Detailed NMR analysis of the heme-protein interactions in component IV Glycera dibranchiata monomeric hemoglobin-CO.

Complete 13C, 15N, and 1H resonance assignments have been obtained for the recombinant, ferrous CO-ligated from of component IV monomeric hemoglobin from Glycera dibranchiata. This 15642 Da myoglobin-like protein contains a large number of glycine and alanine residues (47) and a heme prosthetic group. Coupling constant information has allowed the determination of chi(1) and chi(2) torsion angles, backbone phi angles, as well as 43 of 81 possible assignments to H beta 2/beta 3 pairs. The 13C alpha, 13 beta, 13C', and 1H alpha assignments yield a consensus chemical shift index (CSI) that, in combination with NOE information and backbone torsion angles, defines seven distinct helical regions for the protein's global architecture. Discrepancies between the CSI and NOE/3JHNH alpha-based secondary structure definitions have been attributed to heme ring current shifts on the basis of calculations from a model structure [Alam et al. (1994) J. Protein Chem., 13, 151-164]. The agreement can be improved by correcting the 1H alpha chemical shifts for the ring current contributions. Because the holoprotein was assembled from isotopically enriched globin and natural isotope-abundance heme, data from 13C-filtered/13C-edited and 13C-filtered/13C-filtered 2D NOESY experiments could be used to determine complete heme proton assignments and to position the heme within the protein. The results confirm the unusual presence of Phe31 (B10) and Leu58 (E7) side chains near the heme ligand binding site which may alter the polarity and steric environment and thus the functional properties of this protein.