Structure and ligand binding properties of leucine 29(B10) mutants of human myoglobin.
Site-specific mutants of human myoglobin (Mb) have been prepared, in which Leu29 (B10) is replaced by Ala(L29A) or Ile(L29I), in order to examine the influence of this highly conserved residue in the hydrophobic clusters of the heme distal site on the heme environmental structure and ligand binding properties of Mb. Structural characterizations of these recombinant Mbs are studied by electronic absorption, infrared (IR), one- and two-dimensional proton nuclear magnetic resonance spectroscopies, and ligand-binding kinetics by laser photolysis measurements under ambient and high pressures (up to 2000 bar). Multiple split carbon monoxide (CO) stretch bands in the IR spectra of mutant Mbs exhibit a relative decrease of the 1945 cm-1 band (approximately 50%) which is associated with an upright binding geometry of CO, accompanied by an increase of the tilted CO conformer at 1932 cm-1. On the basis of these results, replacement of Leu29(B10) by Ala or Ile appears to allow bound CO to rotate from a conformation pointing toward the beta meso carbon of the heme group to the one pointing toward the alpha meso carbon atom, presumably filling the space left by removal of the delta 2 carbon atom of Leu29(B10). These substitutions cause the rate constants for CO and O2 association to decrease almost 3-5-fold. Present results show that CO and O2 bindings to the heme iron of Mb are controlled by Leu29(B10) by influencing the structure of close vicinity of the heme and the geometry of iron-bound ligand. Further, mutant Mbs (Leu72(E15)----Ala and Leu104 (G5)----Ala) which have altered residues in another hydrophobic clusters around proximal and distal site are also examined.
Adachi
S
S
Division of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan.
Sunohara
N
N
Ishimori
K
K
Morishima
I
I
D006801:Humans; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D007834:Lasers; D007930:Leucine / Q000737:chemistry*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010782:Photolysis; D013055:Spectrophotometry, Infrared; D013057:Spectrum Analysis
D008024:Ligands; D009211:Myoglobin; D007501:Iron; D007930:Leucine