Distal residues in the oxygen binding site of haemoglobin studied by protein engineering

Nature. 1987 Oct 29-Nov 4;329(6142):858-60. doi: 10.1038/329858a0.

Abstract

The geometries of the Fe-O2 and Fe-CO bonds in myoglobin and haemoglobin differ significantly from those in free porphyrin model compounds. It has been suggested that steric hindrance by Val-E11 and His-E7 and a hydrogen bond between His-E7 and oxygen affect the geometry and electronic state of the Fe-ligand bond, and that these interactions may be important in controlling oxygen affinity. We have produced mutant haemoglobins in E. coli having Val(67 beta)E11 replaced by Ala, Met, Leu or Ile and His(58 beta)E7 by Gln, Val or Gly. We have studied the effect of these mutations on the equilibrium and kinetics of ligand binding. The conformation of the new side chains and their effect on the protein structure have been examined by X-ray crystallography, and the vibrational properties of the Fe-CO bond observed by resonance Raman spectroscopy. We found that the steric hindrance of ligand binding by the E11 residue and the polarity of the E7 residue in the beta subunit are critical for fine-tuning ligand affinity.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Escherichia coli / genetics
  • Genetic Engineering
  • Hemoglobin A / metabolism*
  • Hemoglobin, Sickle / genetics
  • Hemoglobin, Sickle / metabolism*
  • Humans
  • Kinetics
  • Mutation
  • Oxygen / blood
  • Oxyhemoglobins / metabolism*
  • Protein Conformation

Substances

  • Hemoglobin, Sickle
  • Oxyhemoglobins
  • Hemoglobin A
  • oxyhemoglobin A
  • Oxygen