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• W2005280199 abstract "Cytochrome c550 is one of the extrinsic Photosystem II subunits in cyanobacteria and red algae. To study the possible role of the heme of the cytochrome c550 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 +125 mV and –30 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 c550 is not important for Photosystem II activity under normal growth conditions. A mutant lacking the cytochrome c550 was also constructed. It showed a lowered affinity for Cl– and Ca2+ as reported earlier for the cytochrome c550-less Synechocystis 6803 mutant, but it showed a shorter lived S2QB− state, rather than a stabilized S2 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 c550. Cytochrome c550 is one of the extrinsic Photosystem II subunits in cyanobacteria and red algae. To study the possible role of the heme of the cytochrome c550 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 +125 mV and –30 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 c550 is not important for Photosystem II activity under normal growth conditions. A mutant lacking the cytochrome c550 was also constructed. It showed a lowered affinity for Cl– and Ca2+ as reported earlier for the cytochrome c550-less Synechocystis 6803 mutant, but it showed a shorter lived S2QB− state, rather than a stabilized S2 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 c550. Cytochrome c550 (cyt c550), 1The abbreviations used are: cyt c550, cytochrome c550; 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.1The abbreviations used are: cyt c550, cytochrome c550; 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. present in cyanobacteria and red algae, was first discovered by Holton and Myers (1Holton R.W. Myers J. Science. 1963; 142: 234-235Crossref PubMed Scopus (28) Google Scholar) as a soluble monoheme c-type cytochrome. The cyt c550 has a molecular mass of about 15 kDa, His/His coordination and a very low redox potential around –260 mV (for review see Ref. 2Kerfeld C.A. Krogmann D.W. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 397-425Crossref PubMed Google Scholar). Shen et al. (3Shen J.R. Ikeuchi M. Inoue Y. FEBS Lett. 1992; 301: 145-149Crossref PubMed Scopus (118) Google Scholar, 4Shen J.R. Inoue Y. Biochemistry. 1993; 32: 1825-1832Crossref PubMed Scopus (224) Google Scholar, 5Shen J.R. Inoue Y. J. Biol. Chem. 1993; 272: 17821-17826Abstract Full Text Full Text PDF Scopus (67) Google Scholar) showed that cyt c550 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 c550 binds to the luminal PS II surface in the vicinity of the D1 and CP43 proteins (6Zouni A. Witt H.T. Kern J. Fromme P. Krauβ N. Saenger W. Orth P. Nature. 2001; 409: 739-743Crossref PubMed Scopus (1742) Google Scholar, 7Kamiya N. Shen J.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 98-103Crossref PubMed Scopus (983) Google Scholar, 8Ferreira K. Iverson T. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1838Crossref PubMed Scopus (2782) Google Scholar). The phenotype of the ΔpsbV (cyt c550-less) mutant of Synechocystis PCC 6803 was already characterized with respect to PS II activity (9Shen J.R. Vermaas W. Inoue Y. J. Biol. Mol. 1995; 270: 6901-6907Scopus (75) Google Scholar, 10Shen J.R. Burnap R.L. Inoue Y. Biochemistry. 1995; 34: 12661-12668Crossref PubMed Scopus (67) Google Scholar, 11Shen J.R. Qian M. Inoue Y. Burnap R.L. Biochemistry. 1998; 37: 1551-1558Crossref PubMed Scopus (123) Google Scholar). The ΔpsbV and the double ΔpsbV/ΔpsbU (encoding the 12 kDa protein) mutants were unable to grow in the absence of Ca2+ 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 O2 release from the S3 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 c550 deletion (11Shen J.R. Qian M. Inoue Y. Burnap R.L. Biochemistry. 1998; 37: 1551-1558Crossref PubMed Scopus (123) Google Scholar). All these observations led the authors to propose that cyt c550 functions in maintaining the high affinity of PS II for Ca2+ and Cl– and in protecting the Mn-cluster from attack by bulk reductants (9Shen J.R. Vermaas W. Inoue Y. J. Biol. Mol. 1995; 270: 6901-6907Scopus (75) Google Scholar, 10Shen J.R. Burnap R.L. Inoue Y. Biochemistry. 1995; 34: 12661-12668Crossref PubMed Scopus (67) Google Scholar, 11Shen J.R. Qian M. Inoue Y. Burnap R.L. Biochemistry. 1998; 37: 1551-1558Crossref PubMed Scopus (123) Google Scholar). The low midpoint redox potential (E′m) values of the purified cyt c550 (from –250 to –314 mV (1Holton R.W. Myers J. Science. 1963; 142: 234-235Crossref PubMed Scopus (28) Google Scholar, 12Hoganson C.W. Lagenfelt G. Andréasson L.-E. Biochim. Biophys. Acta. 1990; 1016: 203-206Crossref Scopus (13) Google Scholar, 13Navarro J.A. Hervas M. De la Cerda B. De la Rosa M. A Arch. Biochem. Biophys. 1995; 318: 46-52Crossref PubMed Scopus (36) Google Scholar)) seems incompatible with a redox function in PS II electron transfer. However, we have recently demonstrated that the cyt c550 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) (14Roncel M. Boussac A. Zurita J.L. Bottin H. Sugiura M. Kirilovsky D. Ortega J.M. J. Biol. Inorg. Chem. 2003; 8: 206-216Crossref PubMed Scopus (66) Google Scholar). 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 (14Roncel M. Boussac A. Zurita J.L. Bottin H. Sugiura M. Kirilovsky D. Ortega J.M. J. Biol. Inorg. Chem. 2003; 8: 206-216Crossref PubMed Scopus (66) Google Scholar). In conditions more native than isolated PS II complexes, it is possible that the E′m of cyt c550 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 (15Jordan P. Fromme H.T. Witt O Klukas W Saenger N. Krauβ N Nature. 2001; 411: 909-917Crossref PubMed Scopus (2013) Google Scholar) and PS II (6Zouni A. Witt H.T. Kern J. Fromme P. Krauβ N. Saenger W. Orth P. Nature. 2001; 409: 739-743Crossref PubMed Scopus (1742) Google Scholar). As mentioned above, cyt c550 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 c550-like protein located between the psbV1 gene (encoding cyt c550) and the petJ gene (encoding cyt c6, soluble electron donor of PS I) (16Katoh H. Itoh S. Shen J.R. Ikeuchi M. Plant Cell Physiol. 2001; 42: 599-607Crossref PubMed Scopus (44) Google Scholar). We have recently shown that this gene is expressed (17Kerfeld C.A. Sawaya M.R. Bottin H. Tran K.T. Sugiura M. Cascio D. Desbois A. Yeates T.O. Kirilovsky D. Boussac A. Plant Cell Physiol. 2003; 44: 697-706Crossref PubMed Scopus (31) Google Scholar). 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 c550 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 (ΔpsbV2) as a control strain, and a psbV1-disruptant mutant (ΔpsbV1 or cyt c550-less mutant). 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) (18Sugiura M. Inoue Y. Plant Cell Physiol. 1999; 40: 1219-1231Crossref PubMed Scopus (148) Google Scholar) 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 μmol of photons m–2 s–1. Cells were grown in a DTN medium (19Mühlenhoff U. Chauvat F. Mol. Gen. Genet. 1996; 252: 93-100Crossref PubMed Scopus (59) Google Scholar) in a CO2-enriched atmosphere. For maintenance, the cells were grown in the presence of kanamycin (40 μg ml–1) or spectinomycin (25 μg ml–1)/streptomycin (10 μg ml–1). For PS II preparations, the cells were grown in 3-liter conical flasks (1500 ml of culture). For Ca2+ depletion, in the DTN medium, CaCl2 was replaced by NaCl. For Cl– depletion, CaCl2, FeCl3, and NH4Cl were replaced by CaOH2, FeNH4(SO4)2, (NH4)2SO4, 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) (20Golden J.W. Wiest D.R. Science. 1988; 242: 1421-1423Crossref PubMed Scopus (72) Google Scholar), 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′-GAAATTGCTGAGGTGATGCCCAGTCTGCGCAGT-3′ and 5′-GAAATTGCTGAGGTGTGCCCCAGTCTGCGCAGT-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 site-mutated genes were introduced in 43-H T. elongatus cells by electroporation according to Ref. 19Mühlenhoff U. Chauvat F. Mol. Gen. Genet. 1996; 252: 93-100Crossref PubMed Scopus (59) Google Scholar with slight modifications. After washing once with 2 mm Tricine, 2 mm EDTA, and twice with double-distilled water, the cells were resuspended at an OD750 of about 100 (approx 1 × 1011 cells ml–1). 40 μl of this suspension was mixed with 4–8 μl of a 0.5–1 μg μl–1 DNA and chilled on ice. Cells were electroporated in chilled, sterile cuvettes with a 2 mm gap between 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 μg ml–1/6 μg ml–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 μg ml–1 spectinomycin and 10 μg ml–1 streptinomycin before their genomic DNA was analyzed. Genomic DNA was isolated from T. elongatus cells essentially as described by Cai and Wolk (21Cai Y. Wolk C.P. J. Bacteriol. 1990; 172: 3138-3145Crossref PubMed Scopus (396) Google Scholar). To confirm the homoplasmicity of the ΔpsbV2 and ΔpsbV1 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′-CCGGCGCGATCGTCCAGCCCAGCA-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. (14Roncel M. Boussac A. Zurita J.L. Bottin H. Sugiura M. Kirilovsky D. Ortega J.M. J. Biol. Inorg. Chem. 2003; 8: 206-216Crossref PubMed Scopus (66) Google Scholar) with the following modifications: 1) All the buffers used in the preparations contained 1 m glycinebetaine and 10% (v/v) glycerol. 2) The supernatant of the β-d-dodecyl-maltoside treated thylakoids was mixed with 1 volume of Probond™ resin (Invitrogen, Groningen, The Netherlands) and immediately transferred to an empty column and washed. 3) The PS II preparations were resuspended in 40 mm MES, pH 6.5, 15 mm MgCl2, 15 mm CaCl2, 10% (v/v) glycerol, and 1 m glycinebetaine at about 2 mg of Chl·ml–1. The preparations used in this work had an oxygen evolution activity of 2200–3000 μmol of O2·mg Chl–1·h–1. Cytochrome c550 Isolation—Cyt c550 from the ΔpsbV2 mutant was isolated from soluble proteins as described in Ref. 17Kerfeld C.A. Sawaya M.R. Bottin H. Tran K.T. Sugiura M. Cascio D. Desbois A. Yeates T.O. Kirilovsky D. Boussac A. Plant Cell Physiol. 2003; 44: 697-706Crossref PubMed Scopus (31) Google Scholar and from PS II preparations as described in Ref. 23Boussac A. Rappaport F. Carrier P. Verbavatz J-M. Gobin R. Kirilovsky D. Rutherford A.W. Sugiura M. J. Biol. Chem. 2004; 279: 22809-22819Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar. Cyt c550 from H92M and H92C mutants were isolated only from PS II preparations. Incubation of isolated PS II complexes in a solution of 20 mm MES, pH 6.5 containing 10 mm sodium ascorbate for 1 h (4 °C) under daylight induced the release of the cyt c550 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 c550 was concentrated, and then the cyt c550 was purified by HPLC on a Hi-Trap Q Sepharose HP column (Hepes 10 mm, pH 7, sodium ascorbate 10 mm, 0–1 m NaCl 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 μg of Chl ml–1), thylakoid membranes (20 μg of Chl ml–1), and PSII core complexes (5 μg of Chl ml–1) was measured in 40 mm MES, pH 6.5, 15 mm MgCl2, 15 mm CaCl2, 10% (v/v) glycerol, 1 m glycinebetaine, and in the presence of 0.5 mm DCBQ (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 (22Miyao M. Murata N. Lavorel J. Maison B. Boussac A. Etienne A.L. Biochim. Biophys. Acta. 1987; 890: 151-159Crossref Scopus (96) Google Scholar). The short saturating flashes were produced by a xenon flash. The time between flashes was 400 ms. 20 μl of a thylakoid membrane suspension (1 mg of Chl·ml–1) in a buffer containing 40 mm MES, pH 6.5, 15 mm MgCl2, 15 mm CaCl2, 10% (v/v) glycerol, 1 m glycinebetaine 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. 23Boussac A. Rappaport F. Carrier P. Verbavatz J-M. Gobin R. Kirilovsky D. Rutherford A.W. Sugiura M. J. Biol. Chem. 2004; 279: 22809-22819Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar. 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· 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· signal was removed from the spectrum by interactive subtraction to obtain the P700 and Tyr D· 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. 14Roncel M. Boussac A. Zurita J.L. Bottin H. Sugiura M. Kirilovsky D. Ortega J.M. J. Biol. Inorg. Chem. 2003; 8: 206-216Crossref PubMed Scopus (66) Google Scholar. 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 μg of Chl ml–1) or purified cyt c550 (about 0.5–1 μm) were suspended in a buffer containing 40 mm MES, pH 6.5, 15 mm MgCl2, 15 mm CaCl2, 10% (v/v) glycerol, and 1 m glycinebetaine. The buffer for the titrations of purified H92C and H92M cyt c550 contained 10 mm sodium ascorbate. To ensure a good equilibrium between the redox centers and the electrode, a set of suitable redox mediators were added (14Roncel M. Boussac A. Zurita J.L. Bottin H. Sugiura M. Kirilovsky D. Ortega J.M. J. Biol. Inorg. Chem. 2003; 8: 206-216Crossref PubMed Scopus (66) Google Scholar):20 μm duroquinone (E′m, pH 7 = +10 mV), 30 μm 2-methyl-1,4-naphthoquinone (E′m, pH 7 = 0 mV), 30 μm 2,5-dihydroxyl-p-naphthoquinone (E′m, pH 7 = –60 mV), 30 μm anthraquinone (E′m, pH 7 = –100 mV), 30 μm 2-hydroxyl-1,4-naphthoquinone (E′m, pH 7 = –145 mV), 30 μm anthraquinone-1,5-disulfonate (E′m, pH 7 = –170 mV), 30 μm anthraquinone-2,6-disulfonate (E′m, pH 7 = –185 mV) and 30 μm anthraquinone-2-sulfonate (E′m, pH 7 = –225 mV), 30 μm benzyl viologen (1,1′-dibenzyl-4,4′-bipyridylium dichloride) (E′m, pH 7 = –311 mV) and 30 μm methyl viologen (1,1′-dimethyl-4,4′-bipyridylium dichloride) (E′m, pH 7 = –430 mV). In order to calculate E′m values of cyt c550 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 ΔpsbV2 mutant) and 552–560 nm (for His-92 mutants) obtained from difference (PSII core complexes) or absolute spectra (purified cyt c550) 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 non-linear 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 (24Ducruet J-M. J. of Exp. Bot. 2003; 54: 2419-2430Crossref PubMed Scopus (113) Google Scholar). Thylakoid membranes at a Chl concentration of 100 μg ml–1 and PS II complexes at 35 μgml–1 were dark-adapted (at least for half an hour). Cells were centrifuged and resuspended in a 40 mm MES (pH 6.5) buffer containing 15 mm MgCl, 15 mm CaCl, 10% glycerol, and 1 m glycinebetaine at a Chl concentration of 100 μg ml–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 S2Q–B and S3Q–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 S2Q–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 °C to 80 °C. Construction of Gene-interrupted and Site-directed Mutants—To generate mutants of T. elongatus lacking either the cyt c550 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, B and 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 1E shows 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, D and 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 c550, is called the cyt c550-less mutant; the ΔpsbV2/H92M-psbV1 and ΔpsbV2/H92C-psbV1 mutants is called H92M and H92C, respectively. Mutant Cell Growth—The photosynthetic growth rates of control cells and H92M, H92C, and cyt c550-less mutant cells at 45 °C and 80 μmol 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 c550-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 Ca2+-depleted medium, T. elongatus wild type cells only doubled their initial concentration before they completely stopped growing (Fig. 2B and Ref. 27Satoh K. Katoh S. Takamiya A. Plant Cell Physiol. 1972; 13: 885-897Crossref Scopus (13) Google Scholar). The control, H92M and H92C cells presented the same behavior as the wild type cells whereas cyt c550-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 μmol of O2·(mg Chl)–1·h–1) while the activity was significantly lower in the cyt c550-less mutant cells (150–200 μmol of O2·(mg Chl)–1·h–1). We measured the oxygen evolution activity under different light intensities in control and cyt c550-less mutant cells. By plotting the data as the activity versus activity/light intensity (Fig. 3) (25Lumry R. Rieske J.S. Plant Physiol. 1959; 34: 301-305Crossref PubMed Google Scholar, 26Rieske J.S. Lumry R. Spikes J.D. Plant Physiol. 1959; 34: 293-300Crossref PubMed Google Scholar, 27Satoh K. Katoh S. Takamiya A. Plant Cell Physiol. 1972; 13: 885-897Crossref Scopus (13) Google Scholar), straight parallel lines were obtained indicating that the number of active PS II reaction centers decreased in cyt c550-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 μmol O2·(mg Chl)–1·h–1), while in cyt c550-less and H92C thylakoids the activity decreased to 160–200 and 100–130 μmol of O2·(mg Chl)–1·h–1, respectively. Thus, the activity of H92C thylakoids represented 60–75% of the activity of control thylakoids, and the activity of cyt c550-less thylakoids was only 40–48% of control. Highly active PS II complexes (2" @default.
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• W2005280199 title "Cytochrome c550 in the Cyanobacterium Thermosynechococcus elongatus" @default.
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