Flexibility of the Coordination Geometry at the N-Site of Cu(II)₂ Human Serum-Transferrin Induced by the Different Orientations of Arg124

Abstract

The ESR spectra of dicupric human serum-transferrin (serum-Tf) were measured from 20 to 37°C in the liquid state (56% glycerol at pH 7.6). Two coordination geometries (types B-1 and B-2) with different ESR parameters were present at the N-site. The contents of the coordination geometry of type B-1 at the N-site increased as the temperature increased. The equilibrium constant between the coordination geometries of types B-1 and B-2 was determined by ESR spectra. The enthalpy value from type B-2 to B-1 was 5.3 kcal/mol, as obtained from a van't Hoff plot. The two conformational energies of the cluster models of the copper-binding site at the N-site of dicupric human serum-Tf, where the Arg124 residue was oriented in two different directions (conformations I and II), were calculated by Density Functional Theory, and the enthalpy value from conformation II to I was 2.1 kcal/mol. The enthalpy value was similar to that ( 5.3 kcal/ mol) obtained by the coordination geometrical change from type B-2 to B-1 in Cu(II) 2 serum-Tf. In conformations I and II, the residue of Arg124 at the N-site is located either far from or near the copper-binding site, respectively, and in both cases the coordination geometry of the cupric ions at the N-site has changed from a flattened tetrahedron to a trigonal bipyramid. This result implies that the ESR spectral change from type B-2 to B-1 is caused by the presence of two different orientations of Arg124 in the change from conformation II to I. Key words copper transferrin; conformational change energy; coordination geometry change Transferrins (Tfs), a group of homologous iron-binding proteins (serum-transferrin (serum-Tf), ovo-transferrin (ovoTf), and lactoferrin (Lf)), consist of a single polypeptide chain folded into two lobes. Tfs are composed of homologous lobes. The amino-terminal half of these proteins forms one lobe (N-lobe), linked by a short peptide sequence to the carboxyterminal lobe (C-lobe) as shown in Fig. 1. 1) Each lobe is comprised of two domains that open and close with a hinge motion. The metal binding sites are located deep at these clefts in the N-and C-lobes, and the metal binding sites of these lobes are expressed as N-and C-sites, respectively. Crystallographic studies on Fe(III) 2 human serum-Tf, 2) Fe(III) 2 human Lf, 3) and Fe(III) 2 ovo-Tf 4) have demonstrated that the iron coordination is similar for these proteins, with the metal bound to four protein ligands (two Tyr, one Asp, and one His) and a CO 3 2− (bidentate) anion, and that the coordination geometries of the two specific sites in each protein are essentially equivalent. However, Harris 5,6) reported that the metal binding constants of various metal ions at the C-and N-sites are different and that the metal binding constant at the C-site is generally much larger than that of the N-site. A synergistic ion, CO 3 2− , is easily replaced by oxalate at the N-site of serum transferrin or at the C-site of lactoferrin. 7,8) These results show that the two metal binding sites (N-and C-sites) in transferrin are...