Investigations of ligand association and dissociation rates in the "open" and "closed" states of myoglobin.
Kinetic and Raman spectroscopic studies are combined to analyze ligand association and dissociation rates as a function of pH in aqueous solutions of myoglobin. A double-pulse flash photolysis protocol is used to kinetically select a rapidly rebinding (open pocket) fraction of the myoglobin ensemble and determine the timescale for averaging (approximately 1 to 10 microseconds) between the "open" and "closed" distal pocket protein conformations. Since this timescale is fast compared to the rate of ligand migration from the solution to the heme pocket (approximately 10(-4)s), a time-averaged population analysis, rather than a superposition of states, can be used to describe the ligand association and dissociation kinetics. Raman spectroscopy provides the relative populations of the open and closed distal pocket states as a function of pH which, in parallel with kinetics measurements, are used to determine the rates for ligand association and dissociation specific to these states. In aqueous solution at 293 K (1 mM CO) we find kon0 = 5.6 x 10(3) s-1, koff0 = 8.5 x 10(-2) s-1 for the open state and kon1 = 5.0 x 10(2) s-1, koff1 = 1.3 x 10(-2) s-1 for the closed state. The order of magnitude increase in the dissociation and association rates of the open form suggests that it may play a significant role in the ligand binding process, even though it comprises only approximately 5% of the time-averaged population at pH 7. For oxygen binding at 293 K (1.36 mM O2) we find kon0 = 4.6 x 10(4) s-1, koff0 approximately 10(4 +/- 2) s-1 for the open state and kon1 = 2.0 x 10(4) s-1, koff1 = 13 s-1 for the closed state. The dramatic increase in the dissociation rate of the open form is probably due to the loss of the hydrogen bond with the distal histidine, which stabilizes the bound O2 in the closed state. Overall, these results demonstrate that the open conformation plays a significant role in determining the ligand association and dissociation rates and suggest that environmentally induced modulations of the open population could be used as a biomolecular control mechanism for the uptake and delivery of oxygen in muscle cells.
Department of Physics, Northeastern University, Boston, MA 02115.
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D002248:Carbon Monoxide / Q000378:metabolism*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D009211:Myoglobin / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D010782:Photolysis; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman
D008024:Ligands; D009211:Myoglobin; D002248:Carbon Monoxide; D010100:Oxygen