STRUCTURAL AND SPECTROSCOPIC STUDIES OF AZIDE COMPLEXES OF HORSE HEART MYOGLOBIN AND THE HIS-64-]THR VARIANT

The high-resolution X-ray crystallographic structures of horse heart a zidometmyoglobin complexes of the wild-type protein and the His-64 --> Thr variant have been determined to 2.0 and 1.8 Angstrom respectively . Azide binds to wild-type metmyoglobin in a bent configuration with a n Fe-N-1-N-3 angle of 119 degrees and is oriented into the distal crev ice 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 liga nd by the His-64 side chain through hydrogen bonding. In addition, str uctural characterization of wild-type horse heart azidometmyoglobin es tablishes 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 characte rize the myoglobin azide complexes further. EPR spectroscopy revealed that, in contrast with wild-type azidometmyoglobin, two slightly diffe rent 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 g values very similar to those of the azide complex of the wild-type protein. These EPR results together with structural information on this variant indi cate the presence of two distinct conformations of bound azide, with o ne form predominating. The major conformation is comparable to that fo rmed 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.