The high-resolution X-ray crystallographic structures of horse heart azidometmyoglobin complexes of the wild-type protein and the His-64 → Thr variant have been determined to 2.0 and 1.8 Å respectively. Azide binds to wild-type metmyoglobin in a bent configuration with an Fe–N-1–N-3 angle of 119° and is oriented into the distal crevice in the direction of Ile-107. The proximity of the His-64 NE2 atom to the N-1 atom of the bound azide indicates stabilization of the ligand by the His-64 side chain through hydrogen bonding. In addition, structural characterization of wild-type horse heart azidometmyoglobin establishes that the only structural change induced by ligand binding is a small movement of the Leu-29 side chain away from the azide ligand. EPR and Fourier transform infrared spectroscopy were used to characterize the myoglobin azide complexes further. EPR spectroscopy revealed that, in contrast with wild-type azidometmyoglobin, two slightly different low-spin species are formed by azide bound to the His-64 → Thr variant both in solution and in a polycrystalline sample. One of these low-spin species has a greater relative intensity, with gvalues very similar to those of the azide complex of the wild-type protein. These EPR results together with structural information on this variant indicate the presence of two distinct conformations of bound azide, with one form predominating. The major conformation is comparable to that formed by wild-type myoglobin in which azide is oriented into the distal crevice. In the minor conformation the azide is oriented towards the exterior of the protein.

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Author notes

1

Present address: Biochemisches Institut der Universität Zürich, Winterhurestrasse 190, CH-8057 Zürich, Switzerland.