We analyzed the oxygen (O-2) 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 O-2 affinity and discrimination between O-2 and CO of the protein by the intrinsic heme Fe reactivity through the electron density of the heme Fe atom (rho(Fe)). The study revealed that, as in the case of the native protein, the O-2 affinity of the H64L mutant protein is regulated by the rho(Fe) value in such a manner that the O-2 affinity of the protein decreases, due to an increase in the O-2 dissociation rate constant, with a decrease in the rho(Fe) value, and that the O-2 affinities of the mutant and native proteins are affected comparably by a given change in the rho(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 rho(Fe) value, whereas that of the native protein was essentially independent of a change in the rho(Fe) value. As a result, the regulation of the O-2/CO discrimination in the protein through the rho(Fe) value is affected by the distal His64. Thus, the study revealed that the electronic tuning of the intrinsic heme Fe reactivity through the rho(Fe) value plays a vital role in the regulation of the protein function, as the heme environment furnished by the distal His64 does.