title: Iron loading site on the Fe-S cluster assembly scaffold protein is distinct from the active site.
abstract: Iron-sulfur (Fe-S) cluster containing proteins are utilized in almost every biochemical pathway. The unique redox and coordination chemistry associated with the cofactor allows these proteins to participate in a diverse set of reactions, including electron transfer, enzyme catalysis, DNA synthesis and signaling within several pathways. Due to the high reactivity of the metal, it is not surprising that biological Fe-S cluster assembly is tightly regulated within cells. In yeast, the major assembly pathway for Fe-S clusters is the mitochondrial ISC pathway. Yeast Fe-S cluster assembly is accomplished using the scaffold protein (Isu1) as the molecular foundation, with assistance from the cysteine desulfurase (Nfs1) to provide sulfur, the accessory protein (Isd11) to regulate Nfs1 activity, the yeast frataxin homologue (Yfh1) to regulate Nfs1 activity and participate in Isu1 Fe loading possibly as a chaperone, and the ferredoxin (Yah1) to provide reducing equivalents for assembly. In this report, we utilize calorimetric and spectroscopic methods to provide molecular insight into how wt-Isu1 from S. cerevisiae becomes loaded with iron. Isothermal titration calorimetry and an iron competition binding assay were developed to characterize the energetics of protein Fe(II) binding. Differential scanning calorimetry was used to identify thermodynamic characteristics of the protein in the apo state or under iron loaded conditions. Finally, X-ray absorption spectroscopy was used to characterize the electronic and structural properties of Fe(II) bound to Isu1. Current data are compared to our previous characterization of the D37A Isu1 mutant, and these suggest that when Isu1 binds Fe(II) in a manner not perturbed by the D37A substitution, and that metal binding occurs at a site distinct from the cysteine rich active site in the protein.
authors.affiliation: Departments of Biochemistry and Molecular Biology, Wayne State University, Detroit, MI, USA.
authors.forename: Andria V
authors.initials: AV
authors.lastname: Rodrigues
authors.forename: Ashoka
authors.initials: A
authors.lastname: Kandegedara
authors.forename: John A
authors.initials: JA
authors.lastname: Rotondo
authors.forename: Andrew
authors.initials: A
authors.lastname: Dancis
authors.forename: Timothy L
authors.initials: TL
authors.lastname: Stemmler
mesh_terms: D020134:Catalytic Domain; D007501:Iron / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
chemical_list: D007506:Iron-Sulfur Proteins; D029701:Saccharomyces cerevisiae Proteins; D007501:Iron