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 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 (1)H-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(delta)H proton in met-cyano myoglobin is larger than that of Gln-E7 N(epsilon)H 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 approximately 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 approximately 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.
Yamamoto
Y
Y
Department of Chemistry, University of Tsukuba, Tsukuba 305-8571, Japan. yash@sakura.cc.tsukuba.ac.jp
Kurihara
N
N
Egawa
T
T
Shimada
H
H
Ishimura
Y
Y
D000577:Amides / Q000737:chemistry*; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D003486:Cyanides / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008786:Metmyoglobin / Q000031:analogs & derivatives / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011522:Protons
D000577:Amides; D003486:Cyanides; D009211:Myoglobin; D011522:Protons; C043441:S-methylmyoglobin; C007177:cyanometmyoglobin; D008786:Metmyoglobin; D001216:Asparagine; D010100:Oxygen; D005998:Glycine