Cytochrome c550 in the cyanobacterium Thermosynechococcus elongatus: Study of redox mutants

Cytochrome c 550 in the Cyanobacterium Thermosynechococcus elongatus STUDY OF REDOX MUTANTS* Received for publication, July 20, 2004, and in revised form, August 23, 2004 Published, JBC Papers in Press, September 22, 2004, DOI 10.1074/jbc.M408206200 Diana Kirilovsky‡§, Mercedes Roncel¶, Alain Boussac‡, Adje´le´ Wilson‡, Jorge L. Zurita¶, Jean-Marc Ducruet‡, Herve´ Bottin‡, Miwa Sugiura‡, Jose´ M. Ortega¶, and A. William Rutherford‡ From the ‡Service de Bioe´nerge´tique, Departement Biologie Joliot-Curie, URA Consejo Superior de Investigaciones Cientificas 2096, CEA Saclay, 91191 Gif sur Yvette, France and the ¶Instituto de Bioquı´mica Vegetal y Fotosı´ntesis, Universidad de Sevilla y CSIC, Ame´rico Vespucio 49, 41092 Sevilla, Spain Cytochrome c 550 is one of the extrinsic Photosystem II subunits in cyanobacteria and red algae. To study the possible role of the heme of the cytochrome c 550 we constructed two mutants of Thermosynechococcus elongatus in which the residue His-92, the sixth ligand of the heme, was replaced by a Met or a Cys in order to modify the redox properties of the heme. The H92M and H92C mutations changed the midpoint redox potential of the heme in the isolated cytochrome by ⴙ125 mV and ⴚ30 mV, respectively, compared with the wild type. The binding-induced increase of the redox potential observed in the wild type and the H92C mutant was absent in the H92M mutant. Both modified cytochromes were more easily detachable from the Photosystem II compared with the wild type. The Photosystem II activity in cells was not modified by the mutations suggesting that the redox potential of the cytochrome c 550 is not important for Photosystem II activity under normal growth conditions. A mutant lacking the cytochrome c 550 was also constructed. It showed a lowered affinity for Cl ⴚ and Ca 2ⴙ as reported earlier for the cytochrome c 550 -less Synechocystis 6803 mutant, but it showed a shorter lived S 2 Q B ⫺ state, rather than a stabilized S 2 state and rapid deactivation of the enzyme in the dark, which were characteristic of the Synechocystis mutant. It is suggested that the latter effects may be caused by loss (or weaker binding) of the other extrinsic proteins rather than a direct effect of the absence of the cytochrome c 550 . Cytochrome c 550 (cyt c 550 ), 1 present in cyanobacteria and red algae, was first discovered by Holton and Myers (1) as a soluble monoheme c-type cytochrome. The cyt c 550 has a molecular mass of about 15 kDa, His/His coordination and a very low redox potential around ⫺260 mV (for review see Ref. 2). Shen et al. (3–5) showed that cyt c 550 is stoichiometrically bound to the Photosystem II (PS II), activates oxygen evolving activity and allows the binding of the 12 kDa protein, another extrinsic component of the cyanobacterial PS II involved in oxygen evolution. The three-dimensional structure of the PS II from two thermophilic cyanobacteria strains confirmed that cyt c 550 binds to the luminal PS II surface in the vicinity of the D1 and CP43 proteins (6–8). The phenotype of the ⌬psbV (cyt c 550 -less) mutant of Synechocystis PCC 6803 was already characterized with respect to PS II activity (9–11). The ⌬psbV and the double ⌬psbV/⌬psbU (encoding the 12 kDa protein) mutants were unable to grow in the absence of Ca 2⫹ and Cl ⫺ ions, their PS II activity decreased to 40% of the wild type, and they showed a very rapid inactivation of the enzyme in the dark (after 2 h, the activity decreased about 90% whereas in the wild type only 25% of the activity was lost). A slight retardation in O 2 release from the S 3 state was also observed in these mutants. Another effect observed in these Synechocystis PCC 6803 mutants was a large intensity decrease of the B band of thermoluminescence (TL) and an upshift in the temperature maxima of the B and Q bands. The upshift of the TL bands was attributed to the lack of the 12 kDa protein, and the decrease in the intensity of the B band was considered to be the major effect of cyt c 550 deletion (11). All these observations led the authors to propose that cyt c 550 functions in maintaining the high affinity of PS II for Ca 2⫹ and Cl ⫺ and in protecting the Mn-cluster from attack by bulk reductants (9–11). The low midpoint redox potential (E⬘ m ) values of the purified cyt c 550 (from ⫺250 to ⫺314 mV (1, 12, 13)) seems incompatible with a redox function in PS II electron transfer. However, we have recently demonstrated that the cyt c 550 from the thermophilic cyanobacteria Thermosynechococcus elongatus has a significantly higher E⬘ m value when it is bound to the PS II (⫺80/⫺100 mV) compared with its soluble form after its extraction from PS II (⫺240 mV at pH 6) (14). Moreover, while the E⬘ m of the bound form is pH-independent, the E⬘ m of the soluble form varies from ⫺50 mV at pH 4.5 to ⫺350 mV at pH 9–10 (14). In conditions more native than isolated PS II complexes, it is possible that the E⬘ m of cyt c 550 may be even higher than ⫺80/⫺100 mV, and thus a redox function in the water oxidation complex could be conceivable. The thermophilic cyanobacterium T. elongatus has become a new model organism for photosynthesis research since it has provided the first resolved x-ray crystallographic structures of PS I (15) and PS II (6). As mentioned above, cyt c 550 is encoded by the psbV gene, however in the thermophilic strains T. elongatus and T. vulcanus, the genome contains a second gene (psbV2) encoding a cyt c 550 -like protein located between the psbV1 gene (encoding cyt c 550 ) and the petJ gene (encoding cyt c 6 , soluble electron donor of PS I) (16). We have recently shown * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom correspondence should be addressed. Tel.: 33-1-69-08-95- 71; Fax: 33-1-69-08-87-17; E-mail: [email protected]. 1 The abbreviations used are: cyt c 550 , cytochrome c 550 ; PS II, Photosystem II; Chl, chlorophyll; E⬘ m , midpoint redox potential; EPR, electron paramagnetic resonance; PS I, photosystem I; Sp, spectinomycin; Sm, streptinomycin; MES, 2-(N-morpholino) ethanesulfonic acid; DCBQ, 2,6 dichloro-p-benzoquinone; TL, thermoluminescence; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 51, Issue of December 17, pp. 52869–52880, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org 52869 at FAC BIOLOGIA/BIBLIOTECA on April 25, 2016http://www.jbc.org/Downloaded from that this gene is expressed (17). The PsbV2 protein has an apparent molecular mass of about 15.3 kDa and contains a six-coordinated low spin c-type heme, and the sixth ligand seems to be the Tyr-86. The role of the PsbV2 protein is unknown. The present study is an attempt to analyze the possible role of the heme of the cyt c 550 in PS II. To address this problem, two mutants of the thermophilic cyanobacterium T. elongatus were constructed. In these mutants, the His-92, the sixth ligand of the heme, was changed to a methionine or cysteine to modify the redox properties of the heme. In addition, in these two mutants, the psbV2 gene was disrupted by an antibiotic cassette in order to allow its selection. Two other mutants were constructed and studied here: a psbV2-disruptant mutant (⌬psbV2) as a control strain, and a psbV1-disruptant mutant (⌬psbV1 or cyt c 550 -less mutant). EXPERIMENTAL PROCEDURES Strain and Standard Culture Conditions—Cells of the transformed strain of T. elongatus with a histidine tag on the CP43 protein of PS II (43-H strain) (18) and all the other mutants constructed for this work were grown in a rotary shaker (120 rpm) at 45 °C under continuous illumination from fluorescent white lamps giving an intensity of about 80 ␮ mol of photons m ⫺2 s ⫺1 . Cells were grown in a DTN medium (19) in a CO 2 -enriched atmosphere. For maintenance, the cells were grown in the presence of kanamycin (40 ␮ gml ⫺1 )or spectinomycin (25 ␮ gml ⫺1 )/streptomycin (10 ␮ gml ⫺1 ).ForPSII preparations, the cells were grown in 3-liter conical flasks (1500 ml of culture). For Ca 2⫹ depletion, in the DTN medium, CaCl 2 was replaced by NaCl. For Cl ⫺ depletion, CaCl 2, FeCl 3 , and NH 4 Cl were replaced by CaOH 2 , FeNH 4 (SO 4 ) 2 , (NH 4 ) 2 SO 4, respectively. Cloning, Recombinant Plasmids, and in Vitro Mutagenesis—The genome region containing the psbV1 and psbV2 genes was amplified using genomic DNA of T. elongatus as template and two synthesized oligonucleotides: psV1, 5⬘-CGCGGATCCACATGAACAGTGTACGCTGT-3⬘and psV2, 5⬘-CCGGAATTCAACGGAGTTCTCCTTTCAT-3⬘, containing the BamHI and EcoRI restriction sites, respectively, were used as primers. The amplified region of 1.5 kb containing the psbV1,psbV2 genes and the flanking regions was first cloned in the polylinker EcoRV restriction site of a pBC-SK ⫹ chloramphenicol-resistant plasmid (dpV1 plasmid) and then in the BamHI restriction site of a pUC9 ampicillin-resistant plasmid (dpV2 plasmid). Insertional inactivation of the psbV2 gene was carried out by inserting a 2.2-kb DNA fragment containing the aadA gene from Tn7, conferring resistance to spectinomycin and streptinomycin (Sp/Sm) (20), in the unique BstAPI restriction site of the psbV2 gene in the dpV1 plasmid (dpV3 plasmid). Insertional inactivation of the psbV1 gene was achieved with the insertion of the Sp/Sm cassette in the unique BsaAI restriction site of psbV1 in the dpV2 plasmid (dpV4 plasmid). Site-directed mutagenesis of the plasmid containing the interrupted psbV2 gene (dpV3 plasmid) was performed using the QuikChange XL site-directed mutagenesis kit of Stratagene as recommended by the manufacturer. Synthetic mutagenic oligonucleotides: 5⬘-GAAATTGCT- GAGGTGATGCCCAGTCTGCGCAGT-3⬘and 5⬘-GAAATTGCTGAGGT- GTGCCCCAGTCTGCGCAGT-3⬘were used to create in the psbV1 gene the H92M and H92C mutants, respectively. These primers delete the unique ApaLI restriction site of the psbV1 gene. Transformation of T. elongatus Cells and Genetic Analysis of Mutants—The plasmids containing the interrupted genes and the sitemutated genes were introduced in 43-H T. elongatus cells by electroporation according to Ref. 19 with slight modifications. After washing once with 2 mMTricine, 2 mMEDTA, and twice with double-distilled water, the cells were resuspended at an OD 750 of about 100 (approx 1 ⫻10 11 cells ml ⫺1 ). 40 ␮ l of this suspension was mixed with 4–8 ␮ l of a 0.5–1 ␮ g ␮ l ⫺1 DNA and chilled on ice. Cells were electroporated in chilled, sterile cuvettes witha2mmgapbetween the electrodes with a single pulse with a time constant of 5 ms and at field strength of 9 kV/cm. After electroporation, cells were rapidly transferred to 2 ml of DTN and incubated for 48 h in a rotary incubator at 45 °C under low light conditions. Then, the cells in 0.1–0.2 ml aliquots were spread on Sp/ Sm-containing plates (12 ␮ gml ⫺1 /6 ␮ gml ⫺1 ) and incubated at 45 °C, under dim light and humidified atmosphere. Once transformants emerged as green colonies after 2–3 weeks, they were spread at least twice on agar plates containing 25 ␮ gml ⫺1 spectinomycin and 10 ␮ g ml ⫺1 streptinomycin before their genomic DNA was analyzed. Genomic DNA was isolated from T. elongatus cells essentially as described by Cai and Wolk (21). To confirm the homoplasmicity of the ⌬psbV2 and ⌬psbV1 mutants a PCR analysis was carried out using primers psV1 and psV2. To verify that the desired point mutations were present in the transformed T. elongatus cells, a PCR fragment containing the psbV1 gene was obtained using the oligonucleotides psV0, 5⬘-TCCGGCACCGCCCCCAAGGATAAT-3⬘and psV6, 5⬘-CCGGCGC- GATCGTCCAGCCCAGCA-3⬘. The amplified fragment was then digested by the restriction enzyme ApaLI. The mutant PCR fragments were then sequenced to confirm that the correct mutation was the only modification present in the gene. Thylakoids and PS II Core Complexes Preparation—Thylakoids and PS II core complexes were prepared as described by Roncel et al. (14) with the following modifications: 1) All the buffers used in the preparations contained 1 Mglycinebetaine and 10% (v/v) glycerol. 2) The supernatant of the ␤ -D-dodecyl-maltoside treated thylakoids was mixed with 1 volume of Probond TM resin (Invitrogen, Groningen, The Netherlands) and immediately transferred to an empty column and washed. 3) The PS II preparations were resuspended in 40 mMMES, pH 6.5, 15 mM MgCl 2 ,15mMCaCl 2 , 10% (v/v) glycerol, and 1 Mglycinebetaine at about 2mgofChl䡠ml ⫺1 . The preparations used in this work had an oxygen evolution activity of 2200–3000 ␮ mol of O 2 䡠mg Chl ⫺1 䡠h ⫺1 . Cytochrome c 550 Isolation—Cyt c 550 from the ⌬psbV2 mutant was isolated from soluble proteins as described in Ref. 17 and from PS II preparations as described in Ref. 23. Cyt c 550 from H92M and H92C mutants were isolated only from PS II preparations. Incubation of isolated PS II complexes in a solution of 20 mMMES, pH 6.5 containing 10 mMsodium ascorbate for1h(4°C)under daylight induced the release of the cyt c 550 from the PS II. The PS II cores were precipitated by centrifugation (170,000 ⫻g, overnight). The supernatant containing the 33 kDa protein, the 12 kDa protein, and the cyt c 550 was concentrated, and then the cyt c 550 was purified by HPLC on a Hi-Trap Q Sepharose HP column (Hepes 10 mM, pH 7, sodium ascorbate 10 mM, 0–1 MNaCl gradient). Oxygen Evolution Measurements—Oxygen evolution was measured at 25 °C by polarography using a Clark-type oxygen electrode with saturating white light. Oxygen evolution of cells (20 ␮ g of Chl ml ⫺1 ), thylakoid membranes (20 ␮ g of Chl ml ⫺1 ), and PSII core complexes (5 ␮ g of Chl ml ⫺1 ) was measured in 40 mMMES, pH 6.5, 15 mMMgCl 2 ,15 mMCaCl 2 , 10% (v/v) glycerol, 1 Mglycinebetaine, and in the presence of 0.5 mMDCBQ (2,6-dichloro-p-benzoquinone, dissolved in ethanol) as electron acceptor. The amount of oxygen produced per flash during a sequence of saturating flashes was measured at room temperature with a lab-made rate electrode equivalent to that described in (22). The short saturating flashes were produced by a xenon flash. The time between flashes was 400 ms. 20 ␮ l of a thylakoid membrane suspension (1 mg of Chl䡠ml ⫺1 ) in a buffer containing 40 mMMES, pH 6.5, 15 mMMgCl 2 ,15 mMCaCl 2 , 10% (v/v) glycerol, 1 Mglycinebetaine were deposited onto the surface of the platinum electrode and dark adapted for 40 min at room temperature prior to each flash sequence. The analysis of the flash-induced oxygen evolution patterns was done as described in Ref. 23. EPR Measurements—CW-EPR spectra were recorded using a standard ER 4102 (Bruker) X-band resonator with a Bruker ESP300 X-band spectrometer equipped with an Oxford Instruments cryostat (ESR 900). The samples were frozen in the dark to 198 K, degassed at 198 K, and then transferred to 77 K. Determination of g-values was done using an ER032M gaussmeter (Bruker). Illumination of the samples was done with an 800 watt tungsten lamp, in a non-silvered Dewar filled with liquid nitrogen. Light was filtered through water and IR filters. To calculate the PS II/PS I ratio, first an EPR spectrum of Tyr D䡠was recorded using a dark-adapted sample. A second EPR spectrum was recorded after one flash in the presence of ferricyanide (in order to oxidize all the Tyr D and the P700 ⫹ ). Then the Tyr D䡠signal was removed from the spectrum by interactive subtraction to obtain the P700 and Tyr D䡠signals separately. Interactive subtraction is a standard computer-assisted spectrum manipulation in which an unknown spectral component can be extracted from a mixture of two spectra by subtracting a proportion of a second known spectral component. The proportion of the known spectral component in the mixture is gradually varied until the difference spectrum is considered to contain no contribution from the known spectrum. Redox Potential Measurements—Reductive Potentiometric redox titrations were carried out basically as described in Ref. 14. The electrode was calibrated before each titration against a saturated solution of quinhydrone (E⬘ m ⫽⫹280 mV at 20 °C, pH 7). All redox measurements are relative to normal hydrogen electrode. For titration, samples contained PS II core complexes (30–50 ␮ g of Chl ml ⫺1 ) Cytochrome c 550 in T. elongatus 52870 at FAC BIOLOGIA/BIBLIOTECA on April 25, 2016http://www.jbc.org/Downloaded from or purified cyt c 550 (about 0.5–1 ␮ M) were suspended in a buffer containing 40 mMMES, pH 6.5, 15 mMMgCl 2 ,15mMCaCl 2 , 10% (v/v) glycerol, and 1 Mglycinebetaine. The buffer for the titrations of purified H92C and H92M cyt c 550 contained 10 mMsodium ascorbate. To ensure a good equilibrium between the redox centers and the electrode, a set of suitable redox mediators were added (14):20 ␮ M duroquinone (E⬘ m ,pH7⫽⫹10 mV), 30 ␮ M2-methyl-1,4-naphthoqui- none (E⬘ m ,pH7⫽0 mV), 30 ␮ M2,5-dihydroxyl-p-naphthoquinone (E⬘ m ,pH7⫽⫺60 mV), 30 ␮ Manthraquinone (E⬘ m ,pH7⫽⫺100 mV), 30 ␮ M2-hydroxyl-1,4-naphthoquinone (E⬘ m ,pH7⫽⫺145 mV), 30 ␮ M anthraquinone-1,5-disulfonate (E⬘ m ,pH7⫽⫺170 mV), 30 ␮ Manthraquinone-2,6-disulfonate (E⬘ m ,pH7⫽⫺185 mV) and 30 ␮ Manthraquinone-2-sulfonate (E⬘ m ,pH7⫽⫺225 mV), 30 ␮ Mbenzyl viologen (1,1⬘-dibenzyl-4,4⬘-bipyridylium dichloride) (E⬘ m ,pH7⫽⫺311 mV) and 30 ␮ Mmethyl viologen (1,1⬘-dimethyl-4,4⬘-bipyridylium dichloride) (E⬘ m ,pH7⫽⫺430 mV). In order to calculate E⬘ m values of cyt c 550 in PSII core complexes, differential spectra were obtained by subtracting the absolute spectra recorded at each solution redox potential during titrations from the spectra of the fully oxidized state of the cytochrome (at a solution redox potential of about ⫹100 mV). The absorbance differences at 549–560 nm (for ⌬psbV2 mutant) and 552–560 nm (for His-92 mutants) obtained from difference (PSII core complexes) or absolute spectra (purified cyt c 550 ) were normally converted into percentages of reduced cytochrome and plotted versus solution redox potential. E⬘ m values were then determined by fitting percentages plots to an n⫽1 Nernst equation curve using a nonlinear curve-fitting program (Origin 6.0, Microcal Software). Oxidative redox titrations were carried out as controls in order to check for the reproducibility of the experiments; no significant hysteresis effects were observed. Thermoluminescence—Thermoluminescence was measured as described in (24). Thylakoid membranes at a Chl concentration of 100 ␮ g ml ⫺1 and PS II complexes at 35 ␮ gml ⫺1 were dark-adapted (at least for half an hour). Cells were centrifuged and resuspended in a 40 mMMES (pH 6.5) buffer containing 15 mMMgCl, 15 mMCaCl, 10% glycerol, and 1Mglycinebetaine at a Chl concentration of 100 ␮ gml ⫺1 . After dark adaptation (15 min), the cells were frozen at ⫺80 °C. They were maintained for half an hour at ⫺80 °C. After slow thawing, the cell suspension was incubated on ice in darkness. For measurements of S 2 Q B ⫺ and S 3 Q B ⫺ recombination, the samples were incubated for 5 min in the dark at 20 (isolated PS II) or 40 °C (thylakoids and cells) and then flashed one to four times at 1 °C. For measurements of S 2 Q A ⫺ recombination, the dark-adapted samples were flashed once in the presence of DCMU at 20 °C and after 3 min of dark adaptation, one flash was given at ⫺5 °C. For luminescence detection, the samples were warmed at a constant rate (0.5 °C/s) from 1 °C or ⫺5°Cto80°C. RESULTS Construction of Gene-interrupted and Site-directed Mutants—To generate mutants of T. elongatus lacking either the cyt c 550 or the PsbV2 protein, the genome region containing the genes coding for these proteins (Fig. 1A) was amplified by PCR and cloned. Plasmids were constructed in which either the psbV1 or the psbV2 gene was interrupted by insertion of a spectinomycin/streptomycin resistance cassette (Fig. 1, Band C). The plasmids carrying the site-directed mutations H92M- psbV1 and H92C-psbV1 were constructed using the plasmid in which the psbV2 gene had been previously interrupted. Thus, the PsbV2 protein was absent in these mutants. The plasmids were introduced into H-43 T. elongatus cells by electroporation and the interrupted psbV2 and point-mutated psbV1 genes were incorporated into the cyanobacterium genome by homologous double recombination. The construction allowed us to perform mutant selection by growing the cells in the presence of antibiotics. Complete segregation and homoplasmicity of the mutants were tested by PCR analysis. Fig 1Eshows that amplification of the genomic region containing the psbV1 and psbV2 genes using the synthetic oligonucleotides psV1 and psV2 gave a fragment of 3.5 kb in all the mutants containing the Sp/Sm cassette (2.1 kb) instead of a 1.5 kb fragment as was observed in the H-43j T. elongatus strain. No traces of the 1.5 kb PCR fragment were detected in these mutants indicating that complete segregation and total homoplasmicity were achieved in mutant cells. The digestion pattern obtained with the restriction enzyme ApaLI of the 3.5-kb fragment confirmed that the Sp/Sm resistance cassette was incorporated in the BsaAI site of the psbV1 gene in the psbV1-disruptant mutant and in the BstAPI site of psbV2 in the psbV2-disruptant mutant (Fig. 1, Dand F). Since the double recombination could occur between the antibiotic cassette and the point mutations, not all the Sp/Sm resistant mutants contained the site-directed mutations. To select the mutants carrying the proper modified bases, a 1-kb PCR fragment obtained using the psV0 and psV6 oligonucleotides as primers and containing the psbV1 gene was checked by digestion with the restriction enzyme ApaLI. In the absence of a point mutation, the fragment was digested giving two fragments of 0.8 and 0.2 kb (Fig. 1G). The smaller fragment is visible only on overloaded gels (data not shown). With the correct mutation, the amplified DNA fragments lost the restriction site, and no digestion occurred (Fig. 1G). Sequencing of the PCR-amplified fragment confirmed the presence of the proper mutation (data not shown). These PCR analyses were regularly repeated to verify the genotype in the cells used for phenotype characterization. For simplicity, the psbV2-disruptant mutant, lacking the PsbV2 protein, but presenting a phenotype similar to the wild type is called the control strain; the psbV1-disruptant mutant, lacking the cyt c 550 , is called the cyt c 550 -less mutant; the ⌬psbV2/H92M-psbV1 and ⌬psbV2/H92C-psbV1 mutants is called H92M and H92C, respectively. Mutant Cell Growth—The photosynthetic growth rates of control cells and H92M, H92C, and cyt c 550 -less mutant cells at 45 °C and 80 ␮ mol of photons m ⫺2 s ⫺1 were similar (Fig. 2A). The doubling time was about 20 h for the four strains. The T. elongatus cyt c 550 -less mutant cells were unable to grow in DTN medium lacking Cl ⫺ (Fig. 2B). Control, H92M, and H92C cells, however, were able to grow in the chloride-depleted culture medium albeit at slower rate (doubling time ⬇35–37 h). In Ca 2⫹ -depleted medium, T. elongatus wild type cells only doubled their initial concentration before they completely stopped growing (Fig. 2Band Ref. 27). The control, H92M and H92C cells presented the same behavior as the wild type cells whereas cyt c 550 -less mutant cells did not grow at all (Fig. 2B). Oxygen Evolution Activity—The H92M, H92C, and control cells had similar oxygen evolving activities (250–300 ␮ mol of O 2 䡠(mg Chl) ⫺1 䡠h ⫺1 ) while the activity was significantly lower in the cyt c 550 -less mutant cells (150–200 ␮ mol of O 2 䡠(mg Chl) ⫺1 䡠h ⫺1 ). We measured the oxygen evolution activity under different light intensities in control and cyt c 550 -less mutant cells. By plotting the data as the activity versus activity/light intensity (Fig. 3) (25–27), straight parallel lines were obtained indicating that the number of active PS II reaction centers decreased in cyt c 550 -less mutant cells compared with control cells. EPR measurements (see “Experimental Procedures”) indicated that the ratio PS I to total PS II was about 2.5–3 in the four strains (data not shown). The oxygen-evolving activity in H92M and control thylakoids was similar to that measured in whole cells (240–270 ␮ mol O 2 䡠(mg Chl) ⫺1 䡠h ⫺1 ), while in cyt c 550 -less and H92C thylakoids the activity decreased to 160–200 and 100–130 ␮ mol of O 2 䡠(mg Chl) ⫺1 䡠h ⫺1 , respectively. Thus, the activity of H92C thylakoids represented 60–75% of the activity of control thylakoids, and the activity of cyt c 550 -less thylakoids was only 40–48% of control. Highly active PS II complexes (2700–3000 ␮ mol of O 2 䡠(mg Chl) ⫺1 䡠h ⫺1 ) were isolated from control cells as described in Roncel et al. (14). With this method, the PS II complexes isolated from H92M and H92C mutants had much lower activities Cytochrome c 550 in T. elongatus 52871 at FAC BIOLOGIA/BIBLIOTECA on April 25, 2016http://www.jbc.org/Downloaded from (500–800 ␮ mol of O 2 䡠(mg Chl) ⫺1 䡠h ⫺1 ). We observed that during the purification, the cyt c 550 and the 12 kDa protein were completely lost from the PS II complexes (data not shown). PS II complexes with higher activities were obtained from both mutants when the isolation was done using 1 Mglycinebetaine in all the buffers. Glycinebetaine is an osmoprotective plant product known for its stabilizing properties of the oxygenevolving complex (28, 29). Even in the presence of glycinebetaine, the PS II complexes from the mutants were less active than the control PS II complexes: the H92C PS II complexes (1900–2300 ␮ mol of O 2 䡠(mg Chl) ⫺1 䡠h ⫺1 ) were systematically less active than the H92M PS II complexes (2500–2800 ␮ mol of O 2 䡠(mg Chl) ⫺1 䡠h ⫺1 ). The lower activity in isolated PS II from H92C seemed to correspond to a lower amount of cyt c 550 associated with the PS II complex. In Fig. 4 the difference spectra of cyt c 550 and cyt b 559 shows that quantity of cyt c 550 present in isolated PS II complexes was as follows: control ⬎ H92M ⬎H92C mutants. Fig. 4, Band Calso show that the mutations induced an upshift from 549 to 552 nm of the maxima of the ␣ band in the bound and unbound cyt c 550 . Illumination of dark-adapted samples by a train of short saturating flashes produces oxygen with a yield per flash that oscillates with a periodicity of four (30, 31). Fig. 5, Aand Bshow the oxygen emission under flash illumination in thylakoids isolated FIG.1.Mutant construction and test for the presence of mutations. A, gene arrangement of the psbV1 and psbV2 genes and flanking regions. The primers used in PCR amplification are indicated (Band C)psbV1 or psbV2 gene disruption by insertion of the Sp/Sm resistance cassette (aadA) in the BsaAI site in the psbV1 gene and in the BstAPI site in the psbV2 gene. D, size of fragments that must be obtained by digestion with the restriction enzyme ApaLI of the 3.5 kb-amplified PCR fragment of genomic T. elongatus ⌬psbV1 and ⌬psbV2 mutants using as primers the psV1 and psV2 oligonucleotides. E, amplification of genomic DNA from ⌬psbV2 (lane 1), ⌬psbV1 (lane 3), H92M (lane 4), and H92C mutants using as primers psV1 and psV2 oligonucleotides. F, digestion by ApaLI of the 3.5 kb-amplified PCR fragments obtained using as template ⌬psbV2 (lane 1) and ⌬psbV1 (lane 2) DNAs. G, digestion by ApaLI of the 1-kb PCR fragments obtained using as templates H92M (lane 2), H92C (lane 3), and ⌬psbV2 DNAs. Cytochrome c 550 in T. elongatus 52872 at FAC BIOLOGIA/BIBLIOTECA on April 25, 2016http://www.jbc.org/Downloaded from from the four strains. The oscillation was pronounced and clear maxima were observed after the third, seventh, and eleventh flashes (Fig. 5, Aand B). The pattern of the oscillations (Fig. 5, A and B), the miss parameter, and the initial S 0 and S 1 values in control and H92M and H92C mutants were similar (S 0 ⫽10– 15%; S 1 ⫽85–90%; ␣ ⫽0.09–0.12). In cyt c 550 -less mutant thylakoids, the amplitude of oscillations was smaller and the dark concentration of S 0 was higher (25–27% versus 10–15%) than in the other strains (Fig. 5A). In order to examine whether the absence of the cyt c 550 retarded the oxygen release from the S 3 state, we measured the kinetics of oxygen release after the third flash (Fig. 5C). The peak of the oxygen signal for the cyt c 550 -less mutant reproducibly located 1–2 ms later than that for the control (7 versus 5 ms) (Fig. 5C). The t1⁄2for the oxygen release was estimated to be 1.5 ms in the control thylakoids and 2 ms in cyt c 550 -less mutant thylakoids. His-92 mutations did not change the kinetics of oxygen release (data not shown). Characteristics of the Cyt c 550 from control and H92M and H92C Mutants—The cyt c 550 of control cells was isolated 1) from the fraction of soluble proteins and 2) from isolated PS II complexes. The N-terminal amino acid sequence of the cyt c 550 from both sources was AELTPE. This result indicates that the cyt c 550 from both origins had lost the 26 amino acids transit sequence. Moreover, the absorbance spectra and redox properties of cyt c 550 from both sources were identical (data not shown). Cyt c 550 from the H92C and H92M mutants was isolated only from isolated PS II complexes. Although the cyt c 550 was present in the cytoplasm/periplasm fraction of these mutants, it was not possible to isolate it from this fraction. It seemed to be degraded during the different steps of purification. The presence of sodium ascorbate (used as an antioxidant) decreased the rate of denaturation of the mutant cytochromes. However, the H92M cyt c 550 was so unstable that it began to be denaturated during the final step of purification (HPLC) even in the presence of sodium ascorbate. The absorbance spectrum, redox potential measurements and EPR spectra indicated that during the HPLC purification the methionine was lost as the sixth ligand (data not shown). All the data concerning the H92M cyt c 550 shown in this article were obtained without HPLC purification. The redox properties of wild-type cyt c 550 of T. elongatus have been already described in a previous article (14). Fig. 6 shows reductive potentiometric titrations at pH 6.5 of the cyt c 550 of the control and of the H92M and H92C mutants when bound to PS II (closed symbols) and in its soluble form (open symbols). In control cells, the E⬘ m of the bound cytochrome was significantly higher than that of the cytochrome in solution (⫺110 ⫾8mVversus ⫺265 ⫾11 mV). The E⬘ m of the isolated cytochrome was pH-dependent (pH 6: E⬘ m ⫽⫺224 mV, pH 7: E⬘ m ⫽⫺286 mV, data not shown) like in the wild type (23). The mutation of the His to Cys decreased the redox potential of the soluble cyt c 550 to about ⫺300 ⫾7 mV (Fig. 6). The E⬘ m of the bound H92C cyt c 550 was significantly higher (⫺164 ⫾9 mV) than that of the unbound cytochrome (Fig. 6). In contrast, the isolated H92M cyt c 550 and the bound H92M cyt c 550 had similar redox potentials of about ⫺140 ⫾10 mV. Thus, the mutation His to Met induced a significant increase of the redox potential of the isolated cyt c 550 , and the loss of the binding effect on the redox potential. EPR Spectra of Cyt c 550 in Control and H92M and H92C Mutants—EPR spectra of PS II-bound and isolated cyt c 550 are reported in Fig. 7, panels A and B, respectively. In panel A, spectrum awas recorded on control-PS II. In principle, spectrum acould contain both cyt b 559 and cyt c 550 signals. However, in intact PSII, cyt b 559 is mainly in a reduced state in darkness and thus EPR-silent, whereas cyt c 550 is oxidized and thus detectable by EPR (14). The g z (3.02), g y (2.21), and g x (1.44) values of cyt c 550 were similar to those found earlier in the wild type (14). These values are characteristic of a bishistidine hexa-coordinated low spin heme with a rhombicity of 0.48 (14, 17). Spectra band cwere recorded on H92C and H92M PS II mutants, respectively. In both samples, two distinct g z and g y resonances contribute to the spectra (the broadness of the g x resonances makes them more difficult to detect). This suggests either a heterogeneity in the cyt c 550 or the presence of a second oxidized cytochrome. The values of g z resonances at 3.02 and that of the g y resonances at ⬃2.2 suggest that the FIG.2.Growth curves. Growth curves of control (⌬psbV2) (circles), cyt c 550 -less mutant (⌬psbV1) (squares), H92C mutant (triangles), and H92M mutant (rhomboids) cells in complete medium (A) or without Ca 2⫹ (B,open symbols)orCl ⫺ (B,closed symbols). Temperature of growth: 45 °C. FIG.3. Oxygen-evolving activity as function of activity/light intensity of control (squares) and cyt c 550 -less mutant (circles) cells (20 ␮ g of Chl/ml) in the presence of 0.5 mMDCBQ. Cytochrome c 550 in T. elongatus 52873 at FAC BIOLOGIA/BIBLIOTECA on April 25, 2016http://www.jbc.org/Downloaded from minor contributions in both spectra band coriginated from the relaxed high potential form of ferri-cyt b 559 in a fraction of the centers (32). The g z (2.91), g y (2.27), and g x (1.55) resonances are similar in both the H92C and H92M PSII mutants. These values indicate a rhombicity of 0.59 and a tetragonality of 3.36, which correspond to hexa-coordinated low spin heme close to the domain characteristic of thiolate heme ligands in the Peisach and Blumberg diagram (33). Panel B in Fig. 7 shows the EPR spectra of cyt c550 isolated from control-PS II (spectrum a), from H92C-PS II (spectrum b) and H92M-PS II (spectrum c). Cyt c 550 isolated from control-PS II has the EPR characteristics previously reported (14, 17) with g z ⫽2.97, g y ⫽2.24, and g x ⫽1.49. In the isolated H92C cytochrome the g-values were g z ⫽2.94, g y ⫽2.26, and g x ⫽ 1.53 and in the isolated H92M cytochrome the g-values were g z ⫽2.92, g y ⫽2.27, and g x ⫽1.54. Spectra b and c also contain traces of contaminating MnII, which exhibits a 6-line spectrum between ⬇3100 and ⬇3800 gauss. In the isolated cytochromes from both the H92C and H92M mutants the rhombicity (0.56– 0.58) and the tetragonality (3.3–3.4) values are similar to those found in the bound cytochromes. This strongly differs from the situation encountered in the wild type, where the rhombocity value in the isolated cytochrome (0.58) was higher than that in the PS II-bound cytochrome (0.48) (14, 17). Fig. 8 shows the EPR spectra of wild-type cyt c 550 recorded using: (a) the purified cytochrome isolated from cells, (b) whole cells, (c) purified thylakoids, (d) purified PSII. The g z value of the thylakoid-bound cyt c 550 corresponded to that of PS II-bound cyt c 550 and that of the whole cells to that of the isolated cyt c 550 with a shoulder that could be from the bound cyt c 550 . These results suggest the presence of a significant concentration of soluble cyt c 550 in control cells. Comparing the quantity of the cyt c 550 isolated from soluble fraction of 20 liters of cells to the number of PS II present in these cells, we calculated that the soluble fraction could represent between 40 and 60% of the bound population. In His-92 mutant cells the EPR signals from cyt c 550 were very weak but the g z values seemed to correspond better to those of the PS II- bound cytochrome rather than to those of isolated cyt c 550 (data not shown), supporting the idea that the mutant cytochromes are unstable in their soluble form. Thermoluminescence Studies of T. elongatus Mutants—In this section, we will describe the characteristics of the thermoluminescence bands in whole cells, thylakoids and isolated PS II complexes of the four mutants of T. elongatus that were studied. The thermoluminescence (TL) emitted at physiological temperatures arises from charge recombination from S 2/3 with Q B ⫺ (the Q band) and from S 2/3 with Q B ⫺ (the B band) (34, 35). The TL pattern of unfrozen cells was variable due to different values of the dark stable transmembrane proton (and/or electrochemical) gradient, which induces a temperature downshift of the B bands (36), which changed from one culture batch to another. Thus, the comparison between the mutants was done using cells that had been frozen and thawed, a treatment that collapses the transmembrane proton gradient. In these treated cells the pattern of TL was stable, independent of the cell batch and similar to that obtained with cells treated with the protonophore nigericin (data not shown). Fig. 9Acompares the oscillation of the B band intensity in the frozen and thawed cells after a series of flashes. The B band FIG.4.A, redox difference absorption spectra of the cytochromes b 559 and c 550 in PS II-isolated complexes from T. elongatus ⌬psbV2 (control), ⌬psbV1 (cyt c550-less), H92M, and H92C strains. The spectra were obtained by subtracting absolute spectra recorded at ⫺300 mV minus at ⫹458 mV. B, spectra of bound cyt c 550 in PS II-isolated complexes T. elongatus ⌬psbV2 (control), H92M, and H92C strains. The spectra were obtained by subtracting the spectra of cyt c 550 and b 559 of the cyt c 550 -less mutant (shown in A) to those of the other three strains. C, ␣ band of reduced soluble cyt c 550 isolated from control and H92M and H92C mutants. Cytochrome c 550 in T. elongatus 52874 at FAC BIOLOGIA/BIBLIOTECA on April 25, 2016http://www.jbc.org/Downloaded from oscillates with a period of 4 with a first maximum after two flashes corresponding to the maximum amount of S 2 /S 3 .S 3 Q B ⫺ luminescence is 1.7 times greater than S 2 Q B ⫺ (37). TL from the control (⌬psbV2 cells) was identical to that of the wild type (data not shown). The maximum temperature of the B band obtained after one flash (corresponding to S 2 Q B ⫺ recombination) was about 58–60 °C in control and the H92M and H92C mutant cells. The B bands obtained after 2, 3, and 4 flashes were slightly downshifted (56–57 °C) due to the predominance of S 3 Q B ⫺ recombination that occurs at lower temperatures. The intensities of the thermoluminescence B band, and the oscillations were similar in the three strains. In contrast, the intensities of the B bands of the cyt c 550 -less mutant were significantly lower and the maxima of the bands were markedly shifted to lower temperatures (48–50 °C). The same experiments were done in thylakoids and similar results were obtained (data not shown). Since the absence of extrinsic proteins renders the PS II more sensitive to high temperatures (38, 39), the stability of the centers was tested by comparing the B bands obtained after incubating the control and cyt c 550 -less mutant thylakoids for 2 min at 60 °C, for 5 min at 50 °C or for 5 min at 20 °C before flashing. The area and the maximum of the bands were similar (data not shown). Thus, the shift of the B band in the cyt c 550 -less mutant cannot be explained by an increased PS II sensitivity to high temperatures that could give a weaker emission artificially shifted to a lower temperature and must correspond to a real destabilization of the S 2 Q B ⫺ state. The kinetics of S 2 Q B ⫺ deactivation were measured by recording thermoluminescence at increasing time intervals after one flash (Fig. 9C). Dark-adapted cells (40 °C) were flashed once to form S 2 Q B ⫺ . Then, the B band was recorded after various dark periods at 40 °C. Its intensity decreased but its shape and temperature maximum were not significantly altered. The area of the B band obtained just after the flash was taken as 100% of S 2 Q B ⫺ . The S 2 Q B ⫺ decrease could be fitted by one exponential in both strains giving a t1⁄2of about 120 s in control thylakoids and a t1⁄2of about 80 s in the cyt c 550 -less mutant thylakoids A better fit was obtained using a double exponential. In this case the dominant, slower phase, had a t1⁄2of 150 s (cyt c 550 -less) and 100 s (control) whereas the faster phase had a t1⁄2of about 25 s and represented 26 and 10% of the total curve in mutant and control thylakoids respectively. This phase may correspond to the reduction of S 2 to S 1 by TyrD (23). FIG.5.Flash-induced oxygen evolution pattern. Pattern obtained during a sequence of saturating xenon flashes (spaced by 400 ms) using thylakoids of T. elongatus control (⌬psbV2) (squares) and cyt c550-less (⌬psbV1) (circles) mutants (A), thylakoids of T. elongatus H92M (squares) and H92C (circles) mutants (B). C, oxygen release after the third flash given on thylakoids of T. elongatus control (⌬psbV2) (solid line) and cyt c 550 -less mutant (⌬psbV1) (dashed line). The curves were normalized to the maximum of oxygen released. Inset, complete curves without normalization. Cytochrome c 550 in T. elongatus 52875 at FAC BIOLOGIA/BIBLIOTECA on April 25, 2016http://www.jbc.org/Downloaded from We also recorded the thermoluminescence bands obtained after flashing the sample in the presence of DCMU (Fig. 9B). In this case two bands were detected: one related to S 2 Q A ⫺ recombination (Q band) (34) with a maximum at about 26–28 °C and the other related to Tyr䡠Q A ⫺ (C band) (40) with a maximum at 70 °C. No shift to lower or higher temperatures was observed in cells of any mutant. However, in thylakoids a slight (but consistent) upshift (2 °C) of the Q band of the cyt c 550 -less (⌬psbV1) mutant was observed (data not shown). In addition, the Q band of the cyt c 550 -less (⌬psbV1) mutant was always smaller (in cells and thylakoids) (Fig. 10) and that of the H92C was slightly bigger in cells (Fig. 9) but not in thylakoids (data not shown) compared with that of the control (⌬psbV2) and the H92M mutant. Fig. 10 shows the B and Q bands of the isolated PS II complexes. The TL patterns of the control and the H92M PS II isolated centers were similar. Nevertheless, the maxima of the B bands were shifted by about 10 °C to lower temperatures compared with the B bands of cells or thylakoids. This phenomenon was also observed in the wild type (data not shown). This shift could be associated to a change in the lipids due to detergents around the PS II leading to a destabilization of the Q B binding site and/or the S 2 /S 3 states. The fact that a shift to lower temperatures was also detected in the Q band suggests that this effect is caused by an effect on the Mn-cluster rather than on the Q B binding site. In accordance with its lower FIG.6.Reductive potentiometric titrations. Plots of the percentages of reduced cyt c 550 versus solution redox potentials obtained from redox titration of unbound (open circles) and bound (closed circles) cyt c 550 from control, H92M, and H92C. The solid curves represent the best fit of the experimental data to the Nernst equation in accordance with one-electron processes (n⫽1) for one component. E⬘ m values shown in the figure were calculated from the Nernst fitting of the reductive potentiometric titration shown in the figure. Each titration was done three times. The E⬘ m values oscillated about ⫾10 mV between the different experiments. Redox titrations were carried out at pH 6.5 as described under “Experimental Procedures.” FIG.7. EPR spectra of bound (A) and isolated (B) cyt c 550 . CW-EPR spectra of cytochromes bound to PS II (A) and isolated (B) from T. elongatus control, H92C, and H92M strains. Spectrum a, control cytochrome; spectrum b, H92C cytochrome; spectrum c, H92C cytochrome. The amplitudes of the spectra are arbitrary scaled. The center part of the spectra in panel A corresponding to the oxidized tyrosine D radical region, was deleted. Instrument settings: temperature, 20 K; modulation amplitude, 20 G; microwave power, 2 milliwatts; microwave frequency, 9.4 GHz; modulation frequency, 100 kHz. FIG.8.EPR spectra of wild-type cyt c 550 using isolated cytochrome (spectrum a), whole cells (spectrum b), purified thylakoids (spectrum c), and purified PSII (spectrum d). Only the g z region is shown. Amplitudes of the spectra are arbitrarily scaled. Instrument settings: temperature, 20 K; modulation amplitude, 20 G; microwave power, 2 milliwatts; microwave frequency, 9.4 GHz; modulation frequency, 100 kHz. Cytochrome c 550 in T. elongatus 52876 at FAC BIOLOGIA/BIBLIOTECA on April 25, 2016http://www.jbc.org/Downloaded from oxygen evolving activity and its smaller concentration of bound cyt c 550 , the isolated-PS II complexes from H92C presented smaller B and Q bands (Fig. 10). In addition, a slight downshift (46 °C) of the maximum of the B band was observed. The B bands of the cyt c 550 -less PS II presented a shift to lower temperatures and were clearly smaller (Fig. 10) compared with control PS II as seen for the B bands of the cyt c 550 -less thylakoids and cells (Fig. 9). DISCUSSION The Cyt c 550 -less Mutant of T. elongatus—In the present work a cyt c 550 -less (⌬psbV1) mutant of T. elongatus was constructed. Such a mutant has already been reported but the characterization of the phenotype of this mutant was previously limited to oxygen evolution measurements and to the finding that it was unable to grow in the absence of Cl ⫺ ions (16), a finding confirmed in the present study. In the absence of Ca 2⫹ , control cells doubled their concentration while mutant cells did not grow at all suggesting that the absence of the cyt c 550 has an influence not only on Cl ⫺ binding but also Ca 2⫹ binding like in Synechocystis PCC 6803 cells. A further comparison of the cell phenotype of the T. elongatus cyt c 550 -less mutant can be made with literature report of the equivalent mutant in Synechocystis PCC 6803 (11). No information is available about PS II activity in thylakoids or isolated PS II complexes from the cyt c 550 -less Synechocystis PCC 6803 mutant. In cells, the decrease in oxygen evolving activity was less marked (35–40% in T. elongatus versus 60% in Synechocystis PCC 6803), there was no retardation of O 2 release (3 ms in Synechocystis), no upshift of the maximum emission temperature of the S 2 Q A ⫺ and S 2 Q B ⫺ TL bands (10 and 6 °C, respectively, in Synechocystis PCC 6803), and no rapid deactivation of the Mn-cluster in the dark (90% in2hinSynecho- systis PCC 6803). These results suggest that the cyt c 550 -less PS II of the T. elongatus is more stable than that of Synechocystis PCC 6803 at least in whole cells. It is very unlikely that the greater stability of the cyt c 550 - less T. elongatus-PS II compared with that in Synechocystis PCC 6803 is caused by the presence of the PsbV2 protein. This protein has never been detected in association with PS II and is present in very small quantities (only 1% of soluble cyt c 550 ) (17) in the cells. Furthermore when over-expressed in the cyt c 550 -less mutant in Synechocystis, the PsbV2 protein did not reverse the suppression of growth in the absence of Ca 2⫹ or Cl ⫺ ions (16). Most of the defects seen in PS II activity in the cyt c 550 -less Synechocystis mutant cells were also observed in the Synechocystis PCC 6803 mutant lacking the 33 kDa protein (⌬psbO) (41): notably the upshift of TL bands, the retardation of O 2 release and the rapid deactivation of the Mn-cluster in the dark. Thus, the effects attributed to the lack of cyt c 550 in FIG.9. A, thermoluminescence glow curves (B bands) of T. elongatus control (⌬psbV2), cyt c550-less mutant (⌬psbV1), H92M and H92C mutant cells. Cells were maintained on ice in the dark. Before flashing (1–4 flashes) they were darkadapted for 5 min at 40 °C. The flashes were given at 1 °C. B, thermoluminescence glow curves (Q bands) of control (⌬psbV2) cells (close circles), cyt c 550 less (⌬psbV1) (triangles), H92M (open circles), and H92C (open squares) mutant cells recorded in the presence of 10 ␮ MDCMU. The flashes were given at ⫺5 °C. C, kinetics of S 2 Q B ⫺ deactivation measured by thermoluminescence. 100% S 2 ⫽area of B band obtained just after the flash. The thylakoids were dark-adapted for 5 min at 40 °C then a flash was given (at 40 °C). The TL signals were recorded after various periods of dark at 40 °C. Cytochrome c 550 in T. elongatus 52877 at FAC BIOLOGIA/BIBLIOTECA on April 25, 2016http://www.jbc.org/Downloaded from