High-resolution X-ray structures of pig metmyoglobin and two CD3 mutants: Mb(Lys45----Arg) and Mb(Lys45----Ser).

The structure of pig aquometmyoglobin has been refined to a crystallographic R-factor of 19.8% against X-ray diffraction data between 10- and 1.75-A spacing. The final structural model comprises two molecules of pig myoglobin, 233 water molecules, and two sulfate ions. A water molecule is coordinated to each of the heme iron atoms with an average Fe-OH2 bond distance of 2.19 A, and the mean Fe-N epsilon (proximal histidine-93) distance is 2.20 A. In contrast to the structure of sperm whale metmyoglobin, the iron is not significantly displaced from the plane of the heme. At the entrance to the heme pocket, the side-chain amino group of lysine-45 (CD3) is well-defined in the electron density map and forms salt-bridging interactions with the heme 6-propionate and with a sulfate ion. Serine and arginine replacements have been made previously at position 45 to examine the proposal that the CD3 side chain acts as a barrier to ligand entry into the protein. Crystal structures of the arginine-45 and serine-45 mutant metmyoglobins have been solved to 1.9 and 2.0 A resolution, respectively. In both cases the structural changes are confined to the site of mutation. Arginine-45 takes up a conformation closely similar to that observed for this residue in wild-type sperm whale myoglobin, in which it makes more extensive charge-charge and charge-dipole interactions and appears to restrict the movement of the distal histidine away from the ligand. The hydroxyl group of serine-45 is disordered, but it is clear that the effect of the mutation is to open up the solvent-exposed face of the heme pocket.