A distinct environment for iron (III) in the complex with horse spleen apoferritin observed by x-ray absorption spectroscopy.

Cell-specific variations in apoferritin structure correlate with variations in iron metabolism that suggest functional specificity of the protein shell. Using EPR spectroscopy, we previously showed that vanadyl binds to specific sites on apoferritin, and that VO2+ binding is reduced by Fe(II) and Fe(III) (the natural substrates) and by metals known to influence iron storage (Chasteen, N. D., and Theil, E. C. (1982) J. Biol. Chem. 257, 7672-7677). Such observations suggest that the metal-binding site is important to apoferritin function and may define a location where the influence of cell-specific structural features are exerted. To investigate the iron-protein complex further, we have used x-ray absorption spectroscopy and have characterized, for the first time to our knowledge, Fe(III) apparently attached to the protein, after analyzing the x-ray absorption spectrum of an Fe(III)-apoferritin complex (10 Fe/molecule) compared to that of ferritin (polynuclear Fe(III)OOH, about 2000/molecule). The environment of iron in the Fe(III)-protein complex was similar to that in an Fe(III)-oxalate (2:3) hexahydrate complex, both in near edge structure and extended x-ray absorption structure, confirming earlier predictions of carboxylates as protein ligands. The extended x-ray absorption fine structure data for both compounds was fit best by a model in which a second shell of low Z atoms (carbon) was close (0.53-0.55 A) to the first shell of coordinated oxygen. However, small differences between Fe(III)-apoferritin and Fe(III)-oxalate in the Fe-O environment suggest a distorted geometry in the Fe(III)-protein complex and/or the presence of a mixture of atoms, such as nitrogen and oxygen, coordinated to iron. Extension of this approach to other apoferritins and metals will be likely to clarify the role of cell-specific features of the apoprotein in the formation of the iron core.