The behavior of the CtrHb variants brings new insights into the mechanism and limitations of the histidine addition. Ferrous WT CtrHb at neutral pH is reported to be a mixture of 4-, 5-, and 6-coordinate species, with equilibrium favoring the 5-coordinate high-spin complex [29]. In preliminary experiments, this protein was found to lose heme rapidly to apomyoglobin (half-life of ~ 8 min at pH 7.7, data not shown). Optical spectra suggest the same mixture of species in reduced T111H CtrHb and a slight increase in the population of a 6-coordinate species in L75H CtrHb (Supporting Information Fig. S1B). The failure of ferrous T111H and L75H CtrHbs to react therefore appears related to absent or fleeting heme–protein contacts