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.