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• W2160497905 abstract "Article15 February 1998free access The PsaC subunit of photosystem I provides an essential lysine residue for fast electron transfer to ferredoxin Nicolas Fischer Nicolas Fischer Departments of Molecular Biology and Plant Biology, University of Geneva, 30 quai Ernest-Ansermet, 1211 Geneva 4, Switzerland Search for more papers by this author Michael Hippler Michael Hippler Departments of Molecular Biology and Plant Biology, University of Geneva, 30 quai Ernest-Ansermet, 1211 Geneva 4, Switzerland Search for more papers by this author Pierre Sétif Pierre Sétif CEA, Département de Biologie Cellulaire et Moléculaire, CNRS, URA 2096, C.E. Saclay, 91191 Gif-sur-Yvette, Cedex, France Search for more papers by this author Jean-Pierre Jacquot Jean-Pierre Jacquot Université de Paris Sud, IBP-630, 91405 Orsay, Cedex, France Search for more papers by this author Jean-David Rochaix Corresponding Author Jean-David Rochaix Departments of Molecular Biology and Plant Biology, University of Geneva, 30 quai Ernest-Ansermet, 1211 Geneva 4, Switzerland Search for more papers by this author Nicolas Fischer Nicolas Fischer Departments of Molecular Biology and Plant Biology, University of Geneva, 30 quai Ernest-Ansermet, 1211 Geneva 4, Switzerland Search for more papers by this author Michael Hippler Michael Hippler Departments of Molecular Biology and Plant Biology, University of Geneva, 30 quai Ernest-Ansermet, 1211 Geneva 4, Switzerland Search for more papers by this author Pierre Sétif Pierre Sétif CEA, Département de Biologie Cellulaire et Moléculaire, CNRS, URA 2096, C.E. Saclay, 91191 Gif-sur-Yvette, Cedex, France Search for more papers by this author Jean-Pierre Jacquot Jean-Pierre Jacquot Université de Paris Sud, IBP-630, 91405 Orsay, Cedex, France Search for more papers by this author Jean-David Rochaix Corresponding Author Jean-David Rochaix Departments of Molecular Biology and Plant Biology, University of Geneva, 30 quai Ernest-Ansermet, 1211 Geneva 4, Switzerland Search for more papers by this author Author Information Nicolas Fischer1, Michael Hippler1, Pierre Sétif2, Jean-Pierre Jacquot3 and Jean-David Rochaix 1 1Departments of Molecular Biology and Plant Biology, University of Geneva, 30 quai Ernest-Ansermet, 1211 Geneva 4, Switzerland 2CEA, Département de Biologie Cellulaire et Moléculaire, CNRS, URA 2096, C.E. Saclay, 91191 Gif-sur-Yvette, Cedex, France 3Université de Paris Sud, IBP-630, 91405 Orsay, Cedex, France *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:849-858https://doi.org/10.1093/emboj/17.4.849 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info PsaC is the stromal subunit of photosystem I (PSI) which binds the two terminal electron acceptors FA and FB. This subunit resembles 2[4Fe-4S] bacterial ferredoxins but contains two additional sequences: an internal loop and a C-terminal extension. To gain new insights into the function of the internal loop, we used an in vivo degenerate oligonucleotide-directed mutagenesis approach for analysing this region in the green alga Chlamydomonas reinhardtii. Analysis of several psaC mutants affected in PSI function or assembly revealed that K35 is a main interaction site between PsaC and ferredoxin (Fd) and that it plays a key role in the electrostatic interaction between Fd and PSI. This is based upon the observation that the mutations K35T, K35D and K35E drastically affect electron transfer from PSI to Fd, as measured by flash-absorption spectroscopy, whereas the K35R change has no effect on Fd reduction. Chemical cross-linking experiments show that Fd interacts not only with PsaD and PsaE, but also with the PsaC subunit of PSI. Replacement of K35 by T, D, E or R abolishes Fd cross-linking to PsaC, and cross-linking to PsaD and PsaE is reduced in the K35T, K35D and K35E mutants. In contrast, replacement of any other lysine of PsaC does not alter the cross-linking pattern, thus indicating that K35 is an interaction site between PsaC and its redox partner Fd. Introduction In oxygenic photosynthetic organisms, conversion of light energy into chemical energy is achieved by multiprotein-pigment complexes located in the thylakoid membranes. Photosystem I (PSI) performs the light-induced electron transfer from plastocyanin or cytochrome c6 (Cytc) to Fd. PSI complex is composed of at least five chloroplast-encoded subunits (PsaA, -B, -C, -I and -J) and six nuclear-encoded polypeptides (PsaD, -E, -F, -G, -H and -K) (Golbeck, 1992). The primary electron donor (P700) and the intermediate acceptors (A0, A1 and FX) are bound by the two major subunits PsaA and PsaB (Brettel, 1997), whereas PsaC binds the two terminal electron acceptors (FA and FB) (Hayashida et al., 1987; Høj et al., 1987; Oh-Oka et al., 1987). This 8–9 kDa protein is located on the stromal side of PSI and contains two CxxCxxCxxxCP motifs that are the ligands of the [4Fe-4S] clusters FA and FB. The position of these clusters is clearly defined in the PSI crystal structure at 6 and 4 Å resolution, placing one cluster close to FX and the other closer to the stromal surface (Krauss et al., 1993, 1996). Assigning an identity (FA or FB) to the clusters within the PSI structure is still uncertain. PsaC is a ferredoxin-like protein (Dunn and Gray, 1988). However, it contains two domains which are absent from 2[4Fe-4S] ferredoxins: an internal loop between the two [4Fe-4S] cluster binding motifs and a C-terminal extension (Figure 1). These additional sequences have been proposed to interact with the PsaA/B heterodimer and the PsaD subunit, respectively (Naver et al., 1996). PsaC has been shown to be essential for in vivo assembly of PSI in Chlamydomonas reinhardtii. Disruption of the gene or mutagenesis of cysteines liganding the [4Fe-4S] clusters destabilizes the PSI complex in this organism (Takahashi et al., 1991, 1992). The PsaD and PsaE subunits are located on the stromal side of PSI and interact with PsaC (Jansson et al., 1996). PsaD is required for stable assembly of PsaC and PsaE in PSI complex reconstitution assays (Zhao et al., 1990; Li et al., 1991; Naver et al., 1995). Chemical cross-linking revealed a direct interaction between PsaD and Fd (Zanetti and Merati, 1987; Zilber and Malkin, 1988; Andersen et al., 1992a; Lelong et al., 1994). Inactivation of PsaD demonstrated its role for efficient Fd reduction (Chitnis et al., 1989; Xu et al., 1994a). PsaE has also been shown to interact with ferredoxin:NADP+-oxidoreductase as well as Fd and to be required for Fd reduction (Andersen et al., 1992b; Rousseau et al., 1993; Sonoike et al., 1993; Xu et al., 1994a). The PsaF subunit provides a docking site for plastocyanin and Cytc on the lumenal side of PSI, and is essential for their fast photo-oxidation in C.reinhardtii, but not in cyanobacteria (Xu et al., 1994b; Farah et al., 1995; Hippler et al., 1996, 1997). Interaction between the transmembrane domain of PsaF and the stromal subunit PsaE has been reported (Xu et al., 1994b; Jansson et al., 1996). In the current model of PSI three subunits appear to be involved in the reduction of Fd. PsaD and PsaE are involved in the correct docking of Fd to PSI and PsaC coordinates the [4Fe-4S] clusters from which an electron can be transferred to Fd. Figure 1.Schematic representation of the PsaC polypeptide. The two C-C-C-C-P motifs liganding the [4Fe-4S] clusters are indicated. The C-terminal extension and the internal loop are boxed. The wild-type sequence and the possible changes introduced by degenerate oligonucleotide-directed mutagenesis are indicated for the two halves of the internal loop. The positions of the three lysine residues of the PsaC polypeptide of C.reinhardtii are shown by closed triangles. Download figure Download PowerPoint To gain new insights into the function of the internal loop of PsaC which is absent from 2[4Fe-4S] ferredoxins and might therefore be important for the function of this subunit, we performed in vivo degenerate oligonucleotide-directed mutagenesis of this loop in C.reinhardtii. This approach has led to the identification of lysine 35 as a key residue for Fd reduction. Replacement of this lysine with neutral or negatively charged amino acids abolishes fast electron transfer to Fd and limits photosynthetic growth of the mutant strains. Furthermore, we show that Fd can be cross-linked to PsaD, PsaE and PsaC and our data strongly suggest that the cross-linking-site of PsaC is lysine 35. Results Degenerate oligonucleotide-directed mutagenesis of the internal loop of PsaC Because there is no obvious target for site-directed mutagenesis to study the function of the internal loop of PsaC, we chose a mutagenesis approach for the random assortment of defined mutations within a short stretch of amino acids. In this study we defined the additional loop of PsaC as the ten amino acids E27MVPWDGCKA36 and mutated the first five and last five residues separately. Oligonucleotides for each half of the loop were designed with a 2-fold degeneracy of one base of each codon to be mutated (see Materials and methods). In this way, the wild-type or mutant residue was introduced at any given position within the loop. For each half of the loop, the possible changes are shown in Figure 1. Because of the possibility that multiple mutations might often lead to the destabilization of the PSI complex, we designed the oligonucleotides such that psaC genes with single and double mutations should represent about 47% of the mutated psaC copies if one assumes that the oligonucleotides used anneal with the same efficiency. The partially degenerate portion of the oligonucleotides is flanked by 15 bases of homologous sequence on each side to allow polymerase chain reaction amplification (see Materials and methods for details). The amplified DNA, containing a population of psaC genes with different combinations of mutations, was digested with NdeI and BglII and inserted into plasmid pBSEP5.8 aadA [NdeI/BglII]. This plasmid contains a 5.8 kb EcoRI–PstI fragment from the chloroplast DNA fragment R23 (Rochaix, 1978) with two unique NdeI and BglII restriction sites located at the 5′ and 3′ ends of the psaC coding sequence. The aadA expression cassette was inserted at a SalI site 800 bp upstream of psaC (Takahashi et al., 1992). This selectable marker confers spectinomycin and streptomycin resistance to C.reinhardtii (Goldschmidt-Clermont, 1991). The two plasmid libraries of mutated psaC genes corresponding to the two parts of the loop (loop-1 and loop-2) were introduced via biolistic transformation (Boynton et al., 1988) into the chloroplast of a C.reinhardtii strain in which the psaC coding sequence had been deleted (Fischer et al., 1996). Transformants were selected on TAP plates supplemented with 150 μg/ml of spectinomycin and maintained in dim light (5 μE/m2/s). Analysis of the transformants reveals the importance of residue K35 Transformants were restreaked once on selective media and could be directly analysed. As no wild-type copy of the psaC gene is present in the recipient strain, only the mutated version of psaC is expressed and its phenotype can be characterized readily. Forty-eight transformants obtained with each library were tested for their capacity to grow on TAP (permissive for mutants of C.reinhardtii deficient in photosynthesis) or HSM (minimal) plates under different light regimes. Fluorescence transients of cells grown in dim light (5 μE/m2/s) and dark adapted before the experiment were measured to determine if they had a functional PSI complex (Bennoun and Delepelaire, 1982). Transformants with altered growth or fluorescence phenotypes as well as several transformants with wild-type phenotype were identified and further characterized. Total genomic DNA was isolated from these strains and the psaC gene amplified by a two-step asymmetric PCR to generate single stranded DNA (see Materials and methods). The amplified DNA was directly sequenced to identify mutations in the loop region. Table I shows the results of this analysis for 11 transformants of the loop-1 mutagenesis and 14 of the loop-2 mutagenesis. All other transformants examined had either integrated a wild-type psaC or only the aadA selectable marker without psaC. Table 1. In vivo phenotypes of strains carrying mutations in the additional loop of PsaC (A) Loop-1 mutagenesis Growth 27 31 mut. Fluo. TDL TL THL HSMDL HSML HSMHL WT E M V P W 0 WT + + + + + + DC − − − − − − PSI + − − − − − L1–1 E M D S W 2 WT + + + + + + L1–6 E M V S S 2 WT + + + + + + L1–11 E M V S W 1 WT + + + + + + L1–12 V M V S W 2 WT + + + + + + L1–16 E M V S W 1 WT + + + + + + L1–19 V M D S W 3 WT + + + + slow − L1–34 E M D S W 2 WT + + + + + + L1–35 V M D P S 3 WT + + − slow − − L1–37 V M V S S 3 WT + + + + + + L1–39 E K D S W 3 PSI + − − − − − L1–47 E K V S S 3 PSI + − − − − − Growth 32 36 mut. Fluo. TDL TL THL HSMDL HSML HSMHL WT D G C K A 0 WT + + + + + + DC − − − − − − PSI + − − − − − L2–1 V D C K A 2 WT + + + + + + L2–2 V G C T A 2 WT + + + + + − L2–3 D G S K T 2 WT + + + + + + L2–5 V G S K A 2 WT + + + + + + L2–9 V G C T T 3 WT + + + + + − L2–10 D G C T T 2 WT + + + + + − L2–11 V G S K A 2 WT + + + + + + L2–12 V D S T A 4 WT + + slow + + − L2–17 V G S T A 3 WT + + + + + − L2–22 D G S T A 2 WT + + + + + − L2–24 D G S T T 3 WT + + + + + − L2–27 V G C T A 2 WT + + + + + − L2–31 V D S K T 4 WT + + + + + slow L2–32 D D S T A 3 WT + + + + + − DC, deletion of psaC; mut., number of mutations; Fluo., fluorescence phenotype in dim light; T, (TAP) rich medium; HSM, minimal medium; light conditions: DL, 5 μE/m2/s; L, 60 μE/m2/s; HL, 600 μE/m2/s. Results of loop-1 mutagenesis. Transformants L1–39 and L1–47 have characteristic phenotypes of PSI-deficient strains and carry triple mutations M28K/V29D/P30S and M28K/P30S/W31S, respectively. The fact that transformants L1–1 and L1–6 contain two of these three mutations (V29D/P30S and P30S/W31S, respectively), but show no altered growth or fluorescence phenotype, strongly suggests that the mutation M28K is responsible for the PSI-deficient phenotype (Table IA). Western blot analysis of total cell extracts confirmed that in transformants L1–39 and L1–47, the PSI complex was strongly destabilized (data not shown). From a similar subtractive comparison of the different combinations of mutations, it appears that transformants assembling PSI but having a defect in growth phenotype always carry the E27V/V29D mutations. Results of loop-2 mutagenesis. The same type of comparison for this set of mutants showed that all the transformants affected in growth carry the mutation K35T and are unable to grow on HSM under high light conditions. Conversely, in transformant L2–31 all residues of loop-2 are mutated except K35 and this transformant is able to grow on HSM under high light, although its growth rate is reduced compared with wild-type (Table IB). This suggests that the mutation K35T is causing the growth defect. Accordingly, K35 was subjected to further mutagenesis. Random mutagenesis of residue K35 We designed an oligonucleotide with complete degeneracy of the codon corresponding to K35. Only adenosine was excluded in the third position of the codon to avoid the occurrence of two out of three stop codons. Amplification, cloning and transformation of this new library containing psaC genes randomly mutated at position 35 was performed as described above. We analysed 32 transformants and selected four with altered growth and/or fluorescence phenotypes. Fluorescence transients and growth phenotypes on plates as well as the mutation at position 35 for each transformant are shown in Table II. Strains carrying mutations K35T, K35D and K35P displayed fluorescence transients that were very similar to wild-type, suggesting that PSI is functional in these strains (data not shown). Transformants containing the mutation K35T grew significantly slower on HSM plates under high light. The K35P mutation leads to a slightly more severe phenotype as it impairs growth on HSM plates under high light. The most affected transformant contains the mutation K35D. This strain is photosensitive under high light and its growth rate is reduced on HSM at 60 μE/m2/s. We also measured the doubling time of these different strains in liquid HSM media under low light conditions. It can be seen that none of the mutations present at position 35 affects the growth rate (Table II). Western blot analysis was performed on total extracts of cells grown in dim light to measure the accumulation of the PSI complex (Figure 2). It is apparent that the K35T, K35P and K35D mutants accumulate high levels of PSI, indicating that the mutations do not markedly affect the stability of the complex. In contrast, a stop codon at position 35 in one of the transformants leads to a PSI-deficient phenotype (Table II). Western blot analysis showed that truncation of the PsaC polypeptide at position 35 completely destabilizes the PSI complex (data not shown). Figure 2.Immunoblot analysis of total cell extracts of wild-type, the K35T, K35R, K35D, K35E and K35P mutants and of a strain lacking psaC (ΔC). Cells were grown in liquid TAP medium in low light (5 μE/m2/s). 15 μg of total protein were loaded per lane. Proteins were separated by SDS–PAGE, electroblotted on nitrocellulose membrane and probed with antibodies raised against subunits PsaA, PsaD, PsaE and PsaF. Download figure Download PowerPoint Table 2. In vivo phenotypes of K35 mutants Strain Fluo. TDL TL THL HSML HSMHL Doubling time (h) (HSM 10 μE/m2/s) WT WT + + + + + 32 ± 4 DC PSI− + − − − − ∞ K35P WT + + + + − 35 ± 5 K35T WT + + + + slow 31 ± 7 K35D WT + + − slow − 30 ± 6 K35E WT + + − slow − 33 ± 6 K35R WT + + + + + 29 ± 7 K35Stop PSI− + − − − − ∞ DC, deletion of the psaC gene; Fluo., fluorescence phenotype in dim light; T, TAP medium; DL, 5 μE/m2/s; L, 60 μE/m2/s; HL, 600 μE/m2/s. These mutant strains were obtained by random mutagenesis as described above and only one transformant (two in the case of K35T) was obtained for each mutation. The phenotypes observed could be due to additional chloroplast or nuclear mutations that might have occurred during chloroplast transformation. To exclude this possibility, we amplified the psaC genes containing the K35T, K35P and K35D mutations and cloned them into the pBSEP5.8 aadA [NdeI/BglII] transformation vector. After sequencing of the psaC gene to confirm the presence of the mutations at K35 and the absence of any undesired mutation that might have been introduced during amplification, these genes were inserted into the chloroplast genome of C.reinhardtii by transformation. Analysis of several individual transformants for each mutation showed that their growth phenotype was indeed due to the mutation present in the psaC gene. Site-directed mutagenesis of K35 To further test the role of the positive charge provided by K35, we constructed the K35E and K35R mutant strains by site-directed mutagenesis. Amplification with specific oligonucleotides, cloning and transformation of C.reinhardtii was performed as described above. Several transformants for each mutation were obtained and analysed. Clearly, the K35E change leads to a growth deficient phenotype under high light, as observed for the K35D mutant, whereas the K35R mutant grows under all conditions tested (Table II). In both mutants, PSI accumulates to high levels (Figure 2). These results indicate that replacement of K35 by a negatively charged residue severely limits photosynthetic growth under high light, whereas replacement by another positively charged amino acid restores the wild-type growth phenotype. In agreement with an electrostatic role of K35, its replacement by a neutral residue leads to an intermediate phenotype. Fd reduction by PSI Electron transfer from PSI to Fd has already been studied in the cyanobacterium Synechocystis sp. PCC 6803 and in C.reinhardtii (Sétif and Bottin, 1994, 1995; Fischer et al., 1997). Upon P700 oxidation by a saturating laser flash, Fd reduction is accompanied by an absorption change in the 460–600 nm region in the cyanobacterial system. With C.reinhardtii PSI, the measurements were restricted to a single wavelength (580 nm), because its larger antenna size precludes measurements at other wavelengths. In the absence of Fd, an absorption change at 580 nm is also observed (see Materials and methods). This contribution was subtracted from the signals recorded in the presence of Fd so that the kinetic curves shown in Figure 3 correspond solely to electron transfer from [FAFB]− to Fd. When absorption transients were measured at 580 nm with wild-type PSI and Fd, three phases of Fd reduction were observed (Figure 3, curve a): two first order phases, one with t1/2 < 1 μs and a second with t1/2 = 4.5 ± 1.5 μs, and a slower phase due to second order electron transfer, where the rate is proportional to the ferredoxin concentration. The half-times of the first order phases are independent of ferredoxin concentration, but their amplitudes increase with ferredoxin concentration. At 580 nm, the 4.5 μs phase has an amplitude 2-fold larger than that of the submicrosecond phase. The first order components are ascribed to electron transfer within a PSI–Fd complex formed before flash excitation. Linear fitting of the sum of the amplitudes of the first order components as a function of Fd concentration (assuming a simple binding equilibrium between Fd and PSI) allows one to determine the dissociation constant for the wild-type complex (Kd = 6–9 μM, data not shown). A linear fit of the observed rate of the slowest phase as a function of Fd concentration was also performed, providing a second order rate constant of 2.3×108/M/s for the wild-type PSI complex (data not shown). Figure 3.Ferredoxin reduction measured by flash-induced absorption changes at 580 nm with wild-type and mutant PSI complexes. The transients were obtained after subtraction of traces recorded in the absence of ferredoxin from traces recorded in its presence. The contribution of P700+ decay, which is different in the presence or absence of ferredoxin, was also subtracted so that the curves correspond only to electron transfer from [FAFB]− to ferredoxin. PSI complexes were prepared from wild-type (a), K35R (b), K35T (c), K35D (d) and K35E (e) and suspended in 20 mM Tricine pH 8.0, 0.03% β-DM, 30 mM NaCl, 5 mM MgCl2, 2.2 mM sodium ascorbate and 20 μM DCPIP. Traces are the average of 128 measurements with and without ferredoxin. A delay of 3 s was used between two consecutive flashes. The kinetics shown in panels (A) and (B) correspond to 0.14 μM PSI, as measured by absorption changes at 820 nm. Concentrations of ferredoxin from C.reinhardtii are 16.2 μM for K35T, K35D, K35E and K35R, and 17.3 μM for wild-type. The full scales in panels (A) and (B) are 100 μs and 50 ms, respectively. Panel (C) shows the dependence of the observed rate of ferredoxin reduction on the concentration of ferredoxin: K35T (●); K35D (+) and K35E (▴). Note the different scales used. A linear fit of the data is also shown. It corresponds to kobs (s−1) = [Fd]×(4.5×107/M/s) for K35T, kobs (s−1) = [Fd]×(6.3×106/M/s) for K35D and kobs (s−1) = [Fd]×(2.8×106/M/s) for K35E. Download figure Download PowerPoint The same experiments were performed with the K35T, K35D, K35E and K35R PSI complexes. Figure 3A shows that the first order components are completely absent with the K35T, K35D and K35E PSI complexes (curves c, d and e). We estimated the lower limit of the dissociation constants for these mutant complexes: Kd > 300 μM (see Materials and methods for estimation). In contrast, two first order phases with t1/2 similar to wild-type are present in the experiments performed with the K35R PSI complex (Figure 3A, curve b). The ratio of the submicrosecond and the microsecond phases are also similar between wild-type and K35R PSI, whereas the amplitudes of these first order phases are slightly larger in this mutant PSI complex, indicating that the affinity of Fd for this PSI complex is higher (Kd = 4.5–6.5 μM). Clearly, the replacement of K35 by neutral or negatively charged amino acids drastically affects the affinity of Fd for PSI, showing that the positive charge provided by this residue is important for formation of the PSI–Fd complex. The second order component is also altered in the K35T, K35D and K35E PSI complexes (Figure 3B). Linear fitting of the dependence of the observed rates on Fd concentration provides rate constants of 4.5×107/M/s, 6.3×106/M/s and 2.8×106/M/s for K35T, K35D and K35E, respectively (Figure 3C), whereas the second order component observed with the K35R PSI complex is the same as for wild-type. The positive charge provided by K35 appears, therefore, to be important for the precise positioning of Fd on the reducing side of PSI, which allows for fast electron transfer. Removal of this positive charge or its replacement by negative charges leads to 5-fold and 35- to 80-fold decrease of the electron transfer rate, respectively. Properties of the wild-type and mutant PSI-complexes are summarized in Table III. Table 3. Characteristics of wild-type and mutant PSI WT K35T K35D K35E K35R Dissociation constant for PSI-ferredoxin interaction 6.0–9.0 μM >300 μM >300 μM >300 μM 4.5–6.5 μM Half-times of first-order rates of ferredoxin reduction <1 μs 4.5 ± 1.5 μs not observed not observed not observed <1 μs 4.5 ± 1.5 μs Second-order rate constant of ferredoxin reduction 2.3 ± 0.5×108/M/s 4.5×107/M/s 6.3×106/M/s 2.8×106/M/s 2.3 ± 0.5×108/M/s Light induced EPR spectra of PSI at low temperature Stable charge separation between P700+ and [FAFB]− can be achieved in PSI at low temperature. Below 30K, this charge-separated state can be observed by electron paramagenetic resonance (EPR) when the samples are incubated in the dark at room temperature with ascorbate and DPIP to reduce P700+, and frozen in darkness. At low temperature, photoreduction of PSI leads to the formation of FA− or FB−, which can be distinguished by their different EPR g-values (FA−: 2.047, 1.945 and 1.857; FB−: 2.07, 1.932 and 1.878). The difference spectra obtained from EPR measurements after 2 min illumination at 14 K and in darkness are shown in Figure 4. Signals were normalized to the same concentration of slowly decaying P700+ as measured by absorption changes at 820 nm (data not shown). After normalization to P700+, spin quantitation of the three signals gave similar values. The g-values are the same for the five types of PSI, within a margin of error of ±0.002. Vertical arrows (g = 2.11 and 1.911) indicate signals that have been observed only in PSI from C.reinhardtii and which are presumably due to a modified form of FA− (Fischer et al., 1997). These data show that the mutant PSI complexes contain intact FA and FB iron-sulfur clusters and are able to perform stable charge separation between P700+ and [FAFB]−. Figure 4.Light-induced EPR spectra of PSI complexes isolated from wild-type and the K35R, K35T, K35D and K35E mutants. Each of these spectra corresponds to the difference between spectra recorded after 2 min illumination at 14 K and in darkness. The large signal around g ≈ 2.00 (mostly P700+) is not shown. Spectra correspond to approximatively 5 μM PSI. Vertical arrows (g = 2.11 and 1.911) mark signals that have been observed only in PSI from C.reinhardtii, presumably due to a modified form of FA−. EPR conditions: temperature 14 K; microwave power 20 mW; modulation amplitude 1 mT; microwave frequency 9.419 Ghz. The spectra shown are the sum of eight scans before and after illumination. All samples were prepared in Tricine, 20 mM pH 8.0, in the presence of 5 mM sodium ascorbate and 25 μM DCPIP. Tubes were incubated at room temperature for 2 min. in darkness before freezing in darkness. Download figure Download PowerPoint Chemical cross-linking of Fd with C.reinhardtii PSI particles Interaction of Fd with several subunits of PSI has been demonstrated by chemical cross-linking in different organisms. In the cyanobacterium Synechocystis PCC 6803, Fd was shown to cross-link to PsaD, and the cross-linking site was identified (Lelong et al., 1994). In higher plants, cross-linking has been reported between Fd and PsaD in spinach (Zaneti and Merati, 1987; Zilber and Malkin, 1988); in barley, Fd can be cross-linked to the PsaD, PsaE and PsaH subunits (Andersen et al., 1992a). We performed similar experiments to determine which PSI subunits could be cross-linked to Fd in C.reinhardtii. Fd was purified from wild-type cells and incubated with N-ethyl–3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide ester (sulfo-NHS) to activate its carboxyl groups (see Materials and methods). The excess of reagent was then removed by gel filtration, and the activated Fd was eluted. PSI particles purified from wild-type cells were incubated with or without activated Fd. In this way, no internal cross-link between PSI subunits due to free cross-linker can occur and any cross-linking product has to involve activated Fd. The cross-linked sample and the control PSI were separated by SDS–PAGE. Western blot analysis performed with antibodies raised against the PsaD, PsaE and PsaC polypeptides revealed that Fd could be cross-linked to all three subunits (Figure 5, lanes 2 and 7). To our knowledge, this is the first report of a direct interaction between Fd and the PsaC subunit by chemical cross-linking. Figure 5.Western blot analysis" @default.
• W2160497905 created "2016-06-24" @default.
• W2160497905 creator A5047679543 @default.
• W2160497905 creator A5076133005 @default.
• W2160497905 creator A5079916943 @default.
• W2160497905 creator A5080356805 @default.
• W2160497905 creator A5082340647 @default.
• W2160497905 date "1998-02-15" @default.
• W2160497905 modified "2023-10-12" @default.
• W2160497905 title "The PsaC subunit of photosystem I provides an essential lysine residue for fast electron transfer to ferredoxin" @default.
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