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

Yasuhiko Yamamoto, Norimasa Kurihara, Tsuyoshi Egawa, Hideo Shimada, Yuzuru Ishimura

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

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(δ)H proton in met-cyano myoglobin is larger than that of Gln-E7 N(ε)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 ~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. Copyright (C) 1999 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)27-44
Number of pages18
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Volume1433
Issue number1-2
DOIs
StatePublished - Aug 17 1999

Keywords

  • Hydrogen bond
  • Ligand affinity
  • Myoglobin mutant
  • NMR
  • Paramagnetic relaxation
  • Paramagnetic shift

ASJC Scopus subject areas

  • Structural Biology
  • Biophysics
  • Biochemistry
  • Molecular Biology

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