Psb30 contributes to structurally stabilise the Photosystem II complex in the thermophilic cyanobacterium Thermosynechococcus elongatus

Biochimica et Biophysica Acta 1797 (2010) 1546–1554

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a b i o

Psb30 contributes to structurally stabilise the Photosystem II complex in the thermophilic cyanobacterium Thermosynechococcus elongatus Miwa Sugiura a,b,⁎, Sayo Harada b, Takashi Manabe b, Hidenori Hayashi a,b, Yasuhiro Kashino c, Alain Boussac d a

Cell-Free Science and Technology Research Center, and Venture Business Laboratory, Ehime University, Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan Department of Chemistry, Ehime University, Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan Department of Life Science, University of Hyogo, 3-2-1 Kohto, Kamigohri, Ako-gun, Hyogo 678-1297, Japan d iBiTec-S, CNRS URA 2096, CEA Saclay, 91191 Gif sur Yvette, France b c

a r t i c l e

i n f o

Article history: Received 17 February 2010 Received in revised form 23 March 2010 Accepted 24 March 2010 Available online 30 March 2010 Keywords: Photosystem II Psb30 Cytochrome b559 Small subunit Trans-membrane protein Side-path electron transfer Thermosynechococcus elongatus

a b s t r a c t A deletion mutant that lacks the Psb30 protein, one of the small subunits of Photosystem II, was constructed in a Thermosynechococcus elongatus strain in which the D1 protein is expressed from the psbA3 gene (WT*). The ΔPsb30 mutant appears more susceptible to photodamage, has a cytochrome b559 that is converted into the low potential form, and probably also lacks the PsbY subunit. In the presence of an inhibitor of protein synthesis, the ΔPsb30 lost more rapidly the water oxidation function than the WT* under the high light conditions. These results suggest that Psb30 contributes to structurally and functionally stabilise the Photosystem II complex in preventing the conversion of cytochrome b559 into the low potential form. Structural reasons for such effects are discussed. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Photosystem II (PSII) catalyses light-driven water oxidation and plastoquinone reduction in cyanobacteria, algae, and plants. Its minimum structural unit capable of oxygen evolution is called PSII core complex, which consists of 20 protein subunits containing 17 membrane-spanning proteins and three extrinsic proteins. PSII also

Abbreviations: Car, β-carotene; Chl, chlorophyll; ChlD1, ChlD2, monomeric Chls that are bound to D1 and D2, respectively; ChlzD1, ChlzD2, side-path redox-active Chls that are bound to D1 and D2, respectively; CP43, Chl-binding 43 kDa protein; CP47, Chlbinding 47 kDa protein; Cyt, cytochrome; DCBQ, 2,6-dichloro-p-benzoquinone; EPR, electron paramagnetic resonance; LP and HP form, low-potential and high-potential form; MALDI-TOF, matrix-assisted laser desorption/ionisation time of flight; MES, 2(N-morpholino) ethanesulfonic acid; MGDG, monogalactosyl-diacylglycerol; MMOBS, observed molecular mass; MMCALC, calculated molecular mass; OEC, oxygen-evolving complex; Pheo, pheophytin; P680, primary electron donor; PD1, P680 Chl on D1; PD2, P680 Chl on D2; PSII, Photosystem II; QA, primary quinone acceptor; QB, secondary quinone acceptor; Sp, spectinomycin; SQDG, sulfoquinovosyl-diacylglycerol; Sm, streptomycin; WT*, Thermosynechococcus elongatus strain with a His-tag on the C terminus at CP43 and in which the psbA1 and psbA2 genes are deleted; 43-H, T. elongatus strain with a Histag on the C terminus of CP43 ⁎ Corresponding author. Cell-Free Science and Technology Research Center, Ehime University, Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan. Tel.: +81 89 927 9616; fax: +81 89 927 9616. E-mail address: [email protected] (M. Sugiura). 0005-2728/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2010.03.020

involves 35 chlorophyll molecules, 2 pheophytin molecules, 2 hemes, 1 non-heme iron, 4 Mn ions, 1 Ca ion at least, 12 carotenoid molecules, 25 lipids, 2 quinones, and 1 Cl ion [1–5]. Refined three-dimensional Xray structures from 3.5 to 2.9 Å resolution have been obtained using PSII isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus [1–3]. PSII is known to possess two electron transport systems: (1) the photosynthetic electron transport system, which is for conversion of photon energy into chemical energy accompanying water oxidation, and (2) the photo-induced side-path electron transport system, which is rationalised as a photoprotective cycle aimed at removing long-lived P+ 680 under high light illumination and thus limiting oxidative damage. In the photosynthetic electron transport, absorption of a photon by a chlorophyll molecule is followed by the transfer of the exciton to the photochemical trap and the consecutive formation of a radical pair in which the pheophytin molecule, PheoD1, is reduced and the chlorophyll molecule, ChlD1, is oxidised. The positive charge is then stabilised on P680, a weakly coupled chlorophyll dimer [see reviews 6–8]. The PheoD1 anion transfers the electron to a primary plastoquinone, QA. P+ 680, where the cation is shared through a redox equilibrium over PD1 and PD2, is reduced by a tyrosine residue, TyrZ. TyrZ is in turn reduced by the Mn4Ca-cluster and Q− A transfers the electron to a secondary plastoquinone QB. On the other hand, although details of electron pathway of side-path are still on debate, the side-pathway is known to

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involve at least cytochrome b559 (Cyt b559), ChlzD1 and/or ChlzD2, carotenoids and P680 [see reviews 9–11]. All cofactors in charge of the photosynthetic electron transport involving a Mn4Ca cluster, P680 chlorophylls (PD1, PD2, ChlD1, and ChlD2), PheoD1, and the plastoquinones QA and QB ligate to amino acid residues of the D1, D2, and CP43 proteins (mainly to D1). The redox-active Tyr residues, TyrZ and TyrD, correspond to amino acids of D1 and D2, respectively. The cofactors involved in the side-path electron transfer ligate to D1, D2, and Cyt b559. Hence, in both electron transport systems, only D1, D2, CP43, and Cyt b559 out of 20 protein subunits of PSII complex are essential for the fundamental functions. Chlorophyll-binding proteins CP47 and CP43 are functionally important subunits as antenna for absorbing photons. Some of other sixteen protein subunits are known to play a role of the super-complex structure. From X-ray structure of PSII, four small subunits (PsbJ, PsbK, PsbZ, and X1) adjacent to CP43 may facilitate carotenoid binding. PsbM, PsbL, and PsbT, which is located in a boundary of two PSII complex units, may be involved in dimer formation [1–3]. In the psbT deletion mutant of T. elongatus, amount of dimeric PSII was much less than that in wild type [12,13]. However, for knockout mutants lacking PsbM, and PsbL in Synechocystis PCC 6803, it is not clear if they are or not concerned with dimer formation [14,15]. Although the symmetrically related PsbI and PsbX proteins are considered to stabilise the peripheral chlorophylls of the D1 and D2 proteins (ChlzD1 and ChlzD2) [1–3], at least the structure of the PSII complex of the PsbX-knockout mutant of T. elongatus was not greatly affected [16]. Kashino et al. found a membrane-spanning 5 kDa protein, Ycf12 (designated as Psb30 since then), from isolated PSII complex of T. elongatus 43-H strain [17] by analyses of the N-terminal amino acid sequence of the 5 kDa protein band on SDS-polyacrylamide gel [18]. Recently Takasaka et al. [19] assigned the position of Psb30 in PSII to the position of putative PsbN subunit in the structure model of Ref. [1] and X1 subunit (provisional name) in the models of Refs. [2,3]. Psb30 is located in the periphery of PSII dimer and nearby PsbJ, PsbK, and PsbZ transmembrane helices. Particularly, the structure with a 2.9-Å resolution suggested that PsbK and Psb30 could be bridged by a water molecule due to the 5.8 Å spacing [3]. A carotenoid molecule is also nested between these four transmembrane helices [3]. Although the association of some of these subunits has been demonstrated by immunological analyses [20,21] and proteomics analyses of knockout mutants [22–24], the function of these small subunits is still unclear with the exception of the ΔPsbJ knockout mutant (in Synechocystis PCC 6803) in which the efficiency of the electron flow is affected [23]. In Synechocystis PCC 6803, although the cells of Psb30 knockout mutant exhibited ≈ 30% lower oxygen-evolving activity than those in wild type, any significant phenotype in the mutant could not be observed [25]. In this work, to elucidate the function of Psb30 in PSII complex of a thermophilic cyanobacterium T. elongatus, which is the organism for crystal structures, we constructed Psb30-knockout mutant and analysed protein composition of the isolated PSII complexes, the effects of high light on the water oxidation function, and the EPR properties of Cyt b559. 2. Materials and methods 2.1. Construction of ΔPsb30 strain DNA fragments of ≈ 1000 bp of the 5′-flanking region of psb30 (ycf12; gene number tsr1242) were cloned from T. elongatus wild-type genomic DNA by PCR amplification and subcloned into a plasmid vector pBluescript II SK + between KpnI and HincII sites. Then, a separately amplified ≈ 1100 bp DNA fragments of the 3′-flanking region of psb30 were ligated to the downstream of the 5′-flanking region in the subcloned plasmid vector at HincII and EcoRI. A spectinomycin/streptomycin resistance gene cassette (≈ 2100 bp)

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[52] was inserted between the 5′-flanking region and the 3′-flanking region of psb30 at HincII (Fig. 1A). The constructed plasmid DNA was introduced into T. elongatus WT* that has His6-tag on the C-terminus of CP43 and knocked out psbA1 and psbA2 [26] by electroporation (BioRad gene pulser) as described in Ref. [17]. The transformants were selected as single colonies on DTN agar plate containing 25 µg of spectinomycin ml−1, 10 µg of streptomycin ml−1, and 40 µg of kanamycin ml−1 as previously described in Refs. [17,26–29]. Segregation of all genome copies was confirmed by difference in length of amplified DNA by PCR using the Fw primer (5′-TCTCCACGC CCATCACCGACCCATCTTCGG-3′) and Rv primer (5′-CCCCTCTGACCACCCTCATCCTCTCCATTC-3′) as shown in Fig. 1A and B. 2.2. Purification of thylakoids and PSII core complexes The transformed cells were grown in 1 l cultures of DTN in 3-l Erlenmeyer flasks in a rotary shaker with a CO2-enriched atmosphere at 45 °C under continuous light (≈ 60 µmol of photons m−2 s−1). Thylakoids and PSII core complexes were prepared as described earlier in [17,26–29]. The PSII core complexes bound to the Ni2+-resin were eluted with 200 mM imidazole. PSII was concentrated by using Amicon Ultra-15 concentrator devices (Millipore) with a 100-kDa cutoff. Routinely, the total amount of Chl before the breaking of the cells was ≈ 150 mg, and the yield after PSII purification in terms of Chl amounts was ≈ 3–5%. PSII was stored in liquid nitrogen at a concentration of about 2 mg ml−1 in a medium containing 10% glycerol, 1 M betaine, 15 mM CaCl2, 15 mM MgCl2, and 40 mM MES (pH 6.5) until to be used. 2.3. Oxygen evolution measurements Oxygen evolution of isolated PSII complexes and whole cells under continuous illumination was measured at 25 °C by polarography using a Clark-type oxygen electrode (Hansatech) with saturating white light at a Chl concentration of 5 µg of Chl ml− 1 in 40 mM MES buffer (pH6.5) containing 15 mM CaCl2, 15 mM MgCl2, and 100 mM NaCl in both isolated PSII complexes and whole cells. A total of 0.5 mM dichloro-p-benzoquinone (2,6-DCBQ, dissolved in dimethyl sulfoxide) was added as an electron acceptor. 2.4. Oxygen evolution measurements with irradiation of high light The cells were pre-cultivated until OD730 = 0.5 at 45 °C under continuous light (≈ 60 µmol of photons m−2 s−1). Just before the measurements, the cells were diluted to OD730 = 0.3 in the volume of 350 ml in 500-ml Erlenmeyer flasks. In the case of addition of lincomycin, the concentration of lincomycin was adjusted at 200 µg of lincomycin ml− 1 culture. Before oxygen evolution measurements, the cells were illuminated in either the presence or the absence of lincomycin. Illumination was done with fluorescent light tubes (with a stronger illumination between 600 and 700 nm and between 400 and 500, Toshiba Plantlux, Japan) at ≈ 800 µmol of photons m− 2 s− 1 for 4 h. Then the light intensity was decreased to ≈ 60 µmol of photons m− 2 s− 1 for 2 h. For measurements of oxygen-evolving activity, about 30 ml of cells was taken out from the cultures and then harvested by centrifugation. After washing the cells in 40 mM MES buffer (pH6.5) containing 15 mM CaCl2, and 15 mM MgCl2, they were suspended in the same buffer at a concentration of 5 µg of Chl ml− 1. Then oxygen-evolving activity was measured immediately. 2.5. MALDI-TOF mass spectrometric measurements The isolated PSII complexes (30 µg Chl ml− 1) were mixed with the same volume of a saturated matrix (sinapic acid, Fluka) solution, which consists of 60% acetonitrile and 0.1% trifluoroacetic acid. The

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Fig. 1. (A) Map around the psb30 gene (ycf12; gene number tsr1242) in WT* and deletion of psb30 gene from the WT* genome to produce ΔPsb30 strain. A 144-bp fragment including the open reading frame of psb30 was replaced by a 2109-bp fragment containing spectinomycin (Sp)/streptomycin (Sm)-resistant cassette at HincII sites. In both WT* and ΔPsb30 genomes, psbA1 and psbA2 have been deleted [26] and psbC has been extended with an additional DNA fragment encoding 6 consecutive His [17]. Fw and Rv show positions of PCR primers to confirm the length of the psb30 and/or Sp/Sm-resistant cassette. (B) Agarose gel (1%) electrophoresis of amplified products by PCR using Fw and Rv primers. Lanes 1 and 4, 1 kb ladder markers (Toyobo, Japan); lane 2, WT* strain; lane 3, ΔPsb30 strain.

mixed samples were loaded onto the stainless steel target plate and dried. MALDI-TOF mass analysis was performed using a Voyager-DE PRO MALDI-TOF mass spectrometer (Applied Biosystems). The instrument was operated in linear mode at 25 kV accelerating voltage and 400 ns ion extraction delay with the nitrogen laser working at 337 nm and 3 Hz as described in Ref. [30]. One hundred laser flashes were accumulated per spectrum. Internal calibration was performed on the samples premixed with adrenocorticotropic hormone fragment (from human, m/z = 2466.72, Sigma), insulin (from bovine, m/ z = 5734.5910, Sigma), and apomyoglobin (from bovine heart, m/z = 16952.5127, Sigma). 2.6. EPR spectroscopy Cw-EPR spectra were recorded using a standard ER 4102 (Bruker) X-band resonator with a Bruker Elexsys 500 X-band spectrometer equipped with an Oxford Instruments cryostat (ESR 900). Thylakoids samples at ≈ 4–6 mg Chl ml− 1 were loaded in the dark into quartz EPR tubes and further dark adapted for 1 h at room temperature. Then the samples were frozen in the dark to 198 K, degassed at 198 K, and then transferred to 77 K. Illumination of the samples was done at 77 K in liquid nitrogen with a 1000-W Tungsten lamp from which infrared was filtered with water and infrared filters.

similar to that of WT*. This result shows that the removal of Psb30 has no effect on the growth of the cells in ordinary culture conditions.

3.2. Composition of PSII core complexes of ΔPsb30 mutant and water oxidation function To investigate the small peptide content in PSII complex of ΔPsb30, we analysed isolated PSII complex by MALDI-TOF mass spectroscopy. Fig. 3 shows the spectra of ΔPsb30 and WT* in m/z range from 3600 to 6000 (Fig. 3A) and in the range from 5500 to 10,000 (Fig. 3B), and Table 1 is a summary of the assignment of the small polypeptides of PSII complexes form ΔPsb30 and WT*. In isolated ΔPsb30–PSII complex, the Psb30 subunit band (m/z 5067.1) completely disappeared as expected. The PSII complex of ΔPsb30 is made at least of the small subunits PsbT (m/z 3904.0), PsbM and PsbJ (m/z 4011.1), PsbK (m/z 4101.1), PsbX (m/z 4189.2), PsbL (m/z 4298.3), PsbI (m/z 4434.2), PsbF (ß-subunit of Cyt b559 with a m/z 4976.0), PsbZ (m/z

3. Results 3.1. Construction of ΔPsb30 mutant and cell growth To delete Psb30 (Ycf12) from PSII core complex of T. elongatus, an 144-bp DNA fragment including the ORF of psb30 (tsr1242) was replaced by a spectinomycin/streptomycin-resistant cassette gene (2109 bp) in the genome of T. elongatus strain WT*, which expresses only psbA3 after the deletion of psbA1 and psbA2. Complete segregation of the psb30 deletion mutant (strain ΔPsb30) was confirmed by PCR amplification as shown in Fig. 1B. In WT*, a 580-bp DNA fragment including the 144 bp of the psb30 open reading frame was amplified by Fw and Rv primers. In contrast, a 2450-bp fragment including a spectinomycin/streptomycin-resistant cassette was amplified without the 580 bp band in the ΔPsb30 genome. ΔPsb30 cells photosynthetically grew as well as WT* cells. Fig. 2 shows the growth curve of WT* and ΔPsb30 cells under two light intensities; 10 µmol of photons m− 2 s− 1 and 60 µmol of photons m− 2 s− 1. In both conditions, cell growth of the ΔPsb30 strain was quite

Fig. 2. Cell growth of WT* and ΔPsb30 strains under the light condition of 60 µmol of photons m−2 s−1 (circles) and 10 µmol of photons m−2 s−1 (squares). Closed circle and closed square, WT* cells; open circle and open square, ΔPsb30 cells.

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Fig. 3. MALDI-TOF mass spectra of isolated PSII complexes from WT* (solid line) and ΔPsb30 (dotted line). Numerals under the names of PSII subunits indicate m/z values. The peak of m/z at 5734.6 corresponds to bovine insulin (Sigma) as a calibration marker. Peaks in the m/z range from 3600 to 6000 (A) and from 5500 to 10,000 (B).

6792.3), PsbH (m/z 7220.0), and PsbE (α-subunit of Cyt b559 with a m/ z 9441.2). However, a peak of m/z at 4613.6 assigned to the PsbY subunit was also almost totally missing in ΔPsb30–PSII. An unknown weak peak of m/z at 3845.2 was detectable in ΔPsb30–PSII but not in WT*-PSII. This peak could originate either from a partially degraded PsbY peptide or from some contaminant. All other subunit proteins larger than 10 kDa such as PsbU (extrinsic 12 kDa protein), PsbV1 (Cyt c550), PsbA3 (D1), PsbD (D2), PsbO (extrinsic 33 kDa protein), PsbC (CP43), and PsbB (CP47) were detected in ΔPsb30–PSII by means of an SDS-Urea polyacrylamide gel electrophoresis as well as in WT*-PSII (data not shown). To examine the effect of elimination of Psb30 (and as a consequence almost all PsbY) from PSII complex on water oxidation

function, we measured oxygen-evolving activity of isolated PSII complexes from WT* and ΔPsb30 strains under saturating continuous illumination. PSII complex from ΔPsb30 exhibited an oxygen-evolving activity at 3500–4000 µmol O2 Chl−1 h−1 as high as those from WT*. These results indicate that the Psb30 and the PsbY subunits have no influence on the water oxidation mechanism itself. 3.3. Effect of high light irradiation on the ΔPsb30 cells In order to examine effect of high light irradiation on PSII function, the ΔPsb30 and WT* cells were exposed to the high light (≈ 800 µmol of photons m− 2 s− 1) and oxygen-evolving activity was measured every 1 h. As shown in Fig. 4A, high light illumination decreased the

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Table 1 Assignment, observed and calculated molecular masses of small polypeptides of Photosystem II complexes from MALDI-TOF MS of T. elongatus WT* and ΔPsb30. Identification MMOBS MMCALC ΔMM (Da)a (Da) (Da)b

Number of Putative WT* ΔPsb30 Amino PostAcidsc translational Modifications

? PsbT

3844.2 3903.0 3874.7

+28.3 32 (32)

PsbM PsbJ

4010.1 3980.7 4010.1 3973.7

+29.4 36 (36) +36.4 40 (41)

PsbK PsbX PsbL PsbI PsbY

4100.1 4188.2 4297.3 4433.2 4612.6

+0.2 +0.2 +0.3 +28.0 +28.0

PsbF

4975.0 4930.6

+44.4 44 (45)

Psb30 PsbZ PsbH PsbE

5066.1 6791.3 7219.0 9440.2

+28.9 +27.1 +1.1 -1.3

4099.9 4188.0 4297.0 4405.2 4584.6

5037.2 6764.2 7217.9 9441.5

37 (38) 41 (42) 37 (37) 38 (38) 41 (43)

46 (46) 62 (62) 65 (66) 83 (84)

Formylation, -Met1 Formylation Acetylation, -Met1 -Met1 -Met1 Formylation Formylation, -Met1, -Gly2 Acetylation, -Met1 Formylation Formylation -Met1 -Met1

No Yes

Yes Yes

Yes Yes

Yes Yes

Yes Yes Yes Yes Yes

Yes Yes Yes Yes No

Yes

Yes

Yes Yes Yes Yes

No Yes Yes Yes

a

MMOBS = (peak m/z) – 1, assuming the protein molecules are singly charged. MM = MMOBS – MMCALC. Number of amino acids is from mature protein, and number in ( ) indicates number of amino acids deduced from the open reading sequence. b c

O2 activity by ≈ 30% in WT* cells and by ≈ 40% in ΔPsb30 cells. After high light illumination for 4 h, the light intensity was decreased to an intensity similar to that used in normal culture conditions (60 µmol of photons m− 2 s− 1). The activity fully recovered the activity measured before high light illumination in 1 h in both WT* cells and ΔPsb30 cells. Fig. 4B shows an experiment similar to that in Fig. 4A but performed in the presence of lincomycin, an inhibitor of protein synthesis. In these conditions, the activity of ΔPsb30 cells decreased faster than in WT* cells (the half time of the degradation was ≈ 1 h and ≈ 0.6 h in WT* and ΔPsb30, respectively). As expected, the lost activity in both strains could not be recovered even though the light intensity was reduced to the growth light. Since the presence of lincomycin prevents the synthesis of new proteins by inhibiting the translation, the lost activity originates from the degradation of PSII complexes. From the results, PSII complex missing Psb30 (and also PsbY) are functionally and structurally more susceptible to photodamages than WT*. 3.4. Effect of lacking Psb30 on the EPR properties of cytochrome b559 Cyt b559 is known to have at least two states that differ by their redox potential, the high potential form, HP, and the low potential form, LP. In intact PSII, the HP form is predominant, whereas the LP form is found when the PSII structure is affected [10,31,32]. EPR spectroscopy is well suitable to probe the HP or LP form of Cyt b559, since in the oxidised state, these two forms exhibit different EPR signals [10,31]. In addition to Cyt b559, T. elongatus has a second cytochrome (Cyt c550), which is oxidised and therefore detected by EPR in the conditions used in the present study [31,32]. The use of detergents for PSII purification from mutants is susceptible to induce additional damages, which are not necessarily linked to the role of the deleted protein(s). To avoid such side effects, the EPR study of Cyt b559 was performed on thylakoids (Fig. 5). The counterpart of this choice is that only the gz feature is easily detectable in thylakoids, but fortunately, this resonance is very sensitive to the state of Cyt b559. Spectrum a in Fig. 5 shows the gz region in dark-adapted WT* thylakoids. The signal originates from Cyt c550, which is expected to be

Fig. 4. Oxygen-evolving activity of WT* (circle) and ΔPsb30 (square) cells in the absence (A) and presence (B) of lincomycin. The activities were measured using the cells incubated under the high light conditions (HL; 800 µmol of photons m−2 s−1) and growth light conditions (GL; 60 µmol of photons m−2 s−1).

fully oxidised and from Cyt b559 in the centres in which it is oxidised. Spectrum b was recorded after an illumination at 77 K (known to result in the oxidation of Cyt b559 to the detriment of the Mn4Ca cluster) and spectrum c is the light-minus-dark spectrum. The g value of spectrum c is 3.08 and is characteristic of the unrelaxed form of the HP form of Cyt b559 [10,31,32]. Spectrum d was recorded in darkadapted ΔPsb30 thylakoids. The signal is much larger than in spectrum a, which suggests that the Cyt b559 is already oxidised in the dark. Indeed, a further 77 K illumination induced almost no increase in the signal, spectrum e. By subtracting spectrum a (i.e., mainly the Cyt c550 signal) from spectrum d, the resulting signal

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Fig. 5. EPR signals of Cyt b559 in WT* and ΔPsb30 thylakoids. EPR spectra were recorded in WT* thylakoids (spectra a and b) and in ΔPsb30 thylakoids (spectra d and e). Spectra were first recorded in dark adapted samples (spectra a and d) and after illumination at 77 K (spectra b and e). Spectra c and f are the light minus dark spectra in WT* and ΔPsb30 thylakoids, respectively. Spectrum g is spectrum d minus spectrum a. Instrument settings: modulation amplitude, 25 gauss; microwave power, 5 mW; microwave frequency, 9.4 GHz; modulation frequency, 100 kHz. Temperature, 15 K. Amplitude of the spectra was normalised to same chlorophyll concentration.

(spectrum g) approximately corresponds to the Cyt b559 oxidised in the dark-adapted ΔPsb30 thylakoids. This resonance peaks at 2.96 and is characteristic of the relaxed LP form of Cyt b559 in T. elongatus. Clearly, the removal of Psb30 (and PsbY) converts the high potential form of Cyt b559 into the LP form in isolated thylakoids. 4. Discussion Kashino et al. found a 5-kDa polypeptide in isolated PSII complex from T. elongatus 43-H strain [18]. This protein was identified to Ycf12 from the N-terminal sequence and then designated as Psb30. These authors suggested that this polypeptide corresponded to a membrane-spanning helix, which was provisionally assigned as PsbN in the X-ray structure by Ferreira et al. [1]. In the other X-ray crystal structures, Loll et al. named this helix, which is located close to PsbJ, PsbK, and PsbZ, as X1 [2]. The structure by Guskov et al. supposed the correspondence between X1 and Ycf12 (Psb30) [3]. Recently, Takasaka et al. confirmed location of Psb30 as originally proposed [18] by studying the crystal structure of a mutant without Psb30 [19]. In the present work, we further studied the role of Psb30 in PSII complex by using with a psb30-knockout mutant (ΔPsb30) of T. elongatus. The isolated PSII complex from ΔPsb30 cells exhibited an oxygen-evolving activity similar to that isolated from WT*. This result suggests that Psb30 neither has a direct function or a structural influence on the Mn4Ca cluster nor affects the photosynthetic electron transfer. This somewhat disagrees with studies done on similar mutants in Synechocystis PCC 6803 [25] and in T. elongatus [19]. Inoue-Kashino et al. reported that Psb30 in Synechocystis PCC 6803 contributes to the efficient turnover of the acceptor side of PSII, because the isolated PSII from the Psb30 deletion mutant of

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Synechocystis showed 22% lower oxygen-evolving activity than that from wild type measured with the continuous light at 5200 µmol of photons m− 2 s− 1 [25]. Although we measured the activity of isolated ΔPsb30–PSII under the continuous light at 20,000 µmol of photons m− 2 s− 1, the activity was similar to that of isolated WT*PSII. This might be due to different stability of structure of OEC and interaction among helices because of different species and amount of lipids in PSII complexes between the mesophilic and the thermophilic cyanobacteria. Actually, isolated PSII complexes from T. elongatus contain at least 2 or 3 functional plastoquinone QB as probed by thermoluminescence measurements, for example [17,26,29,33–35], whereas PSII isolated from mesophilic cells such as Synechocystis PCC 6803 and Chlamydomonas showed no or a decreased thermoluminescence band derived from the recombination of S2 and Q-B without addition of DCMU [36,37]. More surprisingly, Takasaka et al. [19] reported that the deletion of Psb30 from PSII in T. elongatus reduced the activity by ∼ 30% in contrast to what is observed here. The important difference between the work in Ref. [19] and the present work is the strain in which Psb30 has been deleted. In Ref. [19], the deleted strain was a wild-type strain, which is expected to express mainly psbA1 (or a mixture of psbA1 and psbA3 and less likely psbA2), whereas here we used a deleted strain, which expresses only psbA3. PsbA1 and PsbA3 differ by 21 amino acids [38], and we recently showed that energetic property and oxygen-evolving activity of the PsbA3-PSII of T. elongatus are different from the PsbA1-PSII [26]. The higher activity and the greater stability generally found for PSII with PsbA3 than for PSII with PsbA1 could correlate with a more stable O2 activity found here for the ΔPsb30– PSII deletion mutant in PSII containing PsbA3 than that in PsbA1 containing PSII [19]. When oxygen-evolving activity was measured in whole cells under the high light irradiation conditions, the activity in the ΔPsb30 mutant cells was inhibited more than that in WT* (Fig. 4A). Under the high light conditions, the activity in the mutant was also more rapidly inhibited than that in WT* in the presence of a protein synthesis inhibitor (Fig. 4B). These results suggest that the amount of intact PSII complexes decreased under high light irradiation in the ΔPsb30 than WT*, since the degradation of PSII complex was accelerated and assembly of PSII subunits was decelerated by the absence of Psb30 from the PSII complex. Therefore, the ΔPsb30–PSII complex is less protected against photoinhibition under high light conditions. In addition to the main electron transport pathway in PSII, a secondary electron transfer pathway is proposed for protection against photoinhibition [9–11]. Although there are still some debates on the route of electron transfer and on the species involved [39–42], there is agreement that P+ 680 is reduced by Car, which was reduced by either Chlz and/or Cyt b559 that was reduced by electron donation from PQH2 or QBH2 [39,41,43,44]. Thus, electron donation in the secondary pathway could be modified by changes in the redox potential of Cyt b559. In T. elongatus, the redox potential of the HP form at pH of 6.5 was estimated to be +390 mV, and a lower redox potential form was found to be at +260 mV [32]. It is not clear yet if this form corresponds to the low potential or to an intermediate potential form. Since these potential values are similar to those in Synechocystis (HP: ≈ +310 mV, IP: ≈ 210 mV [45]) and spinach (HP: ≈ +390 mV, IP: ≈ 230 mV [46–48]), the same electron donation via Car12, Car13, Chls as in Synechocystis could occur in T. elongatus. Although the oxygen-evolving activity of isolated ΔPsb30–PSII exhibited similar value to that of WT*-PSII, the mutant cells lost the activity more (Fig. 4A) and faster in the presence of a protein synthesis inhibitor (Fig. 4B) than WT* cells under high light illumination conditions. These results indicate that the absence of Psb30 from PSII complex causes a faster photoinhibition. Light-minus-dark EPR spectra suggested that HP form of Cyt b559 in intact PSII converts into the LP form in ΔPsb30–PSII complex (Fig. 5). Under the high light

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conditions, in ΔPsb30–PSII, the presence of the LP form of Cyt b559 is likely at the origin of the faster photoinhibition. Why did the potential form of Cyt b559 change upon deletion of Psb30? The closest distance between Psb30 and Cyt b559 is ≈ 9 Å, although the N-terminal 18 amino acid residues (actually 17 residues because of the processing of the initial Met) are missing in the X-ray structure of Psb30 (Fig. 6A and C). Especially, the N-terminal region of Psb30 approaches the C-terminal region of ß-subunit of Cyt b559 (PsbF) in the lumenal side of the thylakoid membrane. There is a possibility that some of the N-terminal 17 residues of Psb30 interact directly to PsbF, which therefore would affect the properties of the heme. Berthomieu et al. proposed that LP form of Cyt b559 and HP form of Cyt b559 have different mechanisms of heme oxidation and that in HP form of Cyt b559, both His interact with peptide carbonyls while in LP form of Cyt b559, only one His interacts with a peptide carbonyl and the other His interacts with a peptide side chain [31]. This suggests

that small structural changes of Cyt b559 complex by missing Psb30 modifies interaction among heme, α- and ß-subunit of Cyt b559 (PsbE and PsbF, respectively) or modifies geometry of the heme, so that Cyt b559 changes to LP form in ΔPsb30–PSII. The distance between Psb30 and Car12 is only 3.5 Å, and Psb30 is also in contact with PsbJ, PsbK, and PsbZ (Fig. 6) [3]. Car11, which is close to ChlzD2, is a candidate for the photo-oxidisable Car molecule in the secondary electron transport. Tracewell and Brudvig suggested that Car12 is also a photo-oxidisable species [49]. The short distance between Car12 and Psb30 let us to suggest that the deletion of Psb30 could modify either the location and/or the geometry of this molecule in addition to the modification of the redox potential of Cyt b559. The MALDI-TOF data show that the ΔPsb30–PSII complex also lacks the PsbY, which is a peripheral subunit. PsbY was also missing in the crystal structure of Ferreira et al. [1] made on PSII containing PsbA1. The lack of PsbY in the Ferreira et al. structure was not due to

Fig. 6. Structures around Psb30 subunit in PSII (A) and around the putative Ca2+ (B) from the 2.9 Å model of Guskov et al. [3], and the amino acid sequence of Psb30 deduced from psb30 gene (C). Car12 (red) is with 3.5 Å distance from Psb30, and Car16 (red) is located between 2 helices of PsbZ. The lipids SQDG24 and MGDG18 are near the ß-subunit and αsubunit of Cyt b559, respectively. The putative Ca2+ seems to ligate to Asp23 and Asp19 of PsbK with distance of 3.2 and 2.8 Å, respectively (B). The Ca2+ is 5.8 Å distance from Psb30Gln21. The X-ray structure lacks of 18 amino acid residues (actually 17 residues because of processing the initial Met) of the N-terminal region of Psb30. The missing region is underlined in panel C. Figures were drawn with Swiss Pdb Viewer with PDB 3BZ1.

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the resolution, because the MALDI-TOF mass analysis made on an identical crystal demonstrated the lack of this subunit (see supplementary material). This shows that PsbY is a relatively labile PSII protein. This could explain why PsbY is missing in ΔPsb30–PSII despite the long distance (≈ 25 Å) between these two subunits. Guskov et al. suggested that Psb30 could be bridged to PsbK by a water molecule and possibly a Ca2+ ion with a distance of 5.8 Å [3] (Fig. 6B). The present data show that the lack of Psb30 does not affect the PsbK content. Therefore, the proposed interaction between Psb30 and Ca2+-PsbK could be quite weak. PsbZ is located near Psb30 with the distances of ≈ 3.2 Å [3]. The Cternimal region (Asn45) of Psb30 could make a hydrogen bond with the end of the first helix of PsbZ (Ser29) in the cytoplasmic side. Although PsbK is also close to Psb30, there is no candidate for any hydrogen bonding between these helices. In ΔPsb30–PSII, this work shows that the complex maintained both PsbK and PsbZ. This observation suggests that there is no strong interaction of Psb30 with PsbK and PsbZ, even if they are located at a ≈ 3.5 Å distances. In contrast to the present work, it has been shown that the PsbZ deletion mutant in T. elongatus lacked both Psb30 and PsbK [22], which would suggest that Psb30 strongly interacts with PsbK and PsbZ. This disagreement between subunit interactions might be due a different D1 protein variant. In our work, only psbA3 encodes D1 due to the deletion of both psbA1 and psbA2. In contrast, the ΔPsbZ mutant genome in Ref. [22] had all psbA genes. Since Kós et al. demonstrated that psbA1 gene expresses under the normal conditions in T. elongatus [50], it is assumed that the ΔPsbZ-PSII had PsbA1. PsbA3 differs by 21 amino acids from PsbA1. These differences are expected to cause some differences in PSII architecture [51]. At least, we previously demonstrated that PsbA3-PSII exhibited a significantly higher oxygenevolving activity than PsbA1-PSII [26]. Although D1 is too far to directly interact with Psb30, PsbZ, and PsbK, some amino acids between PsbA1 and PsbA3 might cause differences in the network of the interactions, resulting in a different PSII stability. From these results, although Psb30 is not directly involved in the photosynthetic function including water oxidation catalysis, it contributes to maintain stable structure of PSII complex and efficient assembly of the complex. Acknowledgments We would like to thank Jim Barber and Karim Maghlaoui for kindly providing us with PSII crystals from T. elongatus wild type. We also thank Bill Rutherford, Jim Barber, James Murray, Karim Maghlaoui, Joe Hughes, and Yuki Kato for their helpful discussions. This study was supported by Grant-in-Aid for scientific research from the Ministry of Education, Science, Sports, Culture and Technology (21612007 for M.S.) and the JSPS and CNRS under the Japan-France Research Cooperative Program.

[5]

[6]

[7]

[8]

[9]

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[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

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Appendix A. Supplementary data

[25]

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbabio.2010.03.020.

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