Ligand binding to catalase and metmyoglobin. Direct measurements of proton involvement

1. Spectroscopic measurements of the binding of the weak-acid ligands formate, azide and cyanide to catalase indicate interaction of a primary haem-binding site with the undissociated forms of the ligands between pH 5 and 8. A similar conclusion can be drawn from observations on the pH dependence of catalase activity in the presence of cyanide and formate. 2. Direct measurements of pH changes during catalase interaction with these ligands also indicate stoichiometric uptake of one proton per haem group upon addition of formate or azide at pH 6, but at greater pH values the proton uptake declines, suggesting the involvement of a secondary site in the liganding reaction. This secondary site can be occupied by formate and certain other anions inducing proton dissociation from the protein. The results also suggest a direct displacement of formate or azide from the primary haem site by cyanide. 3. Similar stoichiometric pH measurements on metmyoglobin indicate binding of the formate, azide and cyanide anions to a single (primary) site on the molecule.