The O2-avid hemoglobin from the parasitic nematode Ascaris suum exhibits one of the slowest known O2 off rates. Solution 1H NMR has been used to investigate the electronic and molecular structural properties of the active site for the cyano-met derivative of the recombinant first domain of this protein. Assignment of the heme, axial His, and majority of the residues in contact with the heme reveals a molecular structure that is the same as reported in the A. suum HbO2 crystal structure (Yang, J., Kloek, A., Goldberg, D. E., and Mathews, F. S. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4224-4228) with the exception that the heme in solution is rotated by 180 °about the α,γ-meso axis relative to that in the crystal. The observed dipolar shifts, together with the crystal coordinates of HbO2, provide the orientation of the magnetic axes in the molecular framework. The major magnetic axis, which correlates with the Fe-CN vector, is found oriented ~30 ° away from the heme normal and indicates significant steric tilt because of interaction with Tyr30(B10). The three side chain labile protons for the distal residues Tyr30(B10) and Gln64(E7) were identified, and their relaxation, dipolar shifts, and nuclear Overhauser effects to adjacent residues used to place them in the distal pocket. It is shown that these two distal residues exhibit the same orientations ideal for H bonding to the ligand and to each other, as found in the A. suum HbO2 crystal. It is concluded that the ligated cyanide participates in the same distal H bonding network as ligated O2. The combination of the strong steric tilt of the bound cyanide and slow ring reorientation of the Tyr30(B10) side chain supports a crowded and constrained distal pocket.