Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin

Abstract

A binding site for metal ions has been created on the surface of horse heart myoglobin (Mb) near the heme 6-propionate group by replacing K45 and K63 with glutamyl residues. One-dimensional (1)H NMR spectroscopy indicates that Mn(2+) binds in the vicinity of the heme 6-propionate as anticipated, and potentiometric titrations establish that the affinity of the new site for Mn(2+) is 1.28(4) x 10(4) M(-1) (pH 6.96, ionic strength I = 17.2 microM, 25 degrees C). In addition, these substitutions lower the reduction potential of the protein and increase the pK(a) for the water molecule coordinated to the heme iron of metmyoglobin. The peroxidase [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), ABTS, as substrate] and the Mn(2+)-peroxidase activity of the variant are both increased approximately 3-fold. In contrast to wild-type Mb, both the affinity for azide and the midpoint potential of the variant are significantly influenced by the addition of Mn(2+). The structure of the variant has been determined by x-ray crystallography to define the coordination environment of bound Mn(2+) and Cd(2+). Although slight differences are observed between the geometry of the binding of the two metal ions, both are hexacoordinate, and neither involves coordination by E63.

Figure 1

Ball-and-stick representation of Mb. (A) Detailed view of residues in the wild-type protein in the region near the heme group where the metal ion binding site was designed. (B) The conformation of bound Mn²⁺ and its associated ligand interactions in the new metal ion binding site of the K45E/K63E variant of Mb. Note that H113 is from a related molecule in the crystalline lattice and is expected to be replaced by a water molecule in solution. (C) The conformation of bound Cd²⁺ in the new metal ion binding site of the K45E/K63E Mb variant. (D) A view of the natural metal binding site observed in Mb in structural studies of the Cd²⁺-bound form of K45E/K63E Mb. In related Mn²⁺ studies this site remained unoccupied.

Figure 2

Potentiometric titration of the K45E/K63E variant with MnSO₄ at pH 7.0 and 17.2 mM KCl. The data were fit to one-site (A) and two-site (B) models, the two-site model giving the better fit.

Figure 3

The 200-MHz ¹H NMR spectra of the 100-ppm downfield region of K45E/K63E Mb variant with 0 (trace A), 0.2 (trace B), 0.5 (trace C), and 0.8 (trace D) eq of MnSO₄ [deuterated sodium phosphate buffer, I = 0.1 M, pH 7.0 (uncorrected pH meter reading), 20°C]. The proton resonances that were broadened on addition of Mn²⁺ are indicated by the dotted lines.