1H NMR study of the solution molecular and electronic structure of engineered distal myoglobin His64(E7) Val/Val68(E11) His double mutant. Coordination of His64(E11) at the sixth position in both low-spin and high-spin states.

A genetically engineered human myoglobin (Mb) in which the distal His, His64(E7), and the distal Val, Val68(E11), are replaced by Val and His, respectively, has been expressed in Escherichia coli, for the purpose of assessing the potential role of a E11 residue in providing a hydrogen bond donor to the coordinated ligand. Molecular modeling indicates that such an interaction is possible. The 1H NMR spectrum of the ferric form of the double mutant Mb exhibits large hyperfine shifts and strong paramagnetic relaxation for which the temperature dependence of the hyperfine shifts reveals a thermal equilibrium between a low-spin and high-spin state (70, 30% at 25 degrees C, respectively). Standard sequence specific two-dimensional (2D) NMR assignments of the E and F helical backbones allow the identification of the peptide protons for the proximal His93(F8) and substituted distal His68(E11). Steady-state nuclear Overhauser effect from these peptide protons locate strongly hyperfine shifted His93(F8) and His68(E11) side chain protons which dictate that both the imidazole rings are coordinated to the iron. 2D bond correlation and one-dimensional and 2D dipolar correlation experiments locate and assign the resonances for the heme. The pattern of the heme contact shifts in both the low-spin and high-spin state, together with the nature of the temperature dependence of the His93(F8) and His68(E11) resonances, establish that the two His are ligated in the high-spin as well as low-spin forms. The pattern of heme methyl hyperfine shifts in the low-spin state, and the smaller hyperfine shifts for His68(E11) as compared to His93(F8) in the high-spin state, indicate that the axial bond to the distal His68(E11) is weakened or strained as compared with that for the proximal His93(F8) in both spin states. This weak ligation originates from a tilted iron-His68 bond, the only conformation in which His68 can place its imidazole group sufficiently close to bind to the heme iron in the conventional Mb folding. Not only do these results support the belief that distal His is indispensable for the control of the ligand binding in Mb and hemoglobin, but also reveal the significance of the evolution that the stereochemical disposition of both His64 and Val68 are unique and non-exchangeable for interacting with the bound ligand.