Drosophila Frataxin: an Iron Chaperone During Cellular [2Fe-2S] Cluster Bioassembly

Journal Article · Tue Jan 01 04:00:00 EST 2008 · Biochemistry

DOI:https://doi.org/10.1021/bi800366d· OSTI ID:959834

Kondapalli, K; Kok, N; Dancis, A; Stemmler, T

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 (TM {approx} 59 C) and chemical stability ([urea]50% {approx} 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 (Kd {approx} 6.0 {mu}M) 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 H2O2. 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 Angstroms, 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 (Kd {approx} 0.21 {mu}M) 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 (kobs) of 0.096 min-1.

• Research Organization: Brookhaven National Laboratory (BNL) National Synchrotron Light Source
• Sponsoring Organization: Doe - Office Of Science
• DOE Contract Number: AC02-98CH10886
• OSTI ID: 959834
• Report Number(s): BNL--82820-2009-JA
• Journal Information: Biochemistry, Journal Name: Biochemistry Vol. 47; ISSN 0006-2960; ISSN BICHAW
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
99 GENERAL AND MISCELLANEOUS
ABSORPTION
AFFINITY
BIOSYNTHESIS
BOND LENGTHS
CALORIMETRY
DICHROISM
DNA
DROSOPHILA
ENZYMES
FLUORESCENCE SPECTROSCOPY
HOMEOSTASIS
IN VITRO
IRON
MONOMERS
OXYGEN
PROTEINS
RESIDUES
STABILITY
STOICHIOMETRY
YEASTS
national synchrotron light source