Direct Spectroscopic and Kinetic Evidence for the Involvement of a Peroxodiferric Intermediate during the Ferroxidase Reaction in Fast Ferritin Mineralization

Abstract

Rapid freeze-quench (RFQ) Mössbauer 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 Mössbauer method; a majority (85%) of the ferrous ions was oxidized at a fast rate of approximately 80 s-1 and the remainder at a much slower rate of approximately 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 Mössbauer parameters (DeltaEQ = 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 Mössbauer method match those of a transient blue species (lambdamax = 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 O2 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.

Ferritin reactions: Direct identification of the site for the diferric peroxide reaction intermediate

“…Recombinant L frog ferritin was used as the model ferritin because the kinetic properties of frog ferritins have been particularly favorable for spectroscopic and mechanistic studies (3,14,16,(27)(28)(29)(30). Frog and human ferritins are very similar both structurally (10,11) and mechanistically (14,28,31). However, an early attempt to construct a catalytically active site in human L ferritin led to insoluble protein that had weak mineralization activity when analyzed after solubilization and folding with guanidine in the presence of catalytically active H-ferritin subunits.…”

“…Ferrous substrate (FeSO 4 in 1 mM HCl, 0.05-2 mM) was rapidly mixed with ferritin solutions (4.16 M ϭ 100 M in subunits in 200 mM Mops, pH ϭ 6, 7, or 8) in a stopped-flow UV-visible spectrophotometer (Applied Photophysics, Surrey, U.K.) to achieve molar ratios of ferrous ion to protein from 12 to 480 iron atoms per ferritin molecule (0.5-24 iron per subunit). Data were collected at 650 nm, the absorption maximum of DFP (28), and at Ϸ350 nm, a nonspecific absorbance of ferric oxy species including DFP, Fe 3ϩ -O-Fe 3ϩ F ox products, ferric biomineral, and ferric side reactions in DFP-inactive proteins; absorbance in the range 290-420 nm has often been used to analyze ferritin ferroxidation rates (e.g., refs. 32 and 35-37).…”

Spectroscopic evidence for the presence of a high‐valent Fe(IV) species in the ferroxidase reaction of an archaeal ferritin

“…The molecular structure of the peroxodiferric species in MMO is still unknown 3 and Q is proposed to have a bis‐μ‐oxo diamond core 7. In contrast, it is generally believed that the peroxodiferric species in ferritin decays directly to Fe(III) products 4, 5, 8, 9 (Fig. 1D) and a high‐valent Fe(IV) species has never been reported.…”

“…1A–B): (a) the ferroxidase center has only one coordinating glutamate as bridging ligand, (b) there is a Fe(II) binding site next to the ferroxidase center (site C), which is replaced by a network of hydrogen bonds in the di‐iron cofactor site of dioxygen activating enzymes like methane monooxygenase (MMO) 2, and (c) in ferritins one active site residue that is not conserved (Glu130 in Pfu Ftn) 1, is a conserved histidine coordinating one of the Fe atoms in the di‐iron cofactor site of dioxygen‐activating enzymes. Nevertheless, during catalysis both in the di‐iron cofactor site of dioxygen‐activating enzymes 3 and in the ferroxidase center 4, 5 a peroxodiferric intermediate is formed (Fig. 1C–D).…”

Multiple Pathways for Mineral Core Formation in Mammalian Apoferritin. The Role of Hydrogen Peroxide

“…The above H-chain catalyzed reaction proceeds through a µ-1, 2- (14)(15)(16)(17)(18)(19)(20)(21). The fate of the H 2 O 2 produced in eq 1 has been an open question because some of it is consumed in a subsequent undefined reaction(s) (21,22).…”