Chloroplast monothiol glutaredoxins as scaffold proteins for the assembly and delivery of [2Fe–2S] clusters
2008; Springer Nature; Volume: 27; Issue: 7 Linguagem: Inglês
10.1038/emboj.2008.50
ISSN1460-2075
AutoresSibali Bandyopadhyay, Filipe Gama, Maria Micaela Molina‐Navarro, José M. Gualberto, Ronald Claxton, Sunil G. Naik, Boi Hanh Huynh, Enrique Herrero, Jean‐Pierre Jacquot, Michael K. Johnson, Nicolas Rouhier,
Tópico(s)Redox biology and oxidative stress
ResumoArticle20 March 2008free access Chloroplast monothiol glutaredoxins as scaffold proteins for the assembly and delivery of [2Fe–2S] clusters Sibali Bandyopadhyay Sibali Bandyopadhyay Department of Chemistry, Centre for Metalloenzyme Studies, University of Georgia, Athens, GA, USA Search for more papers by this author Filipe Gama Filipe Gama UMR 1136 IaM, IFR 110, Faculté des Sciences, Nancy University, Vandoeuvre, France Search for more papers by this author Maria Micaela Molina-Navarro Maria Micaela Molina-Navarro Department of Basic Medical Sciences, University of Lleida, Lleida, Spain Search for more papers by this author José Manuel Gualberto José Manuel Gualberto Institut de Biologie Moléculaire des Plantes, CNRS, Strasbourg, France Search for more papers by this author Ronald Claxton Ronald Claxton Department of Chemistry, Centre for Metalloenzyme Studies, University of Georgia, Athens, GA, USA Search for more papers by this author Sunil G Naik Sunil G Naik Department of Physics, Emory University, Atlanta, GA, USA Search for more papers by this author Boi Hanh Huynh Boi Hanh Huynh Department of Physics, Emory University, Atlanta, GA, USA Search for more papers by this author Enrique Herrero Enrique Herrero Department of Basic Medical Sciences, University of Lleida, Lleida, Spain Search for more papers by this author Jean Pierre Jacquot Jean Pierre Jacquot UMR 1136 IaM, IFR 110, Faculté des Sciences, Nancy University, Vandoeuvre, France Search for more papers by this author Michael K Johnson Corresponding Author Michael K Johnson Department of Chemistry, Centre for Metalloenzyme Studies, University of Georgia, Athens, GA, USA Search for more papers by this author Nicolas Rouhier Corresponding Author Nicolas Rouhier UMR 1136 IaM, IFR 110, Faculté des Sciences, Nancy University, Vandoeuvre, France Search for more papers by this author Sibali Bandyopadhyay Sibali Bandyopadhyay Department of Chemistry, Centre for Metalloenzyme Studies, University of Georgia, Athens, GA, USA Search for more papers by this author Filipe Gama Filipe Gama UMR 1136 IaM, IFR 110, Faculté des Sciences, Nancy University, Vandoeuvre, France Search for more papers by this author Maria Micaela Molina-Navarro Maria Micaela Molina-Navarro Department of Basic Medical Sciences, University of Lleida, Lleida, Spain Search for more papers by this author José Manuel Gualberto José Manuel Gualberto Institut de Biologie Moléculaire des Plantes, CNRS, Strasbourg, France Search for more papers by this author Ronald Claxton Ronald Claxton Department of Chemistry, Centre for Metalloenzyme Studies, University of Georgia, Athens, GA, USA Search for more papers by this author Sunil G Naik Sunil G Naik Department of Physics, Emory University, Atlanta, GA, USA Search for more papers by this author Boi Hanh Huynh Boi Hanh Huynh Department of Physics, Emory University, Atlanta, GA, USA Search for more papers by this author Enrique Herrero Enrique Herrero Department of Basic Medical Sciences, University of Lleida, Lleida, Spain Search for more papers by this author Jean Pierre Jacquot Jean Pierre Jacquot UMR 1136 IaM, IFR 110, Faculté des Sciences, Nancy University, Vandoeuvre, France Search for more papers by this author Michael K Johnson Corresponding Author Michael K Johnson Department of Chemistry, Centre for Metalloenzyme Studies, University of Georgia, Athens, GA, USA Search for more papers by this author Nicolas Rouhier Corresponding Author Nicolas Rouhier UMR 1136 IaM, IFR 110, Faculté des Sciences, Nancy University, Vandoeuvre, France Search for more papers by this author Author Information Sibali Bandyopadhyay1,‡, Filipe Gama2,‡, Maria Micaela Molina-Navarro3, José Manuel Gualberto4, Ronald Claxton1, Sunil G Naik5, Boi Hanh Huynh5, Enrique Herrero3, Jean Pierre Jacquot2, Michael K Johnson 1 and Nicolas Rouhier 2 1Department of Chemistry, Centre for Metalloenzyme Studies, University of Georgia, Athens, GA, USA 2UMR 1136 IaM, IFR 110, Faculté des Sciences, Nancy University, Vandoeuvre, France 3Department of Basic Medical Sciences, University of Lleida, Lleida, Spain 4Institut de Biologie Moléculaire des Plantes, CNRS, Strasbourg, France 5Department of Physics, Emory University, Atlanta, GA, USA ‡These authors contributed equally to this work *Corresponding authors: UMR 1136 IaM, IFR 110, Faculté des Sciences, Nancy University, Vandoeuvre 54506, France. Tel.: +33 3 83684225; Fax: +33 3 83684292; E-mail: [email protected] Department of Chemistry, Centre for Metalloenzyme Studies, University of Georgia, Athens, GA, USA. Tel.: +1 706 542 9378; Fax: +1 706 542 9454; E-mail: [email protected] The EMBO Journal (2008)27:1122-1133https://doi.org/10.1038/emboj.2008.50 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Glutaredoxins (Grxs) are small oxidoreductases that reduce disulphide bonds or protein-glutathione mixed disulphides. More than 30 distinct grx genes are expressed in higher plants, but little is currently known concerning their functional diversity. This study presents biochemical and spectroscopic evidence for incorporation of a [2Fe–2S] cluster in two heterologously expressed chloroplastic Grxs, GrxS14 and GrxS16, and in vitro cysteine desulphurase-mediated assembly of an identical [2Fe–2S] cluster in apo-GrxS14. These Grxs possess the same monothiol CGFS active site as yeast Grx5 and both were able to complement a yeast grx5 mutant defective in Fe–S cluster assembly. In vitro kinetic studies monitored by CD spectroscopy indicate that [2Fe–2S] clusters on GrxS14 are rapidly and quantitatively transferred to apo chloroplast ferredoxin. These data demonstrate that chloroplast CGFS Grxs have the potential to function as scaffold proteins for the assembly of [2Fe–2S] clusters that can be transferred intact to physiologically relevant acceptor proteins. Alternatively, they may function in the storage and/or delivery of preformed Fe–S clusters or in the regulation of the chloroplastic Fe–S cluster assembly machinery. Introduction Iron–sulphur (Fe–S) proteins are intimately involved in numerous essential biological processes, such as photosynthesis, respiration and the metabolism of carbon, oxygen, hydrogen, nitrogen and sulphur (Johnson and Smith, 2005). However, little is known concerning the mechanism of Fe–S cluster biogenesis in plants. Much of the current understanding of Fe–S cluster biogenesis stems from investigation of components of the bacterial isc (iron–sulphur cluster assembly), suf (sulfur mobilization) and nif (nitrogen fixation) operons (Johnson et al, 2005) and identification and characterization of homologous ISC-type proteins in yeast and mammalian mitochondria (Lill and Mühlenhoff, 2005). The ISC, SUF and NIF Fe–S cluster assembly machineries share a common basic mechanism involving cysteine desulphurase (IscS, SufS and NifS)-mediated assembly of [2Fe–2S] or [4Fe–4S] clusters on U-type (IscU, SufU and N-terminal domain of NifU), A-type (IscA, SufA and NifIscA) and Nfu-type (corresponding to the C-terminal domain of NifU) scaffold proteins, and subsequent intact cluster transfer into acceptor apo-proteins. In the case of the ISC machinery, [2Fe–2S] cluster transfer from IscU is facilitated by specific molecular co-chaperones (HscA and HscB) in an ATP-dependent reaction (Chandramouli and Johnson, 2006) and [4Fe–4S] cluster assembly on dimeric IscU occurs at the subunit interface via reductive coupling of two [2Fe–2S] clusters (Chandramouli et al, 2007). However, little is currently known concerning the detailed mechanism of Fe–S cluster assembly and transfer involving scaffold proteins. In plants, Fe–S cluster biosynthesis primarily occurs in mitochondria, using the ISC machinery with Isu, IscA and Nfu as potential scaffold proteins, and in chloroplasts using the SUF machinery with SufA, SufB and Nfu proteins as potential scaffold proteins (Balk and Lobreaux, 2005; Ye et al, 2006b; Layer et al, 2007). Glutaredoxins (Grxs) are small proteins that normally function in the reduction of disulphide bridges or glutathionylated proteins. However, recent studies in Saccharomyces cerevisiae and Escherichia coli have indicated specific roles for some Grxs in facilitating Fe–S cluster biosynthesis (Rodríguez-Manzaneque et al, 2002; Mühlenhoff et al, 2003; Achebach et al, 2004). Yeast cells deleted for the GRX5 gene were found to be more sensitive to oxidative stress, to accumulate free iron and to have impaired mitochondrial Fe–S cluster biogenesis and respiratory growth (Rodríguez-Manzaneque et al, 1999, 2002). Other prokaryotic and eukaryotic CGFS Grxs have been shown to be efficient functional substitutes for yeast Grx5 (Cheng et al, 2006; Molina-Navarro et al, 2006). Although the specific function of yeast Grx5 in Fe–S cluster biogenesis remains to be elucidated, 55Fe radiolabelling studies of knockout mutants implicate a role in mediating transfer of clusters preassembled on the Iscu1p scaffold protein into acceptor proteins (Mühlenhoff et al, 2003). The discovery that Grx5 is also required for vertebrate haem synthesis raises the possibility that Grx5 is a key determinant for channelling Fe into haem and Fe–S cluster biosynthesis in mammals (Wingert et al, 2005). The most obvious role for Grxs in Fe–S cluster biogenesis lies in facilitating Fe–S cluster assembly or transfer by reducing disulphides on scaffold or apo forms of Fe–S proteins. However, the discovery that some Grxs can assemble Fe–S clusters suggests the possibility of alternative roles in Fe–S cluster assembly or transfer. Poplar GrxC1 (CGYC active site) and human Grx2 (CSYS active site) are homodimers with a subunit-bridging [2Fe–2S] cluster ligated by one active site cysteine of each monomer and the cysteines of two external glutathione (GSH) molecules (Feng et al, 2006; Johansson et al, 2007; Rouhier et al, 2007). The cluster-containing dimeric form of human Grx2 was proposed to function as a redox sensor for the activation of Grx2 in case of oxidative stress (Lillig et al, 2005). Although this is a viable hypothesis, mutagenesis studies on poplar GrxC1 indicate that incorporation of a [2Fe–2S] cluster is likely to be a general feature of plant Grxs possessing a glycine adjacent to the catalytic cysteine (Rouhier et al, 2007). Hence CGFS Grxs, such as yeast Grx5, might have the capacity to incorporate a Fe–S cluster. Moreover, the requirement of GSH for the export of a Fe–S cluster (or a precursor thereof) from mitochondria to facilitate the assembly of cytosolic Fe–S proteins in S. cerevisiae (Lill and Mühlenhoff, 2005) provides further circumstantial evidence in support of a role for GSH- and Grx-ligated [2Fe–2S] clusters in Fe–S biogenesis. In higher plants, around 30 different Grx isoforms can be classified into three distinct subgroups depending on their active site sequences (Rouhier et al, 2004). The first class, which contains Grxs with C[P/G/S][Y/F][C/S] motifs other than CGFS, is homologous to the classical dithiol Grxs such as E. coli Grx1 and 3, yeast Grx1 and 2 and mammalian Grx1 and 2. The second class has a strictly conserved CGFS active site sequence and includes Grxs homologous to yeast Grx3, 4 and 5 or E. coli Grx4. Plants have generally four members in this group (GrxS14 to S17). The properties of proteins of the third class, which is specific to higher plants and involves a CC[M/L][C/S] active site, are largely unknown. This study presents biochemical, spectroscopic and analytical evidence for the incorporation of [2Fe–2S] clusters in two plant chloroplast CGFS Grxs, GrxS14 and S16, and both in vivo and in vitro evidence for their involvement in the maturation of Fe–S proteins. The results demonstrate that monothiol Grxs have the potential to function as scaffold proteins for de novo synthesis and efficient delivery of [2Fe–2S] clusters, as Fe–S cluster delivery or storage proteins for mediating the transfer of Fe–S clusters from ISC or SUF scaffold proteins to acceptor proteins, or as sensors of the cellular Fe–S cluster status in Fe homoeostasis. Results The plant CGFS Grx subgroup In silico analysis of Grxs from different kingdoms reveals that four or five Grxs with CGFS active site are generally present in higher plants and in Chlamydomonas reinhardtii, whereas only three are present in S. cerevisiae, two in most other fungi and in mammals, one in Synechocystis and in E. coli (Rouhier et al, 2004). In Populus trichocarpa, GrxS14 and S15 are small proteins (171 and 172 amino acids, respectively, including the transit peptide sequence) with a single repeat of the Grx module. GrxS16 is larger (296 amino acids including the transit peptide sequence) with an N-terminal extension linked to the Grx module. GrxS17 is larger (492 amino acids) and displays an N-terminal Trx-like domain with a WCDAS active site followed by three successive Grx modules. A careful examination of amino-acid sequence alignments (Supplementary Figure 1) indicates that although not present in all CGFS Grxs, a second cysteine is found in 60% of the 250 CGFS Grxs found in GenBank at a conserved position closer to the C terminus. It is conserved in GrxS14 and S16 (Cys 87 in GrxS14 and Cys 221 in GrxS16, numbering based on the recombinant mature protein sequences), in the third module of GrxS17 and in GrxC1, whereas it is absent in GrxS15, in the second Grx domain of GrxS17 and in GrxC4 and only partly conserved in the first Grx domain of plant GrxS17. In ScGrx5, these two cysteines are able to form a disulphide bridge in the presence of oxidized GSH (Tamarit et al, 2003). Subcellular localization of the CGFS Grxs We have determined the localization of all the CGFS Grxs that possess an N-terminal transit sequence. GrxS17, predicted to be cytosolic, does not seem to possess such an extension and its localization has not been characterized further. The full-length sequences of the three other Grxs devoid of the stop codon were introduced in frame before the GFP sequence and the construction was used to bombard tobacco leaves. As shown in Figure 1, the fluorescence of GrxS14 and S16 strictly coincides with the one of the chlorophyll, whereas the fluorescence of GrxS15 superimposed well with one of the mitochondrial marker. Therefore, GrxS14 and S16 are chloroplastic and GrxS15 is mitochondrial. Figure 1.Subcellular localization of CGFS Grxs by GFP fusion. (A) GrxS14, (B) GrxS16 and (C) GrxS15. From left to right: visible light, autofluorescence of chlorophyll (red) or mitochondrial marker (white); fluorescence of the constructions and merged images. Only one of the guard cells shows chloroplast-localized GFP, because a small numbers of cells were transfected. As the mitochondrial marker (DsRed) is co-transfected with the GFP construction, it is only visible in the cell that expresses GFP. Download figure Download PowerPoint Some poplar monothiol but not dithiol Grxs rescue the defects of a yeast mutant lacking Grx5 To determine whether the four poplar monothiol Grxs rescue the defects of a yeast Δgrx5 mutant, we targeted the proteins at the mitochondrial matrix (Molina et al, 2004). All the proteins were adequately compartmentalized in the mitochondrial matrix (Figure 2A). Of the two poplar Grxs with a single Grx domain, GrxS14 and S15, only the first one rescued the grx5 mutant defects in respiratory growth (Figure 2B) and its sensitivity to oxidants (Figure 2C). These defects were also efficiently rescued by GrxS16 and S17. To test whether a single Grx domain is sufficient for the function of GrxS17, we fused only the most C-terminal domain of S17 (from amino acid 398 to 492) to the mitochondrial targeting sequence of Grx5 (S17398−492). This protein was also compartmentalized at the mitochondrial matrix (Figure 2A), although a double band appeared. The band with lower mobility probably corresponds to unprocessed precursor still compartmentalizing at yeast mitochondria. S17398−492 suppressed partially the growth phenotypes of the grx5 mutant, in particular growth in respiratory conditions (Figure 2B). The ratio of activities of the mitochondrial enzymes aconitase (containing Fe–S clusters) and malate dehydrogenase (without Fe–S clusters) was used as a measure of the efficiency of the Fe–S cluster assembly in mitochondrial proteins (Molina et al, 2004). This ratio was measured in strains carrying all these constructions in a chromosomal grx5 background (Figure 2D). Ratios were comparable with wild type in strains expressing the mitochondrial forms of S14, S16 and S17, in accordance with the growth phenotypes. In contrast, both S15 and the truncated form of S17 exhibited much lower enzyme ratios (Figure 2D). For all the strains tested, absolute malate dehydrogenase levels were basically similar, and doxycycline addition to growth media lowered aconitase activity to basal levels (data not shown). For the monothiol Grxs analysed, there appears to be a correlation between efficiency to express active aconitase and growth phenotypes, except that poplar mitochondrial S15 did not rescue at all the growth defects of the yeast mutant, although still being able to synthesize low levels of mature aconitase. The anomalous results with S15 and S17398−492 are puzzling and may indicate functional diversity within the general class of monothiol Grxs (see below) or that aconitase maturation does not provide a good measure of the efficiency of general Fe–S cluster biosynthesis in mitochondria. The latter is supported by a very recent study of the requirements for mitochondrial aconitase Fe–S cluster maturation in S. cerevisiae, which indicated a specific requirement for Isa1p, Isa2p and the Iba57, proteins that are not required for general Fe–S cluster biogenesis in mitochondria (Gelling et al, 2008). Figure 2.Rescue of the S. cerevisiae grx5 mutant defects by poplar monothiol glutaredoxins. (A) Compartmentalization of GrxS14, S15, S16, S17 and S17398−492 in the mitochondrial matrix of S. cerevisiae cells. Cultures were grown exponentially in YPLactate medium at 30°C to about 3 × 107 cells ml−1, before mitochondrial isolation and subfractionation. TE, total cell extract; MT, mitochondrial fraction; IMS, intermembrane space; MX, matrix. Proteins (20 μg) were loaded in the TE lanes, and 5 μg was loaded in the other lanes. Anti-HA anti-lipoic acid antibodies were used in the western blot to detect the HA-tagged proteins, and the matrix marker α-ketoglutarate dehydrogenase (α-KGDH). (B) Growth on glucose (YPD plates) or glycerol (YPGly plates), after 3 days at 30°C. (C) Sensitivity to t-BOOH or diamide of the strains after 3 days at 30°C on YPD plates. (D) Ratio between aconitase and malate dehydrogenase activities in exponential cultures at 30°C in YPGalactose medium. Download figure Download PowerPoint To determine whether the capacity to bind a Fe–S cluster is sufficient for grx5 complementation, we then used poplar GrxC1 (CGYC), which incorporates a [2Fe–2S] centre and GrxC1 G32P (CPYC) and GrxC4 (CPYC) which do not (Rouhier et al, 2007). Although all these Grxs were targeted to the matrix (Figure 3A), none of these proteins rescued (i) the inability of a grx5 mutant for respiratory growth, (ii) the sensitivity to oxidants and (iii) the capacity to assembly a Fe–S cluster in aconitase (Figure 3B–D). None of the three dithiol Grxs, even the one binding a Fe–S cluster, is functional in yeast mitochondria for the maturation of Fe–S proteins. To determine whether this is caused by structural incompatibility of the dithiol Grxs with the Fe–S cluster biosynthetic machinery or more specifically by the different active site sequences with either dithiol or monothiol motifs, we modified the CGYC and CPYC active sites of GrxC1 and GrxC4 into CGFS to mimic the active site sequence of Grx5. The resulting GrxC1 CGFS fully substituted for yeast Grx5 with respect to all phenotypes analysed (Figure 3), whereas GrxC4 CGFS did not (data not shown). The GrxC1 CGFS rescuing effects did not occur when its expression from the tet promoter was switched off by doxycycline addition to the growth medium (data not shown). We therefore conclude that the requirement for a monothiol Grx active site could preclude poplar dithiol Grxs from functionally rescuing a grx5 mutant, but in some cases, exemplified by the GrxC4 CGFS derivatives, other sequence or structural requirements are needed. Figure 3.Rescue of the S. cerevisiae grx5 mutant defects by poplar dithiol glutaredoxins. (A) Compartmentalization of GrxC1, C1G32P, C4 and C1CGFS in the mitochondrial matrix of S. cerevisiae cells. Growth conditions and western blot analyses are similar to those described in Figure 2. TE, total cell extract; MT, mitochondrial fraction; IMS, intermembrane space; MX, matrix. (B) Growth on glucose (YPD plates) or glycerol (YPGly plates), after 3 days at 30°C. (C) Sensitivity to t-BOOH or diamide of the strains after 3 days at 30°C on YPD plates. (D) Ratio between aconitase and malate dehydrogenase activities in exponential cultures at 30°C in YPGalactose medium. Download figure Download PowerPoint Purification and spectroscopic characterization of Fe–S cluster-containing poplar GrxS14 and AtGrxS16 The mature form of the three organellar poplar CGFS Grxs was expressed in E. coli to check their ability to incorporate Fe–S clusters. On the basis of our previous experience with GrxC1, GSH, which stabilize and ligate the [2Fe–2S] cluster, was added during the first steps of the purification (Rouhier et al, 2007). Although the presence of a brownish colouration typical of a Fe–S cluster was clearly evident in cells overexpressing poplar and Arabidopsis thaliana (At) GrxS14 and S16, almost no holoprotein was obtained at the end of an aerobic purification, even in the presence of GSH, suggesting that the cluster degrades quickly in air. In contrast, there was no indication of a Fe–S cluster prosthetic group in recombinant poplar or A. thaliana GrxS15. Purification of poplar GrxS14 under strictly anaerobic conditions was undertaken to address the type, stoichiometry and properties of the putative Fe–S centre. The reddish-brown purified samples contained 0.80±0.10 Fe per monomer. The UV–visible absorption and CD spectra of anaerobically purified poplar GrxS14 are shown in Figure 4 and both are characteristic of a [2Fe–2S]2+ centre (Stephens et al, 1978; Dailey et al, 1994). On the basis of the theoretical and experimental ε280 values for the apo protein (9.9 mM−1 cm−1), the ε280 and ε411 values for the [2Fe–2S]2+ centre are estimated to be 3.9 and 4.4 mM−1 cm−1, respectively, and the A411/A280 was found to be 0.31±0.04. In accord with the analytical data, these extinction coefficients are indicative of 0.4–0.5 [2Fe–2S]2+ clusters per monomer. Hence the analytical, absorption and CD data are consistent with approximately one [2Fe–2S]2+ per dimeric GrxS14. Anaerobically purified At GrxS16 contained an analogous [2Fe–2S]2+ centre as judged by very similar UV–visible absorption and CD spectra (Figure 4). Figure 4.Comparison of the UV–visible absorption and CD spectra of [2Fe–2S] cluster-bound forms of poplar GrxS14 (thick line), At GrxS16 (broken line) and poplar GrxC1 (thin line). Download figure Download PowerPoint In vitro reconstitution of aerobically purified apo GrxS14 was attempted under strictly anaerobic conditions in the presence of 5 mM GSH and 2 mM DTT, using Fe(II), L-cysteine and catalytic amounts of E. coli IscS. After chromatographic removal of excess reagents, the resulting cluster-loaded form of GrxS14 was essentially identical to anaerobically purified [2Fe–2S] GrxS14, as judged by Fe analyses and UV–visible absorption and CD spectra (data not shown). GSH was required for successful reconstitution of a [2Fe–2S] cluster on GrxS14. Samples of apo GrxS14 reconstituted using the same procedure in a reaction mixture containing 2 mM DTT, but no GSH, showed no evidence of the presence on a bound Fe–S cluster following repurification. Hence, in vitro Fe–S cluster reconstitution studies confirm the potential of poplar GrxS14 to act as a scaffold for the assembly of [2Fe–2S] clusters in a cysteine desulphurase-mediated reaction and indicate that GSH is required for cluster assembly. Resonance Raman and Mössbauer studies of anaerobically purified poplar GrxS14 confirm the presence of a [2Fe–2S]2+ centre and provide insight into the cluster ligation. Resonance Raman spectra obtained using 457- and 514-nm excitation reveal Fe–S stretching modes at 288, 332, 347, 365, 402 and 424 cm−1 (Figure 5). The vibrational frequencies are generally similar to those of structurally characterized [2Fe–2S] ferredoxins with complete cysteinyl cluster ligation and are readily assigned to vibrational modes of the Fe2Sb2St4 unit (Sb=bridging S and St=terminal or cysteinyl) by direct analogy with published data (Han et al, 1989; Fu et al, 1992). Figure 6 compares the Mössbauer spectra of poplar GrxS14 with those of the all cysteinyl-ligated [2Fe–2S]2+ cluster in poplar GrxC1 and the IscU [2Fe–2S]2+ cluster which has one non-cysteinyl ligand (Agar et al, 2000). Each spectrum is indicative of a S=0 [2Fe–2S]2+ centre that results from antiferromagnetic coupling of two high-spin Fe(III) ions and is simulated as the sum of quadrupole doublets from each Fe site using the parameters listed in the figure legend. The similarity and values of the isomer shift (δ) and quadrupole splitting (ΔEQ) parameters for each Fe site of the [2Fe–2S]2+ clusters in GrxC1 and GrxS14 are consistent with approximately tetrahedral S ligation at each Fe site. Non-cysteinyl ligation is generally manifested by anomalous isomer shifts and quadrupole splittings for the unique Fe site, which results in marked asymmetry in the observed spectrum, as is apparent in the spectrum of [2Fe–2S]2+ centre in IscU (Agar et al, 2000). Hence, the Mössbauer data indicate a [2Fe–2S]2+ cluster as the sole Fe-containing prosthetic group in anaerobically purified poplar GrxS14, and the Mössbauer and resonance Raman data taken together provide support for complete cysteinyl ligation. Figure 5.Comparison of the resonance Raman spectra of [2Fe–2S] cluster-bound forms of poplar GrxS14 (thick line) and GrxC1 (thin line) with 514- and 457-nm laser excitation. Samples were ∼4 mM in Grx and were in the form of a frozen droplet at 17 K. Each spectrum is the sum of 100 scans, with each scan involving counting photons for 1 s each 0.5 cm−1 with 6 cm−1 spectral resolution. Lattice modes of ice have been subtracted. Download figure Download PowerPoint Figure 6.Comparison of the Mössbauer spectra of [2Fe–2S] cluster-bound forms of poplar GrxS14 (blue), poplar GrxC1 (red) and A. vinelandii IscU (green). The GrxS14 and C1 Mössbauer samples were prepared by growing cells on 57Fe-enriched media and the IscU sample was prepared by IscS-mediated reconstitution using 57Fe(II) (Agar et al, 2000). The Mössbauer spectra were recorded at 4.2 K with a magnetic field of 50 mT applied parallel to the γ-beam. Each spectrum is best simulated as the sum of two overlapping quadrupole doublets with the following parameters: ΔEQ=0.56 and δ=0.26 mm s−1 for doublet 1, and ΔEQ=0.76 and δ=0.28 mm s−1 for doublet 2 of GrxS14; ΔEQ=0.54 and δ=0.27 mm s−1 for doublet 1, and ΔEQ=0.76 and δ=0.28 mm s−1 for doublet 2 of GrxC1; ΔEQ=0.66 and δ=0.27 mm s−1 for doublet 1, and ΔEQ=0.94 and δ=0.32 mm s−1 for doublet 2 of IscU. Download figure Download PowerPoint Cluster ligation in GrxS14 and S16 The two structurally characterized [2Fe–2S]2+ centres in dithiol Grxs, human Grx2 (CSYC active site) and poplar GrxC1 (CGYC active site) have very similar absorption and CD spectra and have analogous coordination environments, involving the catalytic cysteine of two Grxs and the cysteines of two GSH (Johansson et al, 2007; Rouhier et al, 2007). On the basis of UV–visible absorption and CD spectra shown in Figure 4, a distinct type of [2Fe–2S]2+ centre may be present in GrxS14 and S16. Marked differences in the excited-state electronic properties and ground-state vibrational properties of the [2Fe–2S]2+ centres in poplar GrxC1 and GrxS14 are evident in comparing the UV–visible absorption/CD and resonance Raman spectra shown in Figures 4 and 5, respectively. Differences in the relative intensities of corresponding Raman bands reflect changes in excitation profiles resulting from perturbation of the excited-state electronic structure. Nevertheless, it is clear that corresponding Fe–S stretching frequencies are upshifted by 2–8 cm−1 in GrxS14 compared with GrxC1, suggesting stronger Fe–S bonds for both terminal and bridging S. As the spectroscopic properties of the [2Fe–2S]2+ centres in monothiol Grxs indicate complete cysteinyl ligation, mutagenesis studies were undertaken to address the possibility that GSH is replaced as a ligand by an intrinsic cysteine residue in GrxS14 and S16. Three cysteine residues are present in GrxS14 at positions 33, 87 and 108 (recombinant poplar GrxS14 numbering). The active site cysteine (Cys33) is conserved in all CGFS Grxs. Cys87 is present in all S14- and S16-type plant Grxs, but not in S15-type plant Grxs, whereas Cys108 is even not conserved in all S14-type plant Grxs (Supplementary Figure 1). Hence, to address the cluster ligation in GrxS14 and check whether the inability for GrxS15 to incorporate a Fe–S cluster is a consequence of the absence of the second cysteine residue, cysteine mutants both on poplar and AtGrxS14 and on poplar GrxS15 have been generated. AtGrxS14 was used for these mutation studies because introducing these mutations in poplar Grx
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