Solution H-1 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 (similar to 65 kDa) and severe relaxation (to T-1 approximate to 3 ms, line width approximate to 1.5 kHz) of two of the protons. The complete distal His E7 ring was assigned in the alpha -chain and alpha -subunit of HbA, and the dipolar shifts and relaxation were analyzed to reveal a disposition intermediate between the positions adopted in HbCO and HbO(2) that is optimal for forming a hydrogen bond with bound cyanide. The lability of the alpha -subunit His E7 N6H is found to be similar to that in sperm whale cyanomet myoglobin. The orientation of the distal His E7 in the P-subunit is found to be consistent with that seen in either HbCO or HbO(2). While the His E7 labile N6H proton signal could not be detected in either the beta -chain or subunit, it is. concluded that this mole likely reflects increased lability over that of the alpha -subunit, and not the absence of a hydrogen bond to the bound ligand. Analysis of the heme mean methyl hyperfine shift, which has been shown to be very sensitive to the presence of distal hydrogen bends to bound cyanide (Nguyen, B. B., Xia, Z.; Cutruzzola, F.; Travaglini Allocatelli, C:., Brancaccio, A.; Brunori:, M.; La Mar. G, N. J. Biol. Chem. 2000, 275, 742-751), directly supports the presence of a distal His E7 hydrogen bond to cyanide in the beta -chain and beta -subunit which is weaker than the same hydrogen bond in the alpha -subunit. The potential for the proximal His hyperfine shifts in serving as indicators of axial strain in the allosteric transition of HbA is discussed.