Biosynthetic Ca2+/Sr2+ Exchange in the Photosystem II Oxygen-evolving Enzyme of Thermosynechococcus elongatus
2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês
10.1074/jbc.m401677200
ISSN1083-351X
AutoresAlain Boussac, Fabrice Rappaport, Patrick Carrier, Jean‐Marc Verbavatz, Renée Gobin, Diana Kirilovsky, A. William Rutherford, Miwa Sugiura,
Tópico(s)Algal biology and biofuel production
ResumoThe thermophilic cyanobacterium, Thermosynechococcus elongatus, has been grown in the presence of Sr2+ instead of Ca2+ with the aim of biosynthetically replacing the Ca2+ of the oxygen-evolving enzyme with Sr2+. Not only were the cells able to grow normally with Sr2+, they actively accumulated the ion to levels higher than those of Ca2+ in the normal cultures. A protocol was developed to purify a fully active Sr2+-containing photosystem II (PSII). The modified enzyme contained a normal polypeptide profile and 1 strontium/4 manganese, indicating that the normal enzyme contains 1 calcium/4 manganese. The Sr2+- and Ca2+-containing enzymes were compared using EPR spectroscopy, UV-visible absorption spectroscopy, and O2 polarography. The Ca2+/Sr2+ exchange resulted in the modification of the EPR spectrum of the manganese cluster and a slower turnover of the redox cycle (the so-called S-state cycle), resulting in diminished O2 evolution activity under continuous saturating light: all features reported previously by biochemical Ca2+/Sr2+ exchange in plant PSII. This allays doubts that these changes could be because of secondary effects induced by the biochemical treatments themselves. In addition, the Sr2+-containing PSII has other kinetics modifications: 1) it has an increased stability of the S3 redox state; 2) it shows an increase in the rate of electron donation from TyrD, the redox-active tyrosine of the D2 protein, to the oxygen-evolving complex in the S3-state forming S2; 3) the rate of oxidation of the S0-state to the S1-state by TyrD. is increased; and 4) the release of O2 is slowed down to an extent similar to that seen for the slowdown of the S3TyrZ. to S0TyrZ transition, consistent with the latter constituting the limiting step of the water oxidation mechanism in Sr2+-substituted enzyme as well as in the normal enzyme. The replacement of Ca2+ by Sr2+ appears to have multiple effects on kinetics properties of the enzyme that may be explained by S-state-dependent shifts in the redox properties of both the manganese complex and TyrZ as well as structural effects. The thermophilic cyanobacterium, Thermosynechococcus elongatus, has been grown in the presence of Sr2+ instead of Ca2+ with the aim of biosynthetically replacing the Ca2+ of the oxygen-evolving enzyme with Sr2+. Not only were the cells able to grow normally with Sr2+, they actively accumulated the ion to levels higher than those of Ca2+ in the normal cultures. A protocol was developed to purify a fully active Sr2+-containing photosystem II (PSII). The modified enzyme contained a normal polypeptide profile and 1 strontium/4 manganese, indicating that the normal enzyme contains 1 calcium/4 manganese. The Sr2+- and Ca2+-containing enzymes were compared using EPR spectroscopy, UV-visible absorption spectroscopy, and O2 polarography. The Ca2+/Sr2+ exchange resulted in the modification of the EPR spectrum of the manganese cluster and a slower turnover of the redox cycle (the so-called S-state cycle), resulting in diminished O2 evolution activity under continuous saturating light: all features reported previously by biochemical Ca2+/Sr2+ exchange in plant PSII. This allays doubts that these changes could be because of secondary effects induced by the biochemical treatments themselves. In addition, the Sr2+-containing PSII has other kinetics modifications: 1) it has an increased stability of the S3 redox state; 2) it shows an increase in the rate of electron donation from TyrD, the redox-active tyrosine of the D2 protein, to the oxygen-evolving complex in the S3-state forming S2; 3) the rate of oxidation of the S0-state to the S1-state by TyrD. is increased; and 4) the release of O2 is slowed down to an extent similar to that seen for the slowdown of the S3TyrZ. to S0TyrZ transition, consistent with the latter constituting the limiting step of the water oxidation mechanism in Sr2+-substituted enzyme as well as in the normal enzyme. The replacement of Ca2+ by Sr2+ appears to have multiple effects on kinetics properties of the enzyme that may be explained by S-state-dependent shifts in the redox properties of both the manganese complex and TyrZ as well as structural effects. The evolution of oxygen as a result of light-driven water oxidation is catalyzed by photosystem II (PSII) 1The abbreviations used are: PSII, photosystem II; PSI, photosystem I; Chl, chlorophyll; EPR, electron paramagnetic resonance; ICP, inductively coupled plasma optical emission spectrometry; EXAFS, extended x-ray absorption fine structure; MES, 2-(N-morpholino)ethanesulfonic acid; CHES, 2-(N-cyclohexylamino)ethanesulfonic acid; PPBQ, phenyl-p-benzoquinone; DCBQ, 2,6-dichlorophenylbenzoquinone; Nd:YAG, neodymium:yttriumaluminum garnet; W, watt(s); J, joule(s); PBS, phosphate-buffered saline; β-DM, n-dodecyl-β-maltoside.1The abbreviations used are: PSII, photosystem II; PSI, photosystem I; Chl, chlorophyll; EPR, electron paramagnetic resonance; ICP, inductively coupled plasma optical emission spectrometry; EXAFS, extended x-ray absorption fine structure; MES, 2-(N-morpholino)ethanesulfonic acid; CHES, 2-(N-cyclohexylamino)ethanesulfonic acid; PPBQ, phenyl-p-benzoquinone; DCBQ, 2,6-dichlorophenylbenzoquinone; Nd:YAG, neodymium:yttriumaluminum garnet; W, watt(s); J, joule(s); PBS, phosphate-buffered saline; β-DM, n-dodecyl-β-maltoside. in which a cluster of 4 manganese ions acts both as a device for accumulating oxidizing equivalents and as the active site. The reaction center of PSII is made up of two membrane-spanning polypeptides (D1 and D2) that bear the redox cofactors involved in the main electron transfer route. Absorption of a photon results in a charge separation between a chlorophyll molecule (P680), and a pheophytin molecule. The pheophytin anion transfers the electron to a quinone, QA, and P680+ is reduced by a tyrosine residue, TyrZ, that in turn is reduced by the Mn4 cluster. During the enzyme cycle, the oxidizing side of PSII goes through five different redox states that are denoted Sn, n varying from 0 to 4. Oxygen is released during the S3 to S0 transition in which S4 is a transient state (reviewed in Refs. 1Debus R.J. Biochim. Biophys. Acta. 1992; 1102: 269-352Crossref PubMed Scopus (1084) Google Scholar, 2Peloquin J.M. Britt R.D. Biochim. Biophys. Acta. 2001; 1503: 96-111Crossref PubMed Scopus (183) Google Scholar, 3Barber J. Curr. Opin. Struct. Biol. 2002; 12: 523-530Crossref PubMed Scopus (117) Google Scholar, 4Robblee J.H. Cinco R.M. Yachandra V.K. Biochim. Biophys. Acta. 2001; 1503: 7-23Crossref PubMed Scopus (182) Google Scholar).Ca2+ ions are known to be required for enzyme activity (1Debus R.J. Biochim. Biophys. Acta. 1992; 1102: 269-352Crossref PubMed Scopus (1084) Google Scholar, 5Yocun C.F. Biochim. Biophys. Acta. 1991; 1059: 1-15Crossref Scopus (161) Google Scholar, 6Ananyev G.A. Zaltsman L. Vasko C. Dismukes G.C. Biochim. Biophys. Acta. 2001; 1503: 52-68Crossref PubMed Scopus (105) Google Scholar, 7Boussac A. Rutherford A.W. Biochemistry. 1988; 27: 3476-3483Crossref Scopus (225) Google Scholar, 8Ghanotakis D.F. Babcock G.T. Yocum C.F. FEBS Lett. 1984; 167: 120-130Crossref Scopus (328) Google Scholar). The role of Ca2+ in PSII oxygen evolution has been the focus of numerous articles in the last 20 years. Most of our knowledge on the role of Ca2+ comes from studies on plant PSII. In PSII from plants, 1 Ca2+ is associated with the chlorophyll-binding protein CP29 (9Jegerschold C. Rutherford A.W. Mattioli T.A. Crimi M. Bassi R. J. Biol. Chem. 2000; 275: 12781-12788Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), a protein that is absent in cyanobacteria, and the second Ca2+ ion is required for water oxidation to take place (1Debus R.J. Biochim. Biophys. Acta. 1992; 1102: 269-352Crossref PubMed Scopus (1084) Google Scholar, 5Yocun C.F. Biochim. Biophys. Acta. 1991; 1059: 1-15Crossref Scopus (161) Google Scholar, 10Boussac A. Rutherford A.W. Photosynth. Res. 1992; 32: 207-209Crossref PubMed Scopus (9) Google Scholar, 11Adelroth P. Lindberg K. Andréasson L.-E. Biochemistry. 1995; 34: 9021-9027Crossref PubMed Scopus (80) Google Scholar, 12Van der Meulen K.A. Hobson A. Yocum C.F. Biochim. Biophys. Acta. 2004; (in press)Google Scholar). A specific binding site appears during the assembly of the Mn4 cluster (6Ananyev G.A. Zaltsman L. Vasko C. Dismukes G.C. Biochim. Biophys. Acta. 2001; 1503: 52-68Crossref PubMed Scopus (105) Google Scholar), and its replacement with Sr2+ perturbs the Mn4 EPR properties (7Boussac A. Rutherford A.W. Biochemistry. 1988; 27: 3476-3483Crossref Scopus (225) Google Scholar). No other metal ions are able to reconstitute significant enzyme activity (1Debus R.J. Biochim. Biophys. Acta. 1992; 1102: 269-352Crossref PubMed Scopus (1084) Google Scholar, 5Yocun C.F. Biochim. Biophys. Acta. 1991; 1059: 1-15Crossref Scopus (161) Google Scholar, 8Ghanotakis D.F. Babcock G.T. Yocum C.F. FEBS Lett. 1984; 167: 120-130Crossref Scopus (328) Google Scholar, 13Vrettos J.S. Brudvig G.W. Phil. Trans. R. Soc. London B. 2002; 357: 1395-1404Crossref PubMed Scopus (48) Google Scholar). The reconstitution of Ca2+-depleted plant PSII with Sr2+ restores approximately 40% of the oxygen evolution activity (7Boussac A. Rutherford A.W. Biochemistry. 1988; 27: 3476-3483Crossref Scopus (225) Google Scholar, 8Ghanotakis D.F. Babcock G.T. Yocum C.F. FEBS Lett. 1984; 167: 120-130Crossref Scopus (328) Google Scholar, 14Boussac A. Sétif P. Rutherford A.W. Biochemistry. 1992; 31: 1224-1234Crossref PubMed Scopus (112) Google Scholar). This decreased activity is because of a slowdown of the S-state transitions (7Boussac A. Rutherford A.W. Biochemistry. 1988; 27: 3476-3483Crossref Scopus (225) Google Scholar, 14Boussac A. Sétif P. Rutherford A.W. Biochemistry. 1992; 31: 1224-1234Crossref PubMed Scopus (112) Google Scholar, 15Westphal K.L. Lydakis-Simantiris N. Cukier R.I. Babcock G.T. Biochemistry. 2000; 39: 16220-16229Crossref PubMed Scopus (44) Google Scholar).When Ca2+ (or Sr2+) is removed from its site, manganese oxidation can still take place allowing formation of S2, but on the following step, the normal S3-state is not formed (16Boussac A. Zimmermann J.-L. Rutherford A.W. Biochemistry. 1989; 28: 8984-8989Crossref PubMed Scopus (246) Google Scholar, 17Boussac A. Zimmermann J.-L. Rutherford A.W. Lavergne J. Nature. 1990; 347: 303-306Crossref Scopus (195) Google Scholar). Instead, an abnormally stable form of the TyrZ. is generated (18Gilchrist M.L. Ball J.A. Randall D.W. Britt R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9545-9549Crossref PubMed Scopus (237) Google Scholar, 19Tang X.S. Randall D.W. Force D.A. Diner B.A. Britt R.D.J. J. Am. Chem. Soc. 1996; 118: 7638-7639Crossref Scopus (111) Google Scholar) that interacts magnetically with the manganese (still in the S2-state) (16Boussac A. Zimmermann J.-L. Rutherford A.W. Biochemistry. 1989; 28: 8984-8989Crossref PubMed Scopus (246) Google Scholar, 17Boussac A. Zimmermann J.-L. Rutherford A.W. Lavergne J. Nature. 1990; 347: 303-306Crossref Scopus (195) Google Scholar). In addition, the rate of oxidation of TyrZ is slowed down by several orders of magnitude when Ca2+ is absent (Ref. 14Boussac A. Sétif P. Rutherford A.W. Biochemistry. 1992; 31: 1224-1234Crossref PubMed Scopus (112) Google Scholar, see also Refs. 20Voelker M. Eckert H.J. Renger G. Biochim. Biophys. Acta. 1987; 890: 66-76Crossref Scopus (31) Google Scholar and 21Haumann M. Junge W. Biochim. Biophys. Acta. 1999; 1411: 121-133Crossref PubMed Scopus (48) Google Scholar), suggesting a role for Ca2+ in the deprotonation of TyrZ. Other suggested roles for Ca2+ include: 1) controlling substrate and Cl– access to the active site (22Rutherford A.W. Trends Biochem. Sci. 1989; 14: 227-232Abstract Full Text PDF PubMed Scopus (264) Google Scholar) and 2) acting as a substrate water site (23Pecoraro V.L. Baldwin M.J. Caudle M.T. Hsieh W.Y. Law N.A. Pure App. Chem. 1998; 70: 925-929Crossref Scopus (295) Google Scholar, 24Vrettos J.S. Limburg J. Brudvig G.W. Biochim. Biophys. Acta. 2001; 1503: 229-245Crossref PubMed Scopus (247) Google Scholar). Such roles imply proximity to manganese ions and the Tyr as first indicated from Ca2+/Sr2+ exchange experiments (7Boussac A. Rutherford A.W. Biochemistry. 1988; 27: 3476-3483Crossref Scopus (225) Google Scholar), and indeed recent EXAFS studies strongly favor a structural model in which Ca2+ is close to manganese or even within its coordination sphere (25Cinco R.M. Robblee J.H. Rompel A. Fernandez C. Yachandra V.K. Sauer K. Klein M.P. J. Phys. Chem. B. 1998; 102: 8248-8256Crossref PubMed Scopus (107) Google Scholar, 26Cinco R.M. Holman K.L.M. Robblee J.H. Yano J. Pizarro S.A. Bellacchio E. Sauer K. Yachandra V.K. Biochemistry. 2002; 41: 12928-12933Crossref PubMed Scopus (117) Google Scholar).The replacement of Ca2+ with Sr2+ provides a relatively rare case in which the enzyme turns over in all the centers but is kinetically limited. This material is thus of particular interest for enzymological and spectroscopic studies. The effects of Ca2+/Sr2+ exchange have been studied by continuous wave-EPR (7Boussac A. Rutherford A.W. Biochemistry. 1988; 27: 3476-3483Crossref Scopus (225) Google Scholar, 15Westphal K.L. Lydakis-Simantiris N. Cukier R.I. Babcock G.T. Biochemistry. 2000; 39: 16220-16229Crossref PubMed Scopus (44) Google Scholar, 16Boussac A. Zimmermann J.-L. Rutherford A.W. Biochemistry. 1989; 28: 8984-8989Crossref PubMed Scopus (246) Google Scholar, 27Boussac A. Sugiura M. Inoue Y. Rutherford A.W. Biochemistry. 2000; 39: 13788-13799Crossref PubMed Scopus (61) Google Scholar) and pulsed-EPR (28Britt R.D. Campbell K.A. Peloquin J.M. Gilchrist M.L. Aznar C.P. Dicus M.M. Robblee J. Messinger J. Biochim. Biophys. Acta. 2004; (in press)PubMed Google Scholar), Fourier transform infrared spectroscopy (29Chu H.-A. Sackett H. Babcock G.T. Biochemistry. 2000; 39: 14371-14376Crossref PubMed Scopus (77) Google Scholar, 30Kimura Y. Hasegawa K. Ono T.-A. Biochemistry. 2002; 41: 5844-5853Crossref PubMed Scopus (41) Google Scholar), and EXAFS (strontium, calcium and manganese EXAFS) (25Cinco R.M. Robblee J.H. Rompel A. Fernandez C. Yachandra V.K. Sauer K. Klein M.P. J. Phys. Chem. B. 1998; 102: 8248-8256Crossref PubMed Scopus (107) Google Scholar, 26Cinco R.M. Holman K.L.M. Robblee J.H. Yano J. Pizarro S.A. Bellacchio E. Sauer K. Yachandra V.K. Biochemistry. 2002; 41: 12928-12933Crossref PubMed Scopus (117) Google Scholar). From Fourier transform infrared spectroscopy measurements, it has been proposed that a Mn-O-Mn cluster vibrational mode is modified upon Ca2+ replacement by Sr2+ (29Chu H.-A. Sackett H. Babcock G.T. Biochemistry. 2000; 39: 14371-14376Crossref PubMed Scopus (77) Google Scholar) and that Ca2+ could be necessary for the formation of the hydrogen bond network involved in the reaction step of water oxidation (30Kimura Y. Hasegawa K. Ono T.-A. Biochemistry. 2002; 41: 5844-5853Crossref PubMed Scopus (41) Google Scholar). From the strontium-EXAFS done on Sr2+-reconstituted Ca2+-depleted PSII, a manganese-strontium distance of 3.4 Å has been proposed (25Cinco R.M. Robblee J.H. Rompel A. Fernandez C. Yachandra V.K. Sauer K. Klein M.P. J. Phys. Chem. B. 1998; 102: 8248-8256Crossref PubMed Scopus (107) Google Scholar) (a value confirmed by calcium-EXAFS done on normal PSII sample (Ref. 26Cinco R.M. Holman K.L.M. Robblee J.H. Yano J. Pizarro S.A. Bellacchio E. Sauer K. Yachandra V.K. Biochemistry. 2002; 41: 12928-12933Crossref PubMed Scopus (117) Google Scholar)). In addition, it was found by mass spectrometry experiments that substitution of Ca2+ by Sr2+ accelerated the slow rate of H 182O exchange by a factor of 3–4 in the S1-, S2-, and S3-states (31Hendry G. Wydrzynski T. Biochemistry. 2003; 42: 6209-6217Crossref PubMed Scopus (114) Google Scholar).These experiments were all performed after appropriate biochemical treatments leading to Ca2+/Sr2+ exchange. In general, these treatments consist of washing PSII in high salt buffers in the light (at pH 6.5, Ca2+/Sr2+ exchange occurred in the S3-state) (7Boussac A. Rutherford A.W. Biochemistry. 1988; 27: 3476-3483Crossref Scopus (225) Google Scholar, 16Boussac A. Zimmermann J.-L. Rutherford A.W. Biochemistry. 1989; 28: 8984-8989Crossref PubMed Scopus (246) Google Scholar) or by treatment at low pHs (≤3) (32Kodera Y. Hara H. Astashkin A.V. Kawamori A. Ono T.-A. Biochim. Biophys. Acta. 1995; 1232: 43-51Crossref Scopus (49) Google Scholar). Doubts have been raised in the past concerning potential secondary effects of the biochemical treatments used, and it has been suggested that some of the phenomena associated with Ca2+ depletion, and by implication Sr2+ replacement, may be because of secondary structural effects (1Debus R.J. Biochim. Biophys. Acta. 1992; 1102: 269-352Crossref PubMed Scopus (1084) Google Scholar, 5Yocun C.F. Biochim. Biophys. Acta. 1991; 1059: 1-15Crossref Scopus (161) Google Scholar, 10Boussac A. Rutherford A.W. Photosynth. Res. 1992; 32: 207-209Crossref PubMed Scopus (9) Google Scholar, 33Pauly S. Schloddler E. Wit H.T. Biochim. Biophys. Acta. 1992; 1099: 203-210Crossref Scopus (24) Google Scholar, 34Shen J.-R. Satoh K. Katoh S. Biochim. Biophys. Acta. 1988; 933: 358-364Crossref Scopus (44) Google Scholar). Although good arguments have been made to counter these doubts (1Debus R.J. Biochim. Biophys. Acta. 1992; 1102: 269-352Crossref PubMed Scopus (1084) Google Scholar, 10Boussac A. Rutherford A.W. Photosynth. Res. 1992; 32: 207-209Crossref PubMed Scopus (9) Google Scholar, 35Han C.-H. Katoh S. Plant Cell Physiol. 1993; 34: 585-593Google Scholar), these have been revived by related work focused on removal and exchange of chloride ions where secondary biochemical effects have been specifically invoked (36Olesen K. Andreasson L.-E. Biochemistry. 2003; 42: 2025-2035Crossref PubMed Scopus (90) Google Scholar).The procedures to remove Ca2+ always result in an inhibition (i.e. the release of the Mn4 cluster) of a small proportion of PSII centers (37Boussac A. Rutherford A.W. Biochim. Biophys. Acta. 1995; 1230: 195-201Crossref Scopus (12) Google Scholar, 38Shen J.-R. Katoh S. Plant Cell Physiol. 1991; 32: 439-446Crossref Scopus (26) Google Scholar). In addition, Sr2+ reconstitution is not necessarily 100% efficient and the Sr2+-reconstituted PSII is very often an unstable material. All of these effects rendered the previous Sr2+-reconstituted PSII an heterogeneous enzyme. These have made imperative the development of a new fully stable and fully active strontium-PSII.The focus of PSII research recently has turned to cyanobacterial PSII because of 1) the availability of mutants (39Diner B.A. Biochim. Biophys. Acta. 2001; 1503: 147-163Crossref PubMed Scopus (172) Google Scholar, 40Debus R.J. Biochim. Biophys. Acta. 2001; 1503: 164-186Crossref PubMed Scopus (183) Google Scholar, 41Sugiura M. Inoue Y. Plant Cell Physiol. 1999; 40: 1219-1231Crossref PubMed Scopus (149) Google Scholar) and 2) a move to study thermophilic species of cyanobacteria that have provided excellent spectroscopic material (27Boussac A. Sugiura M. Inoue Y. Rutherford A.W. Biochemistry. 2000; 39: 13788-13799Crossref PubMed Scopus (61) Google Scholar, 42Noguchi T. Sugiura M. Biochemistry. 2002; 41: 15706-15712Crossref PubMed Scopus (90) Google Scholar, 43Boussac A. Kuhl H. Ghibaudi E. Rögner M. Rutherford A.W. Biochemistry. 1999; 38: 11942-11948Crossref PubMed Scopus (37) Google Scholar, 44Boussac A. Kuhl H. Un S. Rögner M. Rutherford A.W. Biochemistry. 1998; 37: 8995-9000Crossref PubMed Scopus (52) Google Scholar, 45Kretschmann H. Schlodder E. Witt H.T. Biochim. Biophys. Acta. 1996; 1274: 1-8Crossref Scopus (29) Google Scholar) and have provided the first x-ray crystallographic models of PSII (46Zouni A. Witt H.T. Kern J. Fromme P. Krauss N. Saenger W. Orth P. Nature. 2001; 409: 739-743Crossref PubMed Scopus (1754) Google Scholar, 47Kamiya N. Shen J.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 98-103Crossref PubMed Scopus (991) Google Scholar). The role of Ca2+ in cyanobacterial PSII is more poorly understood than in plant systems. From a few studies, a less specific ion requirement has been reported (33Pauly S. Schloddler E. Wit H.T. Biochim. Biophys. Acta. 1992; 1099: 203-210Crossref Scopus (24) Google Scholar, 48Becker D.W. Brand J.J. Plant Physiol. 1985; 79: 552-558Crossref PubMed Google Scholar), and the biochemical procedures developed for Ca2+ ion removal in plants are inappropriate for cyanobacterial systems, because of differences in the extrinsic polypeptides present in cyanobacteria compared with plants (e.g. Refs. 3Barber J. Curr. Opin. Struct. Biol. 2002; 12: 523-530Crossref PubMed Scopus (117) Google Scholar and 49Enami I. Iwai M. Akiyama A. Suzuki T. Okumura A. Katoh T. Tada O. Ohta H.S. Plant Cell Physiol. 2003; 44: 820-827Crossref PubMed Scopus (26) Google Scholar).The measurement of calcium stoichiometries in PSII has proved particularly problematic. Because of the ubiquitous nature of Ca2+, background levels are often close to the concentration found in the biological samples. Many years of wrangling went on before the field stopped arguing over the stoichiometry in plant material (10Boussac A. Rutherford A.W. Photosynth. Res. 1992; 32: 207-209Crossref PubMed Scopus (9) Google Scholar, 11Adelroth P. Lindberg K. Andréasson L.-E. Biochemistry. 1995; 34: 9021-9027Crossref PubMed Scopus (80) Google Scholar, 35Han C.-H. Katoh S. Plant Cell Physiol. 1993; 34: 585-593Google Scholar). To measure the stoichiometry of calcium in PSII of cyanobacteria, we wished to avoid the difficulties encountered in plant PSII. One approach that seemed worth trying was the biosynthetic replacement of Ca2+ with Sr2+ by growing the cyanobacterial cells in Sr2+-containing media. This approach promised the benefit of providing material not only for the measurement of strontium (and hence calcium) stoichiometries, but also for enzymological and spectroscopic studies in cyanobacteria without the necessity of developing the exchange procedures in vitro and without the risk of preparation artifacts.Here we present (a) the results of this biosynthetic replacement study using Thermosynechococcus elongatus, (b) a procedure for the isolation of the Sr2+-containing enzyme, and (c) the results of kinetics and enzymological studies.EXPERIMENTAL PROCEDURESPurification of “Thylakoids” and PSII Core Complexes—T. elongatus (43-H strain) (41Sugiura M. Inoue Y. Plant Cell Physiol. 1999; 40: 1219-1231Crossref PubMed Scopus (149) Google Scholar) were grown in 3-liter Erlenmeyer flasks (1500-ml culture) in a rotary shaker (120 rpm) at 45 °C under continuous illumination from fluorescent white lamps (≈80 μmol of photons·m–2·s–1). The cells were grown in a DTN medium (41Sugiura M. Inoue Y. Plant Cell Physiol. 1999; 40: 1219-1231Crossref PubMed Scopus (149) Google Scholar) containing either 0.8 mm CaCl2 or 0.8 mm SrCl2 in an CO2-enriched atmosphere. Cells were grown until they reached an optical density (OD) close to 1.0 at 800 nm. After harvesting by centrifugation, the cells were washed once with buffer 1 (40 mm MES, pH 6.5, 15 mm MgCl2, 15 mm CaCl2, 10% glycerol, 1.2 m betaine) and resuspended in the same buffer, with 0.2% (w/v) bovine serum albumin, 1 mm benzamidine, 1 mm aminocaproic acid, and 50 μg·ml–1 DNase I added, to a chlorophyll concentration of ≈1.5 mg·ml–1. The cells were ruptured with a French press (≈700 p.s.i.). Unbroken cells were removed by centrifugation (1,000 × g, 5 min). Thylakoids were pelleted by centrifugation at 180,000 × g for 35 min at 4 °C and washed twice with buffer 1. Thylakoids were finally resuspended in buffer 1 and stored in liquid N2 at a Chl concentration of 1 mg·ml–1 before use. Thylakoids obtained from cells grown in the presence of either Ca2+ or Sr2+ will be noted as Ca-thylakoids or Srthylakoids, respectively.PSII were purified from freshly prepared thylakoids essentially as described previously (41Sugiura M. Inoue Y. Plant Cell Physiol. 1999; 40: 1219-1231Crossref PubMed Scopus (149) Google Scholar) but with the following modifications. Thylakoids (1 mg·ml–1, final concentration) were treated with 1% (w/v) n-dodecyl-β-maltoside (β-DM, Biomol, Germany) in buffer 1 supplemented with 100 mm NaCl. After ≈1 min of stirring in the dark at 4 °C the suspension was centrifuged (10 min, 170,000 × g) to remove the non-solubilized material. Then, the supernatant was mixed with an equal volume of Probond resin (Invitrogen, Groningen, The Netherlands) that had been pre-equilibrated with buffer 1. The resulting slurry was transferred to an empty column. After sedimentation of the resin inside the column, the supernatant was removed. The resin was washed with buffer 2 (40 mm MES, 15 mm MgCl2, 15 mm CaCl2, 100 mm NaCl, 15 mm imidazole, 0.03% (w/v) β-DM, 10% (v/v) glycerol, 1.2 m betaine, pH 6.5) until the OD value of the eluate at ≈670 nm decreased below 0.05. Then, PSII core complexes were eluted with buffer 3 (150 mm MES, 15 mm MgCl2, 15 mm CaCl2, 200 mm NaCl, 300 mm imidazole, 0.1% (w/v) β-DM, 10% (v/v) glycerol, 1.2 m betaine, the pH was adjusted to 6.5 by adding concentrated HCl). The eluate was then either precipitated with buffer 1 with 15% (w/v) polyethylene glycol-8000 added by centrifugation (10 min, 170,000 × g) or concentrated and washed using centrifugal filter devices (Ultrafree-15, Millipore). PSII core complexes were finally resuspended in buffer 1 at a Chl concentration of 1–1.5 mg·ml–1 and store in liquid N2 before to be used. The estimate of Chl concentration was done by solubilizing the biological material in methanol and by using an extinction coefficient equal to 79.95 mg–1·ml·cm–1 at 665 nm (51Porra R.J. Scheer H. The Chlorophylls. CRC Press, Boca Raton, FL1991: 31-57Google Scholar). PSII core complexes purified from cells grown in the presence of either Ca2+ or Sr2+ will be noted calcium-PSII or strontium-PSII, respectively.Manganese Depletion—Manganese depletion of PSII was done by a washing of the sample in 1.2 m Tris, pH 9.2, in room light at 4 °C for 1 h. After centrifugation (3 h, 170,000 × g), the pellet was submitted to a second washing in 1 m CaCl2 for 30 min at 4 °C. PSII depleted of the manganese cluster and of the three extrinsic proteins was then pelleted by centrifugation (3 h, 170,000 × g) and resuspended in buffer 1.Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis—Calcium-PSII, strontium-PSII, and manganese-depleted PSII were mixed with 30 mm Tris, pH 8.5, 30 mm dithiothreitol, and 1% lauryl sulfate at a Chl concentration of 0.5 mg·ml–1. Samples were incubated on ice for 10 min and then applied to a 16–22% gradient of SDS-polyacrylamide gel containing 7.5 m urea (52Ikeuchi M. Inoue Y. Plant Cell Physiol. 1988; 29: 1233-1239Google Scholar). Electrophoresis was done under a current of 10 mA. The gel was stained with Coomassie Brilliant Blue as described previously (52Ikeuchi M. Inoue Y. Plant Cell Physiol. 1988; 29: 1233-1239Google Scholar).Oxygen Evolution Measurements—Oxygen evolution under continuous light was measured at 25 °C by polarography using a Clark-type oxygen electrode (Hansatech) with saturating white light. Oxygen evolution of PSII core complexes (5 μg of Chl·ml–1) was measured in buffer 1 in the presence of 0.5 mm DCBQ (2,6-dichloro-p-benzoquinone, dissolved in Me2SO) or 0.5 mm PPBQ (phenyl-p-benzoquinone dissolved in Me2SO) as electron acceptors. For measurements at various pH values, the following buffers were used: MES, pH 5.5, 6.0, and 6.5; HEPES, pH 7.0 and 7.5; CHES, pH 8.0 and 8.5.Oxygen evolution under flashing light was measured with a laboratory-made rate electrode similar to that already described (53Miyao M. Murata N. Lavorel J. Maison B. Boussac A. Etienne A.-L. Biochim. Biophys. Acta. 1987; 890: 151-159Crossref Scopus (96) Google Scholar). Cathylakoids or Sr-thylakoids were used at 1 mg·ml–1 without an added electron acceptor. Illumination was done with a xenon flash. The power of the xenon 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 flash-induced oxygen evolution patterns reported here were obtained as follows; the direct current was recorded with a numerical oscilloscope (see the inset to Fig. 8), and then the derivative versus time was computed mathematically. The derivative step (dt) used was dt = 10 ms for Ca-thylakoids and dt = 20 ms for Sr-thylakoids because the oxygen release was slower in Sr-thylakoids than in Ca-thylakoids. Then, the amplitude of the derivative signal was plotted versus the flash number. Analysis of the flash-induced oxygen evolution patterns was done using the classic equations listed below assuming the miss (α) and double hit (β) parameters to be equal on all flashes.[O2]n=(1−α)[S3]n−1+β[S2]n−1(Eq. 1) [O2]n is the amount of O2 evolved after flash n, and [S3]n–1 and [S2]n–1 are the amount of S3 and S2, respectively, before flash n.[Si]n=(1−α−β)[Si−1]n−1+β[Si−2]n−1+α[Si]n−1(Eq. 2) A scaling factor multiplying the experimental data was introduced into the fitting procedure so that the amount of O2 evolved upon each flash corresponded to the percentage of centers by which it was produced.Continuous Wave-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). Continuous illumination of the Sr-thylakoid samples ([Chl] ≈ 3–4mg·ml–1) was done with an 800-W tungsten lamp, light from which was filtered through water and IR filters, in a nonsilvered Dewar flask filled with ethanol cooled to 198 K with solid CO2. Flash illumination at room temperature was provided by a Nd:YAG laser (532 nm, 550 mJ, 8 ns Spectra Physics GCR-230-10). For measurements of the S2-multiline signal after a given number of flashes, strontium-PSII samples at 1 mg·ml–1 were loa
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