Essential Role of a Single Arginine of Photosystem I in Stabilizing the Electron Transfer Complex with Ferredoxin
2000; Elsevier BV; Volume: 275; Issue: 10 Linguagem: Inglês
10.1074/jbc.275.10.7030
ISSN1083-351X
AutoresPatrick Barth, Isabelle Guillouard, Pièrre Sétif, Bernard Lagoutte,
Tópico(s)Spectroscopy and Quantum Chemical Studies
ResumoPsaE is one of the photosystem I subunits involved in ferredoxin binding. The central role of arginine 39 of this 8-kDa peripheral polypeptide has been established by a series of mutations. The neutral substitution R39Q leads to a 250-fold increase of the dissociation constant K d of the photosystem I-ferredoxin complex, as large as the increase induced by PsaE deletion. At pH 8.0, this K d value strongly depends on the charge of the residue substituting Arg-39: 0.22 μmfor wild type, 1.5 μm for R39K, 56 μm for R39Q, and more than 100 μm for R39D. The consequences of arginine 39 substitution for the titratable histidine were analyzed as a function of pH. The K d value of R39H is increased 140 times at pH 8.0 but only 5 times at pH 5.8, which is assigned to the protonation of histidine at low pH. In the mutant R39Q, the association rate of ferredoxin was decreased 3-fold compared with wild type, whereas an 80-fold increase is calculated for the dissociation rate. We propose that a major contribution of PsaE is to provide a prominent positive charge at position 39 for controlling the electrostatic interaction and lifetime of the complex with ferredoxin. PsaE is one of the photosystem I subunits involved in ferredoxin binding. The central role of arginine 39 of this 8-kDa peripheral polypeptide has been established by a series of mutations. The neutral substitution R39Q leads to a 250-fold increase of the dissociation constant K d of the photosystem I-ferredoxin complex, as large as the increase induced by PsaE deletion. At pH 8.0, this K d value strongly depends on the charge of the residue substituting Arg-39: 0.22 μmfor wild type, 1.5 μm for R39K, 56 μm for R39Q, and more than 100 μm for R39D. The consequences of arginine 39 substitution for the titratable histidine were analyzed as a function of pH. The K d value of R39H is increased 140 times at pH 8.0 but only 5 times at pH 5.8, which is assigned to the protonation of histidine at low pH. In the mutant R39Q, the association rate of ferredoxin was decreased 3-fold compared with wild type, whereas an 80-fold increase is calculated for the dissociation rate. We propose that a major contribution of PsaE is to provide a prominent positive charge at position 39 for controlling the electrostatic interaction and lifetime of the complex with ferredoxin. photosystem I dodecyl β-d-maltoside ferredoxin wild type N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Photosystem I (PSI)1 is a multisubunit complex catalyzing the light-driven electron transfer from reduced plastocyanin to oxidized ferredoxin (Fd). It is found in oxygen-evolving photosynthetic organisms, including cyanobacteria, eukaryotic algae, and higher plants. In cyanobacteria, the PSI complex can be isolated as monomers or trimers (1.Boekema E.J. Dekker J.P. van Heel M.G. Rögner M. Saenger W. Witt I. Witt H.T. FEBS Lett. 1987; 217: 283-286Crossref Scopus (132) Google Scholar). It is partly embedded in the membrane bilayer and constituted of 11 subunits, whose nomenclature (PsaA to PsaF and PsaI to PsaM) is derived from the gene names. The large subunits PsaA and PsaB are organized as a central heterodimeric core that houses most of the chlorophylls involved in light trapping and the redox cofactors required in electron transfer. Numerous structural details on the organization of this core have been gained from the successive improvements in the x-ray structure of PSI trimers from Synechococcus elongatus. The last map was calculated at 4-Å resolution yielding a more precise structural model of the reaction center domain and of the core antenna system (2.Schubert W.-D. Klukas O. Saenger W. Witt H.T. Fromme P. Krauss N. J. Mol. Biol. 1998; 280: 297-314Crossref PubMed Scopus (190) Google Scholar). PsaC, PsaD, and PsaE are easily removed from the core complex by chaotropic agents and are considered as extrinsic subunits (3.Golbeck J.H. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer Academic Publishers Group, Drodrecht, Netherlands1994: 319-360Crossref Google Scholar). The good accessibility of PsaD and PsaE on the stromal side of PSI has been clearly demonstrated in the past by electron microscopy, proteolysis studies, and epitope mapping (4.Lagoutte B. Vallon O. Eur. J. Biochem. 1992; 205: 1175-1185Crossref PubMed Scopus (33) Google Scholar, 5.Zilber A.L. Malkin R. Plant Physiol. (Bethesda). 1992; 99: 901-911Crossref PubMed Scopus (49) Google Scholar, 6.Xu Q. Guikema J.A. Chitnis P.R. Plant Physiol. (Bethesda). 1994; 106: 617-624Crossref PubMed Scopus (26) Google Scholar). These two subunits have been proposed as candidates for the binding and orientation of soluble ferredoxin to PSI. Based on cross-linking experiments, PsaD was the first subunit to be reported as being directly involved in the ferredoxin binding, and associated residues in the functional cross-linked complex were identified (7.Zanetti G. Merati G. Eur. J. Biochem. 1987; 169: 143-146Crossref PubMed Scopus (130) Google Scholar, 8.Zilber A.L. Malkin R. Plant Physiol. (Bethesda). 1988; 88: 810-814Crossref PubMed Google Scholar, 9.Lelong C. Sétif P. Lagoutte B. Bottin H. J. Biol. Chem. 1994; 269: 10034-10039Abstract Full Text PDF PubMed Google Scholar, 10.Lelong C. Boekema E.J. Kruip J. Bottin H. Rögner M. Sétif P. EMBO J. 1996; 15: 2160-2168Crossref PubMed Scopus (69) Google Scholar). First mutagenesis studies established a role of this subunit in the overall reduction process of ferredoxin (11.Xu Q. Jung Y.-S. Chitnis V.P. Guikema J.A. Golbeck J.H. Chitnis P.R. J. Biol. Chem. 1994; 269: 21512-21518Abstract Full Text PDF PubMed Google Scholar, 12.Hanley J. Sétif P. Bottin H. Lagoutte B. Biochemistry. 1996; 35: 8563-8571Crossref PubMed Scopus (42) Google Scholar). PsaC, -D, and -E have now been clearly localized by electron microscopy in a ridge of 30 Å height protruding from the membrane bilayer on the stromal side of PSI (13.Böttcher B. Graber P. Boekema E.J. Biochim. Biophys. Acta. 1992; 1100: 125-136Crossref PubMed Scopus (48) Google Scholar, 14.Kruip J. Chitnis P.R. Lagoutte B. Rögner M. Boekema E.J. J. Biol. Chem. 1997; 272: 17061-17069Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). This ridge is in contact with the electron acceptor proteins, ferredoxin or flavodoxin (10.Lelong C. Boekema E.J. Kruip J. Bottin H. Rögner M. Sétif P. EMBO J. 1996; 15: 2160-2168Crossref PubMed Scopus (69) Google Scholar, 15.Mühlenhoff U. Kruip J. Bryant D. Rögner M. Sétif P. Boekema E.J. EMBO J. 1996; 15: 488-497Crossref PubMed Scopus (43) Google Scholar). PsaC occupies a central position in this ridge. This subunit carries the terminal electron acceptors of PSI, FA and FB([4Fe-4S] clusters), the latter being the direct electron donor to the [2Fe-2S] center of soluble Fd. In electron density maps, PsaC is flanked by two compact regions ascribed to PsaD and PsaE, PsaD facing toward the trimer axis C3 and PsaE being the more distant and located outside (Refs. 2.Schubert W.-D. Klukas O. Saenger W. Witt H.T. Fromme P. Krauss N. J. Mol. Biol. 1998; 280: 297-314Crossref PubMed Scopus (190) Google Scholar and 16.Klukas O. Schubert W.-D. Jordan P. Krauß N. Fromme P. Witt H.T. Saenger W. J. Biol. Chem. 1999; 274: 7351-7360Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar; Fig. 5). Cyanobacterial PsaE is a slightly basic, water-soluble protein that contains 69–75 residues. Although the cyanobacterial proteins are significantly smaller than their mature homologues from higher plants (91–101 amino acids), the common sequence of PsaE proteins is highly conserved (17.Rousseau F. Lagoutte B. FEBS Lett. 1991; 260: 241-244Crossref Scopus (4) Google Scholar, 18.Golbeck J.H. Bryant D.A. Curr. Top. Bioenerg. 1991; 16: 83-177Crossref Google Scholar). No significant homology to other proteins has been found in sequence data bases. NMR studies in solution of the overexpressed PsaE from Synechococcus sp. PCC 7002 have shown that the isolated polypeptide is composed of a rigid, five-stranded β-barrel, with an extended flexible loop between the third and fourth β-strands (19.Falzone C.J. Kao Y.-H. Zhao J. Bryant D.A. Lecomte J.T.J. Biochemistry. 1994; 33: 6052-6062Crossref PubMed Scopus (72) Google Scholar). Recently, the representative NMR model of PsaE was fit into the electron density map of PSI. The superimposition of the NMR and x-ray model structures of PsaE reveals both to be almost identical in the core region (16.Klukas O. Schubert W.-D. Jordan P. Krauß N. Fromme P. Witt H.T. Saenger W. J. Biol. Chem. 1999; 274: 7351-7360Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). PsaE-deleted strains of synechocystis and Synechococcus were originally reported to have no obvious phenotypic alterations (20.Chitnis P.R. Reilly P.A. Miedel M.C. Nelson N. J. Biol. Chem. 1989; 264: 18374-18380Abstract Full Text PDF PubMed Google Scholar). It was later shown that the PsaE subunit was necessary for an efficient reduction of Fd (21.Rousseau F. Setif P. Lagoutte B. EMBO J. 1993; 12: 1755-1765Crossref PubMed Scopus (91) Google Scholar). Subsequently, more careful physiological investigations revealed that the growth rate of the deletion mutant from Synechococcus sp. PCC 7002 was considerably reduced at low light intensity or low carbon dioxide level (22.Zhao J. Snyder W.B. Mühlenhoff U. Rhiel E. Warren P.V. Golbeck J.H. Bryant D.A. Mol. Microbiol. 1993; 9: 183-194Crossref PubMed Scopus (76) Google Scholar). This phenotype suggested that the PsaE subunit might play a role in cyclic electron transport. Apart from these functional implications, different biochemical studies pointed to a stabilizing role of this subunit on the stromal side of PSI (23.Chitnis P.R. Nelson N. Plant Physiol. (Bethesda). 1992; 99: 239-246Crossref PubMed Scopus (27) Google Scholar, 24.Weber N. Strotmann H. Biochim. Biophys. Acta. 1993; 1143: 204-210Crossref PubMed Scopus (40) Google Scholar). Some cross-linking experiments on higher plants PSI indicated a proximity of PsaE and ferredoxin:NADP+oxidoreductase (25.Andersen B. Scheller H.V. Möller L. FEBS Lett. 1992; 311: 169-173Crossref PubMed Scopus (111) Google Scholar), but a high yield cross-linked product between PsaE and Fd was never reported. It becomes clear now that all the three subunits PsaC, PsaD, and PsaE participate to some extent in the formation of the ferredoxin site. A specific role of PsaD and PsaE on the association and dissociation rates of Fd has been proposed from studies on deleted mutants, with a 100-fold decreased affinity for Fd when PsaE is absent (26.Barth P. Lagoutte B. Sétif P. Biochemistry. 1998; 46: 16233-16241Crossref Scopus (48) Google Scholar). More detailed information on some specific residues of PsaD and PsaC (12.Hanley J. Sétif P. Bottin H. Lagoutte B. Biochemistry. 1996; 35: 8563-8571Crossref PubMed Scopus (42) Google Scholar,27.Fischer N. Hippler M. Sétif P. Jacquot J.-P. Rochaix J.-D. EMBO J. 1998; 17: 849-858Crossref PubMed Scopus (75) Google Scholar) involved in this process are also available, but no precise amino acid of PsaE has yet been described as essential for this interaction. In the present work, we clearly show by a series of single site-directed mutations that a unique arginine at position 39 plays a central role in the PsaE-mediated Fd interaction with PSI. Recombinant ferredoxin was overexpressed in Escherichia coli. The fed1 gene ofSynechocystis sp. PCC 6803 was cloned into a pRSet expression vector. Transformed E. coli BL21 (DE3) cells were grown at 30 °C in LB medium supplemented with 100 μg/ml ampicillin. A 48-h growing time is required to get a sufficient yield of Fd. The inducer isopropyl-1-thio-β-d-galactopyranoside has been found dispensable when using the LB medium (28.Jacquot J.-P. Stein M. Suzuki A. Liottet S. Sandoz G. Miginiac-Maslow M. FEBS Lett. 1997; 400: 293-296Crossref PubMed Scopus (45) Google Scholar). Cells were harvested, resuspended in a Tris-HCl buffer (50 mmTris-HCl, 0.1 m NaCl, 1 mm EDTA, 5 mm MgCl2, 0.1% β-mercaptoethanol, pH 8.5), and broken in a French press. Cellular debris were sedimented at 9000 × g for 10 min at 4 °C, and the whole cell extract was stepwise precipitated with ammonium sulfate (40, 55, and 70% saturation). Ferredoxin is purified from the last supernatant as already described (29.Bottin H. Lagoutte B. Biochim. Biophys. Acta. 1992; 1101: 48-56Crossref PubMed Scopus (101) Google Scholar, 30.Lelong C. Sétif P. Bottin H. André F. Neumann J.-M. Biochemistry. 1995; 34: 14462-14473Crossref PubMed Scopus (45) Google Scholar). Ferredoxin from the last purification step was concentrated by ultrafiltration and usually frozen at a concentration of 200 μm in a low ionic strength buffer (5 mm Hepes, pH 7.0). Approximately 2–5 mg of purified ferredoxin was obtained from 1 liter of induced cells. Variant PsaE-PSI were isolated from recombinantSynechocystis. Thylakoid membranes were obtained from French press broken cells after extensive washing with ice-cold 20 mm Tricine, 1 mm EDTA, pH 7.8. PSI was obtained after solubilization with 1% (w/v) β-DM and purified on a sucrose density gradient (31.Rögner M. Nixon P.J. Diner B.A. J. Biol. Chem. 1990; 265: 6189-6196Abstract Full Text PDF PubMed Google Scholar). The upper green band consisting of highly enriched monomeric PSI particles was dialyzed against 20 mmTricine/NaOH, pH 7.8, and 0.03% β-DM and concentrated by ultrafiltration. The chlorophyll concentration was determined in 80% acetone. The last step of the purification procedure was anion-exchange chromatography on a Mono Q column essentially as described (31.Rögner M. Nixon P.J. Diner B.A. J. Biol. Chem. 1990; 265: 6189-6196Abstract Full Text PDF PubMed Google Scholar). The only modification was the substitution of ammonium sulfate instead of magnesium sulfate as eluting salt to avoid any contamination by Mg2+, whose concentration is a critical parameter for the reduction of Fd. The psaE gene was previously cloned into the Bluescript SK+ plasmid to produce pFBsCE (21.Rousseau F. Setif P. Lagoutte B. EMBO J. 1993; 12: 1755-1765Crossref PubMed Scopus (91) Google Scholar). Codon exchanges, leading to amino acid substitutions, were carried out by site-directed mutagenesis by the method of Kunkel (32.Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4892) Google Scholar). For this technique, 20-base oligonucleotide primers were constructed such that a codon change resulted in an amino acid substitution. The desired nucleotide substitution in the genes encoding the mutant proteins was confirmed by dideoxynucleotide sequencing of the complete gene. The strain of Synechocystis sp. PCC 6803 deleted for thepsaE gene (6803 ΔE) was used to raise site-directed mutants of PsaE. Mutants were generated by direct transformation of the 6803 ΔE with the pFBsCE constructs and grown for a 24-h period in liquid BG11. Selection of transformants was carried out by serial dilution in the presence of increasing chloramphenicol concentrations (from 1 to 30 μg/ml). Once resistance to 30 μg/ml chloramphenicol was established, the selection procedure was continued through further 8–10 rounds of liquid subculture. Genomic DNA from liquid cultures of Synechocystisvariants was isolated as described previously (33.Tandeau de Marsac N. Borrias W.E. Kuhlemeier C.J. Castets A.M. van Arkel G.A. van den Hondel C.A.M.J.J. Gene (Amst.). 1982; 20: 11-119Crossref PubMed Scopus (39) Google Scholar) and used for control sequencing. A 360-base pair fragment encompassing the fullpsaE sequence was amplified using the polymerase chain reaction procedure (12.Hanley J. Sétif P. Bottin H. Lagoutte B. Biochemistry. 1996; 35: 8563-8571Crossref PubMed Scopus (42) Google Scholar), and sequencing reactions were carried out by the dideoxy termination method using the cycle sequencing kit (Roche Molecular Biochemicals). Electrophoreses were made on mini-slab gels (Bio-Rad apparatus) using the Tris/Tricine buffer system (34.Schägger H. Von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10452) Google Scholar). The concentration of P700 was measured in all mutated PSI samples, by flash-absorption spectroscopy at 820 nm, and a constant amount of 125 pmol of P700 was loaded for each sample after 1 h dissociation in the loading buffer at 45 °C. Electroblotting of the proteins was made overnight at 4 °C using two layers of Immobilon P membranes (Amersham Pharmacia Biotech) and a constant current of 10 mA. PsaE was probed with a polyclonal antibody (21.Rousseau F. Setif P. Lagoutte B. EMBO J. 1993; 12: 1755-1765Crossref PubMed Scopus (91) Google Scholar) revealed by an alkaline phosphatase anti-mouse IgG conjugate (Sigma) and a luminescent substrate (Immun-star, Bio-Rad). Under these conditions, variable amounts of the PsaE polypeptide were recovered on the two successive membrane layers. This could reflect different behaviors as a result of the different mutations and a possible heterogeneity of the local electric field. It underlines the necessity of a careful survey of the blotting conditions before getting confident quantitations. Quantitation was made by adding the integrated optical densities on the two x-ray films. The yields of recovery for all six mutated PsaE polypeptides were found between 89 and 104% of the WT, which is in the range of cumulated errors of the technique (Table I).Table ICharacteristics of Fd reduction by WT and PsaE site-directed mutants of PSIAmount of PsaE subunit detected by immunoblotSecond-order rate constantk onDissociation constantK dt 1/2 of first-order Fd reduction%10 8 m −1 s −1μmμsWT1002.7a±1.0; this relatively large uncertainty is due to the measuring conditions (1 experiment with [PSI] = [Fd] = 0.24 μm).0.22<1/20/125R42Q92bNot measured.0.41idem wtR39K1013.0a±1.0; this relatively large uncertainty is due to the measuring conditions (1 experiment with [PSI] = [Fd] = 0.24 μm).1.5idem wtR39H970.87cMeasured from a linear fit of k obs of slow phase versus [Fd] (Fig. 2, bottom).31<1/20R39Q910.84cMeasured from a linear fit of k obs of slow phase versus [Fd] (Fig. 2, bottom).56 100dNot measured, amplitude too small.R39E1040.18cMeasured from a linear fit of k obs of slow phase versus [Fd] (Fig. 2, bottom).>100dNot measured, amplitude too small.PsaE minus01.5cMeasured from a linear fit of k obs of slow phase versus [Fd] (Fig. 2, bottom).52<1/16All spectroscopic measurements were performed at pH 8.0.a ±1.0; this relatively large uncertainty is due to the measuring conditions (1 experiment with [PSI] = [Fd] = 0.24 μm).b Not measured.c Measured from a linear fit of k obs of slow phase versus [Fd] (Fig. 2, bottom).d Not measured, amplitude too small. Open table in a new tab All spectroscopic measurements were performed at pH 8.0. Measurements were made as described previously, in cuvettes of either 1-cm or 2-mm optical path length (26.Barth P. Lagoutte B. Sétif P. Biochemistry. 1998; 46: 16233-16241Crossref Scopus (48) Google Scholar, 35.Sétif P. Bottin H. Biochemistry. 1995; 34: 9059-9070Crossref PubMed Scopus (67) Google Scholar). 2-mm cuvettes were used for mutants of low affinity, using high Fd concentrations. Kinetic curves were corrected according to Sétif and Bottin (35.Sétif P. Bottin H. Biochemistry. 1995; 34: 9059-9070Crossref PubMed Scopus (67) Google Scholar), so that they correspond solely to electron transfer from the terminal acceptor of PSI (FA, FB)−, to Fd. For PSIs having a relatively high affinity for Fd (K d ≤ 1.5 μm), it is not possible to obtain second-order rate constants from experiments made under pseudo-first order conditions (with [Fd] ≫ [PSI]). In that case, determination of second-order rate constants was performed with equal concentrations of PSI and Fd (≈0.24 μm), allowing use of an analytical function to fit the data (36.Sétif P. Bottin H. Biochemistry. 1994; 33: 8495-8504Crossref PubMed Scopus (72) Google Scholar). All kinetic measurements were performed at a constant ionic strength of 56 mm, taking into account all contributions from the buffer, sodium ascorbate (1–3 mm) and added salts. Constant buffer and MgCl2 concentrations were used throughout the experiments (20 and 5 mm, respectively). The NaCl concentration (30 mm) was adjusted in order to provide an ionic strength of 56 mm. Dissociation constants were determined from the dependence of the sum of first-order amplitudes upon Fd concentration. For mutants R39D and R39E of PsaE, this was not attempted for two different reasons as follows: first, the first-order amplitude was rather small and became only observable at Fd concentrations above 30 μm. Under these conditions, the second-order process is sufficiently fast and predominant to impede a precise determination of the first-order decay. Second, the first-order amplitude increases only slightly between 32 and 64 μm Fd, which is the maximal Fd concentration that was tested (R39D, from 30 to 32% of total amplitude of Fd reduction; R39E, from 19 to 23%). This suggests that a PSI·Fd complex cannot be formed in 100% of PSI, even at very high Fd concentrations. Although a precise analysis is beyond the precision of our data, it seems that the maximum (asymptotic) proportion of PSI·Fd complexes is less than 50% in both R39D and R39E. Fd reduction by WT PSI has been studied inSynechocystis 6803 by flash-absorption spectroscopy at room temperature in the 460–600-nm region (see under “Experimental Procedures”) (35.Sétif P. Bottin H. Biochemistry. 1995; 34: 9059-9070Crossref PubMed Scopus (67) Google Scholar, 36.Sétif P. Bottin H. Biochemistry. 1994; 33: 8495-8504Crossref PubMed Scopus (72) Google Scholar). The wavelength of 580 nm has been selected in this work to minimize signals not related to Fd reduction on the microsecond time scale. All measurements were made by subtracting a reference signal without Fd, so that the kinetics directly represent Fd reduction (35.Sétif P. Bottin H. Biochemistry. 1995; 34: 9059-9070Crossref PubMed Scopus (67) Google Scholar, 36.Sétif P. Bottin H. Biochemistry. 1994; 33: 8495-8504Crossref PubMed Scopus (72) Google Scholar). Three different first-order phases of Fd reduction have been reported with half-times (t 12) of ≈500 ns and 15 and 110 μs (36.Sétif P. Bottin H. Biochemistry. 1994; 33: 8495-8504Crossref PubMed Scopus (72) Google Scholar). These phases correspond to electron transfer within PSI·Fd complexes preformed before the flash excitation; it may reflect the presence of three, closely related, subclasses of complexes. A second-order rate of Fd reduction has been also previously measured, which is thought to correspond to a diffusion-limited process (k on = 2–5 × 108m−1 s−1) (35.Sétif P. Bottin H. Biochemistry. 1995; 34: 9059-9070Crossref PubMed Scopus (67) Google Scholar). The dissociation constants K d for the PSI·Fd complex were calculated from the dependence of first-order amplitudes upon Fd concentration (35.Sétif P. Bottin H. Biochemistry. 1995; 34: 9059-9070Crossref PubMed Scopus (67) Google Scholar, 36.Sétif P. Bottin H. Biochemistry. 1994; 33: 8495-8504Crossref PubMed Scopus (72) Google Scholar). A recent modification in the final purification step of PSI led to a somewhat decreasedK d of the PSI·Fd complex (0.22 μminstead of ≈0.5 μm at pH 8.0). An equivalent decrease was not observed for the PsaE-deleted mutant (52 μm for both types of preparations). A number of site-directed mutations on positively charged residues were previously made on PsaE. They were more or less drastic, sometimes resulting in the non-integration of PsaE, but none of them led to interesting phenotypes in terms of ferredoxin reduction (21.Rousseau F. Setif P. Lagoutte B. EMBO J. 1993; 12: 1755-1765Crossref PubMed Scopus (91) Google Scholar). Two highly conserved arginines, Arg-39 and Arg-42, escaped this first series of mutations and were first replaced in the present work by the neutral glutamine. Fd reduction by PSI prepared from the R39Q and R42Q mutants is illustrated in Fig. 1. At a Fd concentration of about 1 μm, Fd reduction is very similar in R42Q and WT PSI with a dominating first-order process (traces b and c, respectively). The three first-order phases previously found for WT PSI are recovered in the R42Q mutant (t 12 <1, 20, and 125 μs). TheK d values for the PSI·Fd complex were in the same range for WT and R42Q (0.22 and 0.41 μm, respectively). In contrast with R42Q, Fd reduction is dramatically affected in R39Q. In the presence of ≈1 μm Fd, no first-order phase is present, and only a second-order process of Fd reduction is observed on a slower time scale (not shown). However, at much larger Fd concentrations, a first-order phase becomes visible (trace eof Fig. 1, [Fd] = 16 μm). The first-order components are nevertheless small at this Fd concentration (left partof trace e), and a second-order process is still predominant (right part of trace e). This process is very similar to that previously observed with PsaE-minus PSI (trace f; Ref. 26.Barth P. Lagoutte B. Sétif P. Biochemistry. 1998; 46: 16233-16241Crossref Scopus (48) Google Scholar). This close behavior is further confirmed at higher Fd concentrations up to 64 μm. The K d for R39Q was calculated from the fit of the total first-order amplitude dependence versus Fd concentration (Fig.2, upper part). A value of 56 μm was found, which is very close to theK d value of 52 μm for PsaE-minus PSI. Second-order rate constants k on were also calculated from linear fits of the observed rate of the slowest phase (Fig. 2, lower part): a 3-fold decrease ink on is observed for R39Q (0.84 × 108m−1 s−1) compared with WT (2.7 × 108m−1s−1) (Table I). A decrease of about 2-fold is also found compared with PsaE-minus (1.5 × 108m−1 s−1). The different characteristics of Fd reduction at pH 8.0 are given for WT, PsaE-minus, R39Q, and R42Q in Table I, together with characteristics of the other mutants studied in the present work. The amounts of the PsaE polypeptides, as found by immunoblotting (see under “Experimental Procedures”), are also reported for all mutants in Table I, showing a WT level of integration of the PsaE subunit in all site-directed mutants.Figure 2Characteristics of Fd reduction by PsaE mutants obtained from flash-induced absorbance changes at 580 nm. Upper part, sum of amplitudes of the fast first-order components of Fd reduction as a function of Fd concentration for R39Q (closed circles) and R39H (open triangles) at pH 8.0 (experimental conditions as in Fig. 1; 2-mm cuvette, [PSI] = 1.06 μm). These data were fitted (R39Q, continuous line; R39H, dotted line) assuming a simple binding equilibrium between PSI and Fd and by taking the maximal asymptotic value of Fd reduction as a fixed parameter. These values were obtained from the total amplitudes of first- and second-order components (1.51 × 10−4 and 1.25 × 10−4 for R39Q and R39H, respectively). From these fits, dissociation constants of 56 and 31 μm were calculated for R39Q and R39H, respectively. Lower part, rate of the slower phase of Fd reduction as a function of Fd concentration for PsaE-minus (crosses), R39H (open triangles), R39Q (closed circles), R39D (closed squares), and R39E (closed triangles) PSI (experimental conditions as in Fig.1; 2-mm cuvette, [PSI] ≈1.1 μm; 1-cm cuvette, [PSI] ≈0.2 μm). Linear fits were performed to get the second-order rate constants of Fd reduction (PsaE-minus, 1.5 × 108m−1 s−1; R39H, 0.87 × 108m−1s−1; R39Q, 0.84 × 108m−1 s−1; R39D, 0.27 × 108m−1 s−1; R39E, 0.18 × 108m−1s−1).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Arginine 39 was then changed for residues of the same or reverse charge, and also for histidine, a titratable residue at physiological pH. A kinetic trace of the fast reduction process is shown for mutant R39K in Fig. 1(trace a; [Fd] = 2 μm). In this trace, fast first-order components are observed on a 400-μs time scale, but the signal amplitude is smaller than for WT or R42Q (traces cand b, respectively) and represents only about 50% of ferredoxin reduction (≈50% being due to a slower second-order process, which contributes only marginally on this time scale). A quantitative analysis of R39K kinetics shows a 7-fold decrease in affinity compared with WT (K d = 1.5 μm; Table I) but no modification of the second-order rate constant k on (3.0 × 108m−1 s−1). R39H mutated PSI is much more affected and behaves like R39Q PSI (traces b anda, respectively, upper part of Fig.3) with a 140-fold decrease in Fd affinity (Table I) and a similarly 3-fold decreased second-order rate constant (0.87 × 108m−1s−1). This suggests that His-39 is in a neutral deprotonated form at pH 8.0. A more detailed study of the R39H mutant is described below. When Arg-39 is replaced by an acidic residue (Asp or Glu), Fd reduction becomes even less efficient than in R39Q or R39H mutants (R39D,trace d of Fig. 1, Table I). At all Fd concentrations that were studied (up to 64 μm), the second-order rate constant is largely dominant, suggesting that K dvalue is larger than 100 μm (more than 500-fold decrease in affinity). With both R39D and R39E mutants, a quantitative determination of K d was not attempted (see under “Experimental Procedures”). A semi-quantitative analysis of our data indicates that the maximum (asymptotic) proportion of PSI·Fd complexes is less than 50% in both R39D and R39E. Compared with R39Q and R39H, the second-order rate constants k on of R39D and R39E are also significantly smaller, with values of 0.27 and 0.18 × 108m−1s−1, respectively (lower part of Fig. 2). The kinetics of Fd reduction by R39Q, R39H, and WT PSI at pH values of 5.8 and
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