Frataxin, a small mitochondrial protein linked to the neurodegenerative disease Friedreich ataxia, has recently been proposed as an iron donor for the iron-sulfur cluster assembly. An analogous function has also been attributed to IscA, a key member of the iron-sulfur cluster assembly machinery found in bacteria, yeast, and humans. Here we have compared the iron binding property of IscA and the frataxin ortholog CyaY from Escherichia coli under physiological and oxidative stress conditions. In the presence of the thioredoxin reductase system, which emulates the intracellular redox potential, CyaY fails to bind any iron even at a 10-fold excess of iron in the incubation solution. Under the same physiologically relevant conditions, IscA efficiently recruits iron and transfers the iron for the ironsulfur cluster assembly in a proposed scaffold IscU. In the presence of hydrogen peroxide, however, IscA completely loses its iron binding activity, whereas CyaY becomes a competent ironbinding protein and attenuates the iron-mediated production of hydroxyl free radicals. Hydrogen peroxide appears to oxidize the iron binding thiol groups in IscA, thus blocking the iron binding in the protein. Once the oxidized thiol groups in IscA are re-reduced with the thioredoxin reductase system, the iron binding activity of IscA is fully restored. On the other hand, hydrogen peroxide has no effect on the iron binding carboxyl groups in CyaY, allowing the protein to bind iron under oxidative stress conditions. The results suggest that IscA is capable of recruiting intracellular iron for the iron-sulfur cluster assembly under normal physiological conditions, whereas CyaY may serve as an iron chaperon to sequester redox active free iron and alleviate cellular oxidative damage under oxidative stress conditions.Frataxin is a small mitochondrial protein that has been linked to Friedreich ataxia, an autosomal recessive neurodegenerative disease (1). Most Friedreich ataxia patients are homozygous for a large GAA repeat expansion in the first intron of the frataxin gene which impairs transcription and causes severe reduction in the level of frataxin in mitochondria (1, 2). Frataxin is highly conserved from bacteria to humans (3). Deletion of frataxin results in disruption of iron homeostasis and mitochondrial function in Saccharomyces cerevisiae (4), embryonic lethality in the mouse (5), and developmental arrest in the nematode Caenorhabditis elegans (6). Structural studies of human frataxin (7), yeast frataxin (8), and the bacterial frataxin ortholog CyaY 2 (9, 10) revealed a well conserved three-dimensional structure. However, the specific function of frataxin/CyaY is still not fully understood. Recently, it has been proposed that one of the functions of frataxin/CyaY may be involved in biogenesis of ironsulfur clusters, a group of ubiquitous redox co-factors in cells. This notion is primarily based on the observations that (i) depletion of frataxin in Friedreich ataxia patients is associated with deficiency of iron-sulfur proteins in mitoc...