IRON (II) OXIDATION AND EARLY INTERMEDIATES OF IRON-CORE FORMATION INRECOMBINANT HUMAN H-CHAIN FERRITIN

The paper describes a study of Fe(II) oxidation and the formation of F e(III)-apoferritin complexes in recombinant human H-chain ferritin and its variants. The effects of site-directed changes in the conserved r esidues associated with a proposed ferroxidase centre have been invest igated. A change in any of these residues is shown to reduce the rate of Fe(II) oxidation, confirming the importance of the ferroxidase cent re in the catalysis of Fe(II) oxidation. Mossbauer and u.v.-difference spectroscopy show that in the wild-type protein Fe(II) oxidation give s rise to Fe(III) monomers, dimers and larger clusters. The formation of Fe(III) mu-oxo-bridged dimers occurs at the ferroxidase centre and is associated with fast oxidation: in three variants in which Fe(II) o xidation is especially slow, no Fe(III) dimers are seen. Within the ti me scale 0.5-20 min in wild-type human H-chain ferritin, dimer formati on precedes that of the monomer and the progression dimer --> monomer --> cluster is observed, although not to completion. In a preliminary investigation of oxidation intermediates using a stopped-flow instrume nt, an Fe(III)-tyrosine complex reported by Waldo et al. (1993), is at tributed to Tyr-34, a residue at the ferroxidase centre. The Fe(III)-T yr-34 complex, forms in 0.5 s and then decays, as dimer absorbance inc reases. The relationship between Fe(III)-tyrosinate and the formation of Fe(III) dimers is uncertain.