title: Structural dynamics of myoglobin: effect of internal cavities on ligand migration and binding.
abstract: Using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures, we have studied CO binding to the heme and CO migration among cavities in the interior of sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation. Photoproduct intermediates, characterized by CO in different locations, were selectively enhanced by laser illumination at specific temperatures. Measurements were performed on the wild-type protein and a series of mutants (L104W, I107W, I28F, and I28W) in which bulky amino acid side chains were introduced to block passageways between cavities or to fill these sites. Binding of xenon was also employed as an alternative means of filling cavities. In all samples, photolyzed CO ligands were observed to initially bind at primary docking site B in the vicinity of the heme iron, from where they migrate to the secondary docking sites, the Xe4 and/or Xe1 cavities. To examine the relevance of these internal docking sites for physiological ligand binding, we have performed room-temperature flash photolysis on the entire set of proteins in the CO- and O(2)-bound form. Together with the cryospectroscopic results, these data provide a clear picture of the role of the internal sites for ligand escape from and binding to myoglobin.
authors.affiliation: Department of Biophysics, University of Ulm, 89069 Ulm, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Nienhaus
authors.forename: Pengchi
authors.initials: P
authors.lastname: Deng
authors.forename: Jan M
authors.initials: JM
authors.lastname: Kriegl
authors.forename: G Ulrich
authors.initials: GU
authors.lastname: Nienhaus
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010782:Photolysis; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D014907:Whales
chemical_list: D008024:Ligands; D009211:Myoglobin; D011994:Recombinant Proteins; D006418:Heme; D002248:Carbon Monoxide; D010100:Oxygen