Recent structural investigations have generated uncertainty regarding the protonation state of the exogenous oxo ligand in ferryl derivatives of several heme proteins. We used nuclear resonance vibrational spectroscopy (NRVS) to reveal the complete spectrum of Fe-ligand modes for compound II of myoglobin. Comparison with vibrational DFT predictions allows us to identify vibrations involving FeO tilting, coupled with stretching of the Fe-N bonds to the heme, and stretching of the proximal Fe-His bond, in addition to the previously observed Fe-O stretching vibration. Additional calculations, coupled with measurements on the hydroxyl derivative of metmyoglobin, reveal vibrational signatures for the putative protonated ferryl species. These include a 33 cirri splitting of the FeO tilling modes due to the asymmetrically placed proton, as well as a 250 cm(-1) decrease of the Fe-O stretching frequency. The vibrational data suggest a fully deprotonated oxo ligand in compound II.