PROTON-NMR INVESTIGATION OF THE HEME CAVITY IN THE CYANOMET DERIVATIVE OF THE COOPERATIVE HOMODIMERIC HEMOGLOBIN FROM SCAPHARCA-INAEQUIVALVIS

The active-site structure of the paramagnetic cyanomet complex of the cooperative homodimeric hemoglobin from Scapharca inaequivalvis has be en investigated by solution homonuclear NMR. In spite of the large siz e (32 kDa), the residues on the key proximal F- and distal E-helices c ould be sequence-specifically assigned and placed in the heme pocket i n a manner common to diamagnetic systems. These backbone assignments w ere greatly facilitated by the significant dispersion of backbone chem ical shifts by the highly anisotropic paramagnetic susceptibility tens or of the low-spin ferric state. The remainder of the residues in cont act with the heme are assigned based on unique contacts to the heme pr edicted by the crystal structure and the observations of scalar connec tivities diagnostic for the residues. The magnitude of the dipolar shi fts for non-ligated residues was used to determine the anisotropy and orientation of the paramagnetic susceptibilty tensor, and the major ax is found tilted from the normal in a manner similar to that found for the Fe-CO unit in the crystal structure. The combination of NOESY inte r-residue and heme-residue contacts, paramagnetic-induced relaxation a nd correlation between observed and dipolar shifts provide a descripti on of the heme cavity in cyanomet Hb that is essentially the same as f ound in the carbonmonoxy Hb crystal structure. The pattern of both the heme methyl dominant contact shifts and the heme meso-proton dominant dipolar shifts are shown to be consistent with the orientation of the axial His. It is concluded that the present homonuclear NMR methods a llow effective solution structure determination in the cyanomet form f or dimeric Hb and suggest profitable extension to the tetrameric verte brate hemoglobins.