Electronic Control of Ligand-Binding Preference of a Myoglobin Mutant

The L29F mutant of sperm whale myoglobin (Mb), where the leucine 29 residue was replaced by phenylalanine (Phe), was shown to exhibit remarkably high affinity to oxygen (O₂), possibly due to stabilization of the heme Fe atom-bound O₂ in the mutant protein through a proposed unique electrostatic interaction with the introduced Phe29, in addition to well-known hydrogen bonding with His64 [Carver, T. E.; Brantley, R. E.; Singleton, E. W.; Arduini, R. M.; Quillin, M. L.; Phillips, G. N., Jr.; Olson, J. S. J. Biol. Chem., 1992, 267, 14443–14450]. We analyzed the O₂ and carbon monoxide (CO) binding properties of the L29F mutant protein reconstituted with chemically modified heme cofactors possessing a heme Fe atom with various electron densities, to determine the effect of a change in the electron density of the heme Fe atom (ρ_Fe) on the O₂ versus CO discrimination. The study demonstrated that the preferential binding of O₂ over CO by the protein was achieved through increasing ρ_Fe, and the ordinary ligand-binding preference, that is, the preferential binding of CO over O₂, by the protein was achieved through decreasing ρ_Fe. Thus, the O₂ and CO binding preferences of the L29F mutant protein could be controlled through electronic modulation of intrinsic heme Fe reactivity through a change in ρ_Fe. The present study highlighted the significance of the tuning of the intrinsic heme Fe reactivity through the heme electronic structure in functional regulation of Mb.