Thioredoxin reductase system mediates iron binding in IscA and iron delivery for the iron-sulfur cluster assembly in IscU.
IscA is a key member of the iron-sulfur cluster assembly machinery found in bacteria and eukaryotes. Previously, IscA was characterized as an alternative iron-sulfur cluster assembly scaffold, as purified IscA can host transient iron-sulfur clusters. However, recent studies indicated that IscA is an iron-binding protein that can provide iron for the iron-sulfur cluster assembly in a proposed scaffold IscU (Ding H., Clark, R. J., and Ding, B. (2004) J. Biol. Chem. 279, 37499-37504). To further elucidate the roles of IscA in the biogenesis of iron-sulfur clusters, we reevaluate the iron binding activity of IscA under physiologically relevant conditions. The results indicate that in the presence of the thioredoxin reductase system, Escherichia coli IscA binds iron with an iron association constant of 2.0 x 10(19) M(-1) in vitro. Whereas all three components (thioredoxin 1, thioredoxin reductase and NADPH) in the thioredoxin reductase system are essential for mediating the iron binding in IscA, only catalytic amounts of thioredoxin 1 and thioredoxin reductase are required. In contrast, IscU fails to bind iron in the presence of the thioredoxin reductase system, suggesting that the iron binding in IscA is specific. Nevertheless, the thioredoxin reductase system can promote the iron-sulfur cluster assembly in IscU in the presence of the iron-loaded IscA, cysteine desulfurase (IscS), and L-cysteine, demonstrating a physiologically relevant system for the biogenesis of iron-sulfur clusters. The results provide additional evidence for the hypothesis that IscA is capable of recruiting intracellular "free" iron and delivering the iron for the iron-sulfur cluster assembly in IscU.
Ding
Huangen
H
Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, USA. hding@lsu.edu
Harrison
Karyn
K
Lu
Jianxin
J
D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D019343:Citric Acid / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism; D009249:NADP / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism / Q000502:physiology*
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; C487901:IscA protein, E coli; C411665:IscU protein, E coli; D011994:Recombinant Proteins; D019343:Citric Acid; D009249:NADP; D007501:Iron; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine