Engineered metalloregulation of azide binding affinity and reduction potential of horse heart myoglobin

Abstract

Metal ion binding to a previously reported variant of horse heart myoglobin (Lys45Glu/Lys63Glu) with a metal ion binding site on the surface of the protein that is adjacent to the haem binding site has been shown to influence ligand binding and electrochemical properties of the protein. For example, the K_d (μM) for binding of azide to this variant decreases from 277 ± 9 to 32 ± 3 following addition of a saturating concentration of Mn²⁺ (the value for the wild-type protein under the same conditions is 26 ± 1). Similarly, the midpoint reduction potential E_m (mV vs. standard hydrogen electrode) increases from 9 to 40 in the presence of a saturating concentration of Mn²⁺ (the value for the wild-type protein under the same conditions is 45 ± 2). These results demonstrate the potential value of engineered metal ion binding sites as a means of regulating the functional properties of even simple haem proteins.