled by '*' between 2200 and 2300 gausses). The signal from Tll0287 is located around 2800 gauss. Instrument settings: modulation amplitude, 25 G; microwave power, 5 mW; Temperature, 20 K; microwave frequency, 9.4 GHz; modulation frequency, 100 kHz.

FIGURE 6 .

 6 FIGURE 6. Structural comparison of the Tll0287 protein with other proteins a. Superposition of Tll0287 and PsbV2 from T. elongatus (PDB ID: 4LJI)[START_REF] Suga | Crystal structure at 1.5 Å resolution of the PsbV2 cytochrome from the cyanobacterium Thermosynechococcus elongatus[END_REF]. The colors for the Tll0287 protein and its heme are green and red, respectively, and those for the PsbV2 protein are as follows: heme, yellow; others, orange. b. Superposition of Tll0287 and the sporulation kinase D sensor domain from Bacillus subtilis (blue, A chain of PDB 4JGO; pink, the ligand of this protein, pyruvate)[START_REF] Wu | Insight into the sporulation phosphorelay: crystal structure of the sensor domain of Bacillus subtilis histidine kinase, KinD[END_REF]. c. Superimposition of Tll0287 and a methyl-accepting chemotaxis protein (Mcp) (purple, A chain of PDB 3C8C; cyan, the ligand of this protein, alanine). d. Superimposition of Tll0287 and the dimer of the non-membrane part of GSU0935 (PDB 3B42)[START_REF] Pokkuluri | Structures and solution properties of two novel periplasmic sensor domains with c-type heme from chemotaxis proteins of Geobacter sulfurreducens: Implications for signal transduction[END_REF].

Favoreda

  Statistics for the highest resolution shell are shown in parentheses. b where is the intensity of the reflection and is the mean intensity of a group of equivalent reflection. c R-factor independent of the redundancy. d Percentage of correlation between intensities from two random half-datasets. e Calculated with the MolProbity (35).

Table 2 .

 2 Experimentally determined and calculated molecular masses of Tll0287 by MALDI-TOF/MS.

Figure 1 Figure 2 22 Figure 3 Figure 4 Figure 5 6 Figure 8

 122234568 Figure 1

Table 1 .

 1 Data collection and structural refinement statistics.

	Data collection	Native	Fe-peak
	Wavelength	0.900	1.730
	Space group	P42212	P42212
	Unit cell	a = b = 101.2 Å, c = 33.1	a = b = 100.9 Å, c = 33.1 Å,
		Å,
		 = 90.00°	 = 90.00°
	Resolution (Å)	20-2.00 (2.13-2.00) a	20-3.20 (3.39-3.20)
	Unique reflections	12,169 (2,047)	5,410 (856)
	Redundancy	12.7 (12.9)	25.0 (13.8)
	Completeness	100.0 (100.0)	99.6 (97.5)
	Rmerge b	10.2 (95.5)	8.8 (21.9)
	I/(I)	19.1 (2.8)	36.5 (10.9)
	R-means c (%)	10.6 (99.4)	9.0 (22.7)
	CC(1/2) d	99.9 (96.8)	99.9 (99.0)
	Structural refinement
	Rfactor	0.1765
	Rfree	0.2336
	Number of atoms	1,422
	Proteins	1,294
	Ligands	44
	Water	84
	Number of residues	161
	R.m.s.d. (bond)	0.007
	R.m.s.d. (angle)	1.030
	Average B-factor	36.1
	Ramachandran plot e

Acknowledgements:

The authors thank the staff members at beamlines BL41XU and BL44XU of SPring-8 for their help in data collection, the staff members of CCP4 structure solution workshop for prediction of the extra ligands, and Shinichiro Yonekura for his help in performing the immuno-blot analysis. This work was supported by a Program for Leading Graduate Schools "Next generation picobiology pioneered by photon sciences", a program for promoting the enhancement of research universities at Okayama University, JSPS KAKENHI Grant Nos. JP24000018 (J.-R.S.), JP26840023, JP15H01642, JP16H06162 and JP16H06296 (M. Suga) from MEXT, Japan, an Inamori foundation (M. Suga), a JST-PRESTO program (4018 for M. Sugiura) and the Bioenergy program of CEA-DSV (TLL and AB). Winfried Leibl, Anja Krieger, Frauke Baymann, Ghada Ajlani, Pierre Sétif, Alain Desbois and Fabrice Rappaport are greatly acknowledged for their investment in some periods of this work and for helpful suggestions.

Data deposition: The coordinates for the structure of Tll0287 have been deposited in the Protein Data Bank with the accession number 5B82. The abbreviations used are: CAPS, N-cyclohexyl-3-aminopropanesulfonic acid; β-DDM, n-dodecyl-β-maltoside; LDAO, N, N-dimethyldodecylamine N-oxide; MALDI-TOF/MS, matrix-assisted laser desorption/ionization-time of flight mass spectroscopy; MES, 2-(N-morpholino) ethanesulfonic acid; MW, molecular weight; NHE, normal hydrogen electrode; QA, primary quinone electron acceptor; QB, secondary quinone electron acceptor; PDC and PAS domains, PhoQ-DcuS-CitA and Per-Arnt-Sim domains; PSII, Photosystem II; Tris, Tris(hydroxymethyl)aminomethane; TyrZ, redox-active tyrosine on the D1 subunit (D1-Y161) in photosystem II. FCSD, flavocytochrome c-sulphide dehydrogenase; SQR, sulphide:quinone-oxidoreductase.

Conflict of interest:

The authors declare that they have no conflicts of interest with the contents of this article.

Author contributions: JRS and AB conceived and coordinated the study. TM crystallized the protein; TM and MS collected the X-ray diffraction data and analyzed the structure. AN and TM performed western blot analysis, TK prepared the samples from the WT*2 strain, and MS performed MS analysis. TLL and AB purified the protein; RH performed the redox analysis. WN performed the phylogenetic analysis, and TM performed structural homology analysis. TM, MS, WN, AB and JRS wrote the manuscript, and all the authors reviewed the results and approved the final version of the manuscript.