title: The conserved protein Dre2 uses essential [2Fe-2S] and [4Fe-4S] clusters for its function in cytosolic iron-sulfur protein assembly.
abstract: The cytosolic iron-sulfur (Fe-S) protein assembly (CIA) machinery comprises 11 essential components and matures Fe-S proteins involved in translation and genome maintenance. Maturation is initiated by the electron transfer chain NADPH-diflavin reductase Tah18-Fe-S protein Dre2 that facilitates the de novo assembly of a [4Fe-4S] cluster on the scaffold complex Cfd1-Nbp35. Tah18-Dre2 also play a critical role in the assembly of the diferric tyrosyl radical cofactor of ribonucleotide reductase. Dre2 contains eight conserved cysteine residues as potential co-ordinating ligands for Fe-S clusters but their functional importance and the type of bound clusters is unclear. In the present study, we use a combination of mutagenesis, cell biological and biochemical as well as UV-visible, EPR and Mössbauer spectroscopic approaches to show that the yeast Dre2 cysteine residues Cys(252), Cys(263), Cys(266) and Cys(268) (motif I) bind a [2Fe-2S] cluster, whereas cysteine residues Cys(311), Cys(314), Cys(322) and Cys(325) (motif II) co-ordinate a [4Fe-4S] cluster. All of these residues with the exception of Cys(252) are essential for cell viability, cytosolic Fe-S protein activity and in vivo (55)Fe-S cluster incorporation. The N-terminal methyltransferase-like domain of Dre2 is important for proper Fe-S cluster assembly at motifs I and II, which occurs in an interdependent fashion. Our findings further resolve why recombinant Dre2 from Arabidopsis, Trypanosoma or humans has previously been isolated with a single [2Fe-2S] instead of native [2Fe-2S] plus [4Fe-4S] clusters. In the presence of oxygen, the motif I-bound [2Fe-2S] cluster is labile and the motif II-bound [4Fe-4S] cluster is readily converted into a [2Fe-2S] cluster.
authors.affiliation: Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Koch-Strasse 6, 35032 Marburg, Germany.
authors.forename: Daili J A
authors.initials: DJ
authors.lastname: Netz
authors.affiliation: Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Koch-Strasse 6, 35032 Marburg, Germany.
authors.forename: Heide M
authors.initials: HM
authors.lastname: Genau
authors.affiliation: Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Koch-Strasse 6, 35032 Marburg, Germany.
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Weiler
authors.affiliation: Max-Planck-Institut für Chemische Energiekonversion, Stiftstrasse 34-36, 45470 Mülheim-Ruhr, Germany.
authors.forename: Eckhard
authors.initials: E
authors.lastname: Bill
authors.affiliation: Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Koch-Strasse 6, 35032 Marburg, Germany pierik@chemie.uni-kl.de lill@staff.uni-marburg.de.
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Pierik
authors.affiliation: Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Koch-Strasse 6, 35032 Marburg, Germany LOEWE Zentrum für Synthetische Mikrobiologie SynMikro, Hans-Meerwein-Strasse, 35043 Marburg, Germany pierik@chemie.uni-kl.de lill@staff.uni-marburg.de.
authors.forename: Roland
authors.initials: R
authors.lastname: Lill
mesh_terms: D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015204:Spectroscopy, Mossbauer
chemical_list: C531900:Dre2 protein, S cerevisiae; D007506:Iron-Sulfur Proteins; D029701:Saccharomyces cerevisiae Proteins; D003545:Cysteine
keywords: Dre2; EPR; Mössbauer spectroscopy; biogenesis; cytosolic iron–sulfur protein assembly (CIA) machinery; mutagenesis; spectroscopy