title: Replacement of the proximal ligand of sperm whale myoglobin with free imidazole in the mutant His-93-->Gly.
abstract: The proximal bond between the iron atom of the heme group and the N epsilon of histidine F8 in myoglobin (Mb) and hemoglobin (Hb) is presumed to be an important determinant of heme binding, protein structure, and oxygen binding. Here a system is described in which the proximal ligand is provided intermolecularly by the histidine side chain mimic imidazole. The proximal ligand of sperm whale Mb is replaced with glycine (H93G) using site-directed mutagenesis. The addition of imidazole to Escherichia coli expressing this gene reconstitutes myoglobin function. H93G Mb purified in the presence of imidazole is spectroscopically similar to wild-type Mb in combination with a wide variety of distal ligands. The crystal structure of H93G Mb, determined in the presence of imidazole, reveals that an imidazole molecule is bonded to the heme iron on the proximal side, substituting in trans for the side-chain function of the proximal histidine of wild-type Mb. Although H93G Mb is similar in spectroscopic and gross structural detail to wild-type Mb, subtle differences exist in the orientation of imidazole with respect to the heme group. trans-Complementation of proximal ligand function will allow the proximal bond in hemoproteins to be chemically substituted beyond the limits of the genetic code.
authors.affiliation: Institute of Molecular Biology, University of Oregon, Eugene 97403.
authors.forename: D
authors.initials: D
authors.lastname: Barrick
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D005998:Glycine / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D007093:Imidazoles / Q000737:chemistry*; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D013057:Spectrum Analysis; D014907:Whales
chemical_list: D007093:Imidazoles; D008024:Ligands; D009211:Myoglobin; D006418:Heme; D006639:Histidine; C029899:imidazole; D005998:Glycine