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The Luminal Helix l of PsaB Is Essential for Recognition of Plastocyanin or Cytochrome c 6and Fast Electron Transfer to Photosystem I in Chlamydomonas reinhardtii

2002; Elsevier BV; Volume: 277; Issue: 8 Linguagem: Inglês

10.1074/jbc.m110633200

1083-351X

Frederik Sommer, Friedel Drepper, Michael Hippler,

Light effects on plants

At the lumenal side of photosystem I (PSI) in cyanobacteria, algae, and vascular plants, proper recognition and binding of the donor proteins plastocyanin (pc) and cytochrome (cyt) c 6 are crucial to allow subsequent efficient electron transfer to the photooxidized primary donor. To characterize the surface regions of PSI needed for the correct binding of both donors, loop j of PsaB of Chlamydomonas reinhardtii was modified using site-directed mutagenesis and chloroplast transformation. Mutant strains D624K, E613K/D624K, E613K/W627F, and D624K/W627F accumulated 50% of PSI as compared with wild type. This was sufficient to isolate the altered PSI and perform a detailed analysis of the electron transfer between the modified PSI and the two algal donors using flash-induced spectroscopy. Such an analysis indicated that residue Glu613 of PsaB has two functions: (i) it is crucial for an improved unbinding of the two donors from PSI, and (ii) it orientates the positively charged N-terminal domain of PsaF in a way that allows efficient binding of pc or cyt c 6 to PSI. Mutation of Trp627 to Phe completely abolishes the formation of an intermolecular electron transfer complex between pc and PSI and also drastically diminishes the rate of electron transfer between the donor and PSI. This mutation also hinders binding and electron transfer between the altered PSI and cytc 6. It causes a 10-fold increase of the half-time of electron transfer within the intermolecular complex of cytc 6 and PSI. These data strongly suggest that Trp627 is a key residue of the recognition site formed by the core of PSI for binding and electron transfer between the two soluble electron donors and the photosystem. At the lumenal side of photosystem I (PSI) in cyanobacteria, algae, and vascular plants, proper recognition and binding of the donor proteins plastocyanin (pc) and cytochrome (cyt) c 6 are crucial to allow subsequent efficient electron transfer to the photooxidized primary donor. To characterize the surface regions of PSI needed for the correct binding of both donors, loop j of PsaB of Chlamydomonas reinhardtii was modified using site-directed mutagenesis and chloroplast transformation. Mutant strains D624K, E613K/D624K, E613K/W627F, and D624K/W627F accumulated 50% of PSI as compared with wild type. This was sufficient to isolate the altered PSI and perform a detailed analysis of the electron transfer between the modified PSI and the two algal donors using flash-induced spectroscopy. Such an analysis indicated that residue Glu613 of PsaB has two functions: (i) it is crucial for an improved unbinding of the two donors from PSI, and (ii) it orientates the positively charged N-terminal domain of PsaF in a way that allows efficient binding of pc or cyt c 6 to PSI. Mutation of Trp627 to Phe completely abolishes the formation of an intermolecular electron transfer complex between pc and PSI and also drastically diminishes the rate of electron transfer between the donor and PSI. This mutation also hinders binding and electron transfer between the altered PSI and cytc 6. It causes a 10-fold increase of the half-time of electron transfer within the intermolecular complex of cytc 6 and PSI. These data strongly suggest that Trp627 is a key residue of the recognition site formed by the core of PSI for binding and electron transfer between the two soluble electron donors and the photosystem. In Chlamydomonas reinhardtii, the multiprotein complex photosystem I (PSI) 1PSIphotosystem IcytcytochromepcplastocyaninEeinstein is a light-driven oxidoreductase that transfers electrons from the soluble lumenal donors plastocyanin (pc) and cytochrome (cyt)c 6 to the soluble stromal acceptor ferredoxin. The eukaryotic PSI reaction center is a membrane-bound complex consisting of 13–14 polypeptide subunits (1Chitnis P.R. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 593-626Crossref PubMed Scopus (210) Google Scholar). Depending on the relative availability of copper in the culture medium,Chlamydomonas can replace the type I copper protein pc with a class I c-type cyt c 6 (2Wood P.M. Eur. J. Biochem. 1978; 87: 9-19Crossref PubMed Scopus (186) Google Scholar, 3Merchant S. Bogorad L. Mol. Cell. Biol. 1986; 6: 462-469Crossref PubMed Scopus (140) Google Scholar). The two large PSI subunits, PsaA and PsaB, which carry the photochemical reaction center, each contain 11 transmembrane helices, of which helicesk and m are connected by the lumenal loopj (Fig. 1). The 2.5 Å x-ray crystal structure of PSI from Synechococcus elongatus (4Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauss N. Nature. 2001; 411: 909-917Crossref PubMed Scopus (2082) Google Scholar) reveals that a part of the loop forms an α-helix l that is oriented parallel to the membrane close to the primary donor, P700. photosystem I cytochrome plastocyanin einstein In eukaryotic organisms, docking of the soluble lumenal donors pc and cyt c 6 to PSI depends mainly on two different recognition sites, which are: (i) negative charges on the surface of the donor attracted by the positively charged N-terminal domain of the PsaF subunit of PSI (5Farah J. Rappaport F. Choquet Y. Joliot P. Rochaix J.-D. EMBO J. 1995; 14: 4976-4984Crossref PubMed Scopus (112) Google Scholar, 6Hippler M. Reichert J. Sutter M. Zak E. Altschmied L. Schröer U. Herrmann R.G. Haehnel W. EMBO J. 1996; 15: 6374-6384Crossref PubMed Scopus (127) Google Scholar, 7Hippler M. Drepper F. Haehnel W. Rochaix J.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7339-7344Crossref PubMed Scopus (96) Google Scholar), and (ii) the "northern part" of the donors interacting with an as yet undefined site of PSI (8Haehnel W. Jansen T. Gause K. Klösgen R.B. Stahl B. Michl D. Huvermann B. Karas M. Herrmann R.G. EMBO J. 1994; 13: 1028-1038Crossref PubMed Scopus (128) Google Scholar). The amino acid sequence of helix l is highly conserved among different species (Fig. 1). It is assumed that it could be involved in docking of the soluble donors (9Schubert W. Klukas O. Saenger W. Fromme P. Witt H.T. J. Mol. Biol. 1997; 272: 741-769Crossref PubMed Scopus (227) Google Scholar). Sun et al. (10Sun J. Hervas M. Navarro J.A., De La Rosa M. Chitnis P.R. J. Biol. Chem. 1999; 274: 19048-19054Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) generated site-directed mutants in the lumenal loop of the PsaB protein fromSynechocystis sp. PCC 6803. Indeed, a double mutant (W622C/A623R) was strongly affected in the interaction between the altered PSI and the electron donors pc and cytc 6. In contrast to eukaryotic organisms, in cyanobacteria, efficient binding and electron transfer between PSI and pc or cyt c 6 do not depend on the PsaF subunit because the specific deletion of the psaF gene in cyanobacteria did not affect photoautotrophic growth (11Chitnis P.R. Purvis D. Nelson N. J. Biol. Chem. 1991; 266: 20146-20151Abstract Full Text PDF PubMed Google Scholar), and thein vivo measured electron transfer rate between cytc 553 and PSI was the same as that in wild type (12Xu Q., Xu, L. Chitnis V. Chitnis P. J. Biol. Chem. 1994; 269: 3205-3211Abstract Full Text PDF PubMed Google Scholar). In vitro measurements revealed that even at high concentrations of pc or cyt c 6 no difference in electron transfer rates could be measured between the donor proteins and PSI isolated from the wild type or the PsaF-deficientSynechocystis mutant (6Hippler M. Reichert J. Sutter M. Zak E. Altschmied L. Schröer U. Herrmann R.G. Haehnel W. EMBO J. 1996; 15: 6374-6384Crossref PubMed Scopus (127) Google Scholar). These differences can be explained by the absence of the specific eukaryotic N-terminal recognition site in the cyanobacterial PsaF protein that is required for the binding of pc and cyt c 6 to PSI (6Hippler M. Reichert J. Sutter M. Zak E. Altschmied L. Schröer U. Herrmann R.G. Haehnel W. EMBO J. 1996; 15: 6374-6384Crossref PubMed Scopus (127) Google Scholar, 7Hippler M. Drepper F. Haehnel W. Rochaix J.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7339-7344Crossref PubMed Scopus (96) Google Scholar, 13Hippler M. Drepper F. Rochaix J.D. Muehlenhoff U. J. Biol. Chem. 1999; 274: 4180-4188Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). To elucidate the role of loop j and especially that of helixl of the PsaB protein in a eukaryotic system we performed a combinatorial site-directed mutagenesis approach taking advantage of a PsaB-deficient mutant of C. reinhardtii (14Redding K. MacMillan F. Leibl W. Brettel K. Hanley J. Rutherford A.W. Breton J. Rochaix J.D. EMBO J. 1998; 17: 50-60Crossref PubMed Scopus (85) Google Scholar). Of seven mutant strains, three allowed isolation of PSI sufficient for a further functional characterization. Electron transfer between the altered PSI particles and the donors pc and cyt c 6 has been investigated using flash-induced absorption spectroscopy. The results suggest features of the lumenal surface of PSI that are crucial for the optimized binding equilibria of both donors. Furthermore, differences between both donors in the recognition and requirements for a correct docking and electron transfer can be identified. The data presented here will be discussed in light of the new structural information available for cyanobacterial PSI (4Jordan P. Fromme P. Witt H.T. Klukas O. Saenger W. Krauss N. Nature. 2001; 411: 909-917Crossref PubMed Scopus (2082) Google Scholar). C. reinhardtii wild type and mutant strains were grown as described previously (15Harris E.H. The Chlamydomonas Sourcebook. Academic Press, San Diego, CA1989Google Scholar). Tris acetate phosphate medium and high-salt medium were solidified with 2% Bacto agar (Difco) and supplemented with 150 μg/ml streptomycin (Sigma) when required. Procedures for the preparation of recombinant plasmids and DNA sequencing were performed as described previously (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Escherichia coli DH5α was used as bacterial host. The site-directed change of PsaB Glu613 to Asn and Lys was carried out in a single tube PCR as described by Picardet al. (17Picard V. Ersdal-Badju E., Lu, A. Bock S.C. Nucleic Acids Res. 1994; 22: 2587-2591Crossref PubMed Scopus (216) Google Scholar). We used degenerated mutagenic oligonucleotide N (5′-CAATCCGATAA(C/G)TCGTCTACT-3′) together with two oligonucleotides X (5′-GGTGCTCTAGATGCTCGT-3′) and P (5′-ACCATTAATTAAAAGAGA-3′) complementary to the flanking regions about 150 bp upstream and downstream from the mutagenic site containing cleavage sites XbaI and PacI, respectively. As template DNA, we used pKR162, a kind gift from K. Redding containing the PsaB coding sequence and 5′-untranslated region and also containing an aadA expression cassette conferring spectinomycin and streptomycin resistance to C. reinhardtii(18Goldschmidt-Clermont M. Nucleic Acids Res. 1991; 19: 4083-4089Crossref PubMed Scopus (406) Google Scholar). The resulting DNA fragments were cloned into pBluescript SK, amplified in bacteria, andXbaI/PacI-digested. After gel purification, the fragments were then cloned into pKR162, replacing the original sequence. For the site-directed mutagenesis of PsaB Trp627 to Phe and PsaB Asp624 to Lys, we performed PCR using pKR162 as template, oligonucleotide X, and the degenerated mutagenic oligonucleotides F (5′-ACCATTAATTAATTGAGAAGTT(G/T)AAC(C/A)AT(C/A)GG(G/T)AGTCACGTAACCA-3′) or K (5′-ACCATTAATTAATTGAGAAGAGTTTAACCATAGGTA(G/C)TTACGTAACCA-3′), respectively, containing the PacI cleavage site. The resulting DNA fragments were cloned into pKR162 as described above. For double mutants, PCR was performed with oligonucleotides F or K and X and with previously generated mutagenized pKR162 vectors as templates. All mutations were verified by sequencing using the ABI310 capillary system. Chloroplast transformation in C. reinhardtii was carried out as described previously (19Boynton J.E. Gillham N.W. Methods Enzymol. 1993; 217: 510-536Crossref PubMed Scopus (101) Google Scholar) using a helium-driven PDS-1000/He particle gun (Bio-Rad) with 1100 psi rupture discs (Bio-Rad). M10 tungsten particles (2.5 mg; Bio-Rad) were coated with 2 μg of the appropriate DNA as described and finally washed three times with 500 μl of absolute ethanol and resuspended in 25 μl of absolute ethanol by short sonification; 7 μl of the suspension were used per transformation. ΔPsaB cells lackingpsaB were grown at 25 °C in liquid Tris acetate phosphate medium in the dark, and 4 × 107 cells were dispersed per Tris acetate phosphate plate containing 150 μg/ml spectinomycin. Once the plates were dry, the cells were bombarded with the DNA-coated particles. The bombarded cells were kept under low light (5 μE m2 s) for 2 weeks. The appearing transformants were restreaked on fresh Tris acetate phosphate plates containing spectinomycin and used for further investigations. The isolation of pc and cyt c 6 followed previously published procedures (3Merchant S. Bogorad L. Mol. Cell. Biol. 1986; 6: 462-469Crossref PubMed Scopus (140) Google Scholar, 20Kerfeld C.A. Anwar H.A. Interrante R. Merchant S. Yeates O.T. J. Mol. Biol. 1995; 250: 627-647Crossref PubMed Scopus (92) Google Scholar), with modifications as described in Ref. 21Hippler M. Drepper F. Farah J. Rochaix J.D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (94) Google Scholar. The concentrations of pc and cyt c 6 were determined spectroscopically using an extinction coefficient of 4.9 mm−1 cm−1 at 597 nm for the oxidized form of pc (22Katoh S. Shiratori I. Takamiya A. J. Biochem. (Tokyo). 1962; 51: 32-40Crossref PubMed Scopus (223) Google Scholar) and 20 mm−1cm−1 at 552 nm for the reduced form of cytc 6 (2Wood P.M. Eur. J. Biochem. 1978; 87: 9-19Crossref PubMed Scopus (186) Google Scholar). The isolation of thylakoid membranes purified by centrifugation through a sucrose step gradient and the isolation of PSI particles were as described previously (21Hippler M. Drepper F. Farah J. Rochaix J.D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (94) Google Scholar, 23Chua N.-H. Bennoun P. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2175-2179Crossref PubMed Scopus (409) Google Scholar). Chlorophyll concentrations were determined according to Ref. 24Porra R.J. Thompson W.A. Kriedemann P.E. Biochim. Biophys. Acta. 1989; 975: 384-394Crossref Scopus (4743) Google Scholar. SDS-PAGE (15.5% T, 2.66% C) was carried out according to Ref. 25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar. After the electrophoretic fractionation, the proteins were electroblotted onto nitrocellulose and incubated with antibodies as described previously (26Hippler M. Ratajczak R. Haehnel W. FEBS Lett. 1989; 250: 280-284Crossref Scopus (91) Google Scholar). Immunodetection was carried out according to Ref. 21Hippler M. Drepper F. Farah J. Rochaix J.D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (94) Google Scholar. To quantify the amount of PSI, wild type thylakoids were diluted with thylakoids from theΔpsaB strain (lacking PSI), resulting in fractions that contained 100%, 50%, 25%, and 0% PSI. Equal amounts of thylakoid proteins from these fractions and from mutant thylakoids were separated by SDS-PAGE and analyzed by immunoblotting using PsaF-specific antibodies to estimate the PSI content in the different strains. The blots were also probed with light harvesting complex II antibodies to verify equal loading. Cross-linking was performed as described in Ref. 21Hippler M. Drepper F. Farah J. Rochaix J.D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (94) Google Scholar. Kinetics of flash-induced absorbance changes at 817 nm were measured essentially as described previously (13Hippler M. Drepper F. Rochaix J.D. Muehlenhoff U. J. Biol. Chem. 1999; 274: 4180-4188Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 27Drepper F. Hippler M. Nitschke W. Haehnel W. Biochemistry. 1996; 35: 1282-1295Crossref PubMed Scopus (117) Google Scholar). The measuring light was provided by a luminescence diode (Hitachi HE8404SG; 40 mW; full width at half-maximum, 30 nm) supplied with a stabilized battery-driven current source. The light was filtered through a 817-nm interference filter (full width at half-maximum, 9 nm) and passed through a cuvette containing 200 μl or 50 μl of the sample with an optical pathlength of 1 cm or 3 mm, respectively. To investigate the role of loop j and especially that of helixl of the PsaB protein in binding and electron transfer of the soluble electron donors pc and cyt c 6, we performed a combinatorial site-directed mutagenesis approach. A plasmid containing the altered psaB and the aadA gene (encoding for aminoglycoside adenyl transferase) that can be expressed in the chloroplast to confer resistance to spectinomycin or streptomycin (18Goldschmidt-Clermont M. Nucleic Acids Res. 1991; 19: 4083-4089Crossref PubMed Scopus (406) Google Scholar) was transformed into a PsaB-deficient mutant (14Redding K. MacMillan F. Leibl W. Brettel K. Hanley J. Rutherford A.W. Breton J. Rochaix J.D. EMBO J. 1998; 17: 50-60Crossref PubMed Scopus (85) Google Scholar). In total, seven different mutant strains were obtained after selection on spectinomycin-containing medium (TableI). In spot test analyses, we found that all generated mutants could grow photoautotrophically at low light intensities (Table I). Mutant strains D624K, E613K/D624K, E613K/W627F, and D624K/W627F died under photoautotrophic or heterotrophic conditions at light intensities equivalent to or higher than 60 μE m−2 s −1 or 700 μE m−2 s−1, respectively. In Western blot analyses of SDS-PAGE fractionated thylakoids using PSI-specific antibodies, such as anti-PsaD or anti-PsaF antibodies, the amount of PSI in these strains could be estimated to be 50% PSI as compared with wild type accumulated in the mutant thylakoids. However, strains E613K and W627F died under photoautotrophic or heterotrophic conditions at light intensities equivalent to or higher than 700 μE m−2 s−1 (Table I). These growth phenotypes are comparable with the phenotype observed for the PsaF-deficient mutant. Mutant strain E613N showed a strong light sensitivity under photoautotrophic conditions and light intensities equivalent to or higher than 700 μE m−2 s −1 (Table I). The higher amounts of PSI in these three strains enabled us to isolate the altered PSI complexes and to perform detailed functional studies using flash-induced absorption spectroscopy.Table IGrowth properties of wild type, the PsaF-deficient mutant, and the ΔPsaB transformants with an altered PsaB protein on TAPaTAP, Tris acetate phosphate; HSM, high-salt medium; WT, wild type. and HSM platesStrains10 μE m−2s−160 μE m−2s−1700 μE m−2s−1PSI contentHSMTAPHSMTAPHSMTAPWT++++++++++++100%E613N++++++++−+>50%E613K++++++−−>50%W627F++++++−−>50%E613K/W627F++−+−−>20%D624K++−+−−<10%E613K/D624K++−+−−<10%D624K/W627F++−+−− 50%1-a TAP, Tris acetate phosphate; HSM, high-salt medium; WT, wild type. Open table in a new tab The electron transfer from pc or cytc 6 to PSI isolated from wild type and mutants E613N and E613K was investigated using excitation by single turnover flashes. It should be noted that residue Glu613 is located in the inter-helical-loop region of loop j. Fig.2 shows the absorbance transients at 817 nm induced by a laser flash for PSI particles in the presence of 20 μm cyt c 6. In the cases of wild type and mutant E613N, the time course of the P700+reduction can be deconvoluted into three kinetic components (for wild type, see also Fig. 6). The fast component with a constant half-life of 3–4 μs and a variable amplitude A(1) reflects a first-order electron transfer, the rate of which is independent of the concentration of the donor proteins. This phase can be explained by an electron transfer reaction within a preformed complex between the donor and PSI. The half-time of the fast phase, which was also identified in the kinetics of P700+ reduction using pc as electron donor (data not shown), is found to be the same for wild type and mutant E613N PSI. The intermediate component with an amplitude A(2) shows a half-life that decreases with increasing concentration of reduced donor protein, as known for second-order reactions between soluble reactants (see Fig.3). Amplitude A(1) increases with increasing concentration of reduced donor protein at the expense of A(2) (see Fig. 3). The third very slow component with an amplitude of about 25 and 40% of the total signal for PSI isolated from wild type and mutant E613N, respectively, has an electron transfer rate constant in the range of 7–9 × 105m−1 s−1 for pc or cytc 6, which is comparable with the values found for electron transfer between both donors and PSI from the PsaF-deficient mutant under similar conditions (21Hippler M. Drepper F. Farah J. Rochaix J.D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (94) Google Scholar). In the case of mutant E613K, the time course of the P700+ reduction can be deconvoluted into only one kinetic component, with a second-order rate constant of about 5 × 105m−1 s−1, which is again comparable with the values found for electron transfer between both donors and PSI from the PsaF-deficient mutant. Therefore, the very slow electron transfer between mutant PSI E613K and cytc 6 or pc (Fig. 2, TableII) indicates that binding and electron transfer are independent of PsaF in this mutant.Figure 6Mutation of Trp627 to Phe in PsaB abolishes complex formation of PSI with pc but not with cyt c 6. Absorbance changes at 817 nm induced by a laser flash in PSI particles from wild type and mutant W627F in the presence of 120 μm pc or 120 μm cyt c 6; for conditions, see Fig. 2. The relative amplitudes and half-times of the different kinetic components were as follows: for wild type, A(1) = 0.25 and 0.24 with t12(1) = 3.5 × 10−6 s, A(2) = 0.4 and 0.54 with t12(2) = 6.7 × 10−5 s and 2 × 10−4 s, and A(3) = 0.35 and 0.22 with t12(3) = 1.5 × 10−1 s and 3 × 10−2 s for pc and cyt c 6, respectively, and for mutant PSI W627F, a two exponential decay for pc with A(1) = 0.2 with t12(1) = 6 × 10−4 s, A(2) = 0.73 with t12(2) = 3.4 × 10−1 s, and a triphasic decay with A(1) = 0.09 with t12(1) = 3 × 10−5 s, A(2) = 0.24 with t12(2) = 3.5 × 10−4 s, and A(3) = 0.67 with t12(3) = 1.1 × 10−1s.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Determination of the kinetic constants for the electron transfer between mutant PSI PsaB E613N and the two electron donors. A, amplitude of the fast kinetic component of P700+ reduction as a function of the concentration of donor proteins. B, electron transfer rate constant as a function of the donor concentration. ■, cytc 6; ▪, pc. Measurements were performed at 0.3 mm MgCl2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIProperties of the electron transfer from pc and cyt c to PSI from wild type, the PsaF-deficient mutant and the ΔpsaB transformants with an altered PsaB proteinPlastocyaninCytochromec 6k 22-aThe second-order rate constantk 2 determined from linear regression of the observed rate constant of the donor-dependent kinetic phase as shown in Fig. 3 B and Fig. 7 B also gives an estimate of the on-rate for the formation of the complex. All kinetic constants refer to conditions close to the optimal concentrations of MgCl2 as indicated in parentheses.KDbThe dissociation constant KD of the active complex is estimated from the amplitude of the fast kinetic component as shown in Fig. 3 A and Fig. 7 A.t 1/2(off)2-cThe half-life of the active complex,t 1/2 = ln(2)/k off, where k off is the rate of dissociation of the donor from the photosystem estimated by using the approximate relationk off = KD ×k 2. For a detailed discussion of the limits of these estimates and a comparison of their results to a more refined kinetic analysis, see Ref. (27).k 2KDt 1/2(off)(107m−1s−1)(μm)(ms)(107m−1s−1)(μm)(ms)WT2-dWT, wild type; n.d., not determined.9830.093.4830.25(0.3 mm)(f = 0.69)(0.3 mm)(f = 0.66)PsaB E613N11290.228.27.71.1(0.3 mm)(f = 0.71)(0.3 mm)(f = 0.58)PsaB E613K0.22>1000n.d.0.34>1000n.d.(0.1 m)(0.1m)ΔpsaF0.13>1000n.d.0.25>1000n.d.(1 m)(1m)PsaB W627F0.74>1000n.d.1.61770.24(0.3 mm)(0.3 mm)(f = 0.66)The second-order rate constants k 2 and the dissociation constants KD determined for wild type and the PsaF-deficient strain 3bF are taken from Refs. 7Hippler M. Drepper F. Haehnel W. Rochaix J.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7339-7344Crossref PubMed Scopus (96) Google Scholar and 21Hippler M. Drepper F. Farah J. Rochaix J.D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (94) Google Scholar.2-a The second-order rate constantk 2 determined from linear regression of the observed rate constant of the donor-dependent kinetic phase as shown in Fig. 3 B and Fig. 7 B also gives an estimate of the on-rate for the formation of the complex. All kinetic constants refer to conditions close to the optimal concentrations of MgCl2 as indicated in parentheses.2-b The dissociation constant KD of the active complex is estimated from the amplitude of the fast kinetic component as shown in Fig. 3 A and Fig. 7 A.2-c The half-life of the active complex,t 1/2 = ln(2)/k off, where k off is the rate of dissociation of the donor from the photosystem estimated by using the approximate relationk off = KD ×k 2. For a detailed discussion of the limits of these estimates and a comparison of their results to a more refined kinetic analysis, see Ref. (27Drepper F. Hippler M. Nitschke W. Haehnel W. Biochemistry. 1996; 35: 1282-1295Crossref PubMed Scopus (117) Google Scholar).2-d WT, wild type; n.d., not determined. Open table in a new tab The second-order rate constants k 2 and the dissociation constants KD determined for wild type and the PsaF-deficient strain 3bF are taken from Refs. 7Hippler M. Drepper F. Haehnel W. Rochaix J.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7339-7344Crossref PubMed Scopus (96) Google Scholar and 21Hippler M. Drepper F. Farah J. Rochaix J.D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (94) Google Scholar. For further analysis of PsaF-dependent functional electron transfer, we will consider mainly the two kinetic components A(1) and A(2). Drepper et al. (27Drepper F. Hippler M. Nitschke W. Haehnel W. Biochemistry. 1996; 35: 1282-1295Crossref PubMed Scopus (117) Google Scholar) described a kinetic model for the binding and electron transfer between pc and PSI that also takes into account the redox equilibrium of the electron transfer. In this model, a simple dissociation equilibrium of the complex between the reduced donor protein ([D]) and PSI was used to describe the concentration dependence of the amplitude A(1). An estimate of the dissociation constant (K D ) can be determined using the following equation: A1=f×[D]([D]+KD)Equation 1 where f represents an empirical factor (f < 1) that relates amplitude A(1) observed after the flash to the fraction of PSI in a complex with the reduced donor before the flash (27Drepper F. Hippler M. Nitschke W. Haehnel W. Biochemistry. 1996; 35: 1282-1295Crossref PubMed Scopus (117) Google Scholar). Eq.1 indicates that the relative amplitude A(1) has a hyperbolic dependence on the concentration [D],i.e. shows a half-maximum saturation at a donor protein concentration equal to K D and approaches a maximum value f at infinite concentration. The amplitude of the fast phases of P700+ reduction for kinetic experiments performed with PSI isolated from mutant E613N with various concentrations of pc and cyt c 6 is displayed in Fig. 3 A. The data points can be analyzed by a hyperbolic curve according to Eq. 1. As a result, dissociation constants and the values of f are obtained for pc and cytc 6 as summarized in Table II. Because the concentration of pc and cyt c 6exceeds that of P700 by >1 order of magnitude, the kinetic component A(2) follows an exponential time course. The plots of its rate constant, k = ln2/ t12,versus the donor concentration can be approximated by linear dependences throughout the concentration range used in the experiments (Fig. 3 B). The second-order rate constant,k 2 = ln2/( t12 × [D]), can be determined from the slope of the regression lines in Fig. 3 B. The results for the reactions of cytc 6 and pc with this mutant (E613N PSI) and with wild type PSI are summarized in Table II. The dissociation constants of 29 and 7.7 μm as well as the second-order rate constants of 11 and 8.2 × 107m−1 s−1 for pc and cytc 6, respectively, are significantly smaller for mutant PSI E613N than for the wild type (Table II). The determination of k 2 and K D values for binding of pc and cyt c 6 to PSI isolated from mutant E613N and the dissociation equilibrium of the complex, respectively, implies that the unbinding of the donors from altered PSI is about 3 times slower compared with wild t