Imidazole binding to horse metmyoglobin: dependence upon pH and ionic strength

Area of Science:

• Biochemistry
• Chemical Kinetics
• Protein-Ligand Interactions

Background:

• Metmyoglobin is a key hemoprotein involved in oxygen transport.
• Imidazole and its derivatives are biologically relevant ligands that can interact with heme proteins.
• Understanding ligand binding kinetics is crucial for elucidating protein function and reaction mechanisms.

Purpose of the Study:

• To investigate the reaction kinetics between metmyoglobin and imidazole as a function of pH and ionic strength.
• To compare the binding of imidazole with 1-methylimidazole to understand the role of ligand charge.
• To elucidate the binding mechanisms and determine rate constants for different ligand forms and metmyoglobin states.

Main Methods:

• Kinetic studies of metmyoglobin reactions with imidazole and 1-methylimidazole.
• Experiments conducted across a pH range of 4.2 to 11.5.
• Ionic strength dependence investigated from 0.001 to 1.0 M at integral pH values.

Main Results:

• The initial metmyoglobin-imidazole complex at pH 4.2-11.5 involves neutral imidazole.
• At pH 11.5, the neutral imidazole complex slowly converts to an imidazolate complex.
• Rate constants at neutral pH and 0.1 M ionic strength: imidazole (350 M-1s-1), imidazolium ion (230 M-1s-1).
• Hydroxymetmyoglobin binds imidazole slower (56 M-1s-1) than aquometmyoglobin.
• Protonation of His-97 affects imidazolium ion binding more significantly than imidazole binding.

Conclusions:

• The negatively charged imidazolate ion does not contribute to imidazole binding rates below pH 11.5.
• Metmyoglobin reacts with both imidazole and the imidazolium ion via distinct but similarly rapid pathways.
• The protonation state of heme-linked groups influences the kinetics of ligand binding, particularly for charged ligands.