Ferredoxin-NADP+ Reductase
2005; Elsevier BV; Volume: 280; Issue: 28 Linguagem: Inglês
10.1074/jbc.m503742200
ISSN1083-351X
AutoresNicolas Cassan, Bernard Lagoutte, Pièrre Sétif,
Tópico(s)Porphyrin and Phthalocyanine Chemistry
ResumoThe electron transfer cascade from photosystem I to NADP+ was studied at physiological pH by flash-absorption spectroscopy in a Synechocystis PCC6803 reconstituted system comprised of purified photosystem I, ferredoxin, and ferredoxin-NADP+ reductase. Experiments were conducted with a 34-kDa ferredoxin-NADP+ reductase homologous to the chloroplast enzyme and a 38-kDa N-terminal extended form. Small differences in kinetic and catalytic properties were found for these two forms, although the largest one has a 3-fold decreased affinity for ferredoxin. The dissociation rate of reduced ferredoxin from photosystem I (800 s–1) and the redox potential of the first reduction of ferredoxin-NADP+ reductase (–380 mV) were determined. In the absence of NADP+, differential absorption spectra support the existence of a high affinity complex between oxidized ferredoxin and semireduced ferredoxin-NADP+ reductase. An effective rate of 140–170 s–1 was also measured for the second reduction of ferredoxin-NADP+ reductase, this process having a rate constant similar to that of the first reduction. In the presence of NADP+, the second-order rate constant for the first reduction of ferredoxin-NADP+ reductase was 20% slower than in its absence, in line with the existence of ternary complexes (ferredoxin-NADP+ reductase)-NADP+-ferredoxin. A single catalytic turnover was monitored, with 50% NADP+ being reduced in 8–10 ms using 1.6 μm photosystem I. In conditions of multiple turnover, we determined initial rates of 360–410 electrons per s and per ferredox-in-NADP+ reductase for the reoxidation of 3.5 μm photoreduced ferredoxin. Identical rates were found with photosystem I lacking the PsaE subunit and wild type photosystem I. This suggests that, in contrast with previous proposals, the PsaE subunit is not involved in NADP+ photoreduction. The electron transfer cascade from photosystem I to NADP+ was studied at physiological pH by flash-absorption spectroscopy in a Synechocystis PCC6803 reconstituted system comprised of purified photosystem I, ferredoxin, and ferredoxin-NADP+ reductase. Experiments were conducted with a 34-kDa ferredoxin-NADP+ reductase homologous to the chloroplast enzyme and a 38-kDa N-terminal extended form. Small differences in kinetic and catalytic properties were found for these two forms, although the largest one has a 3-fold decreased affinity for ferredoxin. The dissociation rate of reduced ferredoxin from photosystem I (800 s–1) and the redox potential of the first reduction of ferredoxin-NADP+ reductase (–380 mV) were determined. In the absence of NADP+, differential absorption spectra support the existence of a high affinity complex between oxidized ferredoxin and semireduced ferredoxin-NADP+ reductase. An effective rate of 140–170 s–1 was also measured for the second reduction of ferredoxin-NADP+ reductase, this process having a rate constant similar to that of the first reduction. In the presence of NADP+, the second-order rate constant for the first reduction of ferredoxin-NADP+ reductase was 20% slower than in its absence, in line with the existence of ternary complexes (ferredoxin-NADP+ reductase)-NADP+-ferredoxin. A single catalytic turnover was monitored, with 50% NADP+ being reduced in 8–10 ms using 1.6 μm photosystem I. In conditions of multiple turnover, we determined initial rates of 360–410 electrons per s and per ferredox-in-NADP+ reductase for the reoxidation of 3.5 μm photoreduced ferredoxin. Identical rates were found with photosystem I lacking the PsaE subunit and wild type photosystem I. This suggests that, in contrast with previous proposals, the PsaE subunit is not involved in NADP+ photoreduction. After its reduction by photosystem I (PSI) 1The abbreviations used are: PSI, photosystem I; DCPIP, 2,6-dichlorophenolindophenol; ET, electron transfer; (FA,FB), two [4Fe-4S] clusters, the terminal acceptors of PSI; Fd, ferredoxin; Fdred, reduced ferredoxin; FNR, ferredoxin-NADP+ reductase; FNRox, oxidized form of FNR; FNRsq, semi-reduced state of FNR; FNRred, fully reduced state of FNR; P700, primary donor of photosystem I; TMPD, tetramethyl-p-phenylenediamine dihydrochloride; WT, wild type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; sFNR, short size recombinant FNR. 1The abbreviations used are: PSI, photosystem I; DCPIP, 2,6-dichlorophenolindophenol; ET, electron transfer; (FA,FB), two [4Fe-4S] clusters, the terminal acceptors of PSI; Fd, ferredoxin; Fdred, reduced ferredoxin; FNR, ferredoxin-NADP+ reductase; FNRox, oxidized form of FNR; FNRsq, semi-reduced state of FNR; FNRred, fully reduced state of FNR; P700, primary donor of photosystem I; TMPD, tetramethyl-p-phenylenediamine dihydrochloride; WT, wild type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; sFNR, short size recombinant FNR. in chloroplasts and cyanobacteria, the low potential soluble carrier ferredoxin (Fd) passes its electron to a number of soluble partners that are involved in many metabolic processes such as sulfur and nitrogen assimilation, reduction of NADP+, regulation of the Calvin cycle (1Knaff D.B. Hirasawa M. Biochim. Biophys. Acta. 1991; 1056: 93-125Crossref PubMed Scopus (229) Google Scholar, 2Knaff D.B. Ort D.R. Yocum C. Oxygenic Photosynthesis: The Light Reactions. Kluwer Academic Publishers, Dordrecht, Netherlands1996: 333-361Google Scholar), and cyclic electron transfer (3Bendall D.S. Manasse R.S. Biochim. Biophys. Acta. 1995; 1229: 23-38Crossref Scopus (319) Google Scholar, 4Joliot P. Joliot A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10209-10214Crossref PubMed Scopus (286) Google Scholar). Among the ferredoxin-soluble partners, ferredoxin-NADP+ reductase (FNR, EC 1.18.1.2) is fairly abundant, with a reported molar ratio to PSI of 0.9 and 3 for the cyanobacterium Synechocystis sp. PCC6803 and spinach, respectively (5van Thor J.J. Jeanjean R. Havaux M. Sjollema K.A. Joset F. Hellingwerf K.J. Matthijs H.C.P. Biochim. Biophys. Acta. 2000; 1457: 129-144Crossref PubMed Scopus (77) Google Scholar, 6Böhme H. Eur. J. Biochem. 1978; 83: 137-141Crossref PubMed Scopus (44) Google Scholar). This enzyme catalyzes the reduction of NADP+ to NADPH, using the electrons provided by reduced Fd (Fdred), according to the global reaction: 2Fdred + NADP+ + H+ → 2 Fdox + NADPH. This reaction involves electron transfer from the one-electron donor Fd to the two-electron acceptor NADP+. The FAD cofactor of FNR fulfills this one- to two-electron switch, as is often the case of flavoenzymes. The catalytic process involves the reduction of FAD to the semiquinone form FADH· (FNRsq) followed by its further reduction to FADH– (FNRred), using the protonation pattern during FAD reduction expected at pH 8 (7Corrado M.E. Aliverti A. Zanetti G. Mayhew S.G. Eur. J. Biochem. 1996; 239: 662-667Crossref PubMed Scopus (34) Google Scholar). These reduction steps are performed one by one, with Fdred delivering its electron to FNR at a single binding site. The last catalytic steps are thought to involve hydride transfer from FADH– to NADP+, followed by NADPH release (8Carrillo N. Ceccarelli E.A. Eur. J. Biochem. 2003; 270: 1900-1915Crossref PubMed Scopus (219) Google Scholar, 9Karplus P.A. Faber H.R. Photosynth. Res. 2004; 81: 303-315Crossref PubMed Scopus (45) Google Scholar, 10Medina M. Gomez-Moreno C. Photosynth. Res. 2004; 79: 113-131Crossref PubMed Scopus (79) Google Scholar). The participation of ternary complexes involving FNR, ferredoxin, and NADP+ during catalysis was put forward by several authors (Ref. 11Batie C.J. Kamin H. J. Biol. Chem. 1984; 259 (and references therein): 11976-11985Abstract Full Text PDF PubMed Google Scholar), and this feature is generally considered as being central to the catalytic process of FNR. The FNR/Fd redox system can also function in the reverse direction, using NADPH to reduce Fd. This is the case, for example, in nonphotosynthetic tissues of plants (12Onda Y. Matsumura T. Kimata-Ariga Y. Sakakibara H. Sugiyama T. Hase T. Plant Physiol. 2000; 123: 1037-1045Crossref PubMed Scopus (122) Google Scholar, 13Aliverti A. Faber R. Finnerty C.M. Ferioli C. Pandini V. Negri A. Karplus P.A. Zanetti G. Biochemistry. 2001; 40: 14501-14508Crossref PubMed Scopus (49) Google Scholar) and in heterocysts of cyanobacteria (14Weber-Main A.M. Hurley J.K. Cheng H. Xia B. Chae Y.K. Markley J.L. Martinez-Julvez M. Gomez-Moreno C. Stankovich M.T. Tollin G. Arch. Biochem. Biophys. 1998; 355: 181-188Crossref PubMed Scopus (10) Google Scholar), where isoforms have redox potentials that favor reverse electron flow. Fd and FNR are also found in the apicoplast of apicomplexan parasites (15Vollmer M. Thomsen N. Wiek S. Seeber F. J. Biol. Chem. 2001; 276: 5483-5490Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) where they could also function in the reverse direction with Fdred possibly involved in the biosynthesis of iron-sulfur clusters (16Thomsen-Zieger N. Pandini V. Caprini G. Aliverti A. Cramer J. Selzer P.M. Zanetti G. Seeber F. FEBS Lett. 2004; 576: 375-380Crossref PubMed Scopus (16) Google Scholar). Whether it is reduced by PSI or by FNR/NADPH, Fd has been also reported to be involved in processes such as reduction of protons to hydrogen in some green algae (17Florin L. Tsokoglou A. Happe T. J. Biol. Chem. 2001; 276: 6125-6132Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), chlorophyll catabolism (18Ginsburg S. Schellenberg M. Matile P. Plant Physiol. 1994; 105: 545-554Crossref PubMed Scopus (75) Google Scholar), glycine betaine synthesis (19Rathinasabapathi B. Burnet M. Russell B.L. Gage D.A. Liao P.C. Nye G.J. Scott P. Golbeck J.H. Hanson A.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3454-3458Crossref PubMed Scopus (213) Google Scholar), phytochrome biosynthesis (20Muramoto T. Tsurui N. Terry M.J. Yokota A. Kohchi T. Plant Physiol. 2002; 130: 1958-1966Crossref PubMed Scopus (143) Google Scholar), synthesis of phycobiliproteins in cyanobacteria (21Tu S.L. Gunn A. Toney M.D. Britt R.D. Lagarias J.C. J. Am. Chem. Soc. 2004; 126: 8682-8693Crossref PubMed Scopus (51) Google Scholar), fatty acid desaturase (22Schultz D.J. Suh M.C. Ohlrogge J.B. Plant Physiol. 2000; 124: 681-692Crossref PubMed Scopus (50) Google Scholar), and nitrogen fixation in cyanobacterial heterocysts (23Böhme H. Schrautemeier B. Biochim. Biophys. Acta. 1987; 891: 1-7Crossref Scopus (60) Google Scholar). Other unidentified Fd partners are still being sought in several laboratories. Since it was first isolated in 1956 (24Avron M. Jagendorf A.T. Arch. Biochem. Biophys. 1956; 65: 475-490Crossref PubMed Scopus (108) Google Scholar), the structural and functional properties of FNR have been studied by many groups and have been the subject of an abundant literature. Several recent reviews (8Carrillo N. Ceccarelli E.A. Eur. J. Biochem. 2003; 270: 1900-1915Crossref PubMed Scopus (219) Google Scholar, 9Karplus P.A. Faber H.R. Photosynth. Res. 2004; 81: 303-315Crossref PubMed Scopus (45) Google Scholar, 10Medina M. Gomez-Moreno C. Photosynth. Res. 2004; 79: 113-131Crossref PubMed Scopus (79) Google Scholar, 25Hurley J.K. Morales R. Martinez-Julvez M. Brodie T.B. Medina M. Gomez-Moreno C. Tollin G. Biochim. Biophys. Acta. 2002; 1554: 5-21Crossref PubMed Scopus (75) Google Scholar) summarize our current knowledge on this enzyme. Crystallographic structures from both higher plants and the cyanobacterium Anabaena have been obtained in different redox states and in the presence or absence of the substrate (9Karplus P.A. Faber H.R. Photosynth. Res. 2004; 81: 303-315Crossref PubMed Scopus (45) Google Scholar, 13Aliverti A. Faber R. Finnerty C.M. Ferioli C. Pandini V. Negri A. Karplus P.A. Zanetti G. Biochemistry. 2001; 40: 14501-14508Crossref PubMed Scopus (49) Google Scholar, 26Dorowski A. Hofmann A. Steegborn C. 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Taniguchi I. Hase T. Plant Physiol. 2004; 134: 255-264Crossref PubMed Scopus (117) Google Scholar) but with the [2Fe-2S] cluster of Fd lying close to the FAD moiety of FNR in both cases. Together with the FNR structures, a wide collection of site-directed mutants of the two partners, Fd and FNR, have allowed the residues that are essential for complex formation and for fast electron transfer to be identified (25Hurley J.K. Morales R. Martinez-Julvez M. Brodie T.B. Medina M. Gomez-Moreno C. Tollin G. Biochim. Biophys. Acta. 2002; 1554: 5-21Crossref PubMed Scopus (75) Google Scholar). Residues essential to NADP+ binding and to the specificity of its binding (versus NAD+) have been also identified (see Refs. 32Piubelli L. Aliverti A. Arakaki A.K. Carrillo N. Ceccarelli E.A. Karplus P.A. Zanetti G. J. Biol. Chem. 2000; 275: 10472-10476Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar and 33Tejero J. Martinez-Julvez M. Mayoral T. Luquita A. Sanz-Aparicio J. Hermoso J.A. Hurley J.K. Tollin G. Gomez-Moreno C. Medina M. J. Biol. Chem. 2003; 278: 49203-49214Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In cyanobacteria, two FNR forms of different molecular size have been identified. The shortest 34-kDa form is similar to the usual two-domain FNR from chloroplasts, whereas the largest one contains an additional N-terminal extension (34Schluchter W.M. Bryant D.A. Biochemistry. 1992; 31: 3092-3102Crossref PubMed Scopus (116) Google Scholar). This extension is homologous to the phycocyanin-associated linker polypeptide CpcD found in phycobilisomes and allows FNR binding to phycobilisomes (35van Thor J.J. Gruters O.W.M. Matthijs H.C.P. Hellingwerf K.J. EMBO J. 1999; 18: 4128-4136Crossref PubMed Scopus (49) Google Scholar, 36Gomez-Lojero C. Perez-Gomez B. Shen G.Z. Schluchter W.M. Bryant D.A. Biochemistry. 2003; 42: 13800-13811Crossref PubMed Scopus (46) Google Scholar). Until now, most functional and structural studies were performed on two-domain FNRs, even when originating from cyanobacteria (see Refs. 27Hermoso J.A. Mayoral T. Faro M. Gomez-Moreno C. Sanz-Aparicio J. Medina M. J. Mol. Biol. 2002; 319: 1133-1142Crossref PubMed Scopus (68) Google Scholar and 37Nogues I. Tejero J. Hurley J.K. Paladini D. Frago S. Tollin G. Mayhew S.G. Gomez-Moreno C. Ceccarelli E.A. Carrillo N. Medina M. Biochemistry. 2004; 43: 6127-6137Crossref PubMed Scopus (58) Google Scholar). This prompted us to perform our functional studies on two different N-terminal truncated forms of FNR. Pioneering work on the catalytic mechanism of spinach FNR was performed about 20 years ago by Batie and Kamin (11Batie C.J. Kamin H. J. Biol. Chem. 1984; 259 (and references therein): 11976-11985Abstract Full Text PDF PubMed Google Scholar, 38Batie C.J. Kamin H. J. Biol. Chem. 1981; 256: 7756-7763Abstract Full Text PDF PubMed Google Scholar, 39Batie C.J. Kamin H. J. Biol. 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Later on, the kinetics of FNR reduction by Fd (first reduction in the absence of NADP+) were investigated by the flash photolysis technique, using deazariboflavin as a photosensitizer (41Bhattacharyya A.K. Meyer T.E. Tollin G. Biochemistry. 1986; 25: 4655-4661Crossref PubMed Scopus (41) Google Scholar). Such studies were extended to a large number of mutants of both partners (for a recent work see Ref. 37Nogues I. Tejero J. Hurley J.K. Paladini D. Frago S. Tollin G. Mayhew S.G. Gomez-Moreno C. Ceccarelli E.A. Carrillo N. Medina M. Biochemistry. 2004; 43: 6127-6137Crossref PubMed Scopus (58) Google Scholar and see Ref. 42Hurley J.K. WeberMain A.M. Stankovich M.T. Benning M.M. Thoden J.B. Vanhooke J.L. Holden H.M. Chae Y.K. Xia B. Cheng H. Markley J.L. MartinezJulvez M. GomezMoreno C. Schmeits J.L. Tollin G. Biochemistry. 1997; 36: 11100-11117Crossref PubMed Scopus (97) Google Scholar). In the present work, we studied mixtures of PSI, Fd, and FNR (all from the cyanobacterium Synechocystis sp. PCC6803) by flash-absorption spectroscopy with and without the substrate NADP+. Electron transfer (ET) was initiated by PSI photoexcitation, which was followed by electron transfer to Fd and eventually to FNR (±NADP+). We performed a detailed kinetic study and spectral characterization of many steps involved in the catalytic process performed by FNR by taking advantage of the following: 1) sensitivity of flash-absorption spectroscopy, which is used here with a microsecond time resolution; 2) the possibility of controlling precisely the number of redox equivalents delivered to Fd-FNR-(NADP+) via the amount of PSI. New kinetic data were thus obtained and will be discussed in the context of the available literature. Our approach also represents a step toward the in vitro reconstitution of in vivo systems of higher complexity. In the future, it will be also applied to study mutants of both Fd and FNR and to other soluble partners of Fd. Biological Materials—All experiments were performed with PSI monomers from Synechocystis 6803 and recombinant forms of Fd/FNR from Synechocystis 6803. Recombinant Fd using the fed1 gene of the cyanobacterium Synechocystis 6803 was purified according to Ref. 43Barth P. Guillouard I. Sétif P. Lagoutte B. J. Biol. Chem. 2000; 275: 7030-7036Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar. PSI monomers from Synechocystis 6803 were purified following Ref. 44Rögner M. Nixon P.J. Diner B.A. J. Biol. Chem. 1990; 265: 6189-6196Abstract Full Text PDF PubMed Google Scholar. The plasmid (pET22b series) for the expression of a truncated form of the petH gene (225-bp deletion at the 5′ end) was the gift from Dr. J. van Thor (45van Thor J.J. Geerlings T.H. Matthijs H.C.P. Hellingwerf K.J. Biochemistry. 1999; 38: 12735-12746Crossref PubMed Scopus (45) Google Scholar). The resulting polypeptide has a molecular mass of 38 kDa. Another construct with a longer 336-bp deletion was designed in a different vector (pQE 60) for overproducing a 34-kDa FNR homologous to plant FNR. The appropriate DNA fragment was directly PCR-amplified from genomic DNA. The purification procedure followed a similar scheme as that used for Fd. Overexpressed FNRs were precipitated between 50 and 70% ammonium sulfate saturation. The recovered pellet was solubilized in 20 mm Tricine, pH 7.8, first roughly purified on DE52 (Whatman), and the FNR fraction was further separated by anion exchange chromatography on a Hitrap Q-Sepharose (Amersham Biosciences). The last purification step was by hydrophobic chromatography on a phenyl-Sepharose matrix (HiLoad phenyl-Sepharose 16/10 from Amersham Biosciences). The reverse salt gradient was from 1.6 to 0 m ammonium sulfate in 80 mm Tricine, pH 7.8. FNR fractions were extensively dialyzed against 10 mm HEPES buffer, pH 7.0. N-terminal analysis of the 38- and 34-kDa polypeptides confirmed the sequences LEGDS and TTTPK, respectively. Spectroscopic Measurements—All measurements (flash absorption as well as spectral perturbations experiments) were made in aerobic conditions (open cuvettes) at 295 K, in 20 mm Tricine, pH 8.0, in the presence of 30 mm NaCl, 5 mm MgCl2, and 0.03% β-dodecyl maltoside. The resultant ionic strength was 65 mm. In all experiments except with tetramethyl-p-phenylenediamine dihydrochloride (TMPD), 2 mm sodium ascorbate and 5–25 μm 2,6-dichlorophenolindophenol (DCPIP) were used. Absorption spectra were measured in a Uvikon-XL spectrophotometer. The concentrations of oxidized Fd and FNR were estimated by assuming absorption coefficients of 9.7 mm–1 cm–1 at 422 nm (46Tagawa K. Arnon D.I. Biochim. Biophys. Acta. 1968; 153: 602-613Crossref PubMed Scopus (203) Google Scholar) and of 10.8 mm–1 cm–1 at 460 nm (47Shin M. Methods Enzymol. 1971; 23: 440-447Crossref Scopus (121) Google Scholar), respectively. A coefficient of 6.2 mm–1 cm–1 was used for NADPH at 340 nm. The formation of the Fd-FNR complex was measured by the FAD hyperchromism at 460 nm upon interaction with Fd (48Foust G.P. Mayhew S.G. Massey V. J. Biol. Chem. 1969; 244: 964-970Abstract Full Text PDF PubMed Google Scholar). Aliquots of a highly concentrated Fd sample (1.9 mm) were added to FNR at a concentration of 29 μm in a 1-cm cuvette. The FNR concentration changed from 28.8 to 27.5 μm during the titration. By assuming a simple binding equilibrium between Fd and FNR, a dissociation constant (Kd) of 27.8 μm was obtained from three-dimensional data ([FNR], [Fd], ΔA at 460 nm). Two-dimensional data were fitted as well, assuming a constant FNR concentration of 28.3 μm (average value during the titration), resulting in a Kd of 28 μm. Flash-absorption Spectroscopy—Measurements were made as described previously (49Sétif P. Bottin H. Biochemistry. 1995; 34: 9059-9070Crossref PubMed Scopus (67) Google Scholar), either in 1-cm square cuvettes or in 1-mm cuvettes at 295 K. 1-mm cuvettes were placed at 45° angles to the measuring and exciting beams, leading to an effective light path of 1.2 mm. The laser excitation (wavelength, 695 nm; duration, 6 ns; energy, 30 mJ; repetition rate between 0.02 and 0.25 Hz) was provided by a dye laser (Continuum, Excel Technology France) pumped by a Nd:YAG laser that was frequency-doubled (Quantel, France). Laser excitation was saturating PSI photochemistry. Actinic effects of the measuring light were kept minimal first by opening a shutter 1 ms before flash excitation and second by decreasing the light intensity to a level where no actinic effect was observable. This level was adjusted according to the time scale and the wavelength of the measurement. The time response of the setup was adjusted between 5 and 100 μs, with a varying charge resistor at the output of the measuring photodiode. At each wavelength, two interference filters of 10 nm full width at half the maximum transmission were placed in the measuring beam before and after the sample cuvette. As the present paper is based on quantitative estimates of FNR reduction and catalysis using photoexcited PSI as the source of reducing equivalents, it is essential to quantify PSI as precisely and reliably as possible. This quantitation is generally made by measuring the absorption changes associated to P700 photooxidation in the red or infrared regions. In previous studies from our laboratory (49Sétif P. Bottin H. Biochemistry. 1995; 34: 9059-9070Crossref PubMed Scopus (67) Google Scholar, 50Barth P. Lagoutte B. Sétif P. Biochemistry. 1998; 37: 16233-16241Crossref PubMed Scopus (48) Google Scholar), we used an absorption coefficient of 6500 m–1 cm–1 for P700+ at 820 nm. We reestimated the molar absorption coefficient of P700+ at 800 and 820 nm using TMPD+ as an electron acceptor and TMPD as an electron donor (51Hiyama T. Ke B. Biochim. Biophys. Acta. 1972; 267: 160-171Crossref PubMed Scopus (454) Google Scholar). TMPD concentrations of 200–250 μm were used in the absence of ascorbate and DCPIP. After a few seconds of white illumination, some oxidized TMPD was present in the cuvette. Measurements were performed at 580 nm, which allowed us to observe both the reduction of oxidized TMPD by the terminal PSI acceptors (t½ ≈0.5–2 ms) and reduction of P700+ by reduced TMPD (t½ ≈140–220 ms). Control measurements with methyl viologen (and ascorbate/DCPIP) were also performed at 580 nm for measuring the contribution of the terminal acceptor (FA,FB) to the absorption changes. An absorption coefficient of 11.7 mm–1 cm–1 was taken for oxidized TMPD at 610 nm (51Hiyama T. Ke B. Biochim. Biophys. Acta. 1972; 267: 160-171Crossref PubMed Scopus (454) Google Scholar). The effective absorption coefficient of oxidized TMPD at 580 nm was calculated by convoluting the transmission spectra of the interference filters used in the flash-induced measurements with the absorption spectrum of TMPD. Absorption coefficients of 7740 and 7010 m–1 cm–1 were thus found at 800 and 820 nm, respectively, for P700+ in monomeric PSI isolated from Synechocystis 6803. The absorption changes arising from P700 oxidation were recorded between 740 and 950 nm, with the P700+ spectrum exhibiting its usual shape with a broad maximum around 800–810 nm (data not shown). It appears that using a coefficient of 6500 m–1 cm–1 at 820 nm in previous publications was leading to an 11% overestimation of the Synechocystis 6803 PSI concentration. Moreover, it seems more appropriate to measure the P700+ concentration at 800 nm than at 820 nm, as 800 nm corresponds to an absorption maximum. We also performed this measurement with trimeric PSI from Thermosynechococcus elongatus (not shown). The absorption coefficient of P700+ at 800 nm was similar to that in Synechocystis 6803 within 1% and was identical to that deduced from chlorophyll extraction (52Porra R.J. Thompson W.A. Kriedemann P.E. Biochim. Biophys. Acta. 1989; 975: 384-394Crossref Scopus (4660) Google Scholar) by assuming the presence of 96 chlorophylls a per PSI as observed in crystals of PSI trimers (53Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauss N. Nature. 2001; 411: 909-917Crossref PubMed Scopus (2051) Google Scholar). Control flash-absorption experiments in the visible and near-infrared regions showed that addition of FNR has no effect on PSI in the absence of Fd and that 1 mm NADP+ (concentration used in all experiments when present) has no effect on either PSI or Fd in the absence of FNR. In most cases (data in Figs. 2 and 4, 5, 6, 7), the flash-induced absorption changes of a control cuvette containing wild type PSI, Fd, in some cases NADP+, but no FNR, were subtracted from a similar sample containing FNR. This subtraction procedure is both useful and necessary for the following reasons. 1) It allows us to subtract P700+ decay, which is identical with or without FNR, as checked by flash-absorption measurements in the near-IR region. In the presence of Fd, most parts of the P700+ decay (>95%) are very slow (t½ > 300 ms) and are ascribed to reduction by exogenous donors (ascorbate/DCPIP). A small part (<5%) is faster and is ascribed to damaged PSI reaction centers, in which the terminal acceptor (FA,FB) is not reduced and which exhibit recombination kinetics in the millisecond/submillisecond time range. These reaction centers are not capable of reducing Fd (54Hanley J. Sétif P. Bottin H. Lagoutte B. Biochemistry. 1996; 35: 8563-8571Crossref PubMed Scopus (42) Google Scholar), and the corresponding absorption changes can be eliminated as well by the subtraction procedure. 2) First-order Fd reduction by wild type PSI follows complex kinetics with exponential phases in the submicrosecond and microsecond time ranges (49Sétif P. Bottin H. Biochemistry. 1995; 34: 9059-9070Crossref PubMed Scopus (67) Google Scholar). As these kinetics do not depend on FNR, they are fully eliminated by the subtraction procedure. Therefore, this procedure allows us to selectively record the absorption changes associated with electron transfer from Fd to FNR (and to NADP+ when present). In a few experiments (data in Figs. 8 and 10), only the absorption changes due to P700+ formation and decay were subtracted. This was done by measuring, in the absence of Fd, the differential absorption coefficients of P700+ at the wavelength(s) of interest, λ and at 800 nm, using methyl viologen for fast reoxidation of the terminal PSI acceptor (FA,FB)– and by subtracting the 800 nm kinetics, after normalization to the absorption coefficient at λ. Flash-induced absorption data were fitted with several exponential components either with a home-written software or with the commercial software Origin (version 6.0; Microcal). A global fit of absorption kinetics was performed using the Excel solver (Microsoft). Numerical simulations were performed using the commercial software Mathcad (version 8.0; MathSoft).Fig. 4Dependence of the FNR reduction kinetics upon the FNR concentration. Traces analogous to trace C–B of Fig. 2 were recorded at 630 nm at different FNR concentrations. Experimental conditions were as follows: 0.2 μm PSI and 3 μm Fd in 1-cm cuvettes. Traces A–D, the kinetics were measured with FNR concentrations of 0.8, 1.6, 3.2, and 9.6 μm. Smooth lines correspond to the best simulated fit according to the kinetic model described in the text. This fit was performed by also using the kinetics recorded with 6.4 μm FNR (not shown). The best fit rate constants are as follows: koff = 805 s–1 for dissociation of the PSI-Fdred complex, k
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