TYROSYL RADICAL FORMATION DURING THE OXIDATIVE DEPOSITION OF IRON IN HUMAN APOFERRITIN

The radical chemistry of ferritin is incompletely understood. The pres ent study was undertaken to investigate the production of radicals in H-chain recombinant human ferritin (HuHF) and mixed H/L-chain horse sp leen ferritin (HoSF) and the potential role of radicals in the oxidati ve deposition of iron in these proteins. Radical production follows di stinct pathways for the two proteins; an intact H-chain ferroxidase si te is required for radical generation in both of them, however. With t he H-chain HuHF, an EPR spectrum characteristic of a tyrosyl radical i s seen following Fe2+ oxidation by O-2 and, based on measurements with site-directed variants, is suggested to arise from residue Tyr-34 loc ated in the vicinity of the ferroxidase site. The observation of this radical correlates with the observation of a 400-600 nm absorbance see n in stopped-flow kinetics studies which seems to require the presence of Tyr-34 (Bauminger et al. (1993) Biochem. J. 296, 709-714). The dat a 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. Thes e latter radicals also appear to be centered on aromatic amino acids a nd may be derived from histidine.