Stoichiometric Production of Hydrogen Peroxide and Parallel Formation of Ferric Multimers through Decay of the Diferric−Peroxo Complex, the First Detectable Intermediate in Ferritin Mineralization

Abstract

The catalytic step that initiates formation of the ferric oxy-hydroxide mineral core in the central cavity of H-type ferritin involves rapid oxidation of ferrous ion by molecular oxygen (ferroxidase reaction) at a binuclear site (ferroxidase site) found in each of the 24 subunits. Previous investigators have shown that the first detectable reaction intermediate of the ferroxidase reaction is a diferric-peroxo intermediate, F(peroxo), formed within 25 ms, which then leads to the release of H(2)O(2) and formation of ferric mineral precursors. The stoichiometric relationship between F(peroxo), H(2)O(2), and ferric mineral precursors, crucial to defining the reaction pathway and mechanism, has now been determined. To this end, a horseradish peroxidase-catalyzed spectrophotometric method was used as an assay for H(2)O(2). By rapidly mixing apo M ferritin from frog, Fe(2+), and O(2) and allowing the reaction to proceed for 70 ms when F(peroxo) has reached its maximum accumulation, followed by spraying the reaction mixture into the H(2)O(2) assay solution, we were able to quantitatively determine the amount of H(2)O(2) produced during the decay of F(peroxo). The correlation between the amount of H(2)O(2) released with the amount of F(peroxo) accumulated at 70 ms determined by Mössbauer spectroscopy showed that F(peroxo) decays into H(2)O(2) with a stoichiometry of 1 F(peroxo):H(2)O(2). When the decay of F(peroxo) was monitored by rapid freeze-quench Mössbauer spectroscopy, multiple diferric mu-oxo/mu-hydroxo complexes and small polynuclear ferric clusters were found to form at rate constants identical to the decay rate of F(peroxo). This observed parallel formation of multiple products (H(2)O(2), diferric complexes, and small polynuclear clusters) from the decay of a single precursor (F(peroxo)) provides useful mechanistic insights into ferritin mineralization and demonstrates a flexible ferroxidase site.

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

“…The potential for fewer amino acid substitutions to create an active guest F ox site minimizes the possibility of insoluble protein, as encountered in an earlier experiment with human L ferritin (32). In addition DFP has been mainly characterized in F ox active frog ferritins (14,16,28,34,39), and both active and inactive frog ferritins have been crystallized (10,12,27).…”

“…DFP intermediates in biological reactions of ferrous ions with oxygen are characteristic of both proteins where diiron cofactors react with dioxygen to oxidize organic substrates, and ferritins, where two ferrous ion substrates are coupled by using dioxygen to form diferric oxo͞hydroxo mineral precursors and hydrogen peroxide as the second product (14,16,28,31,39). Coupling and oxidation of the two ferrous ions via oxo͞hydroxo bridges, the result of ferritin F ox activity, apparently overcomes diffusion limits on mineralization rates, and possibly activates protons, because the pK a of water coordinated to iron decreases from Ϸ9 for ferrous to as low as Ϸ3 for ferric.…”

“…3). The cell-specific combinations of F ox -active and inactive ferritin subunits, which affect the rates of DFP formation, release of diferric oxy products (mineral precursors) and hydrogen peroxide (28,31,37,39), may be related to the specific ability of each cell type to manage hydrogen peroxide. In the chimera Lϩ4, pH had a much larger effect on DFP decay and half-life (Fig.…”

DNA and mRNA elements with complementary responses to hemin, antioxidant inducers, and iron control ferritin-L expression

“…2C), which also regulates heme oxygenase 1 (16) and ␤-globin (15), indicates that ferritin may also influence cellular dioxygen metabolism, possibly through the entrapment of dioxygen in the mineral. Ferritin-H transcription, in contrast to ferritin-L, is induced by hydrogen peroxide activation (12), which may relate to the release of hydrogen peroxide associated with ferritin-H catalytic activity (28,29). Other observations emphasizing the importance of ferritin in cell functions beyond storing and concentrating iron include: induction by c-myc and TNF-␣ (ferritin-H) (13,18), RNA interference knockdown effects on cell growth and division (ferritin-L) (30), effects of MARE͞ARE mutation on basal expression (ferritin-L) (Fig.…”

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).…”