AbstractThe ions formally corresponding to protonated heme [FeII–hemeH]+ have been obtained by collision‐induced dissociation from the electrospray ionization of microperoxidase (MP11) and their gas‐phase chemistry has been studied by FTICR mass spectrometry. H/D‐exchange reactions, used as a tool to gain information on the protonation sites in polyfunctional molecules, show that labile hydrogens pertain to the propionyl substituents at the periphery of the protoporphyrin IX. Several conceivable isomers for protonated heme have been evaluated by density functional theory. The most stable among the species investigated is the one corresponding to protonation at the β carbon atom of a vinyl group, yielding a proton affinity (PA) value for [FeII–heme] of 1220 kJ mol−1. This high PA is consistent with the inertness of the hydrogen atoms at the protonation site towards H/D exchange with ND3 and CD3CO2D. Peculiar features of this [FeII–hemeH]+ isomer emerge by analysis of its electronic structure, showing that the vinyl group undergoing formal protonation has gained significant radical character due to electron transfer from the metal center. As a consequence, the iron atom acquires partial iron(III) character and none of the two formal descriptions [FeII–hemeH+] and [FeIII–hemeH.]+ alone may adequately illustrate the protonated heme ion. In agreement with this description, the reactivity of protonated heme presents dual facets, resembling iron(III) in some aspects and iron(II) in others. On the one hand, protonated heme behaves like [FeIII–heme]+ ions in H/D‐exchange reactions. On the other, it shows markedly decreased reactivity towards the addition of ligands with the notable exception of NO, in line with the high affinity shown by iron(II) complexes towards this molecule, NO, of key biological role.