Characterization of the NifS-like Domain of ABA3 from Arabidopsis thaliana Provides Insight into the Mechanism of Molybdenum Cofactor Sulfuration

The molybdenum cofactor sulfurase ABA3 from Arabidopsis thaliana specifically regulates the activity of the molybdenum enzymes aldehyde oxidase and xanthine dehydrogenase by converting their molybdenum cofactor from the desulfo-form into the sulfo-form. ABA3 is a two-domain protein with an NH2-terminal domain sharing significant similarities to NifS proteins that catalyze the decomposition of l-cysteine to l-alanine and elemental sulfur for iron-sulfur cluster synthesis. Although different in its physiological function, the mechanism of ABA3 for sulfur mobilization was found to be similar to NifS proteins. The protein binds a pyridoxal phosphate cofactor and a substrate-derived persulfide intermediate, and site-directed mutagenesis of strictly conserved binding sites for the cofactor and the persulfide demonstrated that they are essential for molybdenum cofactor sulfurase activity. In vitro, the NifS-like domain of ABA3 activates aldehyde oxidase and xanthine dehydrogenase in the absence of the C-terminal domain, but in vivo, the C-terminal domain is required for proper activation of both target enzymes. In addition to its cysteine desulfurase activity, ABA3-NifS also exhibits selenocysteine lyase activity. Although l-selenocysteine is unlikely to be a natural substrate for ABA3, it is decomposed more efficiently than l-cysteine. Besides mitochondrial AtNFS1 and plastidial AtNFS2, which are both proposed to be involved in iron-sulfur cluster formation, ABA3 is proposed to be a third and cytosolic NifS-like cysteine desulfurase in A. thaliana. However, the sulfur transferase activity of ABA3 is used for post-translational activation of molybdenum enzymes rather than for iron-sulfur cluster assembly. The molybdenum cofactor sulfurase ABA3 from Arabidopsis thaliana specifically regulates the activity of the molybdenum enzymes aldehyde oxidase and xanthine dehydrogenase by converting their molybdenum cofactor from the desulfo-form into the sulfo-form. ABA3 is a two-domain protein with an NH2-terminal domain sharing significant similarities to NifS proteins that catalyze the decomposition of l-cysteine to l-alanine and elemental sulfur for iron-sulfur cluster synthesis. Although different in its physiological function, the mechanism of ABA3 for sulfur mobilization was found to be similar to NifS proteins. The protein binds a pyridoxal phosphate cofactor and a substrate-derived persulfide intermediate, and site-directed mutagenesis of strictly conserved binding sites for the cofactor and the persulfide demonstrated that they are essential for molybdenum cofactor sulfurase activity. In vitro, the NifS-like domain of ABA3 activates aldehyde oxidase and xanthine dehydrogenase in the absence of the C-terminal domain, but in vivo, the C-terminal domain is required for proper activation of both target enzymes. In addition to its cysteine desulfurase activity, ABA3-NifS also exhibits selenocysteine lyase activity. Although l-selenocysteine is unlikely to be a natural substrate for ABA3, it is decomposed more efficiently than l-cysteine. Besides mitochondrial AtNFS1 and plastidial AtNFS2, which are both proposed to be involved in iron-sulfur cluster formation, ABA3 is proposed to be a third and cytosolic NifS-like cysteine desulfurase in A. thaliana. However, the sulfur transferase activity of ABA3 is used for post-translational activation of molybdenum enzymes rather than for iron-sulfur cluster assembly. NifS and NifS-like enzymes are present in almost all organisms and fulfill their main functions during iron-sulfur ([Fe-S]) cluster synthesis. Accordingly, they have a cysteine desulfurase activity that is required for the mobilization of sulfur from l-cysteine by simultaneous release of l-alanine. In all NifS-like enzymes, the sulfide is bound as a persulfide to a conserved cysteine residue of the protein, from which it is subsequently transferred to other target proteins such as the scaffold proteins NifU and/or IscU in bacteria to finally assemble [Fe-S] clusters. A pyridoxal phosphate (PLP) 1The abbreviations used are: PLP, pyridoxal phosphate; AO, aldehyde oxidase; DTT, dithiothreitol; 1,5-I-AEDANS, N-(iodoacetyl)-N′-(5-sulfo-1-naphthyl)ethylendiamine; Moco, molybdenum cofactor; XDH, xanthine dehydrogenase. cofactor bound to a conserved lysine residue is essential for this cysteine desulfurase activity (1Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (502) Google Scholar). However, besides [Fe-S] cluster formation, other functions for NifS-like proteins are described as well. The Escherichia coli IscS protein also was found to be involved in the biosynthesis of thiamin and NAD+ and to be able to transfer a sulfur atom to uridine to produce a 4-thiouridine tRNA (2Lauhon C.T. Kambampati R. J. Biol. Chem. 2000; 275: 20096-20110Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). In E. coli, another NifS-like protein, SufS (CsdB), is able to catalyze the elimination of selenium from l-selenocysteine more efficiently than the elimination of sulfur from l-cysteine (3Mihara H. Maeda M. Fujii T. Kurihara T. Hata Y. Esaki N. J. Biol. Chem. 1999; 274: 14768-14772Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). However, its physiological function is connected to its cysteine desulfurase activity, which is up to 50× higher when SufS forms a complex with SufE (4Loiseau L. Ollagnier-de-Choudens S. Nachin L. Fontecave M. Barras F. J. Biol. Chem. 2003; 278: 38352-38359Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). The only NifS-like protein in yeast, Nfs1p, is essential not only for [Fe-S] cluster formation in mitochondria but also for tRNA splicing (5Kolman C. Soll D. J. Bacteriol. 1993; 175: 1433-1442Crossref PubMed Google Scholar). For cell viability, this protein also needs to be localized in the cytosol and to some extent in the nucleus (6Nakai Y. Nakai M. Hayashi H. Kagamiyama H. J. Biol. Chem. 2001; 276: 8314-8320Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Similar to bacteria, Arabidopsis thaliana also possesses more than one NifS-like protein: AtNFS1, which is located in mitochondria (7Kushnir S. Babiychuk E. Storozhenko S. Davey M.W. Papenbrock J. De Rycke R. Engler G. Stephan U.W. Lange H. Kispal G. Lill R. Van Montagu M. Plant Cell. 2001; 13: 89-100Crossref PubMed Scopus (204) Google Scholar), and AtNFS2, which is located in chloroplasts (8Léon S. Touraine B. Briat J.F. Lobreaux S. Biochem. J. 2002; 366: 557-564Crossref PubMed Scopus (98) Google Scholar, 9Pilon-Smits E.A. Garifullina G.F. Abdel-Ghany S. Kato S. Mihara H. Hale K.L. Burkhead J.L. Esaki N. Kurihara T. Pilon M. Plant Physiol. 2002; 130: 1309-1318Crossref PubMed Scopus (111) Google Scholar). However, cysteine desulfurase activity was also shown for ABA3 (10Bittner F. Oreb M. Mendel R.R. J. Biol. Chem. 2001; 276: 40381-40384Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). ABA3 is a molybdenum cofactor (Moco) sulfurase consisting of an N-terminal NifS-like domain that is fused to a C-terminal domain with an as yet unknown function, thereby being distinguished from all other NifS-like proteins described thus far. Although its physiological function is unrelated to [Fe-S] cluster assembly, cysteine desulfurase activity and sulfur transferase activity of ABA3 were shown to be essential for the activation of the two Moco-containing enzymes aldehyde oxidase (AO; EC 1.2.3.1) and xanthine dehydrogenase (XDH; EC 1.1.1.204) that are involved in abscisic acid biosynthesis and degradation of purines, respectively (for review, see Refs. 11Mendel R.R. Haensch R. J. Exp. Bot. 2002; 53: 1689-1698Crossref PubMed Scopus (213) Google Scholar, 12Mendel R.R. Schwarz G. Met. Ions Biol. Syst. 2002; 39: 317-368PubMed Google Scholar, 13Hesberg C. Haensch R. Mendel R.R. Bittner F. J. Biol. Chem. 2004; 279: 13547-13554Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). After having incorporated the Moco, AO and XDH remain inactive unless one of the two oxygen ligands at the molybdenum center is replaced by a sulfur atom that is delivered by ABA3. This sulfuration step catalyzes the final maturation of AO and XDH enzymes that occur in both the sulfo-form and the desulfo-form in the living cell. By this mechanism, the plant is able to rapidly increase the activities of AO and XDH, e.g. for adapting to altering environmental conditions without de novo synthesis of AO and XDH apoproteins. In this work, we examined the mechanism of sulfur mobilization as catalyzed by the NifS-like domain of ABA3 from A. thaliana. We identified the nature of the cofactor and the sulfur bound by ABA3-NifS and characterized the sub-activities of the protein. Finally, we examined the function of the highly conserved lysine 271 and cysteine 430, and we discussed the role of the NifS-like domain during Moco sulfuration. Construction of Expression Vectors—During purification of the N-terminally His6-tagged protein, it became obvious that ABA3 is not only present in full-length with a molecular mass of about 90 kDa but also as a C-terminally truncated cleavage product. The molecular mass of this product was found to correspond well with the calculated mass of the ABA3 NifS-like domain as deduced from sequence comparison with other NifS-like proteins. Both full-length ABA3 and the cleavage product were detected by anti-His tag antibodies in immunoblot analysis. Therefore, the cleavage product as purified by affinity chromatography most likely represents the N-terminal NifS-like domain of ABA3. Hence, we cloned the 5′-region of 1518 bp of the aba3 cDNA open reading frame (GenBank AF325457) encoding a peptide of 506 amino acids with a molecular mass of ∼56 kDa and introduced a TGA termination codon at the 3′-end. The aba3 cDNA construct described previously (10Bittner F. Oreb M. Mendel R.R. J. Biol. Chem. 2001; 276: 40381-40384Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar) was used for expression of the full-length protein and as a template for PCR generation of constructs expressing either the NifS-like domain or the C-terminal domain of ABA3, respectively. Primers for generating ABA3-NifS cDNA fragments were as follows: nifS-start-BamHI/NcoI, 5-TTT-CTT-GGA-TCC-ATG-GAA-GCA-TTT-CTT-AAG-GAA-TTC-3′; and nifS-stop-BamHI, 5′-ATA-TAT-GGA-TTC-TCA-GGT-TCC-ATT-CCC-AGT-CTT-CTT-3′. Primers for generating a 921-bp open reading frame of the C-terminal domain were as follows: C-term-start-BamHI, 5′-TAT-ATA-GGA-TTC-ATG-CAA-CTT-CTT-AGT-GAA-GAC-CTT-GAA-3′; and C-term-stop-BamHI, 5′-CAC-AAG-CGG-ATC-CTT-ATT-CAA-TAT-CTG-GAT-TAA-CTT-CTT-CCC-C-3′. The resulting PCR fragments were digested with BamHI and subcloned into BamHI-pretreated pQE80 (Qiagen, Hilden, Germany), thereby fusing the respective protein's N terminus to a His6 tag. Expression of ABA3, ABA3-NifS, and ABA3-C—Protein expressions of ABA3, ABA3-NifS, and ABA3-C were performed in freshly transformed E. coli DL41 cells. Expression conditions for full-length ABA3 were as described previously (10Bittner F. Oreb M. Mendel R.R. J. Biol. Chem. 2001; 276: 40381-40384Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). For expression of ABA3-NifS, cells were grown at 37 °C to an A600 = 0.5 before a combined induction with 0.2 mm isopropyl β-d-thiogalactopyranoside and 5 g lactose/liter expression culture. After induction, cells were cultured for an additional 22 h at 22 °C. Hyperproduction of the C-terminal domain of ABA3 was performed by growing cells at 37 °C to an A600 = 0.5 before induction with 0.2 mm isopropyl β-d-thiogalactopyranoside and additional culturing for 20 h at 30 °C. Cells were harvested by centrifugation and stored at –70 °C until use. Expression of AOα in Yeast—Overexpression of recombinant His6-tagged Arabidopsis AOα in the yeast Pichia pastoris (kindly provided by Tomokazu Koshiba, Tokyo, Japan) was performed as described previously (14Koiwai H. Akaba S. Seo M. Komano T. Koshiba T. J. Biochem. (Tokyo). 2000; 127: 659-664Crossref PubMed Scopus (37) Google Scholar). Purification of His6-tagged Proteins—Purification of recombinant ABA3, its separately expressed domains, and AOα was performed on a nickel-nitrilotriacetic acid superflow matrix (Qiagen) under native conditions at 4 °C according to the manufacturer's instructions. Proteins were rebuffered to 20 mm Tris/HCl, pH 8.0, containing 2 mm dithiothreitol (DTT) and stored at 4 °C, except for the C-terminal domain of ABA3 that was found to be unstable in low-salt buffers. For further purification, ABA3 proteins were subjected to anion exchange chromatography using a 10-ml Source Q-15 column (Amersham Biosciences) equilibrated with 20 mm Tris/HCl, pH 8.0, 2 mm DTT, and 0.5 mm EDTA (buffer A). Protein samples were applied to the column and eluted with buffer A followed by a linear gradient of 0–1 m NaCl in buffer A. Final purification and size were by chromatography on a size column (Amersham Biosciences) equilibrated with 20 mm Tris/HCl, pH 8.0, containing mm NaCl and 2 mm desulfurase activity of recombinant ABA3 proteins was either as the of l-alanine or as the of sulfide from l-cysteine as described previously L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (502) Google and M. Chem. Scopus Google Scholar, lyase activity was by of with as described previously N. T. H. J. Biol. Chem. Full Text PDF PubMed Google Scholar), except that were performed at 37 °C in mm Tris/HCl, pH containing 2 mm The substrate l-selenocysteine was freshly from its under conditions in the of 5 as described in N. T. H. J. Biol. Chem. Full Text PDF PubMed Google for l-cysteine was a substrate of mm at 37 °C with 20 of ABA3-NifS in 20 mm Tris/HCl, pH by the of for l-selenocysteine was a substrate of mm 5 at 37 °C with of ABA3-NifS in mm Tris/HCl, pH by the of activities were with 20 of ABA3-NifS using 0.5 mm of substrate during at 37 °C. of recombinant AOα by was performed in 20 mm Tris/HCl, pH 8.0, in the of mm for 20 h at 37 °C. After this the was by the protein to 20 mm Tris/HCl, pH 8.0, before In of recombinant AOα by ABA3 was performed in a of of 20 mm Tris/HCl, pH of AOα were with the respective ABA3 protein ABA3 in the of 0.5 mm l-cysteine for h at 37 °C, followed by native with of the and activity with as substrate as described in H. Akaba S. Seo M. Komano T. Koshiba T. J. Biochem. (Tokyo). 2000; 127: 659-664Crossref PubMed Scopus (37) Google of Protein of protein were by of as described in Biochem. PubMed Scopus Google of ABA3 was using an (Amersham of as the was identified by the of a product that is during of and E. Biochem. PubMed Scopus Google Scholar). to be from the protein by combined with acid and by which the protein is addition of and of pH to the of the product by the at at of with a the of a protein of from other NifS-like enzymes and by the of one the sequence of ABA3, binding of one ABA3 protein was The of the protein in was to to into the the molecular mass of the NifS-like domain of ABA3 is a of of purified ABA3-NifS = be Finally, of the protein samples was and and were detected and with of of the the nature of the sulfur bound by ABA3-NifS, we followed a described previously L. White R.H. Cash V.L. Dean D.R. PubMed Scopus Google Scholar). of ABA3-NifS were with for 30 in a of to of the protein with substrate in the was by buffer exchange using a with a molecular ABA3-NifS was with 0.2 mm for at °C. The of was by buffer exchange as described to addition of mm only from the protein that were bound a that from an After mass from the protein the mass was for under A protein that was in the absence of l-cysteine but in the at all other as a of the as A with an lysine conserved NifS-like proteins (1Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (502) Google Scholar), for as a cofactor bound to of protein samples that were as described under were with of and found to be thus that the bound is in comparison of the with a that the calculated of bound by one of ABA3-NifS is in protein samples that were when culturing E. coli in the of 5 mm the in the E. coli also the for the that with is required when ABA3-NifS in E. coli and that of ABA3-NifS binds one of of mm mm mm in a of the as of a bound persulfide intermediate, we used the 1,5-I-AEDANS, which binds to of protein thereby either at or in the of having bound to be from the protein by such as DTT, to the protein by be efficiently in the of by both of the from the protein by be identified by its that a higher was detected in samples that ABA3-NifS with l-cysteine in comparison with samples that were not with l-cysteine it be that during the of ABA3-NifS with a persulfide was that was by was detected when using an protein an of a cysteine residue at that the for binding the persulfide cysteine as an essential of persulfide as by of in a of cysteine desulfurase activity of ABA3 is located the NifS-like domain, we used ABA3-NifS to l-cysteine into l-alanine and elemental sulfur according to L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (502) Google and as described previously for full-length ABA3 (10Bittner F. Oreb M. Mendel R.R. J. Biol. Chem. 2001; 276: 40381-40384Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). ABA3-NifS l-cysteine more efficiently a than of full-length ABA3 of the C-terminal domain to the NifS-like domain rather than cysteine desulfurase activity. were also by the in AO activity in which the sulfur as from l-cysteine and bound to ABA3 or ABA3-NifS, is transferred to the Moco of recombinant The NifS-like domain of ABA3 was found to have the to sulfur for activation of AOα as for cysteine ABA3-NifS was more than full-length ABA3 in activation of the of the C-terminal domain we the of sulfide that was as sulfide in the of the to the of sulfur other than l-cysteine that not release l-alanine. all sulfur only l-cysteine and l-cysteine were by However, the of detected when using l-cysteine as substrate was only to l-cysteine. release of was detected from the other and of for of the ABA3-NifS lysine which to lysine and is to in binding (1Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (502) Google Scholar), with a in of the that is for purified ABA3-NifS protein. This in is by a of the of the protein. the of ABA3-NifS a at but a be in the the activity this a of cysteine desulfurase activity and Moco sulfurase activity and the which to A. cysteine and is proposed to the persulfide L. White R.H. Cash V.L. Dean D.R. PubMed Scopus Google Scholar), with an not the of ABA3 not but the two sub-activities of the protein. A cysteine desulfurase activity of was found when with ABA3-NifS This to sulfur from l-cysteine is by the of to recombinant AOα of NifS-like proteins catalyze not only the formation of elemental sulfur and from l-cysteine but also the formation of elemental selenium and from was found that ABA3-NifS decomposed the selenium substrate more efficiently than the sulfur substrate l-cysteine. 0.5 mm l-cysteine as the activity of the purified was to be protein, as by the of In the activity with l-selenocysteine as substrate was protein, as by the formation of l-alanine. the of ABA3-NifS for l-cysteine is the for l-selenocysteine is that ABA3-NifS l-cysteine However, it be that from was at mm for either thereby the of for activity of ABA3-NifS is to pH and the also on the conditions and of in a which was shown to be to the cofactor and its cysteine desulfurase activity, was found to be in selenocysteine lyase activity as well the to the to l-selenocysteine also was to a activity of about in the protein that was found to be in persulfide Moco catalyze the transfer of a sulfur atom to the center of enzymes to the xanthine oxidase such as AO and XDH, thereby enzymes. Moco the molybdenum of the cofactor is two oxygen one of which to be replaced by this sulfur have shown that the Moco sulfurase ABA3 from A. thaliana is able to AOα from A. thaliana in in the of l-cysteine as sulfur substrate (10Bittner F. Oreb M. Mendel R.R. J. Biol. Chem. 2001; 276: 40381-40384Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). The N-terminal domain of ABA3 similarities to NifS and NifS-like enzymes, a mechanism for sulfur mobilization and In this work, it was shown that the NifS domain of ABA3 binds a cofactor and a persulfide from the decomposition of as was demonstrated previously for NifS from A. (1Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (502) Google Scholar, L. White R.H. Cash V.L. Dean D.R. PubMed Scopus Google Scholar). However, other NifS-like ABA3 not the sulfur for [Fe-S] cluster but it to the Moco of AO and XDH for generation of the of Moco, which is the essential final step for activation of AO and In vitro, the NifS domain of ABA3 possesses not only cysteine desulfurase activity but also the to this activation of AOα be to the formation of it be that protein that sulfide in is able to the Moco of However, in all the NifS-like domain of ABA3 to a of AO and XDH activity (10Bittner F. Oreb M. Mendel R.R. J. Biol. Chem. 2001; 276: 40381-40384Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, M. Plant Physiol. PubMed Scopus Google Scholar, L. M. H. Plant Cell. 2001; 13: PubMed Scopus Google Scholar), thereby the of ABA3. is most likely that in the for ABA3 is bound to its which is different from the of the other A. thaliana cysteine AtNFS1 and AtNFS2, which are located in mitochondria and ABA3 is a cytosolic protein, its target AO Plant Sci. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). well be in addition to the respective the sulfur transfer from the NifS domain to the Moco of AO and XDH also needs the of the C-terminal In vivo, the function of the C-terminal domain is required for activation of AO and XDH, as was shown by of the M. C. R. Plant J. 2002; PubMed Scopus Google Scholar). Although this Moco sulfurase activities of AO and XDH in the it a of AO and XDH activities in the that the function of the C-terminal domain is a cysteine by ABA3-NifS is in the of the C-terminal domain, an of ABA3-NifS and the C-terminal domain during is not such an not able to be shown to and S. Both full-length ABA3 and ABA3-NifS with an molecular mass of and the C-terminal domain is with a mass of S. and M. This that of full-length ABA3 is by its NifS-like domain and that one ABA3 two sulfur as required by AO and XDH, which are of two as well. it well be that the C-terminal domain the sulfur transfer from one of ABA3-NifS to one of AO or XDH, thereby also the of ABA3-NifS during sulfur most of the NifS-like ABA3-NifS also was found to on l-cysteine and of l-selenocysteine by ABA3-NifS was found to be more than cysteine which is for other NifS-like enzymes as well H. Kurihara T. T. Esaki N. J. Biochem. (Tokyo). 2000; 127: PubMed Scopus Google Scholar). However, physiological function is obvious for the selenocysteine lyase activity of ABA3 enzymes were described for higher to this is higher the protein for the formation of l-selenocysteine and thus a mechanism for the decomposition of selenocysteine is required at ABA3-NifS with other NifS-like enzymes that were described to function in [Fe-S] cluster formation, it became obvious that ABA3-NifS all and that are for ABA3-NifS is a and of the full-length ABA3 protein. binds a cofactor that is essential for cysteine desulfurase activity and for sulfur transferase activity, as was shown by binding of one of ABA3-NifS and by of the protein. catalyzes the formation of l-alanine and elemental sulfur by using l-cysteine as substrate to an which was shown by of the ABA3-NifS protein with the and by site-directed mutagenesis of the proposed persulfide binding at cysteine ABA3-NifS possesses sulfur transferase activity, which is required for the persulfide sulfur to the Moco of AO and XDH, and it selenocysteine lyase activity, which also on the of and is on cysteine Although the physiological function of ABA3 is from the function of all NifS proteins described thus that ABA3 be to as a NifS-like protein. Hence, it be that besides AtNFS1 in mitochondria and in ABA3 represents a third and cytosolic NifS-like protein in A. thaliana. We Tomokazu Koshiba for the pastoris and for