Ligand affinities in mutant metmyoglobins.

Ligand binding to the wild-type and a series of mutant porcine myoglobins, expressed and purified from Escherichia coli cells, has been studied using UV-VIS absorption spectroscopy. The proximal pocket mutation, F7 Ser-->Leu (F7), causes an increased affinity for OH- and N3- binding to metmyoglobin. A hydrogen bond between the F7 serine residue and the imidazole side-chain of the proximal histidine has been removed by this mutation. It is suggested that this allows the imidazole group to reorientate, reducing the steric clash between itself and the haem pyrrole nitrogen atoms and leading to a shortening of the bond between the proximal histidine and the haem iron. Other conformational changes further away from the haem pocket have also been induced, but the mutant still crystallizes under the same conditions as for the wild-type protein. A series of distal pocket mutants, E11 Val-->Thr (VT), E7 His-->Val (HV) and a mutant with both of these substitutions (M2) all have greatly reduced the OH- and N3- binding affinity. These effects have been interpreted by considering several factors: the changed stability of the aquometmyoglobin form, hydrogen-bond formation between the ligand and the E7 residue, and electrostatic repulsion between the ligand and the E11 threonine residue.