The ferryl state in globins has previously been reported to undergo a protonation event below pH 5, as assessed using pH jump experiments with stopped-flow UV-Vis spectroscopy. This protonation entails hypsochromic shifts in the α and β bands (~ 20 to 40 nm) and an ~ 10 nm reduction in the energy difference between these two bands. We now report that in Mb this event is also characterized by a hypsochromic shift in the Soret band (~ 5 nm). No similar shifts in Soret, α, and β bands are seen upon the denaturation of ferryl Mb with guanidine-suggesting that the spectroscopic changes in ferryl Mb at pH <
5 are not caused by changes in the solvent exposure or in hydrogen bonding around the ferryl unit. Under the same denaturing conditions (pH jump below pH 5, and/or guanidine), ferric-aqua and ferrous-oxy Mb show no spectral changes of the order seen in the ferryl pH jump experiments. Together, these observations suggest that the protonation event is localized on the iron-bound oxygen atom, as opposed to somewhere on a hydrogen-bonding partner. Time-dependent density functional theory (TD-DFT) calculations were not able to systematically predict the UV-Vis spectra of the heme to the level of detail needed to interpret the experimental findings in this study.