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• W1999394461 abstract "Photosystem II (PSII) oxidizes water to molecular oxygen; the catalytic site is a cluster of four manganese ions. The catalytic site undergoes four sequential light-driven oxidation steps to form oxygen; these sequentially oxidized states are referred to as the S n states, where n refers to the number of oxidizing equivalents stored. The extrinsic manganese stabilizing protein (MSP) of PSII influences the efficiency and stability of the manganese cluster, as well as the rates of the S state transitions. To understand how MSP influences photosynthetic water oxidation, we have employed isotope editing and difference Fourier transform infrared spectroscopy. MSP was expressed in Escherichia coli under conditions in which MSP aspartic and glutamic acid residues label at yields of 65 and 41%, respectively. Asparagine and glutamine were also labeled by this approach. GC/MS analysis was consistent with minimal scrambling of label into other amino acid residues and with no significant scrambling into the peptide bond. Selectively labeled MSP was then reconstituted to PSII, which had been stripped of native MSP. Difference Fourier transform infrared spectroscopy was used to probe the S1QA to S2QA- transition at 200 K, as well as the S1QB to S2QB- transition at 277 K. These experiments show that aspargine, glutamine, and glutamate residues in MSP are perturbed by photooxidation of manganese during the S1 to S2 transition. Photosystem II (PSII) oxidizes water to molecular oxygen; the catalytic site is a cluster of four manganese ions. The catalytic site undergoes four sequential light-driven oxidation steps to form oxygen; these sequentially oxidized states are referred to as the S n states, where n refers to the number of oxidizing equivalents stored. The extrinsic manganese stabilizing protein (MSP) of PSII influences the efficiency and stability of the manganese cluster, as well as the rates of the S state transitions. To understand how MSP influences photosynthetic water oxidation, we have employed isotope editing and difference Fourier transform infrared spectroscopy. MSP was expressed in Escherichia coli under conditions in which MSP aspartic and glutamic acid residues label at yields of 65 and 41%, respectively. Asparagine and glutamine were also labeled by this approach. GC/MS analysis was consistent with minimal scrambling of label into other amino acid residues and with no significant scrambling into the peptide bond. Selectively labeled MSP was then reconstituted to PSII, which had been stripped of native MSP. Difference Fourier transform infrared spectroscopy was used to probe the S1QA to S2QA- transition at 200 K, as well as the S1QB to S2QB- transition at 277 K. These experiments show that aspargine, glutamine, and glutamate residues in MSP are perturbed by photooxidation of manganese during the S1 to S2 transition. Photosystem II (PSII) 1The abbreviations used are: PSII, photosystem II; Chl, chlorophyll; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; FT-IR, Fourier transform infrared; GC/MS, gas chromatography/mass spectrometry; MES, 2-(N-morpholino)ethanesulfonic acid; MSP, manganese stabilizing protein. is the multisubunit membrane protein responsible for the light-driven oxidation of water to molecular oxygen in higher plants, algae, and cyanobacteria (1Britt R.D. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reactions. Vol. 4. Kluwer Academic Publisher, Dordrecht1996: 137-164Google Scholar). PSII contains multiple protein subunits, most of which are hydrophobic and traverse the membrane. Intrinsic PSII proteins ligate the antenna and the reaction center chlorophylls, as well as other cofactors that take part in the oxidation/reduction reactions (2Bricker T.M. Frankel L.K. Photosyn. Res. 2002; 72: 131-146Crossref PubMed Scopus (92) Google Scholar). These intrinsic subunits include the chlorophyll a-binding proteins, CP47 and CP43, the α and β subunits of cytochrome b 559, and the polypeptides known as D1 and D2. D1 and D2 form the heterodimeric core of PSII (3Nanba O. Satoh K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 109-112Crossref PubMed Google Scholar, 4Zouni A. Witt H.-T. Kern J. Fromme P. Krauss N. Saenger W. Orth P. Nature. 2001; 409: 739-743Crossref PubMed Scopus (1776) Google Scholar, 5Kamiya N. Shen J.-R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 98-103Crossref PubMed Scopus (1003) Google Scholar). The catalytic site of the oxygen-evolving complex contains a cluster of four manganese atoms. As water is oxidized, the manganese cluster cycles through five oxidation states, called the S n states (6Joliot P. Kok B. Govindjee Bioenergetics of Photosynthesis. Academic Press, New York1975: 388-412Google Scholar). Water oxidation is initiated by excitation of the primary chlorophyll donor, P680.P680* transfers an electron to pheophytin, which in turn reduces a bound plastoquinone, called QA. QA- reduces a second quinone, QB. QB acts as a two-electron and two-proton acceptor. On the donor side of PSII, P680+ oxidizes a redox active tyrosine, Z, which in turn oxidizes the catalytic site. Redox tyrosine YD and a chlorophyll, ChlZ, are alternate electron donors to P680+. Four sequential photooxidations are required for oxygen production (reviewed in Ref. 1Britt R.D. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reactions. Vol. 4. Kluwer Academic Publisher, Dordrecht1996: 137-164Google Scholar). PSII contains several extrinsic subunits, which are bound to the inner lumenal surface of the reaction center (reviewed in Ref. 7Seidler A. Biochim. Biophys. Acta. 1996; 1277: 35-60Crossref PubMed Scopus (269) Google Scholar). The extrinsic subunits of PSII play key roles in enzymatic activity and stability, but the mechanism of these effects is still unknown. The largest extrinsic subunit is called the manganese stabilizing protein (MSP), and this subunit is found in plants, cyanobacteria, and eukaryotic algae. MSP can be removed from the reaction center with high concentrations of CaCl2 or with low concentrations of urea (8Ono T. Inoue Y. FEBS Lett. 1983; 164: 252-260Crossref Scopus (259) Google Scholar, 9Miyao M. Murata N. FEBS Lett. 1983; 164: 375-378Crossref Scopus (47) Google Scholar). After its removal by these methods, the steady-state rate of oxygen evolution is decreased, and two manganese atoms are easily lost as Mn2+ (10Miyao M. Murata N. FEBS Lett. 1984; 170: 350-354Crossref Scopus (247) Google Scholar). Loss of manganese inactivates the enzyme; these MSP-dependent deactivation reactions can be prevented by addition of high concentrations of chloride to the preparation (10Miyao M. Murata N. FEBS Lett. 1984; 170: 350-354Crossref Scopus (247) Google Scholar, 11Bricker T.M. Biochemistry. 1992; 31: 4623-4628Crossref PubMed Scopus (121) Google Scholar). An Escherichia coli-expressed version of spinach MSP is able to functionally reconstitute oxygen evolution in spinach PSII, from which the native subunit has been removed (12Seidler A. Michel H. EMBO. 1990; 9: 1743-1748Crossref PubMed Scopus (48) Google Scholar, 13Betts S.D. Ross J.R. Hall K.U. Pichersky E. Yocum C.F. Biochim. Biophys. Acta. 1996; 1274: 135-142Crossref PubMed Scopus (42) Google Scholar). MSP exhibits anomalous migration on SDS-PAGE and size exclusion chromatography (see Ref. 14Lydakis-Simantiris N. Betts S.D. Yocum C. Biochemistry. 1999; 38: 15528-15535Crossref PubMed Scopus (35) Google Scholar and references therein). Both hydrodynamic (15Zubrzycki I.Z. Frankel L.K. Russo P.S. Bricker T.M. Biochemistry. 1998; 37: 13553-13558Crossref PubMed Scopus (36) Google Scholar) and small-angle x-ray scattering experiments (16Svensson B. Tiede D.M. Barry B.A. J. Phys. Chem. B. 2002; 106: 8485-8488Crossref Scopus (19) Google Scholar) suggest that MSP is a prolate ellipsoid in solution. Axial ratios of 4.2 (15Zubrzycki I.Z. Frankel L.K. Russo P.S. Bricker T.M. Biochemistry. 1998; 37: 13553-13558Crossref PubMed Scopus (36) Google Scholar) and 4.8 (16Svensson B. Tiede D.M. Barry B.A. J. Phys. Chem. B. 2002; 106: 8485-8488Crossref Scopus (19) Google Scholar) have been reported. Both CD and FT-IR analysis suggest that MSP is primarily a β sheet protein (17Xu Q. Nelson J. Bricker T.M. Biochim. Biophys. Acta. 1994; 1188: 427-431Crossref PubMed Scopus (51) Google Scholar, 18Shutova T. Irrgang K.-D. Shubin V. Klimov V.V. Renger G. Biochemistry. 1997; 36: 6350-6358Crossref PubMed Scopus (69) Google Scholar, 19Lydakis-Simantiris N. Hutchison R.S. Betts S.D. Barry B.A. Yocum C.F. Biochemistry. 1999; 38: 404-414Crossref PubMed Scopus (92) Google Scholar, 20Ahmed A. Tajmir-Riahi H.A. Carpentier R. FEBS Lett. 1995; 363: 65-68Crossref PubMed Scopus (107) Google Scholar, 21Hutchison R.S. Betts S.D. Yocum C.F. Barry B.A. Biochemistry. 1998; 37: 5643-5653Crossref PubMed Scopus (66) Google Scholar). However, FT-IR studies of MSP in solution have suggested some variability in its secondary structure content (19Lydakis-Simantiris N. Hutchison R.S. Betts S.D. Barry B.A. Yocum C.F. Biochemistry. 1999; 38: 404-414Crossref PubMed Scopus (92) Google Scholar, 21Hutchison R.S. Betts S.D. Yocum C.F. Barry B.A. Biochemistry. 1998; 37: 5643-5653Crossref PubMed Scopus (66) Google Scholar), as have protein mapping studies of MSP from different organisms (22Tohri A. Suzuki T. Okuyama S. Kamino K. Motoki A. Hirano M. Ohta H. Shen J.-R. Yamamoto Y. Enami I. Plant Cell Physiol. 2002; 43: 429-439Crossref PubMed Scopus (25) Google Scholar). These results, as well as the high temperature stability of MSP, have been explained by the suggestion that MSP is an intrinsically disordered protein and that this intrinsic disorder is important in facilitating assembly (19Lydakis-Simantiris N. Hutchison R.S. Betts S.D. Barry B.A. Yocum C.F. Biochemistry. 1999; 38: 404-414Crossref PubMed Scopus (92) Google Scholar). Because MSP can be removed from PSII, expressed in E. coli, and then reconstituted to the enzyme, isotope editing was employed to study its functional role in PSII (21Hutchison R.S. Betts S.D. Yocum C.F. Barry B.A. Biochemistry. 1998; 37: 5643-5653Crossref PubMed Scopus (66) Google Scholar, 23Hutchison R.S. Steenhuis J.J. Yocum C.F. Razeghifard R.M. Barry B.A. J. Biol. Chem. 1999; 274: 31987-31995Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). MSP was isotopically edited via expression in an E. coli strain grown on 13C-labeled glucose. 13C-Labeled MSP was purified and bound to PSII. As the only 13C-labeled PSII subunit, MSP could then be detected with vibrational spectroscopy, which is sensitive to isotopic composition. Using this approach, FT-IR experiments showed that a substantial change in MSP secondary structure occurred with reconstitution (21Hutchison R.S. Betts S.D. Yocum C.F. Barry B.A. Biochemistry. 1998; 37: 5643-5653Crossref PubMed Scopus (66) Google Scholar). This result is in agreement with cross-linking studies, which also suggest a change in MSP structure upon PSII binding (24Enami I. Kamo M. Ohta H. Takahashi S. Miura T. Kusayanagi M. Tanabe S. Kamei A. Motoki A. Hirano M. Tomo T. Satoh K. J. Biol. Chem. 1998; 273: 4629-4634Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Difference FT-IR spectroscopy and isotope editing were also employed to identify MSP structural changes that occur during the S1 to S2 transition (23Hutchison R.S. Steenhuis J.J. Yocum C.F. Razeghifard R.M. Barry B.A. J. Biol. Chem. 1999; 274: 31987-31995Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). At 200 K, all transitions in the PSII photocycle are blocked, except the S1 to S2 transition, and electron transfer from QA to QB is also inhibited (see Ref. 25Steenhuis J.J. Barry B.A. J. Phys. Chem. 1997; 101: 6652-6660Crossref Scopus (35) Google Scholar and references therein). When glycerol-containing PSII is illuminated, the multiline form of the S2 state is generated from the dark-stable S1 state (26Dismukes G.C. Siderer Y. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 274-278Crossref PubMed Google Scholar, 27de Paula J.C. Innes J.B. Brudvig G.W. Biochemistry. 1985; 24: 8114-8120Crossref PubMed Scopus (210) Google Scholar). This S state transition corresponds to a manganese oxidation reaction (see Ref. 28Roelofs T.A. Liang W. Latimer M.L. Cinco R.M. Rompel A. Andrews J.C. Sauer K. Yachandra V.K. Klein M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3335-3340Crossref PubMed Scopus (173) Google Scholar and references therein). Thus, it is possible to obtain a S2QA--minus-S1QA FT-IR spectrum at 200 K by continuous illumination (see Ref. 29Steenhuis J.J. Hutchison R.S. Barry B.A. J. Biol. Chem. 1999; 274: 14609-14616Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar and references therein). A different S2QA--minus-S1QA FT-IR spectrum was obtained by laser flash illumination (see Refs. 30Noguchi T. Ono T.-A. Inoue Y. Biochim. Biophys. Acta. 1995; 1232: 65-66Crossref Scopus (55) Google Scholar, 31Noguchi T. Tomo T. Kato C. Biochemistry. 2001; 40: 1497-1502Crossref PubMed Scopus (83) Google Scholar, 32Hillier W. Babcock G. Biochemistry. 2001; 40: 1503-1509Crossref PubMed Scopus (72) Google Scholar, 33Noguchi T. Sugiura M. Biochemistry. 2003; 42: 6035-6042Crossref PubMed Scopus (121) Google Scholar and references therein). Possible reasons for these spectral differences have been discussed (34Barry B.A. Photosyn. Res. 2000; 65: 197-198Crossref PubMed Scopus (4) Google Scholar, 35Halverson K.M. Barry B.A. Biophys. J. 2003; 85: 1317-1325Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 36Halverson K.M. Barry B.A. Biophys. J. 2003; 85: 2581-2588Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). A S2QB--minus-S1QB spectrum has also been reported (36Halverson K.M. Barry B.A. Biophys. J. 2003; 85: 2581-2588Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 37Zhang H. Fischer G. Wydrzynski T. Biochemistry. 1998; 37: 5511-5517Crossref PubMed Scopus (39) Google Scholar). In our previous work, S2QA--minus-S1QA data were acquired under continuous illumination from natural abundance 12C-MSP- and 13C-MSP-reconstituted PSII. A comparison was performed by construction of a double difference, isotope-edited spectrum at 200 K (23Hutchison R.S. Steenhuis J.J. Yocum C.F. Razeghifard R.M. Barry B.A. J. Biol. Chem. 1999; 274: 31987-31995Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). That analysis led to the conclusion that one or more carboxylic acid side chains in MSP contribute to the isotope-edited spectrum (23Hutchison R.S. Steenhuis J.J. Yocum C.F. Razeghifard R.M. Barry B.A. J. Biol. Chem. 1999; 274: 31987-31995Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). These assignments were substantiated by comparison to 13C-labeled model compounds. Thus, it was concluded that one or more aspartic or glutamic acid residues deprotonate upon conversion of the S1 state to the S2 state. To acquire more information about the role of amino acid residues in MSP, this article reports selective 13C labeling of aspartic acid/asparagine or glutamic acid/glutamine. GC/MS analysis of labeled MSP was consistent with little scrambling of label into other amino acid residues. Selectively labeled MSP was then reconstituted to PSII, and difference FT-IR spectroscopy was used to probe the S1 to S2 transition at two different temperatures, with both continuous and flash illumination. These experiments show that aspargine, glutamine, and glutamate residues in MSP are perturbed by photooxidation of manganese. Labeling and Purification of MSP—Selective labeling either of aspartic acid/asparagine or glutamine/glutamic acid residues in MSP was performed via expression in E. coli. MSP was produced from a psbO-containing expression plasmid by induction with isopropyl-1-thio-β-d-galactopyranoside, as previously described (21Hutchison R.S. Betts S.D. Yocum C.F. Barry B.A. Biochemistry. 1998; 37: 5643-5653Crossref PubMed Scopus (66) Google Scholar, 23Hutchison R.S. Steenhuis J.J. Yocum C.F. Razeghifard R.M. Barry B.A. J. Biol. Chem. 1999; 274: 31987-31995Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The host strain for expression was a non-auxotropic strain, BL21(DE3) pLysS(F– ompT rB – mB –). Selective labeling was obtained by growth in a defined media, containing an excess of the 13C-labeled amino acid of choice. This media contained the following components per liter (38Griffey R.H. Redfield A.G. Loomis R.E. Dahlquist F.W. Biochemistry. 1985; 24: 817-822Crossref PubMed Scopus (100) Google Scholar): 0.5 g of MgSO4, 13.2 mg of CaCl2·2H2O, 5 mg of FeSO4·7H2O, 50 mg of nicotinic acid, 50 mg of thiamin, 2 g of sodium acetate, 1 g of ammonium chloride, 10 g of potassium phosphate dibasic, 2 g of succinic acid, 400 mg of alanine, 400 mg of glutamine, 125 mg of guanosine, 125 mg of uracil, 125 mg of cytosine, 50 mg of thymine, 400 mg of arginine, 50 mg of cystine, 400 mg of glycine, 135 mg of histidine-HCl-H2O, 100 mg of isoleucine, 100 mg of leucine, 100 mg of lysine, 250 mg of methionine, 100 mg of proline, 1.6 g of serine, 200 mg of threonine, 50 mg of tryptophan, 100 mg of tyrosine, 100 mg of valine, 50 mg of phenylalanine, and 0.1 mg of biotin. For the selective 13C labeling of aspartic acid/asparagine residues in MSP, 500 mg of l-[4-13C]aspartic acid (Isotec, 99% 13C) were added to 1 liter of the media, along with 800 mg of glutamic acid. For the selective labeling of glutamic acid/glutamine residues in MSP, 800 mg of l-[5-13C]glutamic acid (Isotec, 99% 13C), along with 250 mg of aspartic acid, were added to 1 liter of media. After gentle heating to dissolve all the reagents, the media was filter sterilized. To each liter of sterilized defined media was added 10 ml of 20% glucose (for a final concentration of 0.2%), 1 ml of chloramphenicol (25 mg/ml), and 1 ml of ampicillin (50 mg/ml). One-hundred milliliters of this media was inoculated and incubated overnight at 37 °C in a rotary shaker. The following morning, 20 ml of the cell culture was added to the remaining media, and the 37 °C incubation continued until the A 600 was ∼0.5. This took about 3 h. At this time, 0.2 ml of 0.2 m isopropyl-1-thio-β-d-galactopyranoside was added to induce MSP production. In addition, a 100-ml solution of 10% lactose was added; lactose was found to improve the labeling efficiency (39Ramesh V. Frederick R.O. Syed S.E.H. Gibson C.F. Yang J. Roberts G.C.K. Eur. J. Biochem. 1994; 225: 601-608Crossref PubMed Scopus (34) Google Scholar). After induction, the cells were grown for an additional 4 h and harvested. Natural abundance and globally 13C-labeled MSP were prepared by expression in minimal media, containing either 0.2% 12C- or 0.2% [13C]glucose as the carbon source, as described (21Hutchison R.S. Betts S.D. Yocum C.F. Barry B.A. Biochemistry. 1998; 37: 5643-5653Crossref PubMed Scopus (66) Google Scholar). Inclusion bodies, containing MSP, were isolated, and MSP was solubilized and purified by previously described methods (21Hutchison R.S. Betts S.D. Yocum C.F. Barry B.A. Biochemistry. 1998; 37: 5643-5653Crossref PubMed Scopus (66) Google Scholar, 40Betts S.D. Hachigian T.M. Pichersky E. Yocum C.F. Plant Mol. Biol. 1994; 26: 117-130Crossref PubMed Scopus (43) Google Scholar). Isotope incorporation into MSP was measured using previously employed procedures (21Hutchison R.S. Betts S.D. Yocum C.F. Barry B.A. Biochemistry. 1998; 37: 5643-5653Crossref PubMed Scopus (66) Google Scholar, 23Hutchison R.S. Steenhuis J.J. Yocum C.F. Razeghifard R.M. Barry B.A. J. Biol. Chem. 1999; 274: 31987-31995Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 41Kitson F.G. Larsen B.S. McEwen C.N. Gas Chromatography and Mass Spectrometry, A Practical Guide. Academic Press, San Diego1996Google Scholar). Briefly, the protein was hydrolyzed under anaerobic conditions (42Patterson B.W. Carraro F. Wolfe R.R. Biol. Mass Spectrom. 1993; 22: 518-523Crossref PubMed Scopus (82) Google Scholar). The resulting amino acids were derivatized with tert-butyldimethylsilyl groups via reaction with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide and were subjected to GC/MS. The derivatized amino acids were identified by retention time, which was confirmed using a set of derivatized standards. The extent of labeling in the M-57 fragment was monitored. This fragmentation involves loss of a t-butyl radical and is quite facile; often the molecular ion was not observed. A number of amino acids were monitored for 13C labeling including aspartic acid, glutamic acid, alanine, glycine, valine, leucine, isoleucine, proline, methionine, serine, threonine, lysine, and phenylalanine. The amount of isotope incorporation was determined by comparison to a natural abundance MSP sample, which was subjected to the same procedure. All GC/MS analyses were done in triplicate. PSII Preparations—PSII membranes were isolated from market spinach (43Berthold D.A. Babcock G.T. Yocum C.F. FEBS Lett. 1981; 134: 231-234Crossref Scopus (1653) Google Scholar, 44Anderson L.B. Ouellette A.J.A. Barry B.A. J. Biol. Chem. 2000; 275: 4920-4927Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). This material was treated to remove native MSP, as well as the 18- and 24-kDa extrinsic proteins, while retaining manganese (see method in Ref. 21Hutchison R.S. Betts S.D. Yocum C.F. Barry B.A. Biochemistry. 1998; 37: 5643-5653Crossref PubMed Scopus (66) Google Scholar and references therein). Either natural abundance, globally 13C-, [13C]Asp/Asn- or [13C]Glu/Gln-labeled MSP was rebound to PSII according to established procedures (see Ref. 21Hutchison R.S. Betts S.D. Yocum C.F. Barry B.A. Biochemistry. 1998; 37: 5643-5653Crossref PubMed Scopus (66) Google Scholar and references therein). These PSII membranes were used directly for FT-IR spectroscopy at 277 K and were in a sucrose buffer containing 0.4 m sucrose, 50 mm MES-NaOH, pH 6.0, 60 mm NaCl, and 20 mm CaCl2. PSII complexes, for FT-IR spectroscopy at 200 K, were purified from the membranes via detergent solubilization and ion-exchange chromatography (45MacDonald G.M. Barry B.A. Biochemistry. 1992; 31: 9848-9856Crossref PubMed Scopus (72) Google Scholar). The final buffer contained 25% glycerol, 50 mm MES-NaOH, pH 6.0, 0.05% lauryl maltoside, 7.5 mm CaCl2, and 0.26 m NaCl. Oxygen evolution assays were employed to verify reconstitution. The samples contained 10–15 μg/ml Chl, 20 mm calcium chloride, 0.33 mm recrystallized 2,6-dichloro-p-benzoquinone, 1 mm potassium ferricyanide, 50 mm MES-NaOH, pH 6.0, 60 mm NaCl, and 400 mm sucrose. A Clark-type O2 electrode was employed (YSI 5300, YSI Inc., Yellow Springs, OH) (46Barry B.A. Methods Enzymol. 1995; 258: 303-319Crossref PubMed Scopus (62) Google Scholar). As isolated, the oxygen evolution rates for PSII membranes and PSII complexes were >750 and >1000 μmol of O2 (mg of Chl·h)–1, respectively. The oxygen evolution rates for all MSP reconstituted PSII membranes were between 250 and 500 μmol of O2 (mg of Chl·h)–1. This range of oxygen evolution rates is typical for this type of PSII sample (see Refs. 11Bricker T.M. Biochemistry. 1992; 31: 4623-4628Crossref PubMed Scopus (121) Google Scholar, 21Hutchison R.S. Betts S.D. Yocum C.F. Barry B.A. Biochemistry. 1998; 37: 5643-5653Crossref PubMed Scopus (66) Google Scholar, 23Hutchison R.S. Steenhuis J.J. Yocum C.F. Razeghifard R.M. Barry B.A. J. Biol. Chem. 1999; 274: 31987-31995Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, and 35Halverson K.M. Barry B.A. Biophys. J. 2003; 85: 1317-1325Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar and references therein). The oxygen evolution rates for all MSP-reconstituted PSII complexes were indistinguishable and were between 400 and 600 μmol of O2 (mg of Chl·h)–1. FT-IR Spectroscopy at 200 K—Infrared spectra at 200 K were recorded on a Nicolet 60-SXR FT-IR spectrometer equipped with a liquid nitrogen-cooled MCT/B detector and a KBr beamsplitter (25Steenhuis J.J. Barry B.A. J. Phys. Chem. 1997; 101: 6652-6660Crossref Scopus (35) Google Scholar, 47Steenhuis J.J. Barry B.A. J. Am. Chem. Soc. 1996; 118: 11927-11932Crossref Scopus (21) Google Scholar). Data were recorded under 10 min of illumination, and 2500 mirror scans were added together for each double-sided interferogram. The mirror velocity was 1.57 cm s–1, a Happ-Genzel apodization function was used, and the spectral resolution was 8 cm–1. A PSII sample (3 μl) was placed on a 25-mm germanium window and was concentrated in the dark under a stream of nitrogen for 5 min at 4 °C. Each sample also contained 20 equivalents of potassium ferricyanide. The germanium window was sandwiched with a CaF2 window and placed in the spectrometer. The sample was precooled to 200 K. Upon warming again to 277 K, the sample was preilluminated for 30 s and then allowed to reach the dark-adapted state over an additional 2 min. This procedure equalizes the contributions of the slowly decaying radical, Yd·, and any slowly decaying ChlZ+ to each spectrum (48MacDonald G.M. Bixby K.A. Barry B.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11024-11028Crossref PubMed Scopus (48) Google Scholar, 49MacDonald G.M. Steenhuis J.J. Barry B.A. J. Biol. Chem. 1995; 270: 8420-8428Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 50Kim S. Barry B.A. Biophys. J. 1998; 74: 2588-2600Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The temperature control apparatus and illumination conditions have been described previously (25Steenhuis J.J. Barry B.A. J. Phys. Chem. 1997; 101: 6652-6660Crossref Scopus (35) Google Scholar, 47Steenhuis J.J. Barry B.A. J. Am. Chem. Soc. 1996; 118: 11927-11932Crossref Scopus (21) Google Scholar, 51Steenhuis J.J. Barry B.A. J. Phys. Chem. 1998; 102: 4-8Crossref Scopus (12) Google Scholar). The temperature was continuously monitored and was 200 ± 0.3 K. Red and heat filters were employed. The amide I absorbance for each sample was between 0.2 and 0.6, and all spectra were normalized to an amide II absorbance of 0.5. The normalization corrects for differences in protein concentration and path length. Dark-minus-dark spectra were recorded before illumination at 200 K. FT-IR Spectroscopy at 277 K—Infrared spectra at 277 K were recorded on a Bruker (Billerica, MA) IFS-66v/S FT-IR spectrometer equipped with a liquid nitrogen-cooled MCT detector and a Harrick (Ossining, NY) temperature controller (35Halverson K.M. Barry B.A. Biophys. J. 2003; 85: 1317-1325Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 36Halverson K.M. Barry B.A. Biophys. J. 2003; 85: 2581-2588Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). A Happ-Genzel apodization function and four levels of zero filling were used. The spectral resolution was 8 cm–1. Both the actinic and pre-flashes were provided by a frequency doubled, 532-nm output from a Surelight III Nd:YAG laser (Continuum, Santa Clara, CA). The pulse width was ∼7 ns, and the pulse energy was 20–30 mJ cm–2. A PSII sample (2 μl), containing 10 eq of ferricyanide and 2 eq of 2,6-dichloro-p-benzoquinone, was placed on a 25-mm CaF2 window and was concentrated in the dark under a stream of nitrogen for 7 min at 4 °C. The window was sandwiched with another CaF2 window, placed in the spectrometer, and cooled to 277 K. A germanium filter was used to block the light from the internal laser of the spectrometer. The sample was allowed to equilibrate for 30 min after which it was given a preflash. After an additional 60 min of dark adaptation, an actinic flash was applied, and 200 mirror scans (∼30 s of data) were summed for each double-sided interferogram. The spectra taken after the flash were ratioed to data taken immediately before the flash. Dark-minus-dark spectra were recorded before each actinic flash. In each sample, the amide I absorbances were between 0.6 and 0.9, and each spectrum was normalized to an amide II absorbance of 0.5. A total of 18 data sets were collected for each sample type. Isotopic Labeling—To probe the role of acidic amino acid residues in MSP, aspartic acid/asparagine and glutamic acid/glutamine were selectively labeled. To accomplish this, a strain of E. coli (BL21/DE3), in which the expression of MSP had been previously optimized, was employed. Cultures were grown in defined media containing each essential amino acid, nucleotide, mineral, and vitamin. Ammonium chloride and glucose were employed as additional nitrogen and carbon sources, respectively. Labeled amino acids were present in the media throughout the growth of the culture and during MSP induction. Following purification (21Hutchison R.S. Betts S.D. Yocum C.F. Barry B.A. Biochemistry. 1998; 37: 5643-5653Crossref PubMed Scopus (66) Google Scholar, 40Betts S.D. Hachigian T.M. Pichersky E. Yocum C.F. Plant Mol. Biol. 1994; 26: 117-130Crossref PubMed Scopus (43) Google Scholar), MSP was hydrolyzed and derivatized. The derivatized amino acids were subjected to GC/MS analysis. Acid hydrolysis is expected to deaminate glutamine and asparagine, and the detection of Asn and Gln is difficult with this method (42Patterson B.W. Carraro F. Wolfe R.R. Biol. Mass Spectrom. 1993; 22: 518-523Crossref PubMed Scopus (82) Google Scholar). However, both Asn and Gln are expected to be labeled from 13C-labeled aspartate and glutamate, respectively (52Arnstein H.R.V. Amino Acid and Protein Biosynthesis II, International Review of Biochemistry. 1978; 18 (University Park Press, Baltimore)Google Scholar). MSP was also globally labeled with 13C by methods previously described (21Hutchison R.S. Betts S.D. Yocum C.F. Barry B.A. Biochemistry. 1998; 37: 5643-5653Crossref PubMed Scopus (66) Google Scholar, 23Hutchison R.S. Steenhuis J.J. Yocum C.F. Razeghifard R.M. Barry B.A. J. Biol. Chem. 1999; 274: 31987-31995Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The extent of 13C incorporati" @default.
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• W1999394461 title "Specific Isotopic Labeling and Photooxidation-linked Structural Changes in the Manganese-stabilizing Subunit of Photosystem II" @default.
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