Key words Iron kinetics
Springer Online Journal Archives 1860-2000
Chemistry and Pharmacology
Abstract Ferritins uniquely direct the vectorial transfer of hydrated Fe(II)/Fe(III) ions to a condensed ferric phase in the central cavity of the soluble protein. Secondary, tertiary and quaternary structure are conserved in ferritin, but only five amino acid residues are conserved among all known ferritins. The sensitivity of ferroxidation rates to small differences in primary sequence between ferritin subunits that are cell-specifically expressed or to the conservative replacement of the conserved tyrosine 30 residue was demonstrated by examining recombinant (frog) H-type (red blood cell predominant) and M-type subunit (liver predominant) proteins which are both fast ferritins; the proteins form two differently colored Fe(III)-protein complexes absorbing at 550 nm or 650 nm, respectively. The complexes are convenient reporters of Fe(III)-protein interaction because they are transient in contrast to the Fe(III)-oxy complexes measured in the past at 310–420 nm, which are stable because of contributions from the mineral itself. The A650-nm species formed 18-fold faster in the M-subunit protein than did the 550-nm species in H-subunit ferritin, even though all the ferroxidase residues are the same; the Vmax was fivefold faster but the Hill coefficents were identical (1.6), suggesting similar mechanisms. In H-subunit ferritin, substitution of phenylalanine for conserved tyrosine 30 (located in the core of the subunit four-helix bundle) slowed ferroxidation tenfold, whereas changing surface tyrosine 25 or tyrosine 28 had no effect. The Fe(III)-tyrosinate was fortunately not changed by the mutation, based on the resonance Raman spectrum, and remained a suitable reporter for Fe(III)-protein interactions. Thus, the A550/650 nm can also report on post-oxidation events such as transport through the protein. The impact of Y30F on rates of formation of Fe(III)-protein complexes in ferritin, combined with Mössbauer spectroscopic studies that showed the parallel formation of multiple Fe(III) postoxidation species (three dinuclear oxy and one trinuclear oxy species) (A. S. Periera et al., Biochemistry 36 : 7917–7927, 1997) and the loss of several of the multimeric Fe(III) post-oxidation species in a Y30F alteration of human recombinant H-ferritin (E. R. Bauminger et al., Biochem J. 296 : 709–719, 1993), indicate that at least one of the pathways for Fe oxidation/transfer in ferritin is through the center of the four-helix bundle and is influenced by structural features dependent on tyrosine 30.
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