The proximal and distal pockets of the H93G myoglobin cavity mutant bind identical ligands with different affinities: Quantitative analysis of imidazole and pyridine binding

His93Gly sperm whale myoglobin (H93G Mb) has the proximal histidine ligand removed to create a cavity for exogenous ligand binding, making it a versatile template for the preparation of model heme complexes. In this study, we have measured the first and second ligand binding affinities of imidazole and pyridine to form mono- and bis-ligated ferric and ferrous H93G Mb complexes. Electronic absorption spectroscopy has been utilized to determine the binding affinities for the proximal (K-d1, first ligand) and distal (K-d2, second ligand) pockets of H93G Mb. Magnetic circular dichroism spectroscopy has been used to confirm the identity of the complexes. The binding affinities for the first ligand are one hundred-to one thousand-fold higher than those for the second ligand (K-d1 << K-d2) for the same exogenous ligand. This is entirely opposite to what is seen with free heme in organic solvents where K-d1 >> K-d2. Thus, the proximal pocket is the high affinity binding site. The lower affinity for the distal pocket can be attributed to steric hindrance from the distal histidine. This report provides quantitative evidence for differential ligand binding affinities of the proximal and distal pockets of H93G Mb, a unique property that facilitates generation of heme iron derivatives not easily prepared with other heme model systems.