1 H NMR study of the molecular structure and magnetic properties of the active site for the cyanomet complex of O 2 ‐avid hemoglobin from the trematode Paramphistomum epiclitum

The solution molecular and electronic structures of the active site in the extremely O 2 ‐avid hemoglobin from the trematode Paramphistomum epiclitum have been investigated by 1 H NMR on the cyanomet form in order to elucidate the distal hydrogen‐bonding to a ligated H‐bond acceptor ligand. Comparison of the strengths of dipolar interactions in solution with the alternate crystal structures of methemoglobin establish that the solution structure of wild‐type Hb more closely resembles the crystal structure of the recombinant wild‐type than the true wild‐type met‐hemoglobin. The distal Tyr66(E7) is found oriented out of the heme pocket in solution as found in both crystal structures. Analysis of dipolar contacts, dipolar shift and paramagnetic relaxation establishes that the Tyr32(B10) hydrogen proton adopts an orientation that allows it to make a strong H‐bond to the bound cyanide. The observation of a significant isotope effect on the heme methyl contact shifts confirms a strong contact between the Tyr32(B10) OH and the ligated cyanide. The quantitative determination of the orientation and anisotropies of the paramagnetic susceptibility tensor reveal that the cyanide is tilted ≈ 10° from the heme normal so as to avoid van der Waals overlap with the Tyr32(B10) Oη. The pattern of heme contact shifts with large low‐field shifts for 7‐CH 3 and 18‐CH 3 is shown to arise not from the 180° rotation about the α‐γ‐meso axis, but due to the ≈ 45° rotation of the axial His imidazole ring, relative to that in mammalian globins.