Ligand binding to synthetic mutant myoglobin (His-E7----Gly): role of the distal histidine.
Low-temperature flash photolysis with IR and visible spectroscopy was used to probe the influence of the distal histidine His-64(E7) of sperm-whale myoglobin (Mb) on the orientation of bound carbon monoxide (CO) and on the kinetics of CO rebinding. The synthesis and high-level expression of a sperm-whale myoglobin gene in Escherichia coli permits the efficient substitution of the distal histidine through site-directed mutagenesis. Substitution of His-E7 with glycine [GlyE7]Mb bound with CO (CO[GlyE7]Mb) results in one broad bound-CO IR stretch band, v(C-O), centered at 1973 cm-1 at 10 K, in contrast to three distinct bands for native and synthetic wild-type MbCO at 1966, 1945, and 1929 cm-1. After flash photolysis at 10 K, the unbound state of CO[GlyE7]Mb exhibits two CO stretch bands, whereas MbCO has three. Fourier transform IR spectroscopy measurements of the linear dichroism after photoselective flash photolysis of CO bound to [GlyE7]Mb at 10 K reveals the bound CO to be oriented at an angle of alpha = 20 degrees +/- 2 degrees with respect to the heme normal. Flash photolysis data from 10 to 300 K provide evidence for a larger distal pocket and a smaller enthalpy barrier (by approximately 4 kJ/mol) for [GlyE7]MbCO as compared with wild-type MbCO. These results reinforce the notion that the dominant control of the binding step at the heme iron comes from the proximal side through the protein structure.
Braunstein
D
D
Department of Physics, University of Illinois, Urbana-Champaign 61801.
Ansari
A
A
Berendzen
J
J
Cowen
B R
BR
Egeberg
K D
KD
Frauenfelder
H
H
Hong
M K
MK
Ormos
P
P
Sauke
T B
TB
Scholl
R
R
D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D005998:Glycine; D006639:Histidine; D007700:Kinetics; D008024:Ligands; D009154:Mutation; D009211:Myoglobin / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013816:Thermodynamics; D014907:Whales
D008024:Ligands; D009211:Myoglobin; C018013:carboxymyoglobin; D006639:Histidine; D002248:Carbon Monoxide; D005998:Glycine