Cyanomet T111H CtrHb 1H-15N lr-HMQC data collected before and after DT treatment and compared with WT data indicate the presence of a peculiar imidazole system [52] in the T111H CtrHb-A4 product, exhibiting two downfield shifted 15N signals (at 250.6 and 220.7 ppm) J-coupled to a proton at 8.36 ppm, itself J-connected via TOCSY to a proton at 6.48 ppm. These resonances are attributed to H111 (Table 2).
Restraints defining the position of the side chain include: H111 Hε1 to heme 3-CH3 and F48 Hζ and Hε,ε', and H111 Hδ2 to the heme 3-CH3 and V115 side chain methyls. Both H111 Hε1 and Hδ2 have shared NOEs (strong and weak, respectively) to a methyl group at 3.39 ppm, which is assigned to the modified heme 4-CβH3 group.