Because WT CtrHb does not form a crosslink, H75 is the logical candidate for modification. 1H-15N lr-HMQC spectra collected on cyanomet WT and L75H CtrHbs before and after treatment demonstrate that, in L75H CtrHb-B, signals corresponding to intact H75 are missing and a new set of shifted cross peaks are detected. Both H75 imidazole nitrogens exhibit J-coupling to a proton at 7.12 ppm (Hε1). The ring 15Nε2 is also J-coupled to a proton at 5.42 ppm (Hδ2) and to the heme 4-CβH3 (−0.49 ppm, Fig. 6). NOE connectivities define the position of the modified histidine: H75 Hδ2 has strong dipolar interactions with both the heme 3- and 4-CβH3 groups, whereas H75 Hε1 is oriented towards the 5-CH3 group (data not shown). Together, these data support that the H75 ring undergoes a rotation from its original position
and forms the anticipated crosslink, with R stereochemistry at the heme 4-Cα, as depicted in Supporting Information Fig. S20B. Interestingly, for both T111H and L75H CtrHbs, the engineered histidine reorients in order to react with the heme.