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 surround ed by a shell of 24 protein subunits, which, in mammals, are of two ty pes, H and L. Prior to storage of iron as a hydrous ferric oxide withi n the protein shell, Fe(ll) is catalytically oxidized at dinuclear cen ters within H chains. When 48 Fe(IT) atoms/molecule were added to 1 mu M 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 F e(II) was oxidized within 10 s. A broad visible absorption band (400-8 00 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 whi le Fe(LI) oxidation proceeded to completion and absorbance in the near -UV (300-400 nm) increased. The transient visible species was not obse rved when Tyr-34 was replaced by phenylalanine or when other conserved amino acids at the ferroxidase centers were substituted by residues w hich 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. Re storation of full oxidative activity took over 24 h. The data indicate that the fast oxidation of Fe(II) by apo-HuHF and the transient visib le absorbance associated with it are due to Fe(II) oxidation at the fe rroxidase centers.