Iron (II) oxidation and early intermediates of iron-core formation in recombinant human H-chain ferritin.
The paper describes a study of Fe(II) oxidation and the formation of Fe(III)-apoferritin complexes in recombinant human H-chain ferritin and its variants. The effects of site-directed changes in the conserved residues associated with a proposed ferroxidase centre have been investigated. A change in any of these residues is shown to reduce the rate of Fe(II) oxidation, confirming the importance of the ferroxidase centre in the catalysis of Fe(II) oxidation. Mössbauer and u.v.-difference spectroscopy show that in the wild-type protein Fe(II) oxidation gives 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) oxidation is especially slow, no Fe(III) dimers are seen. Within the time scale 0.5-20 min in wild-type human H-chain ferritin, dimer formation 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 instrument, an Fe(III)-tyrosine complex reported by Waldo et al. (1993), is attributed to Tyr-34, a residue at the ferroxidase centre. The Fe(III)-Tyr-34 complex, forms in 0.5 s and then decays, as dimer absorbance increases. The relationship between Fe(III)-tyrosinate and the formation of Fe(III) dimers is uncertain.
Bauminger
E R
ER
Racah Institute of Physics, Hebrew University of Jerusalem, Israel.
Harrison
P M
PM
Hechel
D
D
Hodson
N W
NW
Nowik
I
I
Treffry
A
A
Yewdall
S J
SJ
D003124:Colorimetry; D005293:Ferritins / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D015204:Spectroscopy, Mossbauer
D011994:Recombinant Proteins; D005293:Ferritins; D007501:Iron