title: Frataxin Accelerates [2Fe-2S] Cluster Formation on the Human Fe-S Assembly Complex.
abstract: Iron-sulfur (Fe-S) clusters function as protein cofactors for a wide variety of critical cellular reactions. In human mitochondria, a core Fe-S assembly complex [called SDUF and composed of NFS1, ISD11, ISCU2, and frataxin (FXN) proteins] synthesizes Fe-S clusters from iron, cysteine sulfur, and reducing equivalents and then transfers these intact clusters to target proteins. In vitro assays have relied on reducing the complexity of this complicated Fe-S assembly process by using surrogate electron donor molecules and monitoring simplified reactions. Recent studies have concluded that FXN promotes the synthesis of [4Fe-4S] clusters on the mammalian Fe-S assembly complex. Here the kinetics of Fe-S synthesis reactions were determined using different electron donation systems and by monitoring the products with circular dichroism and absorbance spectroscopies. We discovered that common surrogate electron donor molecules intercepted Fe-S cluster intermediates and formed high-molecular weight species (HMWS). The HMWS are associated with iron, sulfide, and thiol-containing proteins and have properties of a heterogeneous solubilized mineral with spectroscopic properties remarkably reminiscent of those of [4Fe-4S] clusters. In contrast, reactions using physiological reagents revealed that FXN accelerates the formation of [2Fe-2S] clusters rather than [4Fe-4S] clusters as previously reported. In the preceding paper [Fox, N. G., et al. (2015) Biochemistry 54, DOI: 10.1021/bi5014485], [2Fe-2S] intermediates on the SDUF complex were shown to readily transfer to uncomplexed ISCU2 or apo acceptor proteins, depending on the reaction conditions. Our results indicate that FXN accelerates a rate-limiting sulfur transfer step in the synthesis of [2Fe-2S] clusters on the human Fe-S assembly complex.
authors.forename: Nicholas G
authors.initials: NG
authors.lastname: Fox
authors.forename: Deepika
authors.initials: D
authors.lastname: Das
authors.forename: Mrinmoy
authors.initials: M
authors.lastname: Chakrabarti
authors.forename: Paul A
authors.initials: PA
authors.lastname: Lindahl
authors.forename: David P
authors.initials: DP
authors.lastname: Barondeau
mesh_terms: D002942:Circular Dichroism; D006801:Humans; D007501:Iron / Q000378:metabolism; D033862:Iron-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D013455:Sulfur / Q000378:metabolism; D000096923:Frataxin
chemical_list: D007501:Iron; D033862:Iron-Binding Proteins; D007506:Iron-Sulfur Proteins; D013455:Sulfur; D000096923:Frataxin