Conversion of Fe ions in solution to the solid phase in ferritin concentrates iron required for cell function. The rate of the Fe phase transition in ferritin is tissue specific and reflects the differential expression of two classes of ferritin subunits (H and L). Early stages of mineralization were probed by rapid freeze-quench Mossbauer, at strong fields (up to 8 T), and EPR spectroscopy in an H-type subunit, recombinant frog ferritin; small numbers of Fe (36 moles/mol of protein) were used to increase Fe3+ in mineral precursor forms, At 25 ms, four Fe3+-oxy species (three Fe dimers and one Fe trimer) were identified, These Fe3+-oxy species were found to form at similar rates and decay subsequently to a distinctive superparamagentic species designated the `'young core.'' The rate of oxidation of Fe2+ (1026 s(-1)) corresponded well to the formation constant for the Fe3+- tyrosinate complex (920 s(-1)) observed previously [Waldo, G. S., \& Theil, E. C. (1993) Biochemistry 32, 13261] and, coupled with EPR data, indicates that several or possibly all of the Fe3+-oxy species involve tyrosine. The results, combined with previous Mossbauer studies of Y30F human H-type ferritin which showed decreases in several Fe3+ intermediates and stabilization of Fe2+ [Bauminger, E. R., et al. (1993) Biochem, J. 296, 709], emphasize the involvement of tyrosyl residues in the mineralization of H-type ferritins. The subsequent decay of these multiple Fe3+-oxy species to the superparamagnetic mineral suggests that Fe3+ species in different environments may be translocated as intact units from the protein shell into the ferritin cavity where the conversion to a solid mineral occurs.

Rapid freeze-quench (RFQ) Mossbauer and stopped-flow absorption spectroscopy were used to monitor the ferritin ferroxidase reaction using recombinant (apo) frog M ferritin; the initial transient ferric species could be trapped by the RFQ method using low iron loading (36 Fe2+/ferritin molecule). Biphasic kinetics of ferroxidation were observed and measured directly by the Mossbauer method; a majority (85%) of the ferrous ions was oxidized at a fast rate of similar to 80 s(-1) and the remainder at a much slower rate of similar to 1.7 s(-1). In parallel with the fast phase oxidation of the Fe2+ ions, a single transient iron species is formed which exhibits magnetic properties (diamagnetic ground state) and Mossbauer parameters (Delta E-Q = 1.08 +/- 0.03 mm/s and delta = 0.62 +/- 0.02 mm/s) indicative of an antiferromagnetically coupled peroxodiferric complex. The formation and decay rates of this transient diiron species measured by the RFQ Mossbauer method match those of a transient blue species (lambda(max) = 650 nm) determined by the stopped-flow absorbance measurement. Thus, the transient colored species is assigned to the same peroxodiferric intermediate. Similar transient colored species have been detected by other investigators in several other fast ferritins (H and M subunit types), such as the human H ferritin and the Escherichia coli ferritin, suggesting a similar mechanism for the ferritin ferroxidase step in all fast ferritins. Peroxodiferric complexes are also formed as early intermediates in the reaction of O-2 With the catalytic diiron centers in the hydroxylase component of soluble methane monooxygenase (MMOH) and in the D84E mutant of the R2 subunit of E. coli ribonucleotide reductase. The proposal that a single protein site, with a structure homologous to the diiron centers in MMOH and R2, is involved in the ferritin ferroxidation step is confirmed by the observed kinetics, spectroscopic properties, and purity of the initial peroxodiferric species formed in the frog M ferritin.

Fe XAS data were collected on the oxidized and reduced forms of desulforedoxin from Desulfovibrio gigas, the oxidized form of rubredoxin from Clostridium pasteurianum, and the reduced form of rubredoxin from Pyrococcus furiosus. Analysis of these data is consistent with tetrahedral FeS(4) coordination in both oxidation states, and an expansion of the Fe-S distances from 2.27 to 2.33 Angstrom upon reduction. (C) 1998 Elsevier Science S.A. All rights reserved.