Rapid kinetics of the EPR-active species formed during initial iron uptake in horse spleen apoferritin.
The molecular mechanism of oxidative deposition of iron in ferritin is incompletely understood. In this study, EPR-active species produced during ferritin reconstitution (10-50 Fe/protein) from the apoprotein, Fe2+, and O2 have been investigated using rapid-mixing freeze-quench techniques and EPR spectroscopy. Species studied include a monomeric Fe(3+)-protein complex (g' = 4.3), a mixed-valent Fe(2+)-Fe3+ complex (g' = 1.87), and a newly observed radical with axial symmetry (g parallel = 2.042, g perpendicular = 2.0033), all apparent intermediates formed during the first second of iron oxidation. The monomeric Fe(3+)-protein complex is the principal EPR-observable product of iron(II) oxidation and is produced quantitatively in the first phase of the reaction with the mixed-valent species and the radical formed at slower rates. The initial rate of formation of the monomeric complex (and the radical) is first-order in Fe2+ concentration, consistent with a mechanism in which iron oxidation occurs in a one-electron step(s) with H2O2 being the final product of O2 reduction. A 1:1 relationship between the disappearance of the monomeric Fe(3+)-protein complex and the formation of the mixed-valent Fe(2+)-Fe3+ species was observed in the early phase of the reaction, indicating that the latter is derived from the former and not from the one-electron oxidation of a preformed Fe(2+)-Fe2+ dimer. The g-factors and rapid EPR relaxation properties of the transient radical suggest that it is associated with an Fe2+ (or Fe3+) center but its identity and possible functional role in iron oxidation are unknown.
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Department of Chemistry, University of New Hampshire, Durham 03824.
Chasteen
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D000818:Animals; D001052:Apoferritins / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism; D005615:Freezing; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D013154:Spleen / Q000737:chemistry*; D015032:Zinc / Q000494:pharmacology
D005290:Ferric Compounds; D005296:Ferrous Compounds; D046911:Macromolecular Substances; D001052:Apoferritins; D007501:Iron; D015032:Zinc