Electronic control of discrimination between O₂ and CO in myoglobin lacking the distal histidine residue

We analyzed the oxygen (O₂) and carbon monoxide (CO) binding properties of the H64L mutant of myoglobin reconstituted with chemically modified heme cofactors possessing a heme Fe atom with a variety of electron densities, in order to elucidate the effect of the removal of the distal His64 on the control of both the O₂ affinity and discrimination between O₂ and CO of the protein by the intrinsic heme Fe reactivity through the electron density of the heme Fe atom (ρ_Fe). The study revealed that, as in the case of the native protein, the O₂ affinity of the H64L mutant protein is regulated by the ρ_Fe value in such a manner that the O₂ affinity of the protein decreases, due to an increase in the O₂ dissociation rate constant, with a decrease in the ρ_Fe value, and that the O₂ affinities of the mutant and native proteins are affected comparably by a given change in the ρ_Fe value. On the other hand, the CO affinity of the H64L mutant protein was found to increase, due to a decrease in the CO dissociation rate constant, with a decrease in the ρ_Fe value, whereas that of the native protein was essentially independent of a change in the ρ_Fe value. As a result, the regulation of the O₂/CO discrimination in the protein through the ρ_Fe value is affected by the distal His64. Thus, the study revealed that the electronic tuning of the intrinsic heme Fe reactivity through the ρ_Fe value plays a vital role in the regulation of the protein function, as the heme environment furnished by the distal His64 does.