We analyzed the vibrational frequencies of the Fe-bound carbon monoxide (CO) of myoglobin reconstituted with a series of chemically modified heme cofactors possessing a heme Fe atom with a variety of electron densities. The study revealed that the stretching frequency of Fe-bound CO (谓CO) increases with decreasing electron density of the heme Fe atom (蟻Fe). This finding demonstrated that the 谓CO value can be used as a sensitive measure of the 蟻Fe value and that the 蟺 back-donation of the heme Fe atom to CO is affected by the heme 蟺-system perturbation induced through peripheral side chain modifications.