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A model for ligand binding to hexacoordinate hemoglobins.
J T Trent1, A N Hvitved, M S Hargrove
1Department of Biochemistry, Biophysics, & Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
Biochemistry
|May 16, 2001
Summary
Hexacoordinate hemoglobins feature an internal amino acid side chain coordinating the heme pocket. A new model explains multiphasic ligand binding in rice hexacoordinate hemoglobin (rHb1), revealing a slow-reacting protein conformation.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- Hexacoordinate hemoglobins possess a unique intramolecular coordination of the ligand binding site.
- Nonsymbiotic hemoglobins (nsHbs) are found in plants, with rice nsHb1 (rHb1) serving as a model.
- Understanding ligand binding kinetics is crucial for characterizing protein function.
Purpose of the Study:
- To develop methods for determining rate constants of ligand binding and dissociation in hexacoordinate hemoglobins.
- To present a refined model for ligand binding that accounts for complex reaction kinetics.
- To investigate the role of protein conformation in ligand binding dynamics.
Main Methods:
- Laser flash photolysis to initiate reactions from the pentacoordinate form of rHb1.
- Rapid mixing techniques to study ligand binding kinetics.
- Kinetic modeling to analyze multiphasic reaction time courses.
Main Results:
- Ligand binding to hexacoordinate hemoglobins is a multiphasic reaction, not a simple combination of first and second-order processes.
- A new kinetic model was developed, incorporating a "closed", slow-reacting protein conformation.
- The formation of this "closed" species is independent of hexacoordination.
Conclusions:
- The study refines the understanding of ligand binding mechanisms in hexacoordinate hemoglobins.
- The proposed model accurately describes the complex kinetics observed in rHb1.
- The existence of a non-equilibrium "closed" state influences protein reactivity.