Solution 1H NMR characterization of axial interactions of the proximal and distal His in the cyanomet complexes of the isolated chains and the 65 kDa intact tetramer of human hemoglobin A.

Solution 1H NMR has been used to investigate the axial bonding of the proximal His and the hydrogen-bonding of the distal His to the bound ligand in the isolated chains as well as the subunits of intact, tetrameric, cyanomet human hemoglobin A. The complete proximal His, including all ring protons necessary to monitor bonding in each subunit, could be definitively assigned by 1D/2D methods despite the large size (∼65 kDa) and severe relaxation (to T1 ≈ 3 ms, line width ≈ 1.5 kHz) of two of the protons. The complete distal His E7 ring was assigned in the α-chain and α-subunit of HbA, and the dipolar shifts and relaxation were analyzed to reveal a disposition intermediate between the positions adopted in HbCO and HbO2 that is optimal for forming a hydrogen bond with bound cyanide. The lability of the α-subunit His E7 NeH is found to be similar to that in sperm whale cyanomet myoglobin. The orientation of the distal His E7 in the β-subunit is found to be consistent with that seen in either HbCO or HbO2. Whil...