RAPID KINETICS OF THE EPR-ACTIVE SPECIES FORMED DURING INITIAL IRON UPTAKE IN HORSE SPLEEN APOFERRITIN

The molecular mechanism of oxidative deposition of iron in ferritin is incompletely understood. In this study, EPR-active species produced d uring ferritin reconstitution (10-50 Fe/protein) from the apoprotein, Fe2+, and O-2 have been investigated using rapid-mixing freeze-quench techniques and EPR spectroscopy. Species studied include a monomeric F e3+-protein complex (g' = 4.3), a mixed-valent Fe2+-Fe3+ complex (g' = 1.87), and a newly observed radical with axial symmetry (g(parallel t o) = 2.042, g(perpendicular to) 2.0033), all apparent intermediates fo rmed during the first second of iron oxidation. The monomeric Fe3+-pro tein complex is the principal EPR-observable product of iron(II) oxida tion and is produced quantitatively in the first phase of the reaction with the mixed-valent species and the radical formed at slower rates. The initial rate of formation of the monomeric complex (and the radic al) is first-order in Fe2+ concentration, consistent with a mechanism in which iron oxidation occurs in a one-electron step(s) with H2O2 bei ng the final product of O-2 reduction. A 1:1 relationship between the disappearance of the monomeric Fe3+-protein complex and the formation of the mixed-valent Fe2+ -Fe3+ species was observed in the early phase of the reaction, indicating that the latter is derived from the forme r and not from the one-electron oxidation of a preformed Fe2+-Fe2+ dim er. The g-factors and rapid EPR relaxation properties of the transient radical suggest that ft is associated with an Fe2+ (or Fe3+) center b ut its identity and possible functional role in iron oxidation are unk nown.