An electron paramagnetic resonance study of the iron and copper complexes of transferrin.

The electron paramagnetic resonance spectra of the Cu2+ and Fe3+ complexes of transferrin reveal a heterogeneity of the two specific binding sites of the protein in the pH range 4 to 6 when bicarbonate is absent. Between pH 7 and pH 11, however, or when nitrilotriacetate is present in the iron complex at lower pH, only one dominant species is present in each metal-protein complex, so that under these conditions the binding sites are indistinguishable. In the higher pH range, the spectrum of the bicarbonatefree copper-transferrin complex shows superhyperfine splitting which is unusually well resolved in the 811 region and is best interpreted as being a result of the interaction of the unpaired copper electron (hole) with four equivalent nitrogen nuclei. In the copper-transferrin-bicarbonate complex the superhyperfine splitting gives evidence of only one interacting nitrogen nucleus. Spectrophotometric titrations provide evidence that tyrosine residues also participate in copper binding, as well as in iron binding. It seems probable, therefore, that the metal-binding sites of transferrin have available 4 nitrogen ligands and 2 or 3 oxygen ligands.