Pulsed ELDOR Determination of the Intramolecular Distance between the Metal Binding Sites in Dicupric Human Serum Transferrin and Lactoferrin

Transferrin (Tf) and lactoferrin (Lf) are members of a family of binding proteins that control the level of free iron in biological environments through their tight but reversible binding of iron. Although these proteins are formed of a single polypeptide, they have two very similar domains each of which has a specific metal binding site. Pulsed electron−electron double resonance (ELDOR) has been used to determine the distances between the metal binding sites of copper(II)-substituted human Tf and Lf. The distance distribution was very narrow, and values obtained were 4.16 ± 0.06 nm for Cu 2 Tf and 4.24 ± 0.06 nm for Cu 2 Lf. These distances are within 3% of those obtained by X-ray crystallography. The success of the experiments confirms that Cu(II) may be used as an intrinsic spin label for obtaining structural information in multidomain proteins by pulsed ELDOR.