Structure-function studies of frataxin: An iron chaperone in the mitochondrial iron-sulfur cluster and heme biosynthetic pathways.

摘要

Frataxin is a mitochondrial protein that is involved in regulating cellular iron homeostasis. Frataxin has been suggested to serve as an iron chaperone during cellular Fe-S cluster and heme biosynthesis. In humans, decreased amounts or impaired function of frataxin causes the cardio and neurodegenerative disorder Friedreich's Ataxia. Frataxin is highly conserved from prokaryotes to eukaryotes. Though known to regulate mitochondrial iron homeostasis, the cellular function of frataxin has not been completely characterized. The major goal of the research presented in this report has been to characterize the iron binding ability of frataxin and probe its interaction with partners within the heme and Fe-S cluster biosynthetic pathways to identify how frataxin modulates metal delivery. Research on three orthologs of frataxin (yeast, drosophila and human) is presented in this dissertation.;Yeast frataxin can bind 2 ferrous iron atoms with micromolar binding affinity. A two-site iron-binding model of yeast frataxin was constructed based on the spatial arrangement and conservation of residues identified through iron titration experiments using NMR. Research directed at dissecting the role of individual sites used in metal binding and delivery to frataxin's partner proteins is presented here.;The drosophila model has provided additional insight into the role of frataxin directly within a multicellular eukaryotic organism. A comprehensive characterization of the mature drosophila frataxin's (Dfh) biophysical and metal-binding properties is presented. Furthermore, the protein's ability to transfer Fe(II) to Isu during in vitro Fe-S cluster assembly is also discussed.;The mitochondrial targeting sequence of human frataxin (Hftx) is cleaved in a two-step process. Complications from proteolysis or autodegradation result in an additional, unnatural N-terminal truncation in the mature human frataxin in vitro. Recently the Cowan laboratory at Ohio State University was able to express the full-length protein with a stable N-terminus. Preliminary studies by the Cowan laboratory on the full-length protein indicated that the human protein's N-terminus is structured and modulates the iron binding stoichiometry and the protein's interaction with partners. In this dissertation studies directed at solving the solution structure of the full-length human frataxin are reported.