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

Asn and Gln with an amide group at gamma- and delta-positions, respectively , were substituted for distal His-E7 of bovine myoglobin to establish a sys tem where hydrogen bonding interaction between the distal residue and bound -ligand can be altered by changing donor-acceptor distance. Two mutant myog lobins showed nearly identical H-1-NMR spectral pattern for resolved heme p eripheral side-chain and amino acid proton signals and similar two-dimensio nal NMR connectivities irrespective of cyanide-bound and -unbound states, i ndicating 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 NdeltaH proton in met-cy ano myoglobin is larger than that of Gln-E7 NepsilonH proton by at least tw o orders of magnitude, suggesting a considerable difference in the strength of hydrogen bond between the E7 side-chain and bound-ligand, due to the di fferential donor-acceptor distance between the two mutants. Thus a comparat ive 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 dissociati on of cyano ion from met-myoglobin than the Gln-mutant by over 30-fold. Sim ilarly, oxygen dissociation is faster in the Asn-mutant than in the Gin-mut ant by similar to 100-fold. Association of cyanide anion to the mutant met- myoglobin was accelerated by changing Gin to Asn by a 4-fold. Likewise, oxy gen binding was accelerated by similar to 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 fo r the ligand association. (C) 1999 Elsevier Science B.V. All rights reserve d.