Iron(II) oxidation by H chain ferritin: evidence from site-directed mutagenesis that a transient blue species is formed at the dinuclear iron center.
The iron storage molecule, ferritin, consists of an iron core surrounded by a shell of 24 protein subunits, which, in mammals, are of two types, H and L. Prior to storage of iron as a hydrous ferric oxide within the protein shell, Fe(II) is catalytically oxidized at dinuclear centers within H chains. When 48 Fe(II) atoms/molecule were added to 1 microM recombinant human H chain apoferritin (apo-HuHF), in 0.1 M Mes (pH 6.5), oxidation was 80% complete within about 0.2 s while 99% of the Fe(II) was oxidized within 10 s. A broad visible absorption band (400-800 nm, with a maximum at 650 nm) appeared during the fast phase of Fe(II) oxidation. It reached a plateau at 0.2-0.3 s and then declined while Fe(II) oxidation proceeded to completion and absorbance in the near-UV (300-400 nm) increased. The transient visible species was not observed when Tyr-34 was replaced by phenylalanine or when other conserved amino acids at the ferroxidase centers were substituted by residues which are unable to bind iron or which alter the charge balance. When a second increment of 48 iron atoms was added, 10 min after the first, the visible absorbance was absent and the rate of oxidation slower. Restoration of full oxidative activity took over 24 h. The data indicate that the fast oxidation of Fe(II) by apo-HuHF and the transient visible absorbance associated with it are due to Fe(II) oxidation at the ferroxidase centers.
Treffry
A
A
Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, U.K.
Zhao
Z
Z
Quail
M A
MA
Guest
J R
JR
Harrison
P M
PM
D001665:Binding Sites; D005293:Ferritins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D007502:Iron Chelating Agents; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010618:Phenanthrolines / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D013329:Structure-Activity Relationship
D005296:Ferrous Compounds; D007502:Iron Chelating Agents; D010446:Peptide Fragments; D010618:Phenanthrolines; D011994:Recombinant Proteins; D005293:Ferritins; D010100:Oxygen; C025205:1,10-phenanthroline