DIRECT SPECTROSCOPIC AND KINETIC EVIDENCE FOR THE INVOLVEMENT OF A PEROXODIFERRIC INTERMEDIATE DURING THE FERROXIDASE REACTION IN FAST FERRITIN MINERALIZATION

Rapid freeze-quench (RFQ) Mossbauer and stopped-flow absorption spectr oscopy were used to monitor the ferritin ferroxidase reaction using re combinant (apo) frog M ferritin; the initial transient ferric species could be trapped by the RFQ method using low iron loading (36 Fe2+/fer ritin molecule). Biphasic kinetics of ferroxidation were observed and measured directly by the Mossbauer method; a majority (85%) of the fer rous ions was oxidized at a fast rate of similar to 80 s(-1) and the r emainder at a much slower rate of similar to 1.7 s(-1). In parallel wi th the fast phase oxidation of the Fe2+ ions, a single transient iron species is formed which exhibits magnetic properties (diamagnetic grou nd 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 co upled peroxodiferric complex. The formation and decay rates of this tr ansient diiron species measured by the RFQ Mossbauer method match thos e of a transient blue species (lambda(max) = 650 nm) determined by the stopped-flow absorbance measurement. Thus, the transient colored spec ies is assigned to the same peroxodiferric intermediate. Similar trans ient colored species have been detected by other investigators in seve ral other fast ferritins (H and M subunit types), such as the human H ferritin and the Escherichia coli ferritin, suggesting a similar mecha nism for the ferritin ferroxidase step in all fast ferritins. Peroxodi ferric complexes are also formed as early intermediates in the reactio n of O-2 With the catalytic diiron centers in the hydroxylase componen t of soluble methane monooxygenase (MMOH) and in the D84E mutant of th e 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 con firmed by the observed kinetics, spectroscopic properties, and purity of the initial peroxodiferric species formed in the frog M ferritin.