Presence of endogenous calcium ion in horseradish peroxidase. Elucidation of metal-binding site by substitutions of divalent and lanthanide ions for calcium and use of metal-induced NMR (1H and 113Cd) resonances.

Some divalent (Cd2+, Sr2+, Ba2+) and lanthanide (Ln3+) ions can be substituted for endogenous Ca2+ of horseradish peroxidase (HRP, Ca2+-bound hemoenzyme) and maintain the protein structure in the heme vicinity as well as the enzymatic activity of HRP, as does Ca2+. However, due to lower affinity than Ca2+, the bound Ln3+ was readily replaced by Ca2+ to yield native HRP, in sharp contrast to other typical Ca2+-binding proteins, of which the affinity to Ln3+ is higher than to Ca2+. Addition of paramagnetic Ln3+ to Ca2+-free HRP induced a series of paramagnetically shifted 1H NMR resonances of protein at the first metal binding site in HRP. The pH-dependent spectral changes of the Ln3+-shifted resonances suggest that tyrosyl hydroxyl and glutamate and aspartate carboxyl groups are involved in the binding site of the Ca2+, other divalent ions, or Ln3+. The 113Cd NMR spectra of 113Cd2+-bound HRP also indicated the presence of one metal strongly bound to the oxygen ligands and another site with low affinity for the metal ions, which is consistent with the results from the Ln3+-induced proton NMR. All these results suggest that the binding of one metal ion to HRP is essential for its structural and functional properties.