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).
Although JHN-coupling between H111 15Nε2 and the newly produced 4-CβH3 or 4-CαH was not detected in the long-range data, the observed NOEs, heme methyl 1H chemical shift dispersion pattern [53], propionate shifts, and unusual H111 15N shifts are all consistent with modification occurring at H111 and the heme 4-substituent (Supporting Information Fig. S20A).