The T111H CtrHb spectrum resulting from the treatment (Fig. 3d) contains a multitude of hyperfine shifted lines with intensities suggesting a mixture of at least three species. The new predominant set of resonances corresponds to a major product accounting for ~60% of the sample. The inability for DT to reduce cyanomet T111H CtrHb completely under these NMR conditions can be seen from Supporting Information Fig. S15 and explains the second set of peaks, which corresponds to the cyanomet major heme orientational isomer of the starting material (~ 25%). Finally, a third set corresponds to a minor product that may be due to a different modification of the heme or DT-damaged protein.
Signals corresponding to heme methyl groups of T111H CtrHb-A4 are assigned on the basis of their high intensity and characteristic paramagnetic downfield 1H and upfield 13C shifts. DQF-COSY spectra reveal one vinyl and both propionate spin systems