Monomeric yeast frataxin is an iron-binding protein

Abstract

Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50,000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron.

Figure 1. Oligomeric state and iron to protein stoichiometry for yeast frataxin samples

(A) Size exclusion chromatographs for yeast frataxin with 2, 1 or 0 iron atoms bound shown in (I), (II) and (III) respectively, coupled with protein standards (IV). Samples were run anaerobically on a S-200 Sephacryl size exclusion column at 27° C. (B) Representative comparison of the ICP-MS iron quantitation (squares) versus the protein concentration from a Bradford assay (circles) for 1:1 iron loaded yeast frataxin on data collected on a high resolution S-100 Sephacryl column under identical conditions as when we ran the S-200 column.

Figure 2. Raw isothermal titration calorimetry (A) and binding isotherm data (B) for ferrous iron to yeast frataxin

Grey line in panel (A) shows baseline and in panel (B) shows the simulated fit to the binding isotherm data. Data were collected anaerobically at 30° in 20 mM HEPES (pH 7.0) and 10 mM MgSO₄ . These data were collected by injecting small aliquots of a 0.8 mM ferrous iron solution into a 50 μM Yfh1 sample. Spacing between injections was 10 minutes. Syringe stirring speed was kept at 500 rpm.

Figure 3. Frataxin residues perturbed at the amide position by the presence of iron

(A) ¹⁵N filtered HSQC spectra for 500 μM apo- (blue) and 2Fe-Yfh1 (red) in 20 mM HEPES buffer (pH 7.0), 10 mM MgSO₄ at 600 MHz. NMR data collected anaerobically at 30°C. Inset: expanded region marked in dashed lines with residues identified, “§” indicates side chain or unassigned peaks. (B) Summary of yeast frataxin normalized amide chemical shift changes in the presence and absence of 2 equivalents of iron. Residues identified in black correspond to paramagnetically broadened amide signals. Residues identified in red correspond to amide signals with chemical shift changes. Residues indicated with a “*” in the x-axis indicate Pro positions. Horizontal black line indicates chemical shift cutoff, which corresponds to 2x data resolution. Top: secondary structural elements for yeast frataxin with helices and strands labeled as H and S, respectively. (C) Location of residues on apo-yeast frataxin structure with amide backbone resonances that are significantly line broadened (yellow) or have substantial chemical shift changes (red) in the presence of Fe(II). Note: the protein's structure represents an updated PDB file for yeast frataxin (PDB accession number 2GA5), currently available for download.

Figure 4. XANES comparison of iron-yeast frataxin with ferrous and ferric models

Full XANES spectra for yeast frataxin with 1 iron (solid) and 2 iron bound (bold, solid) with ferrous ammonium sulfate (dashed line) and ferric ammonium sulfate (dotted line). Inset: expansion of background subtracted 1s→3d region of the XANES spectra for all samples.

Figure 5. EXAFS and Fourier transforms of iron loaded yeast frataxin XAS data

EXAFS spectra in black for yeast frataxin with 1 iron bound (A) and 2 iron bound (C), along with the corresponding Fourier transforms (B and D, respectively). Simulations for EXAFS and FT data are shown in grey.

Figure 6. Location of yeast frataxin residues strongly perturbed by the presence of iron

A) Apo-protein structure with helix-1 amino acid side chain atoms from line broadened amide resonances in the yeast frataxin NMR iron titration. Expanded view to the right of arrows. B) Apo-protein structure with β-sheet amino acid side chain atoms from line broadened amide resonances in the yeast frataxin NMR iron titration and D50. Expanded view to the right of the arrow. Note: the protein's structure represents an updated PDB file for yeast frataxin (PDB accession number 2GA5), currently available for download.