Hydrogen bonding interaction of the amide group of Asn and Gln at distal E7 of bovine myoglobin with bound-ligand and its functional consequences

Abstract

Asn and Gln with an amide group at γ- and δ-positions, respectively, were substituted for distal His-E7 of bovine myoglobin to establish a system where hydrogen bonding interaction between the distal residue and bound-ligand can be altered by changing donor–acceptor distance. Two mutant myoglobins showed nearly identical 1H-NMR spectral pattern for resolved heme peripheral side-chain and amino acid proton signals and similar two-dimensional NMR connectivities irrespective of cyanide-bound and -unbound states, indicating that the heme electronic structure and the molecular structure of the active site are not affected by a difference in one methylene group at the E7 position. Chemical exchange rate of Asn-E7 N δH proton in met-cyano myoglobin is larger than that of Gln-E7 N ϵH proton by at least two orders of magnitude, suggesting a considerable difference in the strength of hydrogen bond between the E7 side-chain and bound-ligand, due to the differential donor–acceptor distance between the two mutants. Thus a comparative study between the two proteins provides an ideal system to delineate a relationship between the stabilization of bound-ligand by the hydrogen bond and myoglobin’s ligand affinity. The Asn-mutant showed a faster dissociation of cyano ion from met-myoglobin than the Gln-mutant by over 30-fold. Similarly, oxygen dissociation is faster in the Asn-mutant than in the Gln-mutant by ∼100-fold. Association of cyanide anion to the mutant met-myoglobin was accelerated by changing Gln to Asn by a 4-fold. Likewise, oxygen binding was accelerated by ∼2-fold by the above substitution. The present findings confirm that hydrogen bonding with the distal residue is a dominant factor for determining the ligand dissociation rate, whereas steric hindrance exerted by the distal residue is a primary determinant for the ligand association.