Alteration of axial coordination by protein engineering in myoglobin. Bisimidazole ligation in the His64 → Val/Val68 → His double mutant

Y. Dou, S. J. Admiraal, M. Ikeda-Saito, S. Krzywda, A. J. Wilkinson, T. Li, J. S. Olson, R. C. Prince, I. J. Pickering, G. N. George

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70 Citations (Scopus)


Pig and human myoglobin have been engineered to reverse the positions of the distal histidine and valine (i.e. His64(E7) → Val and Val68(E11) → His). Spectroscopic and ligand binding properties have been measured for human and pig H64V/V68H myoglobin, and the structure of the pig H64V/V68H double mutant has been determined to 2.07-Å resolution by x-ray crystallography. The crystal structure shows that the N(ε) of His68 is located 2.3 Å away from the heme iron, resulting in the formation of a hexacoordinate species. The imidazole plane of His68 is tilted relative to the heme normal; moreover it is not parallel to that of His93, in agreement with our previous proposal (Qin, J., La Mar, G. N., Dou, Y., Admiraal, S. J., and Ikeda-Saito, M. (1994) J. Biol. Chem. 269, 1083-1090). At cryogenic temperatures, the heme iron is in a low spin state, which exhibits a highly anisotropic EPR spectrum (g1 = 3.34, g2 = 2.0, and g3 < 1), quite different from that of the imidazole complex of metmyoglobin. The mean iron- nitrogen distance is 2.01 Å for the low spin ferric state as determined by x-ray spectroscopy. The ferrous form of H64V/V68H myoglobin shows an optical spectrum that is similar to that of b-type cytochromes and consistent with the hexacoordinate bisimidazole heroin structure determined by the x-ray crystallography. The double mutation lowers the ferric/ferrous couple midpoint potential from +54 mV of the wild-type protein to -128 mV. Ferrous H64V/V68H myoglobin binds CO and NO to form stable complexes, but its reaction with O2 results in a rapid autooxidation to the ferric species. All of these results demonstrate that the three-dimensional positions of His64 and Val68 in the wild-type myoglobin are as important as the chemical nature of the side chains in facilitating reversible O2 binding and inhibiting autooxidation.

Original languageEnglish
Pages (from-to)15993-16001
Number of pages9
JournalJournal of Biological Chemistry
Issue number27
Publication statusPublished - 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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