Solution Structure Determination of the Heme Cavity in the E7 His → Val Cyano-Met Myoglobin Point Mutant Based on the ¹H NMR Detected Dipolar Field of the Iron: Evidence for Contraction of the Heme Pocket

The ¹H NMR spectrum of the cyanomet complex of the sperm whale His[E7] Val myoglobin (Mb) point mutant has been analyzed by 2D methods to yield the assignments for the active site residues, including the substituted Val E7. The dipolar shifted proximal residues are used to quantitatively locate the magnetic axes for the paramagnetic susceptibility tensor in the molecular framework. The orientation of the major axis, which correlates with the ligand tilt, is ∼ 15° from the heme normal, as found in wild-type (WT) Mb, but is tilted in a direction rotated ∼40° toward the heme γ-meso position with respect to WT and similar to that in the His[E7]Gly mutant [Rajarathnam, K., La Mar, G. N., Chiu, M., & Sligar, S. G. (1992) J. Am. Chem. Soc. 114, 9048–9058]. The altered direction of an unchanged tilt angle for the Fe⁺³-CN unit is shown to be qualitatively consistent with earlier computations of the potential energy surface for MbCO [Kuriyan, J., Wilz, S., Karplus, M., & Petsko, G. A. (1986) J. Mol. Biol. 192, 133–154]. It is concluded that His E7 does not significantly contribute to the ligand tilt but strongly influences the direction of tilt. Deviations between observed and predicted dipolar shifts for the E-helix backbone protons and perturbed patterns of their respective nuclear Overhauser effect between the E-helix and the heme 1,8-methyls are separately analyzed for movement of the E-helix and agree on a translation of the E-helix of the order of 0.8 Å in a direction toward the iron. The discrepancy between observed and predicted dipolar shifts for Phe CD1 indicates a ∼0.5-Å movement by the ring parallel to the heme and towards the E-helix. The E-helix and Phe CD1 movements are consistent with a contraction of the pocket to fill the space created by the His → Val substitution. The correlation between the observed dipolar shifts of the substituted Val E7 side chain and those calculated as a function of rotation of the residue with and without movement of the E-helix confirm the movement of the E-helix and allow a quantitative description of the Val orientation. It is concluded that the dipolar field of the paramagnetic susceptibility tensor provides an important quantitative constraint for defining the heme cavity structure in cyanomet complexes of distal point mutants of myoglobin and hemoglobin.