DETAILED NMR ANALYSIS OF THE HEME-PROTEIN INTERACTIONS IN COMPONENT-IV GLYCERA-DIBRANCHIATA MONOMERIC HEMOGLOBIN-CO

Complete C-13, N-15, and H-1 resonance assignments have been obtained for the recombinant, ferrous CO-ligated form of component IV monomeric hemoglobin from Glycera dibranchiata. This 15642 Da myoglobin-like pr otein contains a large number of glycine and alanine residues (47) and a heme prosthetic group. Coupling constant information has allowed th e determination of chi(1) and chi(2) torsion angles, backbone phi angl es, as well as 43 of 81 possible assignments to H-beta 2/beta 3 pairs. The C-13(alpha), C-13(beta) , C-13', and H-1(alpha) assignments yield a consensus chemical shift index (CSI) that, in combination with NOE information and backbone torsion angles, defines seven distinct helica l regions for the protein's global architecture. Discrepancies between the CSI and NOE/(3)J(HNH alpha)-based secondary structure definitions have been attributed to heme ring current shifts on the basis of calc ulations from a model structure [Alam et al. (1994) J. Protein Chem., 13, 151-164]. The agreement can be improved by correcting the H-1(alph a) chemical shifts for the ring current contributions. Because the hol oprotein was assembled from isotopically enriched globin and natural i sotope-abundance heme, data from C-13-filtered/C-13-edited and C-13-fi ltered/C-13-filtered 2D NOESY experiments could be used to determine c omplete heme proton assignments and to position the heme within the pr otein. The results confirm the unusual presence of Phe(31)(B10) and Le u(58)(E7) side chains near the heme ligand binding site which may alte r the polarity and steric environment and thus the functional properti es of this protein.