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 (v(co)) increases with decreasing electron density of the heme Fe atom (p(Fe)). This finding demonstrated that the vco value can be used as a sensitive measure of the p(Fe), value and that the a back-donation of the heme Fe atom to CO is affected by the heme pi-system perturbation induced through peripheral side chain modifications.