Cytochrome c550 in the Cyanobacterium Thermosynechococcus elongatus

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 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{S}_{2}Q_{B}^{-}\) \end{document} 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.

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 2ϩ and Cl Ϫ 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 2ϩ and Cl Ϫ and in protecting the Mn-cluster from attack by bulk reductants (9 -11).
The low midpoint redox potential (EЈ m ) values of the purified cyt c 550 (from Ϫ250 to Ϫ314 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 EЈ m value when it is bound to the PS II (Ϫ80/Ϫ100 mV) compared with its soluble form after its extraction from PS II (Ϫ240 mV at pH 6) (14). Moreover, while the EЈ m of the bound form is pH-independent, the EЈ m of the soluble form varies from Ϫ50 mV at pH 4.5 to Ϫ350 mV at pH 9 -10 (14). In conditions more native than isolated PS II complexes, it is possible that the EЈ m of cyt c 550 may be even higher than Ϫ80/Ϫ100 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 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 Ϫ2 s Ϫ1 . 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 g ml Ϫ1 ) or spectinomycin (25 g ml Ϫ1 )/streptomycin (10 g ml Ϫ1 ). For PS II preparations, the cells were grown in 3-liter conical flasks (1500 ml of culture). For Ca 2ϩ depletion, in the DTN medium, CaCl 2 was replaced by NaCl. For Cl Ϫ 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, 5Ј-CGCGGATCCACATGAACAGTGTACGCTGT-3Ј and psV2, 5Ј-CCGGAATTCAACGGAGTTCTCCTTTCAT-3Ј, 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 ϩ 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: 5Ј-GAAATTGCT-GAGGTGATGCCCAGTCTGCGCAGT-3Ј and 5Ј-GAAATTGCTGAGGT-GTGCCCCAGTCTGCGCAGT-3Ј 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 mM Tricine, 2 mM EDTA, and twice with double-distilled water, the cells were resuspended at an OD 750 of about 100 (approx 1 ϫ 10 11 cells ml Ϫ1 ). 40 l of this suspension was mixed with 4 -8 l of a 0.5-1 g l Ϫ1 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 g ml Ϫ1 /6 g ml Ϫ1 ) 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 g ml Ϫ1 spectinomycin and 10 g ml Ϫ1 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, 5Ј-TCCGGCACCGCCCCCAAGGATAAT-3Ј and psV6, 5Ј-CCGGCGC-GATCGTCCAGCCCAGCA-3Ј. 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 M glycinebetaine 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 mM MES, pH 6.5, 15 mM MgCl 2 , 15 mM CaCl 2 , 10% (v/v) glycerol, and 1 M glycinebetaine at about 2 mg of Chl⅐ml Ϫ1 . The preparations used in this work had an oxygen evolution activity of 2200 -3000 mol of O 2 ⅐mg Chl Ϫ1 ⅐h Ϫ1 .
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 mM MES, pH 6.5 containing 10 mM sodium ascorbate for 1 h (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 ϫ 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 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 g of Chl ml Ϫ1 ), thylakoid membranes (20 g of Chl ml Ϫ1 ), and PSII core complexes (5 g of Chl ml Ϫ1 ) was measured in 40 mM MES, pH 6.5, 15 mM MgCl 2 ,15 mM CaCl 2 , 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 (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 Chl⅐ml Ϫ1 ) in a buffer containing 40 mM MES, pH 6.5, 15 mM MgCl 2 , 15 mM CaCl 2 , 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. 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 ⅐ 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 ϩ ). Then the Tyr D ⅐ signal was removed from the spectrum by interactive subtraction to obtain the P700 and Tyr D ⅐ 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.
Thermoluminescence-Thermoluminescence was measured as described in (24). Thylakoid membranes at a Chl concentration of 100 g ml Ϫ1 and PS II complexes at 35 g ml Ϫ1 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 g ml Ϫ1 . After dark adaptation (15 min), the cells were frozen at Ϫ80°C. They were maintained for half an hour at Ϫ80°C. After slow thawing, the cell suspension was incubated on ice in darkness. For measurements of S 2 Q B Ϫ and S 3 Q B Ϫ 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 Ϫ 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 Ϫ5°C. For luminescence detection, the samples were warmed at a constant rate (0.5°C/s) from 1°C or Ϫ5°C to 80°C.

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, 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 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 Ϫ2 s Ϫ1 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 Ϫ (Fig. 2B). Control, H92M, and H92C cells, however, were able to grow in the chloride-depleted culture medium albeit at slower rate (doubling time Ϸ35-37 h). In Ca 2ϩ -depleted medium, T. elongatus wild type cells only doubled their initial concentration before they completely stopped growing ( Fig. 2B and 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 ⅐(mg Chl) Ϫ1 ⅐h Ϫ1 ) while the activity was significantly lower in the cyt c 550 -less mutant cells (150 -200 mol of O 2 ⅐(mg Chl) Ϫ1 ⅐h Ϫ1 ). 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)(26)(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 ⅐(mg Chl) Ϫ1 ⅐h Ϫ1 ), while in cyt c 550 -less and H92C thylakoids the activity decreased to 160 -200 and 100 -130 mol of O 2 ⅐(mg Chl) Ϫ1 ⅐h Ϫ1 , 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 ⅐(mg Chl) Ϫ1 ⅐h Ϫ1 ) 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 (500 -800 mol of O 2 ⅐(mg Chl) Ϫ1 ⅐h Ϫ1 ). 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 M glycinebetaine 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 ⅐(mg Chl) Ϫ1 ⅐h Ϫ1 ) were systematically less active than the H92M PS II complexes (2500 -2800 mol of O 2 ⅐(mg Chl) Ϫ1 ⅐h Ϫ1 ). 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 Ͼ H92M ϾH92C mutants. Fig. 4, B and C also 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, A and B show the oxygen emission under flash illumination in thylakoids isolated from the four strains. The oscillation was pronounced and clear maxima were observed after the third, seventh, and eleventh flashes (Fig. 5, A and 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 ϭ 10 -15%; S 1 ϭ 85-90%; ␣ ϭ 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 ⁄2 for 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 EЈ m of the bound cytochrome was significantly higher than that of the cytochrome in solution (Ϫ110 Ϯ 8 mV versus Ϫ265 Ϯ 11 mV). The EЈ m of the isolated cytochrome was pH-dependent (pH 6: EЈ m ϭ Ϫ224 mV, pH 7: EЈ m ϭ Ϫ286 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 Ϫ300 Ϯ 7 mV (Fig. 6). The EЈ m of the bound H92C cyt c 550 was significantly higher (Ϫ164 Ϯ 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 Ϫ140 Ϯ 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 a was recorded on control-PS II. In principle, spectrum a could 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 b and c were 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 ϳ2.2 suggest that the 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 ϭ 2.97, g y ϭ 2.24, and g x ϭ 1.49. In the isolated H92C cytochrome the g-values were g z ϭ 2.94, g y ϭ 2.26, and g x ϭ 1.53 and in the isolated H92M cytochrome the g-values were g z ϭ 2.92, g y ϭ 2.27, and g x ϭ 1.54. Spectra b and c also contain traces of contaminating MnII, which exhibits a 6-line spectrum between Ϸ3100 and Ϸ3800 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 IIbound 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 Ϫ (the Q band) and from S 2/3 with Q B Ϫ (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). 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 Ϫ luminescence is 1.7 times greater than S 2 Q B Ϫ (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 Ϫ 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 Ϫ 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 Ϫ state. The kinetics of S 2 Q B Ϫ 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 Ϫ . 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 Ϫ . The S 2 Q B Ϫ decrease could be fitted by one exponential in both strains giving a t1 ⁄2 of about 120 s in control thylakoids and a t1 ⁄2 of 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 ⁄2 of 150 s (cyt c 550 -less) and 100 s (control) whereas the faster phase had a t1 ⁄2 of 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). 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 Ϫ recombination (Q band) (34) with a maximum at about 26 -28°C and the other related to Tyr ⅐ Q A Ϫ (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  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. 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 Ϫ ions (16), a finding confirmed in the present study. In the absence of Ca 2ϩ , 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 Ϫ binding but also Ca 2ϩ 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 Ϫ and S 2 Q B Ϫ TL bands (10 and 6°C, respectively, in Synechocystis PCC 6803), and no rapid deactivation of the Mn-cluster in the dark (90% in 2 h in Synechosystis 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 550less 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 2ϩ or Cl Ϫ 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 Ϫ deactivation measured by thermoluminescence. 100% S 2 ϭ 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.
Synechocystis could at least in part be due to a weaker binding of the 33 kDa (and maybe of the 12 kDa) to the PS II complex even in the cells. It is well known that the binding of the extrinsic proteins is very labile in Synechocystis PCC 6803. The isolation of active PS II complexes, even from wild-type cells, is not trivial. While it was possible to isolate active thylakoids from the cyt c 550 -less T. elongatus mutant, changes in PS II properties were induced: notably a slight retardation of O 2 release, a slight upshift of the maximum of the TL Q band and decreased oxygen activity compared with cells. These changes were attributable to the loss or loosening of extrinsic proteins, particularly the 33 kDa.
As already mentioned, when the cyt c 550 -less mutant from Synechocystis PCC 6803 was studied with TL, the S 2 Q B Ϫ and the S 2 Q A Ϫ TL bands were found to be upshifted (11). In contrast, the cyt c 550 -less T. elongatus mutant cells showed no-shift of the S 2 Q A Ϫ TL band and a downshift of the maximum of the S 2 Q B Ϫ recombination TL band. This corresponded to a more rapid decay of S 2 Q B Ϫ in the mutant. Thermoluminescence was reported previously from isolated PS II complexes of T. vulcanus, another thermophilic species (4), in which the extrinsic proteins were removed and reconstituted. In PS II with the 33 kDa reconstituted but lacking the cyt c 550 and the 12 kDa, a shift to lower temperature was seen for the S 2 Q B Ϫ band, in agreement with the present work. Moreover, the upshift of the Q band disappeared after reconstitution with the 33 kDa protein alone, suggesting that the stabilization of the S 2 Q A Ϫ state was related to the lack (or weaker binding) of the 33 kDa protein and not to the lack of the 12 kDa protein as suggested by Shen et al. (11). The destabilization of the S 2 Q B Ϫ state could be due to a higher instability of either the S 2 state or Q B Ϫ , or both. The S 2 Q A Ϫ state appears to be unaffected by the lack of cyt c 550 . Thus there are two possible explanations that would rationalize the TL results. 1) Q B Ϫ is destabilized while S 2 and Q A Ϫ are unaffected, or 2) Q B Ϫ is unaffected, S 2 is destabilized and Q A Ϫ is stabilized thus canceling out the effect of S 2 destabilization. Both situations are not without precedence, with donor-side influences on the acceptor side (e.g. [42][43][44] and vice versa (45), both are present in the literature.
Another minor modification in the PS II attributable to the absence of the cyt c 550 was the increased concentration of S 0 in dark-adapted cells (see oxygen per flash results). The cyt c 550less mutant may allow access of reductants to the Mn-cluster, either directly or because of weaker binding of the extrinsic proteins when the cytochrome is absent. Since the slow reduction of the Mn-cluster resulting in formation of S 0 and lower S states will be counteracted by their reoxidation by TyrD ⅐ (46), it seems reasonable that higher than normal concentrations of S 0 and lower than normal amounts of TyrD ⅐ could occur together under such conditions. The fast phase of S 2 Q B Ϫ decay attributable to electron donation from TyrD is indeed increased in the cyt c 550 -less mutant.
In conclusion the absence of the cyt c 550 results in 1) a lowered affinity for Cl Ϫ and most probably Ca 2ϩ , 2) a destabilization of the S 2 Q B Ϫ because of donor and/or acceptor side effects, 3) a modification in reactions between the TyrD/TyrD ⅐ and the Mn complex, which could be because the Mn-cluster was less well insulated from attack by exogenous reductants, caused by a weaker binding of the other extrinsic proteins.
The Control and the Site-directed Mutated Cyt c 550 -Shen and Inoue (5) suggested that all the cyt c 550 present in the cells was attached to the PS II. Here, however, we presented data suggesting that in control whole cells, in addition to the PS II-bound cyt c 550 , a significant fraction of cyt c 550 is present in the soluble form. EPR experiments clearly showed that the g z -value of the signal corresponding to cyt c 550 in whole cells was similar to that of isolated cyt c 550 . The role and cellular localization of this soluble cyt c 550 is still unknown. However, the fact that the cyt c 550 isolated from the soluble fraction had lost the transit sequence could suggest that it was present in the lumen and not in the cytoplasm as suggested by its very low redox potential.
In the present work, we substituted the His-92, the sixth axial ligand of the heme iron in cyt c 550 with a cysteine and a methionine. EPR spectra demonstrated that both mutations resulted in the heme as six-coordinated. However, the g-values of the mutated cytochromes were different from those of the wild-type cyt c 550 , indicating that the environment of the heme was modified. The mutated cytochromes were less stable and more weakly bound to the PS II than the wild-type cyt c 550 . The change of His to Met in cyt c 550 led to an increase of the redox potential of about 130 mV. In contrast, in the H92C mutant, the change of His to Cys downshifted the already low EЈ m (Ϫ270 mV) of the cyt c 550 about 30 mV. The marked difference in the EЈ m of the hemes seen in the isolated mutant cytochromes (H92C ϭ Ϫ300 mV, H92M ϭ Ϫ140 mV) was much less apparent when they were bound to PS II. (H92C ϭ Ϫ165 mV, H92M ϭ Ϫ145 mV). In addition, the bound mutated cytochromes presented quite similar EPR spectra (the g-values were almost identical), both different from that of the wild-type cyt c 550 .
The binding of the wild-type cytochrome led to an EЈ m increase of about 180 -200 mV compared with the isolated cyt c 550 (14). It was proposed that this large increase in EЈ m could be due to a much lower solvent accessibility of the heme when the cyt is bound to the PS II (14). In addition we have proposed that Tyr-82 is responsible for the pH dependence of the redox potential of the soluble cyt c 550 (14). This pH dependence disappears when cyt c 550 is bound to PS II. This could be due to either a binding-induced change of the pK a of the Tyr-82 or a loss of solvent accessibility to this group. Such a low solvent exposure would allow the ionizing group to remain protonated, inducing the more positive EЈ m also observed in the soluble cyt c 550 at low pH values. The recent resolution of the PS II structure at 3.5 Å (8) shows that when bound to PS II the heme pocket is occluded by amino acids chains from the 12 kDa and the CP43 proteins, confirming the lower exposure of the heme to the solvent in the bound cyt c 550 . Moreover, it is possible that the oxygen of Tyr-82 forms a hydrogen bond with the C-terminal Ile-66 of the 12 kDa protein (8).
The binding of the H92C-mutated cyt c 550 also resulted in an increase in the EЈ m , although this increase was somewhat smaller than in control (135 versus 180/200 mV). In contrast, the H92M-mutated cyt c 550 did not show a binding-induced effect on the EЈ m : the redox potential of the bound and unbound cytochrome was around Ϫ140 mV. This could be explained by a mutation-induced change in the binding that prevents the usual increase in the EЈ m . Alternatively the phenomenon triggered by the binding (e.g. a decrease in solvent access or a change in a pK a of the dominant ionizable group) could already be present in the soluble mutated cytochrome. If the Tyr-82 does play a role, as suggested, it is possible to imagine several scenarios in which the phenomena can be explained, including modifications of its electrostatic environment by exclusion of solvent or other binding effects.
Effect of Point Mutations on PS II Activity-The mutation of the His-92 to Met or Cys did not modify PS II activity. In whole mutant cells and in thylakoids, the oxygen evolving activity, light saturation curves (data not shown), the pattern of oxygen evolved per flash, the thermoluminescence B and Q bands, and the cell growth in the absence of Ca 2ϩ or Cl Ϫ were all identical to those in our control strain, the ⌬psbV2 mutant. None of the characteristics of the cyt c 550 -less mutant were present in the His-92 mutants. This is also true for the characteristics of the isolated PS II complexes from H92M mutant, which were almost identical to those of the control strain. In the case of the H92C mutant, however, characteristics of the cyt c 550 -less strain appeared in the isolated PS II complexes, and those were almost certainly caused by the fractional loss of cyt c 550 implying that the mutated cytochrome was more weakly bound.
Even though the large changes in the redox potential of the mutated unbound cyt c 550 were damped out to a large extent by the binding of the cytochrome to PSII, smaller but still significant shifts in potential (30 -50 mV) were detected in the bound mutated cyt c 550 . This difference in the redox potential had no influence on PSII activity. This argues against a redox role for this heme in PSII function or in cell growth under the conditions tested. The absence of the cyt c 550 had more influence on PSII activity but all the effects observed could be explained in terms of a binding role: contributing to the stability and binding of the other extrinsic polypetides, responsible at least in part for maintaining the affinity for the Cl Ϫ and Ca 2ϩ ions and possibly acting directly as a barrier to reductive attack on the Mn 2ϩ . The role of the heme itself remains enigmatic.