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• W2092856305 abstract "The C-terminal alanine 344 (Ala-344) in the D1 protein of photosystem II is conserved in all of the organisms performing oxygenic photosynthesis. A free α-COO- of Ala-344 has been proposed to be responsible for ligating the Mn cluster. Here, we constructed a mutant having D1 in which D1-Ala-344 was replaced with glycine (Gly) in cyanobacterium Synechocystis sp. PCC 6803. The effects of this minimal change in the side group from methyl to hydrogen on the properties of the oxygen-evolving complex were comprehensively investigated using purified core particles. The mutant grew photoautotrophically, and little change was observed in the protein composition of the oxygen-evolving core particles. The Gly-substituted oxygen-evolving complex showed small but normal S2 multiline and enhanced g = 4.1 electron spin resonance signals and S2-state thermoluminescence bands with slightly elevated peak temperature. The Gly substitution resulted in distinct but relatively small changes in a few bands arising from the putative carboxylate ligand for the Mn cluster in the mid-frequency (1800-1000 cm-1) S2/S1 Fourier transform infrared difference spectrum. In contrast, the low frequency (670-350 cm-1) S2/S1 Fourier transform infrared difference spectrum was markedly changed by the substitution. The results indicate that the internal structure of the Mn cluster and/or the interaction between the Mn cluster and its ligand are considerably altered by a simple change in the side group, from methyl to hydrogen, at the C-terminal of the D1 protein. The C-terminal alanine 344 (Ala-344) in the D1 protein of photosystem II is conserved in all of the organisms performing oxygenic photosynthesis. A free α-COO- of Ala-344 has been proposed to be responsible for ligating the Mn cluster. Here, we constructed a mutant having D1 in which D1-Ala-344 was replaced with glycine (Gly) in cyanobacterium Synechocystis sp. PCC 6803. The effects of this minimal change in the side group from methyl to hydrogen on the properties of the oxygen-evolving complex were comprehensively investigated using purified core particles. The mutant grew photoautotrophically, and little change was observed in the protein composition of the oxygen-evolving core particles. The Gly-substituted oxygen-evolving complex showed small but normal S2 multiline and enhanced g = 4.1 electron spin resonance signals and S2-state thermoluminescence bands with slightly elevated peak temperature. The Gly substitution resulted in distinct but relatively small changes in a few bands arising from the putative carboxylate ligand for the Mn cluster in the mid-frequency (1800-1000 cm-1) S2/S1 Fourier transform infrared difference spectrum. In contrast, the low frequency (670-350 cm-1) S2/S1 Fourier transform infrared difference spectrum was markedly changed by the substitution. The results indicate that the internal structure of the Mn cluster and/or the interaction between the Mn cluster and its ligand are considerably altered by a simple change in the side group, from methyl to hydrogen, at the C-terminal of the D1 protein. Photosynthetic water oxidation takes place in an oxygen-evolving complex (OEC) 1The abbreviations used are: OEC, oxygen-evolving complex; PS, photosystem; Chl, chlorophyll; QA, primary quinone acceptor of photosystem II; QB, secondary quinone acceptor of photosystem II; FTIR, Fourier transform infrared; ESR, electron spin resonance; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; MES, 2-morpholinoethanesulfonic acid. in which the catalytic center is composed of a tetranuclear Mn cluster located on the lumenal side of the D1/D2 heterodimer. Two water molecules are oxidized to an oxygen molecule through five intermediates labeled Sn (n = 0-4), where n denotes the number of oxidizing equivalents stored. In a dark-adapted sample, a thermally stable S1 state predominates. The Sn state advances to the Sn+1 state by absorbing each photon to reach the highest oxidation state, S4, which spontaneously relaxes to the lowest oxidation state, S0, concomitant with the release of an oxygen molecule (1Joliot P. Barbieri G. Chabaud R. Photochem. Photobiol. 1969; 10: 309-329Crossref Scopus (523) Google Scholar, 2Kok B. Forbush B. McGloin M.P. Photochem. Photobiol. 1970; 11: 457-475Crossref PubMed Scopus (1818) Google Scholar). Studies using chemical modifiers (3Tamura N. Noda K. Wakamatsu K. Kamachi H. Inoue H. Wada K. Plant Cell Physiol. 1997; 38: 578-585Crossref Scopus (17) Google Scholar) and electron spin echo envelope modulation (4Tang X.-S. Diner B.A. Larsen B.S. Gilchrist M.L. Lorigan G.A. Britt R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 704-708Crossref PubMed Scopus (122) Google Scholar) and FTIR spectroscopy (5Noguchi T. Ono T.-A. Inoue Y. Biochim. Biophys. Acta. 1995; 1228: 189-200Crossref Scopus (164) Google Scholar, 6Noguchi T. Inoue Y. Tang X.-S. Biochemistry. 1999; 38: 10187-10195Crossref PubMed Scopus (124) Google Scholar) suggested that histidine and/or acidic amino acids are involved in the ligation of the Mn cluster. Several residues of the D1 protein have been proposed based on site-directed mutagenesis studies mainly using cyanobacterium Synechocystis sp. PCC 6803 as potential candidates for the ligands to the Mn cluster, (reviewed in Refs. 7Debus R.J. Biochim. Biophys. Acta. 1992; 1102: 269-352Crossref PubMed Scopus (1085) Google Scholar, 8Diner B.A. Biochim. Biophys. Acta. 2001; 1503: 147-163Crossref PubMed Scopus (173) Google Scholar, 9Debus R.J. Biochim. Biophys. Acta. 2001; 1503: 164-186Crossref PubMed Scopus (183) Google Scholar). They are Asp-170, Glu-189, His-190, His-332, Glu-333, His-337, Asp-342, and Ala-344 (10Nixon P.J. Diner B.A. Biochemistry. 1992; 31: 942-948Crossref PubMed Scopus (226) Google Scholar, 11Nixon P.J. Trost J.T. Diner B.A. Biochemistry. 1992; 31: 10859-10871Crossref PubMed Scopus (195) Google Scholar, 12Chu H.-A. Nguyen A.P. Debus R.J. Biochemistry. 1994; 33: 6137-6149Crossref PubMed Scopus (137) Google Scholar, 13Nixon P.J. Diner B.A. Biochem. Soc. Trans. 1994; 22: 338-343Crossref PubMed Scopus (61) Google Scholar, 14Chu H.-A. Nguyen A.P. Debus R.J. Biochemistry. 1995; 34: 5839-5858Crossref PubMed Scopus (183) Google Scholar, 15Chu H.-A. Nguyen A.P. Debus R.J. Biochemistry. 1995; 34: 5859-5882Crossref PubMed Scopus (117) Google Scholar), some of which were arranged in close proximity to the Mn cluster in x-ray structural models of photosystem (PS) II (16Zouni A. Witt H.T. Kern J. Fromme P. Krauss N. Saenger W. Orth P. Nature. 2001; 409: 739-743Crossref PubMed Scopus (1762) Google Scholar, 17Fromme P. Kern J. Loll B. Biesiadka J. Saenger W. Witt H.T. Krauss N. Zouni A. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2002; 357: 1337-1345Crossref PubMed Scopus (38) Google Scholar, 18Kamiya N. Shen J.-R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 98-103Crossref PubMed Scopus (994) Google Scholar, 19Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1837Crossref PubMed Scopus (2850) Google Scholar). However, the properties of OEC have not been characterized using isolated PS II preparations with the exception of a few mutants. The studies using the O2-evolving PS II core particles from the D1-D170H mutant showed that the mutation leads to little change of the S1 and S2 multiline ESR, S2 multiline electron spin echo envelope modulation signals, and the mid-frequency S2/S1 FTIR difference spectrum but does lead to some changes of the low frequency (650-500 cm-1) S2/S1 FTIR difference spectrum (20Chu H.-A. Debus R.J. Babcock G.T. Biochemistry. 2001; 40: 2312-2316Crossref PubMed Scopus (71) Google Scholar, 21Debus R.J. Aznar C. Campbell K.A. Gregor W. Diner B.A. Britt R.D. Biochemistry. 2003; 42: 10600-10608Crossref PubMed Scopus (42) Google Scholar). The PS II cores from D1-H332E showed no O2 evolution but retained the Mn cluster with an altered S2 multiline ESR signal in which the electron spin echo envelope modulation spectrum showed no nitrogen modulation (22Debus R.J. Cambell K.A. Peloquin J.M. Pham D.P. Britt R.D. Biochemistry. 2000; 39: 470-478Crossref PubMed Scopus (46) Google Scholar, 23Debus R.J. Cambell K.A. Cregor W. Li Z.-L. Burnap R.L. Britt R.D. Biochemistry. 2001; 40: 3690-3699Crossref PubMed Scopus (82) Google Scholar). The PS II cores from D1-E189D, D1-E189N, D1-E189H, D1-E189G, and D1-E189S showed no oxygen evolution and neither S1 nor S2 multiline ESR signal but did reveal a YZS2-state split signal. In contrast, D1-E189Q and D1-E189L mutants grew photoautotrophically, and their PS II cores showed the normal multiline signals (24Debus R.J. Cambell K.A. Pham D.P. Hays A.-M.A. Britt R.D. Biochemistry. 2000; 39: 6275-6287Crossref PubMed Scopus (39) Google Scholar). The D1 protein is synthesized with a short C-terminal extension with the exception of Euglena, assembled into the PS II complex (25Marder J.B. Goloubinoff P. Edelman M. J. Biol. Chem. 1984; 259: 3900-3908Abstract Full Text PDF PubMed Google Scholar), and subsequently cleaved on the carboxyl side of Ala-344 by D1 C-terminal-processing protease (26Shestakov S.V. Anbudurai R.R. Stanbekova G.E. Gadzhiev A. Lind L.K. Pakrasi H.B. J. Biol. Chem. 1994; 269: 19354-19359Abstract Full Text PDF PubMed Google Scholar). The processing is prerequisite to the light-dependent assembly of the Mn cluster (27Taylor M.A. Packer J.C.L. Bowyer J.R. FEBS Lett. 1988; 237: 229-233Crossref Scopus (57) Google Scholar, 28Diner B.A. Ries D.F. Cohen B.N. Metz J.G. J. Biol. Chem. 1988; 263: 8972-8980Abstract Full Text PDF PubMed Google Scholar), but the mutant with no extension by substituting the stop codon at D1-345 for the amino acid codon (D1-345stop) showed normal photoautotrophic growth and O2 evolution capability (11Nixon P.J. Trost J.T. Diner B.A. Biochemistry. 1992; 31: 10859-10871Crossref PubMed Scopus (195) Google Scholar, 29Lers A. Heifetz P.B. Boynton J.E. Gillham N.W. Osmond C.B. J. Biol. Chem. 1992; 267: 17494-17497Abstract Full Text PDF PubMed Google Scholar). Replacement of D1-Ala-344 with Gly, Met, Ser, or Val in the D1-345stop (D1-Ala-344-stop) strain did not affect photoautotrophic growth. However, Tyr or Lys substitution led to a marked decrease in O2 evolution and loss of the capability for photoautotrophic growth (11Nixon P.J. Trost J.T. Diner B.A. Biochemistry. 1992; 31: 10859-10871Crossref PubMed Scopus (195) Google Scholar). None of D1 C-terminal-truncated mutants evolved oxygen (11Nixon P.J. Trost J.T. Diner B.A. Biochemistry. 1992; 31: 10859-10871Crossref PubMed Scopus (195) Google Scholar). Therefore, the free C-terminal (α-COO-) of the D1 Ala-344 has been proposed to ligate one or more Mn ions (11Nixon P.J. Trost J.T. Diner B.A. Biochemistry. 1992; 31: 10859-10871Crossref PubMed Scopus (195) Google Scholar). The PS II core particles isolated from wild-type Synechocystis cells labeled with l-[1-13C]alanine showed that several bands in the mid-frequency S2/S1 FTIR difference spectrum are affected by the labeling (30Chu H.-A. Hiller W. Debus R.J. Biochemistry. 2004; 42: 3152-3166Crossref Scopus (140) Google Scholar). This result indicates that the isotope-affected bands can be ascribed to the α-carboxylate group of D1-Ala-344 as a ligand for the Mn cluster, although an indirect structural coupling between the Mn cluster and D1-Ala-344 cannot be excluded. Ligation of the D1-Ala-344 carboxylate to the Mn cluster was proposed based on the 3.6 Å (17Fromme P. Kern J. Loll B. Biesiadka J. Saenger W. Witt H.T. Krauss N. Zouni A. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2002; 357: 1337-1345Crossref PubMed Scopus (38) Google Scholar) and 3.7 Å (18Kamiya N. Shen J.-R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 98-103Crossref PubMed Scopus (994) Google Scholar) x-ray structural model. The C-terminal carboxylate was arranged in close proximity to a Ca ion in a recent 3.5-Å model in which a cubane-like Mn3CaO4 linked to a fourth Mn by a mono-μ-oxo bridge was proposed, although the C-terminal carboxylate was disordered and not visible in the electron density map (19Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1837Crossref PubMed Scopus (2850) Google Scholar). It may be worthwhile to note in this context that no individual metal ion in the OEC was resolved in any reported x-ray electron density maps. The D1-A344G mutant has been reported to grow photoautotrophically (11Nixon P.J. Trost J.T. Diner B.A. Biochemistry. 1992; 31: 10859-10871Crossref PubMed Scopus (195) Google Scholar). Although the replacement of alanine with glycine is accompanied with only the change of a side group from methyl to hydrogen, the functional and/or structural properties of the Mn cluster may be affected even by this minimal change if the C-terminal free carboxylate is involved in the ligation of the Mn cluster. In the present study, we have constructed D1-Ala-344-stop (= D1-Ser-345stop) and D1-A344G-stop mutants on a Synechocystis sp. PCC 6803 strain with histidine-tagged CP47 and have isolated active PS II core particles from them. The replacement of the C-terminal Ala with Gly had little effect on biochemical properties of OEC but led to considerable changes in functional/structural properties of the Mn cluster. These results are compatible with the proposal that the C-terminal α-COO- carboxylate of D1-Ala-344 is crucial for maintaining the intrastructure of the Mn cluster, possibly by ligating the Mn cluster. Construction of a Host Strain for Mutation—The genomic DNA from Synechocystis sp. PCC 6803 was amplified by PCR with a specific primer set corresponding to the psbA1, psbA2, or psbA3 gene. For psbA1, a 1727-bp DNA containing 1080 bp psbA1 plus 414 bp 5′ and 233 bp 3′ flanking DNA was cloned into the plasmid pBluescript II SK+. A 0.45-kb HincII/NsiI fragment containing 40% psbA1 was replaced by a 1.4-kb fragment from the plasmid pACYC184 conferring resistance to chloramphenicol to generate the plasmid pNΔA1. For psbA2, a 1972-bp DNA containing 1053 bp 3′ 97% psbA2 plus 919 bp 3′flanking DNA was cloned into pUC19 to generate the plasmid pNA2. A 0.9-kb HincII/StuI fragment corresponding to 83% psbA2 and a part of its downstream gene, slr1312, was replaced by a 2-kb fragment of the plasmid pRL463 (a kind gift from Prof. T. Omata, Nagoya University) conferring resistance to spectinomycin to generate the pNΔA2 plasmid. For psbA3, a 1691-bp DNA containing 1080 bp psbA3 plus 326 bp 5′ and 285 bp 3′flanking DNA was cloned into the plasmid pUC19. A 0.4-kb KpnI/KpnI fragment was replaced by a 1.7-kb fragment of pACYC184 conferring resistance to tetracycline to generate the plasmid pNΔA3. The plasmids pNA1, pNA2, and pNA3 were successively transformed into the glucose-tolerant wild-type strain of Synechocystis. The resulting strain (NΔAA) was resistant to chloramphenicol, tetracycline, and spectinomycin and lacked all three psbA genes. A hexahistidine tag was introduced to the C terminus of CP47 of the wild-type and NΔAA strain as described previously (31Kimura Y. Mizusawa N. Ishii A. Yamanari T. Ono T.-A. Biochemistry. 2003; 42: 13170-13177Crossref PubMed Scopus (68) Google Scholar) to generate the wild*-type and B-His/NΔAA strain. The B-His/NΔAA strain was used for further site-directed mutagenesis. Construction of Site-directed Mutants—A 1.3-kb fragment from the plasmid pUC4K conferring resistance to kanamycin (Km) was inserted into the StuI site located 288 bp downstream of the psbA2 gene in pNA2, thus generating the plasmid pNA219. Mutations were introduced into the psbA2 gene in pNA219 using a commercial oligonucleotide-mediated mutagenesis kit (QuikChange site-directed mutagenesis kit; Stratagene). For the Ala-344-stop (= Ser-345stop) strain, the Ser-345 codon TCT was changed to a stop codon TGA. For the A344G-stop strain, the Ala-344 codon GCG and the Ser-345 codon TCT were changed to glycine GGG and the stop codon TGA, respectively. Plasmids bearing the mutations were transformed into the host strain (B-His/NΔAA), and single colonies were selected for their photoautotrophic growth ability on solid BG-11 medium containing 5 μg/ml kanamycin. Culture Conditions and Preparation of PSII Core Particles—The Synechocystis cells were photoheterotrophically grown in liquid BG-11 medium supplemented with 5 mm glucose at 30 °C under 30-50 μmol photons/m2/s in an 8-liter Clearboy (NALGENE), bubbling with air up to 7-8 μg of Chl/ml unless otherwise noted. The PS II core particles were prepared as previously described (31Kimura Y. Mizusawa N. Ishii A. Yamanari T. Ono T.-A. Biochemistry. 2003; 42: 13170-13177Crossref PubMed Scopus (68) Google Scholar). Briefly, the harvested cells were disrupted using a Bead beater (Bio-Spec Products). The resulting thylakoid membranes were solubilized using n-dodecyl-β-d-maltoside, and then the PS II particles were affinity purified with a nickel-nitrilotriacetic acid column (Quiagen). The purified core particles were washed with a medium (medium A) containing 400 mm sucrose, 20 mm NaCl, 20 mm CaCl2, and 20 mm Mes-NaOH (pH 6.0) supplemented with 10% (w/v) polyethylene glycol 6000 and suspended in medium A after extensive washing with medium A. Protein Composition—The PS II core particles were solubilized using 1% SDS and then electrophoresed in an SDS-PAGE with a 16-22% gradient gel containing 7.5 m urea (32Ikeuchi M. Inoue Y. Plant Cell Physiol. 1988; 29: 1233-1239Google Scholar). A sample corresponding to 0.8 μg of Chl was applied to each lane. Peptide bands were visualized by staining with Coomassie Brilliant Blue R-250. The apparent molecular mass of a resolved protein was estimated by comigrating a molecular mass standard (Bio-Rad). Measurements—Mid-frequency (1800-1000 cm-1) FTIR spectra were recorded on a Bruker IFS 66v/S spectrophotometer equipped with a mercury cadmium telluride detector (EG&G Optoelectronics D316/6). Low frequency (650-350 cm-1) FTIR spectra were recorded on a Bomen MB102 spectrophotometer equipped with a Si bolometer (Infrared, HDL-5) as previously described (31Kimura Y. Mizusawa N. Ishii A. Yamanari T. Ono T.-A. Biochemistry. 2003; 42: 13170-13177Crossref PubMed Scopus (68) Google Scholar). The PS II core suspension (∼40 μg of Chl) was mixed with 1 μl of sodium ferricyanide solution (100 mm stock) as an electron acceptor. The sample suspensions deposited on either a 20-mmϕ BaF2 disk (mid-frequency) or an AgCl disk (low frequency) were partially desiccated and then rehydrated. The sample temperature was maintained within ± 0.03 °C using a homemade cryostat. The sample cores were illuminated with a flash provided from a frequency-doubled Nd3+:YAG laser (Spectra Physics INDI-50, 532 nm, pulse width 6-7 ns) with flash energy of ∼10 mJ/cm2 at the sample surface. Single beam spectra were accumulated at 4-cm-1 resolution for 15 s in the mid-frequency (20 scans) or in the low frequency region (10 scans) before and after excitation, and light - dark spectra were calculated. 116-145 mid-frequency difference spectra or 271-284 low frequency difference spectra were averaged. Low temperature X-band ESR spectra were measured using a Bruker E580 spectrometer equipped with an Oxford-900 cryostat and a temperature controller (Oxford, ITC4). The sample cores (4 mg of Chl/ml) in a Spracil quartz ESR sample tube were illuminated at 213 K for 3 s with a cold light (Hayashi, LA-150TX) passing through a long pass filter (≥680 nm). Thermoluminescence was measured using a homemade apparatus (33Ono T.-A. Inoue Y. Biochim. Biophys. Acta. 1986; 850: 380-389Crossref Scopus (132) Google Scholar). The cells were suspended in medium A at 250 μg of Chl/ml in the presence or absence of 0.1 mm DCMU. The sample suspension was illuminated at 0 °C with a saturating xenon flash. The O2 evolution activity was measured using a Clark-type oxygen electrode in medium A at 25 °C under saturating light conditions supplemented with exogenous electron acceptors, 1 mm 2,5-dimethyl-1,4-benzoquinone, and 2 mm potassium ferricyanide for cells or 4 mm potassium ferricyanide for core particles. Physiological and Biochemical Properties—As shown in Table I, the histidine-tagged wild-type (wild*-type) and the C-terminal extension-truncated Ala-344-stop cells grew photo-autotrophically under both low and high light conditions and evolved oxygen at rates similar to the wild-type cells. The affinity-purified PS II core particles from wild*-type and Ala-344-stop cells showed high O2 evolution capability, although the activity of the Ala-344-stop cores was slightly lower than that of the wild*-type cores, following the trend observed for O2 evolution in the cells. The A344G-stop cells grew photoautotrophically under low light conditions as previously reported (11Nixon P.J. Trost J.T. Diner B.A. Biochemistry. 1992; 31: 10859-10871Crossref PubMed Scopus (195) Google Scholar) and evolved oxygen at ∼90% of the rate of the Ala-344-stop cells but did not grow photoautotrophically under high light conditions. The PS II core particles from the low light-grown A344G-stop cells preserved high O2 evolution activity, but it was relatively lower (∼60%) than that from the Ala-344-stop cells. The activity was not enhanced by the further supplementation of the Ca2+ and/or Cl- to medium A.Table IPhotoautotrophic growth and PSII activities in wild-type, wild*-type, Ala-344-stop, and A344G-stop strains of Synechocystis sp. PCC 6803StrainsPhotoautotrophic growthOxygen evolutionaμmol of O2 (mg of Chl)−1 h−1.Low lightb50 μmol photons m−2 s−1.High lightc200 μmol photons m−2 s−1.CellsPSII coresWild-type+++++++410 (100)dNumbers in parentheses represent the relative O2-evolving activity in relative %.n.d.Wild*-type+++++++410 (100)2500 (100)Ala-344-stop+++++++395 (96)2400 (96)A344G-stop++−350 (85)1450 (58)a μmol of O2 (mg of Chl)−1 h−1.b 50 μmol photons m−2 s−1.c 200 μmol photons m−2 s−1.d Numbers in parentheses represent the relative O2-evolving activity in relative %. Open table in a new tab Fig. 1 shows SDS-PAGE profiles of the PS II core particles from wild*-type (lane a), Ala-344-stop (lane b), and A344G-stop (lane c). All the core particles showed very similar protein composition in terms of the major intrinsic proteins, including CP47, CP43, D2, D1, and α subunit of cytochrome b559, and the three extrinsic proteins, including 33 kDa, cytochrome c550, and 12 kDa. The particles also showed very similar profiles in the low molecular mass-region bands (<10 kDa), although each band was diffusive and could not be individually defined. The results demonstrated that neither deletion of the C-terminal extension nor replacement of the C-terminal alanine with glycine affects the protein composition of OEC. A protein band at ∼15 kDa (lane c, asterisk) was sometimes detected with a slightly higher amount in the A344G-stop than the other cores, although the identity of the band was not defined in this study. Redox Properties of OEC—The effects of mutations on the redox properties of OEC were studied by measuring the thermoluminescence glow curve in the presence (panel A) or absence (panel B) of DCMU as shown in Fig. 2. The wild-type cells (a) showed a 37 °C (-DCMU) and a 10 °C band (+DCMU) because of the charge recombination of the S2QB- and S2QA- pair, respectively. Very similar thermoluminescence glow curves were observed in the wild*-type (b) and Ala-344-stop (c) cells. The results indicate that the attachment of the histidine tag and the deletion of the C-terminal extension do not affect the redox potential of the S2-state OEC and, thus, do not affect the Mn cluster. In contrast, the peak temperatures of the respective bands in the A344G-stop cells (d) were upshifted by ∼5 °C for the S2QB- and S2QA- pair, respectively, indicating that the redox potential of the Mn cluster for the S2/S1 couple in the A344G-stop OEC is lower than that of the control OEC. Structural Properties of the Mn Cluster—Fig. 3 shows the light-induced ESR spectra in the PS II core particles from the Ala-344-stop (a) and A344G-stop (b) cells. The sample cores were illuminated at 213 K, a temperature at which the accumulation of the S2 state is allowed. The Ala-344-stop particles showed a prominent g = 2 S2 multiline, a much smaller g = 4.1 S2 signal, and a Fe2+QA- signal at g = 1.9. The spectral features of the observed S2 ESR signals were very similar to those in the normal OEC. The A344G-stop spectrum showed a smaller multiline and much larger g = 4.1 signals compared with the Ala-344-stop spectrum, although the hyperfine structure of the multiline signal and the width and position of the g = 4.1 signal were almost identical to those of the Ala-344-stop spectrum. These results indicate that the Gly substitution induces changes in the Mn cluster that facilitate the g = 4.1 state formation. Fig. 4 shows the mid-frequency (1800-1000 cm-1) S2/S1 FTIR difference spectra of the PS II core particles from the wild*-type (a), Ala-344-stop (b, blue line), and A344G-stop (b, red line) cells. The wild*-type and Ala-344-stop spectra showed largely identical S2/S1 vibrational features, including the symmetric (1450-1300 cm-1) and asymmetric (1600-1500 cm-1) stretching modes from the putative carboxylate ligands for the Mn cluster as well as the amide I (1700-1600 cm-1) and II (1600-1500 cm-1) modes from polypeptide backbones (5Noguchi T. Ono T.-A. Inoue Y. Biochim. Biophys. Acta. 1995; 1228: 189-200Crossref Scopus (164) Google Scholar, 31Kimura Y. Mizusawa N. Ishii A. Yamanari T. Ono T.-A. Biochemistry. 2003; 42: 13170-13177Crossref PubMed Scopus (68) Google Scholar). In contrast, the A344G-stop spectrum (red line) showed a small but distinctive difference from the Ala-344-stop one (blue line) as can be clearly observed in the double difference spectrum (c) obtained by subtracting the A344G-stop from the Ala-344-stop spectrum. Several of the bands in the symmetric carboxylate stretching region (1450-1300 cm-1) were shifted in the A344G-stop S2/S1 spectrum to yield the bands at 1403(-), 1394(+), 1360(+), and 1345(-) cm-1, in spite of no change of the S2/S1 bands at 1437(+) and 1417(-) cm-1. In the 1600-1500-cm-1 region, the bands were yielded at 1565(-), 1552(+), 1521(-), and 1503(+) cm-1, which are ascribed to the changes in the asymmetric carboxylate stretching modes and/or amide II modes of the polypeptide backbones. The S2/S1 bands at 1652(+), 1642(-), and 1620(+) cm-1 in the amide I region decreased in their intensity by Gly substitution to yield the 1653(+)-, 1642(-)-, and 1623(+)-cm-1 bands in the double difference spectrum. The negative band at 1113 cm-1 assigned to the CN stretching mode of the putative histidine ligand for the Mn cluster (6Noguchi T. Inoue Y. Tang X.-S. Biochemistry. 1999; 38: 10187-10195Crossref PubMed Scopus (124) Google Scholar) was considerably enhanced in the A344G-stop spectrum, as indicated by a distinct positive band in the double difference spectrum. Notably, no mode for the YD tyrosine was included in the double difference spectrum, indicating little contribution of the Mn-depleted PSII to the A344G-stop spectrum. Fig. 5 shows the low frequency (670-350 cm-1) S2/S1 FTIR difference spectra of the PS II core particles from the Ala-344-stop (a, blue line) and A344G-stop (a, red line) cells and the double difference spectrum (b). The Ala-344-stop S2/S1 spectrum showed prominent bands at 629(+), 617(-), 606(+), 590(+), 577(-), 403(-), and 388(-)cm-1 as well as many other medium to low intensity bands. The 590(+)- and ∼400(-)-cm-1 bands were ascribed to the vibrational modes of ferrocyanide and ferricyanide, respectively (31Kimura Y. Mizusawa N. Ishii A. Yamanari T. Ono T.-A. Biochemistry. 2003; 42: 13170-13177Crossref PubMed Scopus (68) Google Scholar). The spectrum was most comparable with the previously reported wild*-type spectrum measured under different sample conditions, but bands at 660(+), 652(-), 642(+), 374(+), 368(-), and 359(+) cm-1 had not been well resolved because of the large absorption of water (31Kimura Y. Mizusawa N. Ishii A. Yamanari T. Ono T.-A. Biochemistry. 2003; 42: 13170-13177Crossref PubMed Scopus (68) Google Scholar). The spectrum of A344G-stop (a, red line) was markedly different from that of Ala-344-stop. The double difference spectrum (b) showed prominent bands at 663(+), 648(+), 625(-), 615(-), 606(+), 384(+), 368(-), and 361(+) cm-1 as well as several minor bands at 532 cm-1 and in the 500-420-cm-1 region. Based on a study using 18O water, it has been suggested that the Mn-O-Mn cluster modes in the S2 and S1 states are responsible for the 606(+)- and 625(-)-cm-1 bands (34Chu H.-A. Sackett H. Babcock G.T. Biochemistry. 2000; 39: 14371-14376Crossref PubMed Scopus (78) Google Scholar). Therefore, the results indicate that the interactions between Mn ions in the cluster are considerably influenced by the Gly substitution. However, the Gly substitution was not observed to affect the bands at 577(-), 565(+), 554(+), and 542(+) cm-1. The 577(-)-cm-1 band has been ascribed to the skeletal vibration of the Mn cluster or the Mn-ligand(oxygen) interaction (31Kimura Y. Mizusawa N. Ishii A. Yamanari T. Ono T.-A. Biochemistry. 2003; 42: 13170-13177Crossref PubMed Scopus (68) Google Scholar). Therefore, the primary structure of the Mn cluster is thought to be little affected by the Gly substitution. The present results showed that the A344G-stop mutant grew photoautotrophically with a high O2 evolution activity and revealed no biochemical difference in the isolated PS II core complex. However, the mutant cells could not grow photoautotrophically under high light conditions. The elevated peak temperature of the S2-state thermoluminescence bands in the A344G-stop cells indicates that the oxidation potential of the S2-state Mn cluster is lower in the mutant than in the control cells. This may account for the increased susceptibility of the A344G-stop cells to high light because this change lowers the efficiency for the oxidation of the substrate water and, consequently, leads to the increase in the charge recombination probability in PS II, promoting the generation of the reactive oxygen species. The Ala-344-stop - A344G-stop double difference spectrum (Fig. 4c) showed the bands at 1403-1345 and 1565-1503 cm-1 in the carboxylate symmetric and asymmetric stretching regions, respectively. The band change induced by the Gly substitution is explained by assuming a shift of a single carboxylate group. As shown in Fig. 6, the observed double difference spectrum was reproduced by assuming the downshift of an Ala-344-stop S2/S1 band pair at 1360/1403 cm-1 (a, blue line)to 1346/1395 cm-1 (a, red line) or the downshift of an S2/S1 band pair at 1392/1404 cm-1 (b, blue line) to 1345/1360 cm-1 (b, red line). The latter (scheme b) is less likely because the presumed S2 bands were much smaller relative to the presumed S1 bands. This is, however, quite difficult to rationally explain. Alternatively, the observed band change was reproduced by the upshift of an S2/S1 band pair from 1395/1346 (or 1360/1345) cm-1 to 1403/1360 (or 1404/1392) cm-1, although the presumed shift opposes the expected trends of the ligand (5Noguchi T. Ono T.-A. Inoue Y. Biochim. Biophys. Acta. 1995; 1228: 189-200Crossref Scopus (164) Google Scholar, 35Smith J.C. Gonzalez-Vergara E. Vincent J.B. Inorg. Chim. Acta. 1997; 255: 99-103Crossref Scopus (32) Google Scholar). It was indicated that the symmetric stretching modes of the α-carboxylate group of the D1 C-terminal Ala-344 appear at ∼1356 cm-1 for the S1 state and at ∼1320 cm-1 for the S2 state (30Chu H.-A. Hiller W. Debus R.J. Biochemistry. 2004; 42: 3152-3166Crossref Scopus (140) Google Scholar), respectively. The downshift of the Ala-344 band has been interpreted to indicate the ligation of C-terminal carboxylate to the Mn ion, which is oxidized during the S1 to S2 transition. The lack of the C-terminal bands in the double difference spectrum shown in Fig. 4 indicates that the Gly substitution induces little change in the modes for the C-terminal carboxylate but results in a change of the other carboxylate ligand. A possible candidate of the Gly-affected modes is the carboxylate side chain mode of D1-Asp-342. This residue was assigned as a ligand for a Mn ion in the 3.5-Å x-ray structural model (19Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1837Crossref PubMed Scopus (2850) Google Scholar) in which the CH3 group of Ala-344 is arranged toward the Mn cluster. Therefore, the replacement of CH3 (Ala) with H (Gly) can influence the strength of the coordination bond between the Asp-342 carboxylate and a Mn ion. The absence of the Gly substitution effect on the C-terminal carboxylate modes may be related to the finding that the C-terminal carboxylate was disordered and not visible in the x-ray electron density map (19Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1837Crossref PubMed Scopus (2850) Google Scholar). Possibly, the C-terminal ligand is relatively flexible to be able to move to some extent with little change in the ligation geometry. It is of note in this context that the observed frequency difference between the asymmetric and symmetric modes (∼160 cm-1) for the putative Asp-342 carboxylate is similar to the value empirically found for the bidentate coordinate (5Noguchi T. Ono T.-A. Inoue Y. Biochim. Biophys. Acta. 1995; 1228: 189-200Crossref Scopus (164) Google Scholar, 30Chu H.-A. Hiller W. Debus R.J. Biochemistry. 2004; 42: 3152-3166Crossref Scopus (140) Google Scholar, 38Socrates G. Infrared and Raman Characteristic Group Frequencies. 3rd Ed. John Wiley & Sons, Chichester, UK1994Google Scholar), although the x-ray model indicated a unidentate coordinate of Asp-342 (19Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1837Crossref PubMed Scopus (2850) Google Scholar). According to Fig. 6a, the putative Asp-342 carboxylate band is downshifted by 43 cm-1 upon the S2-state formation, suggesting oxidation of the ligating Mn ion or a change in ligation structure during the S1 to S2 transition. Taking into account the possible ligation of the Ala-344 C-terminal carboxylate to the Mn ion that changes in oxidation state during the S1 to S2 transition (30Chu H.-A. Hiller W. Debus R.J. Biochemistry. 2004; 42: 3152-3166Crossref Scopus (140) Google Scholar), it may be assumed that the carboxylate side chain of Asp-342 and the α-carboxylate of Ala-344 participate in the ligation of the Mn ion that is oxidized upon the S2 formation. Most of the bands in the low frequency S2/S1 difference spectrum were changed upon Gly substitution as seen in Fig. 5. The affected bands possibly included the S2/S1 modes of the Mn-O-Mn stretching vibration of the Mn cluster at 606(+)/625(-) cm-1 (34Chu H.-A. Sackett H. Babcock G.T. Biochemistry. 2000; 39: 14371-14376Crossref PubMed Scopus (78) Google Scholar), Mn-COO- bending modes of putative carboxylate ligands (36Chu H.-A. Gardner M.T. O'Brien J.P. Babcock G.T. Biochemistry. 1999; 38: 4533-4541Crossref PubMed Scopus (49) Google Scholar), the ring torsion mode of histidine residues (37Hasegawa K. Ono T.-A. Noguchi T. J. Phys. Chem. B. 2000; 104: 4253-4265Crossref Scopus (166) Google Scholar), the amide IV (40% O = C-N bending) and amide VI (C = O bending) modes of polypeptide backbone (38Socrates G. Infrared and Raman Characteristic Group Frequencies. 3rd Ed. John Wiley & Sons, Chichester, UK1994Google Scholar), and ligand-dependent Mn-O modes at 532-420 cm-1 (31Kimura Y. Mizusawa N. Ishii A. Yamanari T. Ono T.-A. Biochemistry. 2003; 42: 13170-13177Crossref PubMed Scopus (68) Google Scholar, 39Visser H. Dubé C.E. Armstrong W.H. Sauer K. Yachandra V.K. J. Am. Chem. Soc. 2002; 124: 11008-11017Crossref PubMed Scopus (39) Google Scholar). The Gly substitution resulted in marked changes in the bands in the 400-350-cm-1 region, frequencies that include the stretching vibrations between the Mn ion and the ligands (O and/or N) (40Chu H.-A. Hillier W. Law N.A. Babcock G.T. Biochim. Biophys. Acta. 2001; 1503: 69-82Crossref PubMed Scopus (97) Google Scholar). These marked spectral changes upon Gly substitution suggest a gross structural change in the Mn cluster. Nevertheless, there are relatively insignificant changes in the mid-frequency region of the spectrum except for the putative Asp-342 mode. Interestingly, the D1-D170H OEC Synechocystis showed prominent changes in the low frequency S2/S1 spectrum (660-500 cm-1) but limited changes in the mid-frequency (1800-1200 cm-1) spectrum (20Chu H.-A. Debus R.J. Babcock G.T. Biochemistry. 2001; 40: 2312-2316Crossref PubMed Scopus (71) Google Scholar). A possible explanation for the different manifestation of the Gly substitution in the midand low frequency spectra is that the mid-frequency bands include the modes, which are not the direct ligands to the Mn cluster. Such indirect structural coupling may be less sensitive to intrastructural changes of the Mn cluster. The observed structural changes of the Mn cluster induced by the Gly substitution suggest that Ala-344 is crucial for maintaining the intrastructure of the Mn cluster as a ligand. Nevertheless, the present FTIR results do not preclude the possibility that the C-terminal Ala-344 carboxylate is located close to the Ca ion of the Mn3CaO4 cubane-like cluster core as proposed by the recent x-ray structural model (19Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1837Crossref PubMed Scopus (2850) Google Scholar). In this case, the present results suggest that Ca may participate in controlling the structural changes of the Mn cluster during the S1 to S2 transition and the C-terminal Ala is involved in this Ca-dependent function, although it is not clear how it is achieved at present. As shown in Fig. 3, ∼70% of the total A344G-stop OEC existed in the S = 5/2 g = 4.1 state, whereas more than 90% OEC existed in the S = 1/2 multiline state in the control Ala-344-stop. Some difference in the electronic structure within the Mn cluster has been proposed to be responsible for the appearance of these two states (41Kusunoki M. Murata N. Research in Photosynthesis. 2. Kluwer Academic Publishers, Dordrecht, The Netherlands1992: 297-300Google Scholar, 42Boussac A. Girerd J.-J. Rutherford A.W. Biochemistry. 1996; 35: 6984-6989Crossref PubMed Scopus (134) Google Scholar). The change in the intrastructure of the Mn cluster may induce considerable alterations in the low frequency modes of the Mn cluster with much smaller or little change in the mid-frequency modes indirectly coupled with the Mn cluster. The Sr2+-substituted OEC with an enhanced g = 4.1 signal showed the normal-like mid-frequency S2/S1 difference spectrum (43Kimura Y. Hasegawa K. Ono T. Biochemistry. 2002; 41: 5844-5853Crossref PubMed Scopus (42) Google Scholar) and the markedly altered low frequency spectrum (34Chu H.-A. Sackett H. Babcock G.T. Biochemistry. 2000; 39: 14371-14376Crossref PubMed Scopus (78) Google Scholar). Furthermore, little change of the mid-frequency S2/S1 spectrum has been reported upon conversion from the multiline to the g = 4.1 state by IR illumination (44Onoda K. Mino H. Inoue Y. Noguchi T. Photosyn. Res. 2000; 63: 47-57Crossref PubMed Scopus (32) Google Scholar). A possible difference between these two S2 ESR states is a valence exchange between strongly antiferro-magnetic Mn(IV) and Mn(III) (41Kusunoki M. Murata N. Research in Photosynthesis. 2. Kluwer Academic Publishers, Dordrecht, The Netherlands1992: 297-300Google Scholar, 42Boussac A. Girerd J.-J. Rutherford A.W. Biochemistry. 1996; 35: 6984-6989Crossref PubMed Scopus (134) Google Scholar). The valence exchange may alter the Mn-Mn and/or Mn-ligand interactions that affect the low frequency modes but scarcely influence the mid-frequency modes." @default.
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• W2092856305 title "Impact of Replacement of D1 C-terminal Alanine with Glycine on Structure and Function of Photosynthetic Oxygen-evolving Complex" @default.
• W2092856305 cites W1484450422 @default.
• W2092856305 cites W1503334333 @default.
• W2092856305 cites W1533581019 @default.
• W2092856305 cites W1961714136 @default.
• W2092856305 cites W1966876943 @default.
• W2092856305 cites W1968355282 @default.
• W2092856305 cites W1973378860 @default.
• W2092856305 cites W1983755158 @default.
• W2092856305 cites W1984489267 @default.
• W2092856305 cites W1996414124 @default.
• W2092856305 cites W1999632228 @default.
• W2092856305 cites W2000179616 @default.
• W2092856305 cites W2001467311 @default.
• W2092856305 cites W2002432567 @default.
• W2092856305 cites W2004707892 @default.
• W2092856305 cites W2014043594 @default.
• W2092856305 cites W2025691656 @default.
• W2092856305 cites W2033918244 @default.
• W2092856305 cites W2034923700 @default.
• W2092856305 cites W2035830004 @default.
• W2092856305 cites W2043260446 @default.
• W2092856305 cites W2048675843 @default.
• W2092856305 cites W2049211895 @default.
• W2092856305 cites W2049332437 @default.
• W2092856305 cites W2054826561 @default.
• W2092856305 cites W2057863409 @default.
• W2092856305 cites W2064128549 @default.
• W2092856305 cites W2072824838 @default.
• W2092856305 cites W2087448330 @default.
• W2092856305 cites W2088730308 @default.
• W2092856305 cites W2090990712 @default.
• W2092856305 cites W2094243059 @default.
• W2092856305 cites W2096781815 @default.
• W2092856305 cites W2098472363 @default.
• W2092856305 cites W2100021706 @default.
• W2092856305 cites W2105125469 @default.
• W2092856305 cites W2132108816 @default.
• W2092856305 cites W2400429696 @default.
• W2092856305 cites W420083224 @default.
• W2092856305 cites W4250220621 @default.
• W2092856305 cites W45895803 @default.
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