title: The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster Scaffold Is an ATPase.
abstract: Nbp35 and Cfd1 are prototypical members of the MRP/Nbp35 class of iron-sulfur (FeS) cluster scaffolds that function to assemble nascent FeS clusters for transfer to FeS-requiring enzymes. Both proteins contain a conserved NTPase domain that genetic studies have demonstrated is essential for their cluster assembly activity inside the cell. It was recently reported that these proteins possess no or very low nucleotide hydrolysis activity in vitro, and thus the role of the NTPase domain in cluster biogenesis has remained uncertain. We have reexamined the NTPase activity of Nbp35, Cfd1, and their complex. Using in vitro assays and site-directed mutagenesis, we demonstrate that the Nbp35 homodimer and the Nbp35-Cfd1 heterodimer are ATPases, whereas the Cfd1 homodimer exhibited no or very low ATPase activity. We ruled out the possibility that the observed ATP hydrolysis activity might result from a contaminating ATPase by showing that mutation of key active site residues reduced activity to background levels. Finally, we demonstrate that the fluorescent ATP analog 2'/3'-O-(N'-methylanthraniloyl)-ATP (mantATP) binds stoichiometrically to Nbp35 with a KD = 15.6 μM and that an Nbp35 mutant deficient in ATP hydrolysis activity also displays an increased KD for mantATP. Together, our results demonstrate that the cytosolic iron-sulfur cluster assembly scaffold is an ATPase and pave the way for interrogating the role of nucleotide hydrolysis in cluster biogenesis by this large family of cluster scaffolding proteins found across all domains of life.
authors.affiliation: From the Department of Chemistry, Boston University, Boston, Massachusetts 02215.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Camire
authors.affiliation: From the Department of Chemistry, Boston University, Boston, Massachusetts 02215.
authors.forename: John D
authors.initials: JD
authors.lastname: Grossman
authors.affiliation: From the Department of Chemistry, Boston University, Boston, Massachusetts 02215.
authors.forename: Grace J
authors.initials: GJ
authors.lastname: Thole
authors.affiliation: From the Department of Chemistry, Boston University, Boston, Massachusetts 02215.
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Fleischman
authors.affiliation: From the Department of Chemistry, Boston University, Boston, Massachusetts 02215 dperl@bu.edu.
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Perlstein
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D055503:Protein Multimerization / Q000502:physiology; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C520224:Cfd1 protein, S cerevisiae; D007506:Iron-Sulfur Proteins; D046912:Multiprotein Complexes; C102265:NBP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D019204:GTP-Binding Proteins
keywords: ATPase; Cfd1; Michaelis-Menten; Nbp35; cytosolic iron-sulfur cluster assembly (CIA); enzyme kinetics; fluorescence anisotropy; iron metabolism; iron-sulfur protein