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• W2012861398 abstract "Photosystem I (PS I) is a transmembraneal multisubunit complex that mediates light-induced electron transfer from plactocyanine to ferredoxin. The electron transfer proceeds from an excited chlorophyll a dimer (P700) through a chlorophyll a (A0), a phylloquinone (A1), and a [4Fe-4S] iron-sulfur cluster FX, all located on the core subunits PsaA and PsaB, to iron-sulfur clusters FA and FB, located on subunit PsaC. Earlier, it was attempted to determine the function of FX in the absence of FA/B mainly by chemical dissociation of subunit PsaC. However, not all PsaC subunits could be removed from the PS I preparations by this procedure without partially damaging FX. We therefore removed subunit PsaC by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp. PCC 6803. Cells could not grow under photosynthetic conditions when subunit PsaC was deleted, yet the PsaC-deficient mutant cells grew under heterotrophic conditions and assembled the core subunits of PS I in which light-induced electron transfer from P700 to A1 occurred. The photoreduction of FX was largely inhibited, as seen from direct measurement of the extent of electron transfer from A1 to FX. From the crystal structure it can be seen that the removal of subunits PsaC, PsaD, and PsaE in the PsaC-deficient mutant resulted in the braking of salt bridges between these subunits and PsaB and PsaA and the formation of a net of two negative surface charges on PsaA/B. The potential induced on FX by these surface charges is proposed to inhibit electron transport from the quinone. In the complete PS I complex, replacement of a cysteine ligand of FX by serine in site-directed mutation C565S/D566E in subunit PsaB caused an ∼10-fold slow down of electron transfer from the quinone to FX without much affecting the extent of this electron transfer compared with wild type. Based on these and other results, we propose that FX might have a major role in controlling electron transfer through PS I. Photosystem I (PS I) is a transmembraneal multisubunit complex that mediates light-induced electron transfer from plactocyanine to ferredoxin. The electron transfer proceeds from an excited chlorophyll a dimer (P700) through a chlorophyll a (A0), a phylloquinone (A1), and a [4Fe-4S] iron-sulfur cluster FX, all located on the core subunits PsaA and PsaB, to iron-sulfur clusters FA and FB, located on subunit PsaC. Earlier, it was attempted to determine the function of FX in the absence of FA/B mainly by chemical dissociation of subunit PsaC. However, not all PsaC subunits could be removed from the PS I preparations by this procedure without partially damaging FX. We therefore removed subunit PsaC by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp. PCC 6803. Cells could not grow under photosynthetic conditions when subunit PsaC was deleted, yet the PsaC-deficient mutant cells grew under heterotrophic conditions and assembled the core subunits of PS I in which light-induced electron transfer from P700 to A1 occurred. The photoreduction of FX was largely inhibited, as seen from direct measurement of the extent of electron transfer from A1 to FX. From the crystal structure it can be seen that the removal of subunits PsaC, PsaD, and PsaE in the PsaC-deficient mutant resulted in the braking of salt bridges between these subunits and PsaB and PsaA and the formation of a net of two negative surface charges on PsaA/B. The potential induced on FX by these surface charges is proposed to inhibit electron transport from the quinone. In the complete PS I complex, replacement of a cysteine ligand of FX by serine in site-directed mutation C565S/D566E in subunit PsaB caused an ∼10-fold slow down of electron transfer from the quinone to FX without much affecting the extent of this electron transfer compared with wild type. Based on these and other results, we propose that FX might have a major role in controlling electron transfer through PS I. Photosystem I (PS I) 1The abbreviations used are: PS I, photosystem I; Chl, chlorophyll; DCIP, 2,6-dichlorophenolindophenol; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.1The abbreviations used are: PS I, photosystem I; Chl, chlorophyll; DCIP, 2,6-dichlorophenolindophenol; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. is a multisubunit complex located in the thylakoid membranes of chloroplasts and cyanobacteria. It mediates light-induced electron transfer from plastocyanin or cytochrome c553 to ferredoxin (1Chitnis P.R. Nelson N. Bogoras L. Vsil I.K. Photosynthetic Apparatus: Molecular Biology and Operation. Academic Press, New York1991: 177-224Google Scholar, 2Golbeck J.H. Bryant D.A. Lee C.P. Current Topics in Bioenergetics. Academic Press, New York1991Google Scholar). In cyanobacteria, the complex consists of at least 12 polypeptides, some of which bind light-harvesting chlorophyll molecules. The reaction center core complex is made up of the heterodimeric PsaA and PsaB subunits, containing the primary electron donor P700, which transfers an electron through the sequential carriers A0, A1, and FX. The final acceptors FA and FB are located on another subunit, PsaC. P700 is a chlorophyll a dimer, which undergoes light-induced charge separation. The electron carriers A0, A1, FX, FA, and FB represent a monomeric chlorophyll a, a phylloquinone, and three [4Fe-4S] iron sulfur centers, respectively. The crystalline structure of PS I from Synechococus elongatus resolved to 2.5 Å (3Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauss N. Nature. 2001; 411: 909-917Crossref PubMed Scopus (2013) Google Scholar) reveals that 11 transmembranal helices are contributed by each of the heterodimeric core subunits PsaA and PsaB. FX is located in the center of the core heterodimer at the interface of subunit PsaC. FX is bound at this interface to interhelical loops (hi; nomenclature according to Ref. 3Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauss N. Nature. 2001; 411: 909-917Crossref PubMed Scopus (2013) Google Scholar) of both subunits PsaA and PsaB. The cysteine residues in the two subunits of the heterodimer, which are in an identical amino acid sequence CDGPGRGGTC (4Fish L.E. Bogorad L. J. Biol. Chem. 1986; 261: 8134-8139Abstract Full Text PDF PubMed Google Scholar), bind the iron-sulfur cluster. The two cysteines, Cys-556 and Cys-565, in PsaB of Synechocystis sp. PCC 6803 that are analogous to Cys-565 and Cys-574 in S. elongatus, are the ligands of FX. Indeed there was no assembly of PS I when they were modified to histidine or aspartate (5Smart L.B. Warren P.V. Golbeck J.H. McIntosh L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1132-1136Crossref PubMed Scopus (39) Google Scholar). Thus FX is positioned as an intermediate carrier between the initial electron mediators A0 and A1 and the final electron acceptors of PS I FA and FB. Crystallographic determination of PS I placed FX between A1 and FA with edge to edge distance of 6.8 Å between A1 and FX and 10.1 Å between FX and FA (3Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauss N. Nature. 2001; 411: 909-917Crossref PubMed Scopus (2013) Google Scholar). The midpoint potential of FX/FX– is about –700 mV (6Evans M.C.W. Reeves S.G. Cammack R. FEBS Lett. 1974; 49: 111-114Crossref PubMed Scopus (100) Google Scholar), the lowest among the iron-sulfur clusters known in biology. It therefore has the properties required for a carrier that mediates electrons between A1, with an estimated redox potential of –800 to –700 mV (7Setif P. Bottin H. Biochemistry. 1989; 28: 2689-2697Crossref Scopus (76) Google Scholar) and FA with –530 mV (8Evans M.C.W. Heathcote P. Biochim. Biophys. Acta. 1980; 590: 89-96Crossref PubMed Scopus (51) Google Scholar). Forward electron transfer was directly shown to proceed through FX at room temperature by optical spectroscopy (9Luneberg J. Fromme P. Jekow P. Schlodder E. FEBS Lett. 1994; 338: 197-202Crossref PubMed Scopus (59) Google Scholar) and by EPR (10van der Est A. Bock C. Golbeck J. Brettel K. Setif P. Stehlik D. Biochemistry. 1994; 33: 11789-11797Crossref PubMed Scopus (75) Google Scholar). An approximate lifetime of 200 ns by EPR (10van der Est A. Bock C. Golbeck J. Brettel K. Setif P. Stehlik D. Biochemistry. 1994; 33: 11789-11797Crossref PubMed Scopus (75) Google Scholar, 11Moenne-Loccoz P. Heathcote P. Maclachlan D.J. Berry M.C. Davis I.H. Evans M.C.W. Biochemistry. 1994; 33: 10037-10042Crossref PubMed Scopus (71) Google Scholar) and two components of 10–30 and ∼300 ns by optical method (12Setif P. Brettel K. Biochemistry. 1993; 32: 7846-7854Crossref PubMed Scopus (88) Google Scholar, 13Schlodder E. Falkenberg K. Gergeleit M. Brettel K. Biochemistry. 1998; 37: 9466-9476Crossref PubMed Scopus (125) Google Scholar, 14Guergova-Kuras M. Boudreaux B. Joliot A. Joliot P. Redding K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4437-4442Crossref PubMed Scopus (262) Google Scholar, 15Joliot P. Joliot A. Biochemistry. 1999; 38: 11130-11136Crossref PubMed Scopus (158) Google Scholar) for the oxidation of A1– by FX were determined. The presence of two kinetic components for the oxidation of A1– may reflect the use two similar but not identical electron transfer branches in PS I (14Guergova-Kuras M. Boudreaux B. Joliot A. Joliot P. Redding K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4437-4442Crossref PubMed Scopus (262) Google Scholar, 15Joliot P. Joliot A. Biochemistry. 1999; 38: 11130-11136Crossref PubMed Scopus (158) Google Scholar), although other explanations are still under discussion (16Brettel K. Biochim. Biophys. Acta. 1997; 1318: 322-373Crossref Scopus (429) Google Scholar). Electron transport from A1– through FX to FA and FB appears to be modulated by a variety of environmental factors (for review see Ref. 16Brettel K. Biochim. Biophys. Acta. 1997; 1318: 322-373Crossref Scopus (429) Google Scholar). A quantum yield of close to one was determined in the forward electron transport in PS I (17Sun A.S. Sauer K. Biochim. Biophys. Acta. 1971; 234: 399-414Crossref PubMed Scopus (63) Google Scholar), indicating that the light energy conversion in PS I was an extremely efficient process. Only when electron transfer between the carriers is either inefficient or inhibited will charge recombination between intermediate carriers and the primary donor occur. The rate of recombination increases with decreasing distance of the electron carrier from the primary donor and can be used to determine the efficiency of the reduction of a given carrier. The rate of recombination therefore is expected to increase if the electron transfer to FX became less efficient in response to the redox state of FA/B, to the absence of these iron-sulfur clusters, to temperature, and possibly to the subunit interaction in PS I. Thus, at low temperature, the single turnover flash reduction of FX was either partially or totally inhibited (12Setif P. Brettel K. Biochemistry. 1993; 32: 7846-7854Crossref PubMed Scopus (88) Google Scholar, 18Crowder M.S. Bearden A.J. Biochem. Biophys. Res. Commun. 1983; 722: 23-35Google Scholar, 19Brettel K. Biochim. Biophys. Acta. 1989; 976: 246-249Crossref Scopus (26) Google Scholar). In the absence of an extrinsic acceptor, following a single turnover flash the t½ of 45 ms for the reduction of P700+ was assigned to the recombination of P700+ FA/B– (20Hiyama T. Ke B. Arch. Biochem. Biophys. 1971; 147: 99-108Crossref PubMed Scopus (97) Google Scholar). When FA/B were prereduced by dithionite, a recombination t½ of 250 μs was determined (21Sauer K. Mathis P. Acker S. van Best J.A. Biochim. Biophys. Acta. 1978; 503: 120-134Crossref PubMed Scopus (115) Google Scholar). Although it was expected that when FA/B were pre-reduced the recombination would occur between P700+ and FX–, spectral analysis of this reaction in cyanobacterial PS I indicated that A1– was the dominant electron donor in this recombination; thus electron transfer from A1– to FX appeared to be largely inhibited under such conditions (19Brettel K. Biochim. Biophys. Acta. 1989; 976: 246-249Crossref Scopus (26) Google Scholar). Removal of FA/B by dissociation of subunit PsaC with urea treatment resulted in t½ of 1.2 ms for most of P700+ reduction. In the absence of final iron-sulfur clusters of PS I it was reasonable to attribute the reduction of P700+ to recombination with FX– (22Parrett K.G. Mehari T. Warren P.G. Golbeck J.H. Biochim. Biophys. Acta. 1989; 973: 324-332Crossref PubMed Scopus (132) Google Scholar, 23Vassiliev I.R. Jung Y.S. Mamedov M.D. Semenov A.Y. Golbeck J.H. Biophys. J. 1997; 72: 301-315Abstract Full Text PDF PubMed Scopus (81) Google Scholar). Indeed under such conditions the rate of electron transfer from A1– to FX was reported to be the same as in untreated PS I (9Luneberg J. Fromme P. Jekow P. Schlodder E. FEBS Lett. 1994; 338: 197-202Crossref PubMed Scopus (59) Google Scholar). Yet in PS I lacking PsaC by genetic deletion of the subunit (24Yu J. Smart L.B. Jung Y.S. Golbeck J. McIntosh L. Plant Mol. Biol. 1995; 29: 331-342Crossref PubMed Scopus (53) Google Scholar) or as a result of mutation in the ligands of FA/B (25Yu J. Vassiliev I.R. Jung Y.S. Golbeck J.H. McIntosh L. J. Biol. Chem. 1997; 272: 8032-8039Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), only partial reduction of FX was observed. Although electron transfer to FX was not directly measured in these works, a significant part of the total reduction of P700+ proceeded at a rate that was faster than the rate of P700+/FX– recombination. This indicated that complete removal of subunit PsaC, and the iron-sulfur clusters FA and FB partially inhibited forward electron transport from A1– to FX. In this work we report the effect of the interruption of the psaC gene, in the cyanobacterium Synechocystis sp. PCC 6803, on electron transport in PS I. The intra- and intersubunit interactions were studied in PS I mutated in a cysteine ligand of FX in PsaB and in psaC– mutants, respectively. PS I was assembled in the absence of subunit PsaC and the associated iron-sulfur clusters FA and FB. Single turnover flash experiments showed that a 10-fold slow down and an inhibition of the forward electron transfer to FX occurred in PS I prepared from the cysteine to serine mutant in psaB and the psaC– mutant, respectively. Based on these and other results, we propose that FX has a major role in controlling electron transfer through PS I. The redox potential of FX is influenced by electronic interaction with FA and FB. Reduction or removal of FA and FB may lower the redox potential of FX and thus block electron transfer from A1– to FX. The inhibition of FX reduction resulted in charge recombination of A1– with P700+. We suggest that this control switch has physiological importance in prevention of photooxidation damage that could result from reduction of oxygen by reduced FX and FA/B. Photooxidation damage to FA/B was shown to happen during selective activation of PS I under high light intensity (26Tjus S.E. Scheller H.V. Andersson B. Moller B.L. Plant Physiol. 2001; 125: 2007-2015Crossref PubMed Scopus (76) Google Scholar). In Vitro Insertional Inactivation of the psaC2 Gene—Plasmid p61–2.4 containing a 2.26-kb fragment of the psaC2 gene from Synechocystis (GenBankTM accession number D63999) (27Steinmuller K. Plant Mol. Biol. 1992; 20: 997-1001Crossref PubMed Scopus (9) Google Scholar) was used to construct a vector for inactivation of the psaC2 gene. Plasmid p61–2.4 was a kind gift from Dr. Klaus Steinmuller (Universitat Dusseldorf, Dusseldorf, Germany). A 2-kb Ω fragment from the pHP45Ω plasmid, containing the streptomycin and spectinomycin resistance-conferring gene (28Prentki P. Krisch H.M. Gene. 1984; 29: 303-313Crossref PubMed Scopus (1337) Google Scholar), was digested with SmaI and subcloned in the filled-in XbaI site of psaC2 gene in p61–2.4 plasmid resulting in plasmid pAC2 (Fig. 1). Synechocystis cells from wild type and mutant C565S/D566E in PsaB (29Zeng M. Sagi I. Evans M.C.W. Nelson N. Carmeli C. Gabar G. Pusztai J. Photosynthesis: Mechanisms and Effects. Kluwer Academic Publishers, Dordrecht, The Netherlands1999: 643-646Google Scholar) were transformed with the pAC2 plasmid for inactivation of the psaC2 gene, essentially as described (30Williams J.G.K. Methods Enzymol. 1988; 167: 766-778Crossref Scopus (842) Google Scholar). After transformation, selections of spectinomycin colonies were carried out under light-adapted heterotrophic growth conditions (5Smart L.B. Warren P.V. Golbeck J.H. McIntosh L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1132-1136Crossref PubMed Scopus (39) Google Scholar) for PsaC mutants. Single spectinomycin colonies were plated and sequentially transferred in increasing spectinomycin concentrations from 5 to 20 μg/ml, respectively, until full segregation was obtained. Cyanobacterial Strains and Growth Conditions—Synechocystis sp. PCC 680 cells, a glucose-tolerant strain, were grown in BG-11 medium supplemented with 5 mm TES, pH 8.0, and 5 mm glucose at 30 °C (31Rippka R. Deruelles J. Waterbury J.B. Herdman M. Stanier R.Y. J. Gen. Microbiol. 1979; 111: 1-16Crossref Google Scholar). Mixotrophic cultures were grown in continuous light at photon flux density of 40 μmol·m–2s–1. Heterotrophic cultures were grown in dim light (photon flux density of 2 μmol·m–2s–1) or under light-adapted heterotrophic growth conditions: in complete darkness, except for 10 min of light (photon flux density of 40 μmol·m–2s–1) every 24 h (5Smart L.B. Warren P.V. Golbeck J.H. McIntosh L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1132-1136Crossref PubMed Scopus (39) Google Scholar). For photoautotrophic growth, cells were grown in the medium without addition of glucose in continuous light (photon flux density of 40 μmol·m–2s–1). PsaB and PsaC mutants were grown in medium supplemented with kanamycin (25 μg/ml) and spectinomycin (20 μg/ml), respectively, under heterotrophic growth conditions. For growth on plates, BG-11 medium was supplemented with 1.5% (w/v) Bacto-agar and 0.3% sodium thiosulfate (32Anderson S.L. McIntosh L. J. Bacteriol. 1991; 173: 2761-2767Crossref PubMed Google Scholar). Growth of cells was monitored by measurement of absorption changes at 730 nm. Manipulation of DNA—Most of the techniques for DNA analysis and manipulation used in this study were according to standard procedures (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Synechocystis genomic DNA, used as a template for psaB and psaC2 genes, was extracted essentially as described (30Williams J.G.K. Methods Enzymol. 1988; 167: 766-778Crossref Scopus (842) Google Scholar) and purified by using a GENECLEAN Kit (BIO 101). DNA sequencing was performed on double-stranded plasmid or was PCR amplified DNA by the dideoxy sequencing method (34Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52251) Google Scholar) with a Sequenase Version 2.0 kit (USB). A GCG program (Wisconsin Package Version 9.1; Genetics Computer Group) was used to design oligonucleotides as primers and analyze DNA sequences. Isolation of Thylakoid Membranes and PS I Complexes—Harvested cells were washed and broken in a French pressure cell at 500 p.s.i., and the thylakoids were isolated. PS I was solubilized by the detergent n-dodecyl β-d-maltoside and purified on DEAE-cellulose columns and on a sucrose gradient as described (35Nechushtai R. Muster P. Binder A. Liveanu V. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 1179-1183Crossref PubMed Scopus (64) Google Scholar). SDS-polyacrylamide gel electrophoresis and Western blotting were done as described previously (36Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar, 37Tindall K.R. Kunkel T.A. Biochemistry. 1988; 27: 6008-6013Crossref PubMed Scopus (621) Google Scholar). Protein in the membranes was determined after solubilization in 1% SDS as described (38Lowry O.H. Rosenbrough N.L. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Chlorophyll concentration and P700 chemical oxidation and photooxidation were determined according to published methods (39Arnon D.I. Plant Physiol. 1949; 24: 1-15Crossref PubMed Google Scholar, 40Hiyama T. Ke B. Biochim. Biophys. Acta. 1978; 257: 160-171Google Scholar). Determination of Acid-labile Sulfide and Iron in the Photosystem I Complexes—The iron content in iron-sulfur clusters in PS I was determined by an adaptation of an earlier reported method (41Golbeck J.H. Lien S. San Pietro A. Arch. Biochem. Biophys. 1977; 178: 140-150Crossref PubMed Scopus (57) Google Scholar, 42Lovenberg W. Buchanan B.B. Rabinowitz J.C. J. Biol. Chem. 1963; 238: 3899-3913Abstract Full Text PDF PubMed Google Scholar). Acid-labile sulfide was determined by modification of published methods (43King T.E. Morris R.O. Methods Enzymol. 1967; 10: 634-641Crossref Scopus (145) Google Scholar, 44Golbeck J.H. San Pietro A. Anal. Biochem. 1976; 73: 539-542Crossref PubMed Scopus (30) Google Scholar). In the modified procedure the amount of acid-labile sulfide was determined after the extraction of chlorophyll and measurement of absorption of the dye at 750 nm in the presence of 4 m KCl. It was found that there is less interference from chlorophyll breakdown products left after extraction, when determination was carried out by measurement of the absorption at 750 nm rather than at 640 nm used in the standard assay. Ferredoxin (molecular mass 6.2 kDa) from Clostridium pasteurianum (45Rabinowitz J.C. Methods Enzymol. 1972; 24: 431-446Crossref PubMed Scopus (67) Google Scholar, 46Bertini I. Donaire A. Feinberg B.A. Luchinat C. Piccioli M. Yuan H. Eur. J. Biochem. 1995; 232: 192-205Crossref PubMed Scopus (85) Google Scholar) was used as standard. For each preparation, the mean values of three independent experiments, each in triplicate, were calculated to obtain 95% statistical confidence. The values were normalized on the basis of the chlorophyll content in the samples. Isolation and Reconstitution of the PS I Core Complexes and the Subunits saC, PsaD, and PsaE—Preparation of PS I core and isolation of subunits PsaC, PsaD, and PsaE were carried out essentially according to the methods as described (47Parrett K.G. Mehari T. Golbeck J.H. Biochim. Biophys. Acta. 1990; 1015: 341-352Crossref Scopus (61) Google Scholar, 48Mehari T. Parrett K.G. Warren P.G. Golbeck J.H. Biochim. Biophys. Acta. 1991; 1056: 139-148Crossref Scopus (34) Google Scholar). PS I was dissociated by treatment with 6.8 m urea for 10–30 min until the P700+ reduction reached t of 1 ms. Following removal of the urea by dialysis, the dissociated peripheral low molecular mass polypeptides PsaC, PsaD, and PsaE were separated from the core by ultrafiltration and concentrated by the same method. Reconstitution of iron-sulfur clusters in PS I core preparation and in the PS I complexes from the PsaC-deficient mutant strains was adapted from the previously reported studies (47Parrett K.G. Mehari T. Golbeck J.H. Biochim. Biophys. Acta. 1990; 1015: 341-352Crossref Scopus (61) Google Scholar, 48Mehari T. Parrett K.G. Warren P.G. Golbeck J.H. Biochim. Biophys. Acta. 1991; 1056: 139-148Crossref Scopus (34) Google Scholar). Essentially PS I core preparations in the absence or the presence of the isolated small subunits obtained from urea dissociation were suspended in N2-purged solutions 0.5% β–mercaptoethanol to which FeCl3 and Na2S solutions were added and incubated for 12–18 h. The solution was concentrated to near dryness by ultrafiltration, washed twice with a solution containing 0.05% β-mercaptoethanol. The reconstituted PS I complexes were stored at –80 °C in the presence of 15% glycerol. Spectroscopic Measurements—The kinetics of P700+ reduction were measured by monitoring flash-induced transient absorption changes at 700 and 820 nm. Measurements at 820 nm were performed on the set-up as described earlier (49Faller P. Debus R.J. Brettel K. Sugiura M. Rutherford A.W. Boussac A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14368-14373Crossref PubMed Scopus (105) Google Scholar). In brief, the sample was excited by 532-nm laser flashes of 300-ps duration and an energy of ∼0.7 mJ/cm2. Continuous measuring light from an 820-nm laser diode passed through the sample (path length, 10 mm) and was monitored by a silicon photodiode, amplified, and digitized. The electronic bandwidth of the detection system was usually limited to 20 MHz. Typically, 16 transients were averaged at a repetition rate of 1 Hz. Measurements at 700 nm and measurements at 820 nm on reconstituted samples used a modified flash photolysis setup as described earlier (50Zeng M.T. Gong X.M. Evans M.C. Nelson N. Carmeli C. Biochim. Biophys. Acta. 2002; 1556: 254-264Crossref PubMed Scopus (8) Google Scholar), which included a 10-ns flash from a Quantal Nd-YAG laser at 532 nm. A measuring beam was provided by a tungsten-iodide source passed through a monochromator. The absorption changes at 820 and 700 nm were monitored by a photomultiplier, interfaced by Tektronix TDS 520A digitizing oscilloscope, and recorded on a personal computer. The kinetics of A1– oxidation in time scales shorter than 5 μs were measured by monitoring flash-induced transient absorption changes at 380 nm on a set-up similar to the one described earlier (51Brettel K. Leibl W. Liebl U. Biochim. Biophys. Acta. 1998; 1363: 175-181Crossref PubMed Scopus (43) Google Scholar). The sample was excited by 53-nm laser flashes of 300-ps duration and an energy of ∼0.5 mJ/cm2. The measuring light was provided by the relatively flat top of a 50-μs xenon flash and was filtered by a combination of interference and colored glass filters placed in the measuring beam before the sample and in front of the detector (silicon photodiode FND 100Q from EG&G), yielding a spectrum of 8-nm width centered at 380 nm. The detector output to 50 ohms was amplified (30 dB, 500 Hz-1.7 GHz) and recorded by a digitizing oscilloscope (DSA 602A with amplifier plug-in 11A52 from Tektronix). The time resolution of the detection system was 2 ns. The baseline was recorded separately with mechanically blocked excitation flash and subtracted from the transient recorded with excitation. Deviating from previous methodology (51Brettel K. Leibl W. Liebl U. Biochim. Biophys. Acta. 1998; 1363: 175-181Crossref PubMed Scopus (43) Google Scholar, 52Brettel K. Gabar G. Pusztai J. Photosynthesis: Mechanisms and Effects. Kluwer Academic Publishers, Dordrecht, The Netherlands1999: 611-614Google Scholar), the sample was contained in a quartz cell of 20-mm width, 30-mm height, and 2-mm path length for the measuring light. This cell was allowed to spread the measuring light on a larger cross-section than previously and to diminish a potential actinic effect of the measuring light. Typically, 2048 transients were averaged at a repetition rate of 1 Hz. The same set-up was used to monitor absorption changes at 430 nm (essentially because of oxidation and re-reduction of P700) under identical conditions of excitation and time resolution. The spectral bandwidth of the measuring light was 3 nm, and only 256 transients were averaged. Absorption changes at 380 nm in a time scale up to 500 μs were recorded essentially as described previously (53Shen G. Antonkine M.L. van der E.A. Vassiliev I.R. Brettel K. Bittl R. Zech S.G. Zhao J. Stehlik D. Bryant D.A. Golbeck J.H. J. Biol. Chem. 2002; 277: 20355-20366Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The excitation energy was ∼0.3 mJ/cm2 in a 300-ps flash at 532 nm. The measuring light from a continuous 100-watt tungsten halogen lamp was filtered as above. A shutter placed in the measuring light beam in front of the sample was opened 3 ms before the excitation flash and closed 5 ms after the flash to diminish exposure of the sample to the measuring light. The time resolution of the detection system was ∼1 μs. The sample was contained in a 10 × 10-mm standard cuvette. 256 transients were averaged at a repetition rate of 2 Hz. For measurements at 380 nm in a 10-ms time scale, the measuring light was continuous. The sample was contained in a quartz cell of 20-mm width, 30-mm height, and 2-mm path length, allowing to spread the measuring light to a larger cross-section and to diminish its actinic effect. The time resolution of the detection system was limited to 5 μs. 512 transients were averaged at a repetition rate of 1 Hz. Unless specified otherwise, samples contained 25 mm Tris, pH 8.3, 10 mm sodium ascorbate, 500 μm DCIP, and PS I complexes as indicated in the figure legends. All spectroscopic measurements were performed at room temperature. Absorption change transients were analyzed by fitting with a multiexponential decay using Marquardt least-squares algorithm programs (KaleidaGraph 3.5 from Synergy Software, Reading, PA, and DECO, kindly provided by P. Sétif, Commissariat à l'Energie Atomique, Saclay, France). Assembly of PS I in the PsaC-deficient Mutants—The segregated cells of the PsaC-deficient mutants, psaC– and psaC– C565S/D575E, failed to grow either mixotrophically or autotrophically under light intensity of 40 μmol·m–2s–1 but could grow heterotrophically at wild type growth rate under a low light intensity of 2 μmol·m–2s–1. The absorption of the chlorophyll/cell at 440 and 680 nm in the psaC– cells was smaller than in the wild type cells suggesting that photosynthetic complexes were assembled at a reduced level in the mutants. A semi-quantitative evaluation of the assemblies of the PS I complexes in the PsaC-deficient mutants was done by Wester" @default.
• W2012861398 created "2016-06-24" @default.
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• W2012861398 title "Control of Electron Transport in Photosystem I by the Iron-Sulfur Cluster FX in Response to Intra- and Intersubunit Interactions" @default.
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