Mapping iron binding sites on human frataxin: implications for cluster assembly on the ISU Fe–S cluster scaffold protein
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Frataxin is an iron binding mitochondrial matrix protein that has been shown to mediate iron delivery during iron–sulfur cluster and heme biosynthesis. There is a high degree of structural homology for frataxin proteins from diverse sources, and all possess an anionic surface defined by acidic residues. In the human protein these residues principally lie on a surface defined by the α1 helix and β1 sheet and the impact of multiple substitutions of these carboxylate residues on iron binding is described. Full-length human frataxin has previously been shown to undergo self-cleavage to produce a truncated form both in vitro and in vivo. This truncated protein has been shown to bind approximately seven iron centers that are presumably associated with the acidic patch. Relative to this native protein, the stoichiometry decreases according to the number and sites of mutations. Nevertheless, the iron-dependent binding affinity of each frataxin derivative to the iron–sulfur cluster scaffold protein ISU is found to be similar to that of native frataxin, as defined by isothermal titration calorimetry experiments, requiring only one iron center to promote nanomolar binding. While frataxins from various cell types appear to bind differing numbers of iron centers, the physiologically relevant number of bound irons appears to be small, with significantly higher binding affinity following complex formation with partner proteins (micromolar compared with nanomolar binding). By contrast, in reconstitution assays for frataxin-promoted [2Fe–2S]2+ cluster assembly on ISU, one derivative does display a modestly lower reconstitution rate. The overall consensus from these data is to consider a pool of potential sites that can stably bind an iron center when bridged to a variety of physiological targets.
KeywordsFrataxin Iron binding Fe–S cluster biosynthesis ISU
We thank Grazia Isaya for the gift of vectors that facilitated preparation of the frataxin derivatives used in these studies. This work was supported by a grant the National Science Foundation, CHE-0111161.
- 3.Campuzano V, Montermini L, Molto MD, Pianese L, Cossee M, Cavalcanti F, Monros E, Rodius F, Duclos F, Monticelli A, Zara F, Canizares J, Koutnikova H, Bidichandani SI, Gellera C, Brice A, Trouillas P, De Michele G, Filla A, De Frutos R, Palau F, Patel PI, Di Donato S, Mandel JL, Cocozza S, Koenig M, Pandolfo M (1996) Science 271:1423–1427PubMedCrossRefGoogle Scholar
- 13.Bencze KZ, Yoon T, Millán-Pacheco C, Bradley PB, Pastor N, Cowan JA, Stemmler TL (2007) Chem Commun 1798–1800Google Scholar
- 37.Liu Y, Cowan JA (2007) Chem Commun 3192–3194Google Scholar