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Acidic residues of yeast frataxin have an essential role in Fe-S cluster assembly.

ABSTRACT: Friedreich ataxia is caused by decreased levels of frataxin, a mitochondrial acidic protein that is assumed to act as chaperone in the assembly of Fe-S clusters on the scaffold Isu protein. Frataxin has the in vitro capacity to form iron-loaded multimers, which also suggests an iron storage function. It has been reported that alanine substitution of residues in an acidic ridge of yeast frataxin (Yfh1) elicits loss of iron binding in vitro but has no effect on Fe-S cluster synthesis in vivo. Here, we show that a marked change in the electrostatic properties of a specific region of Yfh1 surface - by substituting two or four acidic residues by lysine or alanine, respectively - impairs Fe-S cluster assembly, weakens the interaction between Yfh1 and Isu1, and increases oxidative damage. Therefore, the acidic ridge is essential for the Yfh1 function and is likely to be involved in iron-mediated protein-protein interactions.

SUBMITTER: Foury F

PROVIDER: S-EPMC1796776 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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Acidic residues of yeast frataxin have an essential role in Fe-S cluster assembly.

Foury Françoise F Pastore Annalisa A Trincal Mathieu M

EMBO reports 20061222 2

Friedreich ataxia is caused by decreased levels of frataxin, a mitochondrial acidic protein that is assumed to act as chaperone in the assembly of Fe-S clusters on the scaffold Isu protein. Frataxin has the in vitro capacity to form iron-loaded multimers, which also suggests an iron storage function. It has been reported that alanine substitution of residues in an acidic ridge of yeast frataxin (Yfh1) elicits loss of iron binding in vitro but has no effect on Fe-S cluster synthesis in vivo. Here ...[more]

PMID: 17186026

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Project description:Frataxin, a mitochondrial protein that is directly involved in regulating cellular iron homeostasis, has been suggested to serve as an iron chaperone during cellular Fe-S cluster biosynthesis. In humans, decreased amounts or impaired function of frataxin causes the autosomal recessive neurodegenerative disorder Friedreich's ataxia. Cellular production of Fe-S clusters is accomplished by the Fe cofactor assembly platform enzymes Isu (eukaryotes) and IscU (prokaryotes). In this report, we have characterized the overall stability and iron binding properties of the Drosophila frataxin homologue (Dfh). Dfh is highly folded with secondary structural elements consistent with the structurally characterized frataxin orthologs. While the melting temperature ( T M approximately 59 degrees C) and chemical stability ([urea] 50% approximately 2.4 M) of Drosophila frataxin, measured using circular dichroism (CD) and fluorescence spectroscopy, closely match values determined for the human ortholog, pure Dfh is more stable against autodegradation than both the human and yeast proteins. The ferrous iron binding affinity ( K d approximately 6.0 microM) and optimal metal to protein stoichiometry (1:1) for Dfh have been measured using isothermal titration calorimetry (ITC). Under anaerobic conditions with salt present, holo-Dfh is a stable iron-loaded protein monomer. Frataxin prevents reactive oxygen species-induced oxidative damage to DNA when presented with both Fe(II) and H 2O 2. Ferrous iron bound to Dfh is high-spin and held in a partially symmetric Fe-(O/N) 6 coordination environment, as determined by X-ray absorption spectroscopy (XAS). Extended X-ray absorption fine structure (EXAFS) simulations indicate the average Fe-O/N bond length in Dfh is 2.13 A, consistent with a ligand geometry constructed by water and carboxylate oxygens most likely supplied in part by surface-exposed conserved acidic residues located on helix 1 and strand 1 in the structurally characterized frataxin orthologs. The iron-dependent binding affinity ( K d approximately 0.21 microM) and optimal holo-Dfh to Isu monomer stoichiometry (1:1) have also been determined using ITC. Finally, frataxin mediates the delivery of Fe(II) to Isu, promoting Fe-S cluster assembly in vitro. The Dfh-assisted assembly of Fe-S clusters occurs with an observed kinetic rate constant ( k obs) of 0.096 min (-1).

Dataset Information

Acidic residues of yeast frataxin have an essential role in Fe-S cluster assembly.

Publications

Acidic residues of yeast frataxin have an essential role in Fe-S cluster assembly.

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