Determination of the Orientation of the Magnetic Axes of the Cyano-MetMb Complexes of Point Mutants of Myoglobin by Solution ¹H NMR: Influence of His E7 → Gly and Arg CD3 → Gly Substitutio

A method is described for obtaining the axes of the diagonal paramagnetic susceptibility tensor for the low-spin cyanide complexes of distal point mutants of ferric sperm whale myoglobin (metMbCN). It relies on using the crystal coordinates of the wild-type (WT) protein for that portion of the molecule unperturbed by the point mutation, together with the experimental dipolar shifts, to search for the Euler rotation that correctly converts the crystal coordinates to the magnetic axes. The complete set of ¹H NMR dipolar shifts is shown to lead to the determination of the magnetic anisotropies as well as the orientation of the magnetic axes for WT metMbCN. Various sets of input ¹H NMR dipolar shifts for protons not only emphasizing the proximal side of the heme but also considering distal backbone protons and the structurally conserved Phe CD1 are shown to lead to well-defined magnetic axes for WT metMbCN with closely clustered Euler angles. The tilt of the major magnetic axis from the heme normal, the projection of this tilt on the heme plane, and the position of the rhombic axies projected on the heme plane range only over 1.5°, ∼ 10°, and ∼ 10°, respectively, for nine different data sets comprising as many as 37 to as few as five input dipolar shifts. The ¹H NMR spectra of the metMbCN complexes of a strongly perturbed (His E7 → Gly) and a minimally perturbed (Arg CD3 → Gly) point mutant are analyzed to yield the assignments necessary to define the magnetic axes. Using a variety of input data sets of dipolar shifts limited to the residues expected to be unperturbed by distal point mutation, the magnetic axes were determined by a three parameter least-square search for both the His E7 → Gly and Arg CD3 → Gly mutants. For the E7 Gly mutant, the major magnetic axis tilt is minimally altered, but the projection of the tilt is rotated by ∼ 45°; the CD3 Gly mutant yields a magnetic axes orientation within the range defined by different data sets of WT metMbCN. However, simulation of the predicted dipolar shift based on systematic changes is used to show that the axes of the CD3 Gly mutant differ from those of the WT by a very small (2°) rotation of the projection of the tilt of the major axis, rather than from a change in tilt. Inasmuch as the orientation of the magnetic axes can be related to distal steric tilt of the isostructural Fe‒CO unit in WT MbCO, the present demonstration that magnetic axes can be determined for point mutants has significant implications for the elucidation of steric constraints on bound ligands in a variety of low-spin hemoproteins.