Reaction paths of iron oxidation and hydrolysis in horse spleen and recombinant human ferritins.

UV-visible spectroscopy, electrode oximetry, and pH stat were used to study Fe(II) oxidation and hydrolysis in horse spleen ferritin (HoSF) and recombinant human H-chain and L-chain ferritins (HuHF and HuLF). Appropriate test reactions and electrode responses were measured, establishing the reliability of oxygen electrode/pH stat for kinetics studies of iron uptake by ferritin. Stoichiometric ratios, Fe(II)/O2 and H+/Fe(II), and rates of oxygen uptake and proton production were simultaneously measured as a function of iron loading of the protein. The data show a clear distinction between the diiron ferroxidase site and mineral surface catalyzed oxidation of Fe(II). The oxidation/hydrolysis reaction attributed to the ferroxidase site has been determined for the first time and is given by 2Fe2+ + O2 + 3H2O --> [Fe2O(OH)2]2+ + H2O2 + 2H+ where [Fe2O(OH)2]2+ represents the hydrolyzed dinuclear iron(III) center postulated to be a mu-oxo-bridged species from UV spectrometric titration data and absorption band maxima. The transfer of iron from the ferroxidase site to the mineral core has been now established to be [Fe2O(OH)2]2+ + H2O --> 2FeOOH(core) + 2H+. Regeneration of protein ferroxidase activity with time is observed for both HoSF and HuHF, consistent with their having enzymatic properties, and is facilitated by higher pH (7.0) and temperature (37 degreesC) and by the presence of L-subunit and is complete within 10 min. In accord with previous studies, the mineral surface reaction is given by 4Fe2+ + O2 + 6H2O --> 4FeOOH(core) + 8H+. As the protein progressively acquires iron, oxidation/hydrolysis increasingly shifts from a ferroxidase site to a mineral surface based mechanism, decreasing the production of H2O2.

Tyrosyl radical formation during the oxidative deposition of iron in human apoferritin.

The radical chemistry of ferritin is incompletely understood. The present study was undertaken to investigate the production of radicals in H-chain recombinant human ferritin (HuHF) and mixed H/L-chain horse spleen ferritin (HoSF) and the potential role of radicals in the oxidative deposition of iron in these proteins. Radical production follows distinct pathways for the two proteins; an intact H-chain ferroxidase site is required for radical generation in both of them, however. With the H-chain HuHF, an EPR spectrum characteristic of a tyrosyl radical is seen following Fe2+ oxidation by O2 and, based on measurements with site-directed variants, is suggested to arise from residue Tyr-34 located in the vicinity of the ferroxidase site. The observation of this radical correlates with the observation of a 400-600 nm absorbance seen in stopped-flow kinetics studies which seems to require the presence of Tyr-34 (Bauminger et al. (1993) Biochem. J. 296, 709-714). The data are inconsistent, however, with the Tyr-34 radical being critically important in the protein-catalyzed mechanism of iron oxidation. Unlike HuHF, the radicals observed in L-chain-rich HoSF appear to arise from hydroxyl radical damage to the protein through Fenton chemistry. These latter radicals also appear to be centered on aromatic amino acids and may be derived from histidine.