Physical evidence that yeast frataxin is an iron storage protein.
Frataxin is a conserved mitochondrial protein required for iron homeostasis. We showed previously that in the presence of ferrous iron recombinant yeast frataxin (mYfh1p) assembles into a regular multimer of approximately 1.1 MDa storing approximately 3000 iron atoms. Here, we further demonstrate that mYfh1p and iron form a stable hydrophilic complex that can be detected by either protein or iron staining on nondenaturing polyacrylamide gels, and by either interference or absorbance measurements at sedimentation equilibrium. The molecular mass of this complex has been refined to 840 kDa corresponding to 48 protein subunits and 2400 iron atoms. Solution density measurements have determined a partial specific volume of 0.58 cm(3)/g, consistent with the amino acid composition of mYfh1p and the presence of 50 Fe-O equivalents per subunit. By dynamic light scattering, we show that the complex has a radius of approximately 11 nm and assembles within 2 min at 30 degrees C when ferrous iron, not ferric iron or other divalent cations, is added to mYfh1p monomer at pH between 6 and 8. Iron-rich granules with diameter of 2-4 nm are detected in the complex by scanning transmission electron microscopy and energy-dispersive X-ray spectroscopy. These findings support the hypothesis that frataxin is an iron storage protein, which could explain the mitochondrial iron accumulation and oxidative damage associated with frataxin defects in yeast, mouse, and humans.
Gakh
Oleksandr
O
Departments of Pediatric & Adolescent Medicine, Biochemistry & Molecular Biology, and Pharmacology, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
Adamec
Jiri
J
Gacy
A Marquis
AM
Twesten
Ray D
RD
Owen
Whyte G
WG
Isaya
Grazia
G
D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005296:Ferrous Compounds / Q000737:chemistry*; D006706:Homeostasis / Q000502:physiology; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D033862:Iron-Binding Proteins; D017348:Microscopy, Electron, Scanning Transmission; D008928:Mitochondria / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism*; D011108:Polymers / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012542:Scattering, Radiation; D013052:Spectrometry, X-Ray Emission; D014461:Ultracentrifugation; D015003:Yeasts / Q000737:chemistry; D000096923:Frataxin
D005296:Ferrous Compounds; D007501:Iron; D033862:Iron-Binding Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D011108:Polymers; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; D000096923:Frataxin; C038178:ammonium ferrous sulfate