The ferroxidase reaction of ferritin reveals a diferric mu-1,2 bridging peroxide intermediate in common with other O-2-activating non-heme diiron proteins

Ferritins are ubiquitous proteins that concentrate, store, and detoxify int racellular iron through oxidation of Fe2+ (ferroxidation), followed by tran slocation and hydrolysis to form a large inorganic mineral core. A series o f mutagenesis, kinetics, and spectroscopic studies of ferritin led to the p roposal that the oxidation/translocation path involves a diiron protein sit e. Recent stopped-flow absorption and rapid freeze-quench Mossbauer studies have identified a single peroxodiferric species as the initial transient i ntermediate formed in recombinant frog M ferritin during rapid ferroxidatio n [Pereira, S, A., Small, W,, Krebs, C,, Tavares, P,, Edmondson, D. E., The il, E. C., and Huynh, B. H. (1998) Biochemistry 37, 9871-9876], To further characterize this transient intermediate and to establish unambiguously the peroxodiferric assignment, rapid freeze-quenching was used to trap the ini tial intermediate for resonance Raman investigation. Discrete vibrational m odes are observed for this intermediate, indicating a single chromophore in a homogeneous state, in agreement with the Mossbauer conclusions. The freq uency at 851 cm(-1) is assigned as nu(O-O) of the bound peroxide, and the p air of frequencies at 485 and 499 cm(-1) is attributed, respectively, to nu (s) and nu(as) of Fe-O-2-Fe. Identification of the chromophore as a mu-1,2 bridged diferric peroxide is provided by the isotope sensitivity of these R aman bands. Similar peroxodiferric intermediates have been detected in a mu tant of the R2 subunit of ribonucleotide reductase from Escherichia coil an d chemically reduced Delta(9) stearoyl-acyl carrier protein desaturase (Del ta 9D), but in contrast, the ferritin intermediate is trapped from the true reaction pathway of the native protein. Differences in the Raman signature s of these peroxide species are assigned to variations in Fe-O-O-Fe angles and may relate to whether the iron is retained in the catalytic center or r eleased as an oxidized product.