Title: Biosynthetic Exchange of Bromide for Chloride and Strontium for Calcium in the Photosystem II Oxygen-evolving Enzymes
Abstract: The active site for water oxidation in photosystem II goes through five sequential oxidation states (S0 to S4) before O2 is evolved. It consists of a Mn4Ca cluster close to a redox-active tyrosine residue (TyrZ). Cl- is also required for enzyme activity. To study the role of Ca2+ and Cl- in PSII, these ions were biosynthetically substituted by Sr2+ and Br-, respectively, in the thermophilic cyanobacterium Thermosynechococcus elongatus. Irrespective of the combination of the non-native ions used (Ca/Br, Sr/Cl, Sr/Br), the enzyme could be isolated in a state that was fully intact but kinetically limited. The electron transfer steps affected by the exchanges were identified and then investigated by using time-resolved UV-visible absorption spectroscopy, time-resolved O2 polarography, and thermoluminescence spectroscopy. The effect of the Ca2+/Sr2+ and Cl-/Br- exchanges was additive, and the magnitude of the effect varied in the following order: Ca/Cl < Ca/Br < Sr/Cl < Sr/Br. In all cases, the rate of O2 release was similar to that of the S3TyrZ. to S0TyrZ transition, with the slowest kinetics (i.e. the Sr/Br enzyme) being ≈6-7 slower than in the native Ca/Cl enzyme. This slowdown in the kinetics was reflected in a decrease in the free energy level of the S3 state as manifest by thermoluminescence. These observations indicate that Cl- is involved in the water oxidation mechanism. The possibility that Cl- is close to the active site is discussed in terms of recent structural models. The active site for water oxidation in photosystem II goes through five sequential oxidation states (S0 to S4) before O2 is evolved. It consists of a Mn4Ca cluster close to a redox-active tyrosine residue (TyrZ). Cl- is also required for enzyme activity. To study the role of Ca2+ and Cl- in PSII, these ions were biosynthetically substituted by Sr2+ and Br-, respectively, in the thermophilic cyanobacterium Thermosynechococcus elongatus. Irrespective of the combination of the non-native ions used (Ca/Br, Sr/Cl, Sr/Br), the enzyme could be isolated in a state that was fully intact but kinetically limited. The electron transfer steps affected by the exchanges were identified and then investigated by using time-resolved UV-visible absorption spectroscopy, time-resolved O2 polarography, and thermoluminescence spectroscopy. The effect of the Ca2+/Sr2+ and Cl-/Br- exchanges was additive, and the magnitude of the effect varied in the following order: Ca/Cl < Ca/Br < Sr/Cl < Sr/Br. In all cases, the rate of O2 release was similar to that of the S3TyrZ. to S0TyrZ transition, with the slowest kinetics (i.e. the Sr/Br enzyme) being ≈6-7 slower than in the native Ca/Cl enzyme. This slowdown in the kinetics was reflected in a decrease in the free energy level of the S3 state as manifest by thermoluminescence. These observations indicate that Cl- is involved in the water oxidation mechanism. The possibility that Cl- is close to the active site is discussed in terms of recent structural models. Light-driven water oxidation by the photosystem II (PSII) 3The abbreviations used are: PSII, photosystem II; P680, primary electron donor; Chl, chlorophyll; DCBQ, 2,6-dichloro-p-benzoquinone; PPBQ, phenyl-p-benzoquinone; EXAFS, extended X-ray absorption fine structure; Nd:YAG, neodymium-yttrium aluminum garnet; Pheo, pheophytin; QA and QB, primary and secondary quinone acceptors; MES, 4-morpholineethane-sulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. enzyme is responsible for the O2 on Earth and is at the origin of the production of most of the biomass. Refined three-dimensional x-ray structures at 3.5 and 3.0 Å resolution have been obtained by using PSII isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus (1Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1838Crossref PubMed Scopus (2855) Google Scholar, 2Loll B. Kern J. Saenger W. Zouni A. Biesiadka J. Nature. 2005; 438: 1040-1044Crossref PubMed Scopus (1611) Google Scholar). PSII is made up of more than 20 membrane protein subunits, 35-36 chlorophyll molecules, more than 10 carotenoid molecules, several lipids, two hemes, and the cofactors involved in the electron transfer reactions (1Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1838Crossref PubMed Scopus (2855) Google Scholar, 2Loll B. Kern J. Saenger W. Zouni A. Biesiadka J. Nature. 2005; 438: 1040-1044Crossref PubMed Scopus (1611) Google Scholar). Absorption of a photon by chlorophyll is followed by the formation of a radical pair in which the pheophytin molecule, PheoD1, is reduced and the accessory chlorophyll molecule, ChlD1, is oxidized (3Diner B.A. Schlodder E. Nixon P.J. Coleman W.J. Rappaport F. Lavergne J. Vermaas W.F. Chisholm D.A. Biochemistry. 2001; 40: 9265-9281Crossref PubMed Scopus (196) Google Scholar, 4Groot M.L. Pawlowicz N.P. van Wilderen L.J. Breton J. van Stokkum I.H. van Grondelle R. Proc. Natl. Acad. Sci. U. S., A. 2005; 102: 13087-13092Crossref PubMed Scopus (163) Google Scholar, 5Holzwarth A.R. Muller M.G. Reus M. Nowaczyk M. Sander J. Rögner M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6895-6900Crossref PubMed Scopus (256) Google Scholar). The cation is then stabilized on P680, a weakly coupled chlorophyll dimer (see e.g. Refs. 6Rappaport F. Diner B.A. Coord. Chem. Rev. 2007; 252: 259-272Crossref Scopus (214) Google Scholar and 7Renger G. Photosynth. Res. 2007; 92: 407-425Crossref PubMed Scopus (75) Google Scholar for energetic considerations). The pheophytin anion transfers the electron to a quinone, QA, which in turn reduces a second quinone, QB.P680+.. oxidizes a tyrosine residue of the D1 polypeptide, TyrZ, which in turn oxidizes the Mn4Ca cluster. The Mn4Ca cluster acts as a device for accumulating oxidizing equivalents and as the active site for water oxidation. During the enzyme cycle, the oxidizing side of PSII goes through five sequential redox states, denoted as Sn, where n varies from 0 to 4 upon the absorption of four photons (8Kok B. Forbush B. McGloin M.P. Photochem. Photobiol. 1970; 11: 457-475Crossref PubMed Scopus (1819) Google Scholar). Upon formation of the S4 state, two molecules of water are rapidly oxidized, the S0 state is regenerated, and O2 is released. The mechanism by which water is oxidized and O2 produced is still largely unknown (9Yano J. Kern J. Sauer K. Latimer M.J. Pushkar Y. Biesiadka J. Loll B. Saenger W. Messinger J. Zouni A. Yachandra V.K. Science. 2006; 314: 821-825Crossref PubMed Scopus (700) Google Scholar, 10McEvoy J.P. Brudvig G.W. Chem. Rev. 2006; 106: 4455-4483Crossref PubMed Scopus (1317) Google Scholar, 11Rutherford A.W. Boussac A. Science. 2004; 303: 1782-1784Crossref PubMed Scopus (116) Google Scholar, 12Debus R.J. Coord. Chem. Rev. 2007; 252: 244-258Crossref Scopus (143) Google Scholar, 13Hillier W. Wydrzynski T. Phys. Chem. Chem. Phys. 2004; 6: 4882-4889Crossref Scopus (119) Google Scholar, 14Messinger J. Phys. Chem. Chem. Phys. 2004; 6: 4764-4771Crossref Scopus (176) Google Scholar, 15Betley T.A. Surendranath Y. Childress M.V. Alliger G.E. Fu R. Cummins C.C. Nocera D.G. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2008; 263: 1293-1303Crossref Scopus (2) Google Scholar, 16Sproviero E.M. Shinopoulos K. Gascón J.A. McEvoy J.P. Brudvig G.W. Batista V.S. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2008; 363: 1149-1156Crossref PubMed Scopus (1) Google Scholar, 17Siegbahn P.E.M. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2008; 363: 1221-1228Crossref PubMed Scopus (54) Google Scholar). The geometry and ligand environment of the Mn4Ca cluster in the crystal structure is not clearly defined because the x-ray beam reduces the native high valence manganese cluster back to the MnII state (18Yano J. Kern J. Irrgang K.D. Latimer M.J. Bergmann U. Glatzel P. Pushkar Y. Biesiadka J. Loll B. Sauer K. Messinger J. Zouni A. Yachandra V.K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12047-12052Crossref PubMed Scopus (542) Google Scholar, 19Grabolle M. Haumann M. Muller C. Liebisch P. Dau H. J. Biol. Chem. 2006; 281: 4580-4588Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The transition from S (or S3TyrZ., which is kinetically indistinguishable) to S0 probably involves several reaction intermediates. These have largely escaped detection for the following reasons: 1) the rate constant of this transition is rapid (t½ ≈ 1 ms); 2) the reduction of TyrZ. is the limiting step for water oxidation in the native enzyme; and 3) experimental methods for trapping potential intermediate states are lacking (see Refs. 20Clausen J. Junge W. Nature. 2004; 430: 480-483Crossref PubMed Scopus (108) Google Scholar and 21Clausen J. Junge W. Dau H. Haumann M. Biochemistry. 2005; 44: 12775-12779Crossref PubMed Scopus (39) Google Scholar for a recent elegant thermodynamic approach). One strategy that could allow intermediates to be detected is to modify the enzyme, maintaining its capacity for turnover but impairing its kinetics. A change in the rate-limiting step could allow one or more intermediates to become detectable. Recently (22Boussac 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 (135) Google Scholar), we have shown that growing the thermophilic cyanobacterium T. elongatus in the presence of Sr2+ instead of Ca2+ resulted in the exchange of Ca2+ by Sr2+, and this produced a significant slowdown of the oxygen evolution rate. Among all of the cations tested, only Sr2+ can substitute for Ca2+ (23Ghanotakis D.F. Babcock G.T. Yocum C.F. FEBS Lett. 1984; 167: 127-130Crossref Scopus (328) Google Scholar, 24Debus R.J. Biochim. Biophys. Acta. 1992; 1102: 269-352Crossref PubMed Scopus (1085) Google Scholar, 25McEvoy J.P. Brudvig G.W. Phys. Chem. Chem. Phys. 2004; 6: 4754-4763Crossref Scopus (191) Google Scholar, 26Yocum C.F. Coord. Chem. Rev. 2008; 252: 296-305Crossref Scopus (211) Google Scholar). The effects of Ca2+/Sr2+ exchange have been studied by EPR (22Boussac 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 (135) Google Scholar, 27Boussac A. Sugiura M. Lai T.-L. Rutherford A.W. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2008; 363: 1203-1210Crossref PubMed Scopus (32) Google Scholar, 28Boussac A. Rutherford A.W. Biochemistry. 1988; 27: 3476-3483Crossref Scopus (228) Google Scholar, 29Boussac A. Sugiura M. Inoue Y. Rutherford A.W. Biochemistry. 2000; 39: 13788-13799Crossref PubMed Scopus (62) Google Scholar), Fourier transform infrared spectroscopy (30Strickler M.A. Walker L.M. Hillier W. Debus R.J. Biochemistry. 2005; 44: 8571-8577Crossref PubMed Scopus (93) Google Scholar, 31Kimura Y. Hasegawa K. Yamanari T. Ono T.-A. Photosynth. Res. 2005; 84: 245-250Crossref PubMed Scopus (19) Google Scholar, 32Suzuki H. Taguchi Y. Sugiura M. Boussac A. Noguchi T. Biochemistry. 2006; 45: 13454-13464Crossref PubMed Scopus (48) Google Scholar), EXAFS spectroscopy (33Cinco R.M. Robblee J.H. Messinger J. Fernandez C. Holman K.L.M. Sauer K. Yachandra V.K. Biochemistry. 2004; 43: 13271-13282Crossref PubMed Scopus (53) Google Scholar, 34Pushkar Y. Yano J. Sauer K. Boussac A. Yachandra V.K. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 1879-1884Crossref PubMed Scopus (158) Google Scholar), mass spectrometry experiments monitoring the water substrate exchange rates (35Hendry G. Wydrzynski T. Biochemistry. 2003; 42: 6209-6217Crossref PubMed Scopus (114) Google Scholar), and time-resolved UV-visible spectroscopy (22Boussac 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 (135) Google Scholar). Ca2+/Sr2+ exchange has a slight effect on the geometry of the manganese cluster as detected by EPR (27Boussac A. Sugiura M. Lai T.-L. Rutherford A.W. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2008; 363: 1203-1210Crossref PubMed Scopus (32) Google Scholar, 28Boussac A. Rutherford A.W. Biochemistry. 1988; 27: 3476-3483Crossref Scopus (228) Google Scholar). The kinetics of O2 release and the S3TyrZ. to S0TyrZ transition are slowed down to the same extent (22Boussac 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 (135) Google Scholar). The affinity of the slowest exchangeable water substrate molecule bound in the S3 state is decreased (35Hendry G. Wydrzynski T. Biochemistry. 2003; 42: 6209-6217Crossref PubMed Scopus (114) Google Scholar). These studies indicate the involvement of Ca2+ (or Sr2+) in the catalytic cycle (10McEvoy J.P. Brudvig G.W. Chem. Rev. 2006; 106: 4455-4483Crossref PubMed Scopus (1317) Google Scholar, 36Barber J. Ferreira K. Maghlaoui K. Iwata S. Phys. Chem. Chem. Phys. 2004; 6: 4737-4742Crossref Scopus (95) Google Scholar, 37Meelich K. Zaleski C.M. Pecoraro V.L. Phil. Trans. R. Soc. B. 2008; 363: 1271-1281Crossref PubMed Scopus (23) Google Scholar). When Ca2+ is removed from its site, manganese oxidation can still take place, allowing the formation of the S2 state, but in the following step, the normal S3 state is not formed. Instead, an alternative, abnormally stable form appears to be induced in which the Mn4 cluster is in the same redox state as it was in S2 state but in magnetic interaction with a radical (spin = 1/2) (38Boussac A. Zimmermann J.-L. Rutherford A.W. Biochemistry. 1989; 28: 8984-8989Crossref PubMed Scopus (247) Google Scholar), likely TyrZ. (39Tang X.S. Randall D.W. Force D.A. Diner B.A. Britt R.D. J. Am. Chem. Soc. 1996; 118: 7638-7639Crossref Scopus (111) Google Scholar, 40Un S. Boussac A. Sugiura M. Biochemistry. 2007; 46: 3138-3150Crossref PubMed Scopus (30) Google Scholar), giving rise to a characteristic EPR signal that is known as the split signal. Choride is also an essential ion for PSII activity (24Debus R.J. Biochim. Biophys. Acta. 1992; 1102: 269-352Crossref PubMed Scopus (1085) Google Scholar, 26Yocum C.F. Coord. Chem. Rev. 2008; 252: 296-305Crossref Scopus (211) Google Scholar, 41Homann P.H. Photosynth. Res. 2002; 73: 169-175Crossref PubMed Scopus (38) Google Scholar, 42Wincencjusz H. Yocum C.F. van Gorkom H.J. Biochemistry. 1999; 38: 3719-3725Crossref PubMed Scopus (78) Google Scholar, 43Wincencjusz H. Yocum C.F. van Gorkom H.J. Biochemistry. 1998; 37: 8595-8604Crossref PubMed Scopus (49) Google Scholar, 44Hasegawa K. Kimura Y. Ono T.-A. Biochemistry. 2002; 41: 13839-13850Crossref PubMed Scopus (53) Google Scholar, 45Haumann M. Barra M. Loja P. Loscher S. Krivanek R. Grundmeier A. Andreasson L.-E. Dau H. Biochemistry. 2006; 45: 13101-13107Crossref PubMed Scopus (54) Google Scholar, 46vanVliet P. Rutherford A.W. Biochemistry. 1996; 35: 1829-1839Crossref PubMed Scopus (78) Google Scholar, 47Boussac A. Sétif P. Rutherford A.W. Biochemistry. 1992; 31: 1224-1234Crossref PubMed Scopus (113) Google Scholar, 48Boussac A. Chem. Phys. 1995; 194: 409-418Crossref Scopus (17) Google Scholar, 49Homann, P. H., and Inoue, Y. (1986) in Ion Interactions in Energy Transfer Biomembranes (Papageorgiou, G., Barber, J., and Papa, S., eds) pp. 291-302, Plenum Press, New YorkGoogle Scholar, 50Wincencjusz H. van Gorkom H.J. Yocum C.F. Biochemistry. 1997; 36: 3663-3670Crossref PubMed Scopus (129) Google Scholar, 51Lindberg K. Vänngård T. Andréasson L.-E. Photosynth. Res. 1993; 38: 401-408Crossref PubMed Scopus (66) Google Scholar, 52Lindberg K. Andréasson L.-E. Biochemistry. 1996; 35: 14259-14267Crossref PubMed Scopus (89) Google Scholar, 53Ono T. Zimmermann J.-L. Inoue Y. Rutherford A.W. Biochim. Biophys. Acta. 1986; 851: 193-201Crossref Scopus (144) Google Scholar). In plant PSII, removal of Cl- inhibits oxygen evolution and perturbs the Mn4 cluster to a variable extent depending on the precise Cl--depletion method used (24Debus R.J. Biochim. Biophys. Acta. 1992; 1102: 269-352Crossref PubMed Scopus (1085) Google Scholar, 26Yocum C.F. Coord. Chem. Rev. 2008; 252: 296-305Crossref Scopus (211) Google Scholar, 46vanVliet P. Rutherford A.W. Biochemistry. 1996; 35: 1829-1839Crossref PubMed Scopus (78) Google Scholar). The removal of Cl- by incubation in Cl--free buffer increased the proportion of the Mn4 cluster in the high-spin state (g = 4 EPR signal), but the enzyme continues to work at a reduced rate (46vanVliet P. Rutherford A.W. Biochemistry. 1996; 35: 1829-1839Crossref PubMed Scopus (78) Google Scholar). When Cl- is removed by a high pH shock, the high-spin state is formed, and the S-state cycle is blocked after S2 formation in the majority of centers, whereas treatment with SO 2-4 inhibits the enzyme but allows radical (TyrZ.) formation in the presence of S2, giving rise to the split EPR signal, at least in a fraction of the centers (46vanVliet P. Rutherford A.W. Biochemistry. 1996; 35: 1829-1839Crossref PubMed Scopus (78) Google Scholar, 47Boussac A. Sétif P. Rutherford A.W. Biochemistry. 1992; 31: 1224-1234Crossref PubMed Scopus (113) Google Scholar, 53Ono T. Zimmermann J.-L. Inoue Y. Rutherford A.W. Biochim. Biophys. Acta. 1986; 851: 193-201Crossref Scopus (144) Google Scholar, 54Baumgarten M. Philo J.S. Dismukes G.C. Biochemistry. 1990; 29: 10814-10822Crossref PubMed Scopus (93) Google Scholar). In the SO 2-4-treated enzyme the electron donation rate from TyrZ to P680+. is not greatly affected on the first two flashes (47Boussac A. Sétif P. Rutherford A.W. Biochemistry. 1992; 31: 1224-1234Crossref PubMed Scopus (113) Google Scholar), but Cl- is required to progress through the S2 to S3 (46vanVliet P. Rutherford A.W. Biochemistry. 1996; 35: 1829-1839Crossref PubMed Scopus (78) Google Scholar, 47Boussac A. Sétif P. Rutherford A.W. Biochemistry. 1992; 31: 1224-1234Crossref PubMed Scopus (113) Google Scholar, 50Wincencjusz H. van Gorkom H.J. Yocum C.F. Biochemistry. 1997; 36: 3663-3670Crossref PubMed Scopus (129) Google Scholar) and S3 to S0 transitions (50Wincencjusz H. van Gorkom H.J. Yocum C.F. Biochemistry. 1997; 36: 3663-3670Crossref PubMed Scopus (129) Google Scholar). When the first PSII structural models from crystallography appeared, the resolution was not good enough to detect the Ca2+ ion (55Zouni A. Witt H.T. Kern J. Fromme P. Krauss N. Saenger W. Orth P. Nature. 2001; 409: 739-743Crossref PubMed Scopus (1762) Google Scholar, 56Kamiya N. Shen J.-R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 98-103Crossref PubMed Scopus (994) Google Scholar), and the question arose whether Ca2+ really was intimately associated with the Mn4 cluster in cyanobacteria as was thought to be the case in plants. This prompted us to develop a method for biosynthetic Ca2+/Sr2+ exchange in T. elongatus (22Boussac 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 (135) Google Scholar). We showed that there is one Sr2+ per PSII in fully active cyanobacterial PSII (22Boussac 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 (135) Google Scholar). At the same time, a structure with 3.5 Å resolution was reported in which a Ca2+ ion in close interaction with the Mn4 cluster was identified based on anomalous diffraction data (1Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1838Crossref PubMed Scopus (2855) Google Scholar). Recently, x-ray crystallography and EXAFS spectroscopy using the Sr2+-containing enzyme confirmed that Sr2+ was associated with the Mn4 cluster and was located in a position similar to that of Ca2+ (34Pushkar Y. Yano J. Sauer K. Boussac A. Yachandra V.K. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 1879-1884Crossref PubMed Scopus (158) Google Scholar, 57Kargul J. Maghlaoui K. Murray J.W. Deak Z. Boussac A. Rutherford A.W. Vass I. Barber J. Biochim. Biophys. Acta. 2007; 1767: 404-413Crossref PubMed Scopus (34) Google Scholar). The situation for chloride is less clear. No chloride ions are defined in the current three-dimensional structural models of the enzyme (1Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1838Crossref PubMed Scopus (2855) Google Scholar, 2Loll B. Kern J. Saenger W. Zouni A. Biesiadka J. Nature. 2005; 438: 1040-1044Crossref PubMed Scopus (1611) Google Scholar). This is partly because the resolution is insufficient but also because the structure of the cluster is perturbed by the x-ray beam, which reduces the high valence manganese cluster to the MnII state (18Yano J. Kern J. Irrgang K.D. Latimer M.J. Bergmann U. Glatzel P. Pushkar Y. Biesiadka J. Loll B. Sauer K. Messinger J. Zouni A. Yachandra V.K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12047-12052Crossref PubMed Scopus (542) Google Scholar, 19Grabolle M. Haumann M. Muller C. Liebisch P. Dau H. J. Biol. Chem. 2006; 281: 4580-4588Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The role and location of chloride as a cofactor in PSII from cyanobacteria thus remain open. For this reason, Cl-/Br- exchange experiments are potentially interesting. Previously reported Cl-/Br- exchange studies were done in chloride-depleted PSII. Depending on the Cl--depletion procedure (e.g. sulfate treatment at high pH versus extrinsic polypeptide depletion), the Br- reconstitution may have different effects. These differences can be explained by the presence of more than one chloride-binding site in PSII, the activity of which would depend on the binding of chloride to a high affinity site; and this activity could be modulated by the binding of another chloride to a low affinity binding site (46vanVliet P. Rutherford A.W. Biochemistry. 1996; 35: 1829-1839Crossref PubMed Scopus (78) Google Scholar, 48Boussac A. Chem. Phys. 1995; 194: 409-418Crossref Scopus (17) Google Scholar). An alternative interpretation has been suggested, namely that many of the effects induced by Cl- depletion are depletion-induced artifacts and that there is a single Cl--binding site that has only an indirect effect on enzyme function. Indeed, in this interpretation Cl- is not considered necessary for enzyme activity (51Lindberg K. Vänngård T. Andréasson L.-E. Photosynth. Res. 1993; 38: 401-408Crossref PubMed Scopus (66) Google Scholar, 52Lindberg K. Andréasson L.-E. Biochemistry. 1996; 35: 14259-14267Crossref PubMed Scopus (89) Google Scholar). As in the case of Ca2+/Sr2+, the biosynthetic substitution of Br- for Cl- could provide a "bromide phenotype" that does not suffer from the ambiguities associated with biochemical exchange procedures. The study of the bromide exchanged PSII could yield evidence for the involvement of Cl- in the water-splitting process and provide new insights on the role of chloride in the water oxidation mechanism. Here we present the results of this biosynthetic Cl-/Br- replacement study using T. elongatus with either Ca2+ or Sr2+ in the active site. Fully intact PSII preparations containing Ca/Cl, Ca/Br, Sr/Cl, and Sr/Br were analyzed using a combination of spectroscopic and enzymological studies. Culture of the Cells and Purification of Thylakoids and PSII— Thylakoids, and thence PSII, were purified from a T. elongatus strain that had a His6 tag on the CP43 subunit (58Sugiura M. Inoue Y. Plant Cell Physiol. 1999; 40: 1219-1231Crossref PubMed Scopus (149) Google Scholar) and in which the psbA1 and psbA2 genes were deleted (WT*) (59Sugiura, M., Boussac, A., Noguchi, T., and Rappaport, F. (2008) Biochim. Biophys. Acta, 1777, 331-342Google Scholar). The D1 protein in PSII can potentially be encoded by three genes, psbA1, psbA2, and psbA3, each differing slightly. Because in this WT* T. elongatus only the psbA3 gene is present, the enzyme is not affected by the possible heterogeneity in D1 that would result from the expression of more than one of the three psbA genes. Such a situation could occur, for example, because of regulatory mechanisms triggered when changing light intensity during batch culture (60Kós P.B. Deák Z. Cheregi O. Vass I. Biochim. Biophys. Acta. 2008; 1777: 74-83Crossref PubMed Scopus (90) Google Scholar). The WT* cells were grown in 1 liter of culture medium (58Sugiura M. Inoue Y. Plant Cell Physiol. 1999; 40: 1219-1231Crossref PubMed Scopus (149) Google Scholar) in 3-liter Erlenmeyer flasks in a rotary shaker with a CO2-enriched atmosphere at 45 °C under continuous light (80 μmol of photons·m-2·s-1). The culture medium was supplemented with either 0.8 mm CaX2 or SrX2 (X was either bromide or chloride depending on the experiments). The grade of the chemicals used was ≥99.999% for CaBr2, SrCl2, and SrBr2 and ≥99.99% for CaCl2. The chloride content was ≤0.001% in glycerol, ≤0.02% in betaine, and ≤0.005% in MES. In the culture medium and before the addition of the CaX2 or SrX2 salts, the Ca2+ contamination was measured by ICP (22Boussac 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 (135) Google Scholar) to be less than 1.5 μm. The Cl- contamination can be estimated from the specifications given by the suppliers of the chemicals to be ≤40 μm (essentially from the Tricine buffer). Thylakoids and PSII were prepared as described earlier (61Sugiura M. Rappaport F. Brettel K. Noguchi T. Rutherford A.W. Boussac A. Biochemistry. 2004; 43: 13549-13563Crossref PubMed Scopus (63) Google Scholar) with the exception that no polyethylene glycol was used to concentrate the samples. Instead, the samples were concentrated by using Amicon Ultra-15 concentrator devices (Millipore) with a 100-kDa cut-off. Routinely, the total amount of Chl after the breaking of the cells was ≈180 mg, and the yield after PSII purification in terms of Chl amounts was ≈4-5%. Thylakoids and PSII were stored in liquid nitrogen at a concentration of about 1.5-2 mg Chl/ml in a medium containing 10% glycerol, 1 m betaine, 15 mm CaX2, 15 mm MgX2, and 40 mm MES, pH 6.5 (pH adjusted with NaOH), until they were used. Oxygen Evolution under Continuous Light—Oxygen evolution of PSII under continuous light was measured at 25 °C by polarography using a Clark-type oxygen electrode (Hansatech) with saturating white light at a Chl concentration of 5 μg of Chl·ml-1 in the media described above. A total of 0.5 mm DCBQ (2,6-dichloro-p-benzoquinone, dissolved in dimethyl sulfoxide) was added as an electron acceptor. The betaine, DCBQ, and QB react in the time range of minutes in the presence of O2. For that reason, measurements of PSII activity were done at ≤1 min after the addition of DCBQ. Flash-induced Oxygen Evolution—Oxygen evolution under flashing light was measured with a laboratory-made rate electrode (22Boussac 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 (135) Google Scholar). Two combinations of electrodes were used: either Pt/Ag/AgCl or Pt/Ag/AgBr, depending on the conditions. The Ag/AgCl and Ag/AgBr electrodes were prepared by using 1-2 m HCl or HBr acids, respectively. Thylakoids were used at 1.2 mg Chl·ml-1. Typically, 25 μl of a thylakoid suspension was put onto the platinum electrode. The volume of the circulating medium was ≈250 ml and contained 10% glycerol, 1 m betaine, 15 mm CaX2, 15 mm MgX2, 50 mm KX, and 40 mm MES, pH 6.5 (pH adjusted with NaOH). Illumination was done with a xenon flash (PerkinElmer Optoelectronics). The intensity of the flash was adjusted so that the light intensity was saturating (i.e. the miss parameter was minimum). Measurements were done at room temperature (20-25 °C). The amplified amperometric signal resulting from the flash-induced oxygen evolution was recorded with a numerical oscilloscope. UV-visible Absorption Change Spectroscopy—Absorption changes were measured with a laboratory-built spectrophotometer where the absorption changes were sampled at discrete times by short flashes (62Béal D. Rappaport F. Joliot P. Rev. Sci. Instrum. 1999; 70: 202-207Crossref Scopus (80) Google Scholar). These flashes were provided by a neodymium-yttrium aluminum garnet (Nd:YAG) pumped (355 nm) optical parametric oscillator, which produces monochromatic flashes (1 nm full-width at half-maximum) with a duration of 6 ns. Excitation at 685 nm was provided by a dye laser pumped by a frequency-doubled Nd:YAG laser. The path length of the cell was 2.5 mm. PSII was used at 25 μg of Chl·ml-1 in 10% glycerol, 1 m betaine, 15 mm CaX2, 15 mm MgX2, and 40 mm MES, pH 6.5 (pH adjusted with NaOH). After dark adaptation for 1 h at room temperature (20-22 °C), 0.1 mm PPBQ dissolved in dimethyl sulfoxide was added as an electron acceptor. PPBQ was used here instead of DCBQ because of the length of the experiment (see above). Thermoluminescence Experiments—Thermoluminescence glow curves were measured with a laboratory-built apparatus (63Ducruet J.-M. J. Exp. Bot. 2003; 54: 2419-2430Crossref PubMed Scopus (113) Google Scholar). PSII samples at 10 μg Chl·ml-1 were first dark-adapted at room temperature for 1 h. Illumination was done by using saturating xenon flashes (PerkinElmer Optoelectronics) at 5 °C. Then the samples were frozen to -10 °C in 5 s. After an additional 5 s at -10 °C, the frozen samples were heated at a constant rate (0.33 °C/s). From the lifetime of the S2 and S3 states at room temperature (22Boussac 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 (135) Google Scholar), these states can be considered as st