Iron binding by phosvitin and its conformational consequences.

Abstract

With a view to the potential biological significance of iron binding by the phosphoprotein phosvitin, the interaction of these two electrostatically complementary constituents of egg yolk particles was studied by ultrafiltration, circular dichroism, and sedimentation. Ferric complexes of phosvitin are strong and stable; ferrous complexes are weak and dissociate readily. When saturated, pairs of the approximately 135 phosphate groups of a phosvitin molecule appear to bind 1 iron atom each. These findings confirm and extend previous reports regarding the ferric complex and characterize the ferrous complex for the first time. The iron binding sites are not equivalent. Contrary to previous speculations, iron binding is not accompanied by a conformational change from an unordered structure to one of the beta-type. Apparently, neutralization of negative charges, while necessary, is not a sufficient condition of this transition. Nevertheless, iron affects phosvitin structure. Above pH 2, where the protein is unordered but adjusts its average conformation to changes in its net charge as the pH of its solution is varied, iron mimics the effect of protons quantitatively. Near pH 2, where the beta-type conformation is readily acquired by the protein in the absence of iron, the consequences of iron binding upon conformation are determined by the manner in which the iron-phosvitin interaction is brought about. Either the extent of the transition to the "normal" beta-structure becomes limited or the nature of the resulting conformation becomes modified. It is noteworthy that ellipticity changes in the presence of iron do not necessarily occur in parallel at about 200 and 215 nm as they do when the transconformation is produced by pH changes alone. The binding of iron appears to be mostly intramolecular. Intermolecular cross-links become dominant only if most binding sites are filled and then only as a secondary event subsequent to binding and the initial conformational adjustment.