A series of proximal side mutants of sperm whale metmyoglobin (metMb) that involves residues which provide hydrogen bonds to the axial His and heme have been prepared, and the CO binding and solution molecular and electronic structure has been investigated by 1H NMR. These include Ser92(F7), whose O(γ) serves as a hydrogen-bond acceptor to the axial His ring N(δ)H and whose O(γ)H serves as hydrogen-bond donor to the 7-propionate carboxylate, and His97(FG3) whose ring provides the other hydrogen-bond donor to the 7- propionate carboxylate. 2D NMR data on the S92A-metMbCN, S92P-metMbCN and H97F-metMbCN show that the distal structure is completely conserved and that proximal side structural changes are highly localized. For the S92A-metMbCN, altered dipolar contacts to the F-helix backbone show that the axial His imidazole has rotated clockwise by ~10°relative to a stationary heme, while in H97F-metMbCN, the altered heme-E helix backbone contacts reveal that the heme has rotated counterclockwise by ~3°relative to a conserved axial His. The pattern of axial His rotation was qualitatively predicted by energy minimization calculations. The assignments and conserved structural elements allow the determination of a set of magnetic axes whose major magnetic axis is unchanged with respect to WT and confirms that local distal, and not proximal, interactions control the orientation of the major magnetic axis and, by inference, the degree and direction of tilt of the Fe-CN from the heme normal. The rhombic magnetic axes in S92A-metMbCN are rotated ~10°in the opposite direction from the established ~10°rotation for the axial His ring as expected. It is shown, moreover, that the pairwise α-, γ-meso vs β-, δ-meso-H hyperfine shift differences are well predicted by the change in the location of the rhombic magnetic axes. Carbon monoxide ligation rates experience minor but systematic perturbation for the S92A substitutions which confirms an influence (albeit very small) for axial His orientation on ligand affinity.
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