Imidazole is a sensitive probe of steric hindrance in the distal pockets of oxygen-binding heme proteins

Sheref S. Mansy, John S. Olson, Gonzalo Gonzalez, Marie A. Gilles-Gonzalez

Research output: Contribution to journalArticlepeer-review

40 Scopus citations


The FixL heme-based sensor, despite its low affinity for oxygen, is much more reactive than myoglobin toward the large polar ligand imidazole. To determine which features of a myoglobin heme pocket favor binding of imidazole, we have measured binding of this ligand to the FixL heme domain, elephant myoglobin, wild-type sperm whale myoglobin, and sperm whale myoglobins having alanine, valine, threonine, glutamine, leucine, phenylalanine, or tryptophan substitutions of the distal (E7) histidine residue. Except for histidine, the association rate constants dropped more than 3000-fold as the volume of the E7 side chain, at position 64, was expanded from alanine (106 M-1 s-1) to phenylalanine (103 M-1 s- 1). There was inhibition of imidazole binding due to displacement of coordinated water from H64 and H64Q sperm whale myoglobins, where the E7 side chain hydrogen bonds directly to the bound ligand. The imidazole dissociation rate constants varied less dramatically and less consistently with any single factor, though they were measurably decreased by hydrogen bonding to an E7 glutamine or histidine. On the whole, the results for the sperm whale myoglobin E7 substitutions show that the rate constants for imidazole binding are useful and sensitive indicators of steric hindrance and polar interactions in the distal pockets of myoglobins. The combined effects of the glutamine 64 and phenylalanine 29 in elephant myoglobin largely account for its increased imidazole association and dissociation rate constants, respectively, compared to those of sperm whale myoglobin. An unhindered distal pocket not competent to stabilize positive poles is indicated by the large imidazole association (≤ 104 M-1 s-1) and dissociation (≤50 s- 1) rate constants, parameters that are characteristic of FixL.

Original languageEnglish (US)
Pages (from-to)12452-12457
Number of pages6
Issue number36
StatePublished - Sep 8 1998

ASJC Scopus subject areas

  • Biochemistry


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