Structural dynamics of myoglobin: effect of internal cavities on ligand migration and binding.
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.
Nienhaus
Karin
K
Department of Biophysics, University of Ulm, 89069 Ulm, Germany.
Deng
Pengchi
P
Kriegl
Jan M
JM
Nienhaus
G Ulrich
GU
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
D008024:Ligands; D009211:Myoglobin; D011994:Recombinant Proteins; D006418:Heme; D002248:Carbon Monoxide; D010100:Oxygen