ALTERATION OF AXIAL COORDINATION BY PROTEIN ENGINEERING IN MYOGLOBIN - BISIMIDAZOLE LIGATION IN THE HIS(64)-]VAL/VAL(68)-]HIS DOUBLE MUTANT

Pig and human myoglobin have been engineered to reverse the positions of the distal histidine and valine (i.e. His(64)(E7) --> Val and Val(68)(E11) --> His). Spectroscopic and ligand binding properties have been measured for human and pig H64V/V68H myoglobin, and the structure of the pig H64V/V68H double mutant has been determined to 2.07-Angstrom resolution by x-ray crystallography. The crystal structure shows that the N-epsilon of His(68) is located 2.3 Angstrom away from the heme iron, resulting in the formation of a hexacoordinate species, The imidazole plane of His(68) is tilted relative to the heme normal; moreover it is not parallel to that of His(93), in agreement with our previous proposal (Qin, J,, La Mar, G. N., Dou, Y,, Admiraal, S. J., and Ikeda-Saito, M. (1994) J. Biol. Chem. 269, 1083-1090). At cryogenic temperatures, the heme iron is in a low spin state, which exhibits a highly anisotropic EPR spectrum (g(1) = 3.34, g(2) = 2.0, and g(3) < 1), quite different from that of the imidazole complex of metmyoglobin. The mean iron-nitrogen distance is 2.01 Angstrom for the low spin ferric state as determined by x-ray spectroscopy, The ferrous form of H64V/V68H myoglobin shows an optical spectrum that is similar to that of b-type cytochromes and consistent with the hexacoordinate bisimidazole hemin structure determined by the x-ray crystallography, The double mutation lowers the ferric/ferrous couple midpoint potential from +54 mV of the wild-type protein to -128 mV. Ferrous H64V/V68H myoglobin binds CO and NO to form stable complexes, but its reaction with O-2 results in a rapid autooxidation to the ferric species. All of these results demonstrate that the three-dimensional positions of His(64) and Val(68) in the wild-type myoglobin are as important as the chemical nature of the side chains in facilitating reversible O-2 binding and inhibiting autooxidation.