STRUCTURAL FACTORS GOVERNING AZIDE AND CYANIDE BINDING TO MAMMALIAN METMYOGLOBINS

The structural factors governing azide and cyanide binding have been e xamined by measuring the effects of 46 mutations at key topological po sitions in the distal pocket in sperm whale, pig, and human myoglobin. Replacement of His(64)(E7) with smaller amino acids results in dramat ic increases in the association rate constant for azide binding primar ily due to relief of steric hindrance imposed by the imidazole side ch ain. Gln(64) and His(64) (native) metmyoglobins have abnormally low ra te constants for azide dissociation (0.1-0.3 s(-1)) due to direct hydr ogen bonding between the N-epsilon atoms of these residues and the bou nd ligand. Mutations at positions 67(E10) and 68(E11) produce large bu t complex changes in the azide binding parameters as a result of both steric and electrostatic effects, which alter water coordination, infl uence the rate of anion movement into the distal pocket, and affect th e stability of the Fe-N-3 bond. Replacement of Phe(46) 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 k(N3) but produce little ohange in overal l azide affinity. All of these results suggest strongly that azide ent ers the distal pocket of native myoglobin through a polar channel that is regulated by a His(64) ''gate.'' In contrast to azide binding, the rate constant for cyanide association decreases 4-300-fold when the d istal histidine is replaced with apolar residues. His(64), Gln(64), an d distal pocket water molecules appear to facilitate deprotonation of HCN, which is the major kinetic barrier to cyanide binding at neutral pH.