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). A ppropriate test reactions and electrode responses were measured, estab lishing the reliability of oxygen electrode/pH stat for kinetics studi es of iron uptake by ferritin. Stoichiometric ratios, Fe(II)/O-2 and H +/Fe(II), and rates of oxygen uptake and proton production were simult aneously measured as a function of iron loading of the protein. The da ta show a clear distinction between the diiron ferroxidase site and mi neral surface catalyzed oxidation of Fe(II). The oxidation/hydrolysis reaction attributed to the ferroxidase site has been determined for th e first time and is given by 2Fe(2+) + O-2 + 3H(2)O --> [Fe2O(OH)(2)]( 2+) + H2O2 + 2H(+) where [Fe2O(OH)(2)](2+) represents the hydrolyzed d inuclear iron(III) center postulated to be a mu-oxo-bridged species fr om UV spectrometric titration data and absorption band maxima. The tra nsfer of iron from the ferroxidase site to the mineral core has been n ow 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 propert ies, and is facilitated by higher pH (7.0) and temperature (37 degrees C) and by the presence of L-subunit and is complete within 10 min. In accord with previous studies, the mineral surface reaction is given b y 4Fe(2+) + O-2 + 6H(2)O --> 4FeOOH((core)) + 8H(+). As the protein pr ogressively acquires iron, oxidation/hydrolysis increasingly shifts fr om a ferroxidase site to a mineral surface based mechanism, decreasing the production of H2O2.