Iron−Sulfur Cluster Biosynthesis. Characterization of Frataxin as an Iron Donor for Assembly of [2Fe-2S] Clusters in ISU-Type Proteins

• Published in: Journal of the American Chemical Society Vol. 125; no. 20; pp. 6078 - 6084
• Main Authors: Yoon, Taejin, Cowan, J. A
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 21.05.2003
• Subjects: Analytical biochemistry: general aspects, technics, instrumentation; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Escherichia coli - genetics; Escherichia coli - metabolism; Fluorometry; Frataxin; Fundamental and applied biological sciences. Psychology; Humans; Iron-Binding Proteins - chemistry; Iron-Binding Proteins - genetics; Iron-Binding Proteins - metabolism; Iron-Sulfur Proteins - biosynthesis; Iron-Sulfur Proteins - metabolism; Kinetics; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Thermodynamics; Characterization; Biochemistry; Biosynthesis; Iron; Sulfur; Protein
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja027967i

ISU (eukaryotes) and IscU (prokaryotes) are a homologous family of proteins that appear to provide a platform for assembly of [2Fe-2S] centers prior to delivery to an apo target protein. The intermediate [2Fe-2S] ISU-bound cluster is formed by delivery of iron and sulfur to the apo ISU, with the latter delivered through an IscS-mediated reaction. The identity of the iron donor has thus far not been established. In this paper we demonstrate human frataxin to bind from six to seven iron ions. Iron binding to frataxin has been quantitated by iron-dependent fluorescence measurements [K D(Fe3+) ≈ 11.7 μM; (K D(Fe2+) ≈ 55.0 μM] and isothermal titration calorimetry (ITC) [K D(Fe3+) ≈ 10.2 μM]. Enthalpies and entropies for ferric ion binding were determined from calorimetric measurements. Both fluorescence (K D 0.45 μM) and ITC measurements (K D 0.15 μM) demonstrate holo frataxin to form a complex with ISU with sub-micromolar binding affinities. Significantly, apo frataxin does not bind to ISU, suggesting an important role for iron in cross-linking the two proteins and/or stabilizing the structure of frataxin that is recognized by ISU. Holo frataxin is also shown to mediate the transfer of iron from holo frataxin to nucleation sites for [2Fe-2S] cluster formation on ISU. We have demonstrated elsewhere [J. Am. Chem. Soc. 2002, 124, 8774−8775] that this iron-bound form of ISU is viable for assembly of holo ISU, either by subsequent addition of sulfide or by NifS-mediated sulfur delivery. Provision of holo frataxin and inorganic sulfide is sufficient for cluster assembly in up to 70% yield. With NifS as a sulfur donor, yields in excess of 70% of holo ISU were obtained. Both UV−vis and CD spectroscopic characteristics were found to be consistent with those of previously characterized ISU proteins. The time course for cluster assembly was monitored from the 456 nm absorbance of holo ISU formed during the [2Fe-2S] cluster assembly reaction. A kinetic rate constant k obs ≈ 0.075 min-1 was determined with 100 μM ISU, 2.4 mM Na2S, and 40 μM holo frataxin in 50 mM Tris-HCl (pH 7.5) with 4.3 mM DTT. Similar rates were obtained for NifS-mediated sulfur delivery, consistent with iron release from frataxin as a rate-limiting step in the cluster assembly reaction.