Structural factors governing azide and cyanide binding to mammalian metmyoglobins.

Abstract

The structural factors governing azide and cyanide binding have been examined by measuring the effects of 46 mutations at key topological positions in the distal pocket in sperm whale, pig, and human myoglobin. Replacement of His64 (E7) with smaller amino acids results in dramatic increases in the association rate constant for azide binding primarily due to relief of steric hindrance imposed by the imidazole side chain. Gln64 and His64 (native) metmyoglobins have abnormally low rate constants for azide dissociation (0.1-0.3 s-1) due to direct hydrogen bonding between the N epsilon atoms of these residues and the bound ligand. Mutations at positions 67(E10) and 68(E11) produce large but complex changes in the azide binding parameters as a result of both steric and electrostatic effects, which alter water coordination, influence the rate of anion movement into the distal pocket, and affect the stability of the Fe-N3 bond. Replacement of Phe46 with Leu or Val and substitution of Arg(Lys)45 with Glu and Ser cause disorder in the position of the distal histidine side chain and result in 4-700-fold increases in both k'N3 and kN3 but produce little change in overall azide affinity. All of these results suggest strongly that azide enters the distal pocket of native myoglobin through a polar channel that is regulated by a His64 "gate." In contrast to azide binding, the rate constant for cyanide association decreases 4-300-fold when the distal histidine is replaced with apolar residues. His64, Gln64, and distal pocket water molecules appear to facilitate deprotonation of HCN, which is the major kinetic barrier to cyanide binding at neutral pH.