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

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 H2O2 and formation of ferric mineral precursors. The stoichiometric relationship between F-peroxo, H2O2, 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 H2O2. By rapidly mixing apo M ferritin from frog, Fe2+, 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 H2O2 assay solution, we were able to quantitatively determine the amount of H2O2 produced during the decay of F-peroxo. The correlation between the amount of H2O2 released with the amount of F-peroxo accumulated at 70 ms determined by Mossbauer spectroscopy showed that F-peroxo decays into H2O2 with a stoichiometry of 1 F-peroxo:H2O2. When the decay of F-peroxo was monitored by rapid freeze-quench Mossbauer 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 (H2O2, 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.