The nature of the Fe2+ and Fe3+ binding sites on the protein shell of ferritin and their role in the accumulation of iron within this storage protein are poorly understood. An ESEEM (electron spin-echo envelope modulation) study is reported which provides new insight into the nature of these sites in the horse spleen protein. ESEEM spectra have been obtained of complexes of horse spleen apoferritin with VO2+−an ion that binds apoferritin competitively with both Fe2+ and Fe3+; samples prepared both in 1H20 and 2H2O and at both pH ~5.5 and at ~7.4, which correspond respectively to the α and β species of Chasteen and Theil (J. Biol. Chem. 1982, 257, 7672–7677), have been studied. The ESEEM spectra clearly reveal the presence of endogenous nitrogen in the environment of VO2+ in both types of complexes. This nitrogen most likely derives from an imidazole ligand of a histidine residue coordinated cis to the vanadyl oxo group. ESEEM is also observed from a proton whose dipolar coupling to the VO2+ center is characteristic of a hydrogen attached to the coordinated atom of a cis ligand. This hydrogen undergoes exchange with aqueous solvent; the VO2+ binding site is thus solvent-accessible. The ratio of the amplitude of the ESEEM peak associated with this proton in the β vs the α complex is approximately 0.5. Attribution of these ESEEM features to the hydrogens of a cis-coordinated aquo ligand that deprotonates to hydroxide at pH =6.5 is consistent with these observations. The ESEEM results are best interpreted in terms of a binding site that accommodates VO2+ with one aquo and one histidine ligand—and with other coordination positions filled by protein carboxylate donors. The possible location of this VO2+ binding site and its significance to iron accumulation in ferritin are discussed.
ASJC Scopus subject areas
- Colloid and Surface Chemistry