Journal of Biomolecular NMR, 11: 119–133, 1998. KLUWER/ESCOM © 1998 Kluwer Academic Publishers. Printed in Belgium. 119 Detailed NMR analysis of the heme–protein interactions in component IV Glycera dibranchiata monomeric hemoglobin-CO Steve L. Alam a , Brian F. Volkman b , John L. Markley b and James D. Satterlee a,∗ a Department of Chemistry, Washington State University, Pullman, WA 99164-4630, U.S.A. b National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin-Madison, 420 Henry Mall, Madison, WI 53706, U.S.A. Received 6 August 1997; Accepted 10 September 1997 Key words: complete assignments, heme, heme protein, secondary structure Abstract Complete 13 C, 15 N, and 1 H 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 protein contains a large number of glycine and alanine residues (47) and a heme prosthetic group. Coupling constant information has allowed the determination of χ 1 and χ 2 torsion angles, backbone φ angles, as well as 43 of 81 possible assignments to H β2/β3 pairs. The 13 C α , 13 C β , 13 C ′ , and 1 H α 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/ 3 J HNHα -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 1 H α chemical shifts for the ring current contributions. Because the holoprotein was assembled from isotopically en- riched globin and natural isotope-abundance heme, data from 13 C-filtered/ 13 C-edited and 13 C-filtered/ 13 C-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 Phe 31 (B10) and Leu 58 (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. Abbreviations: GMH4, component IV Glycera dibranchiata monomeric hemoglobin; GMH4CO, CO-ligated form of component IV Glycera dibranchiata monomeric hemoglobin; Hb, hemoglobin; Mb, myoglobin; GMH, Glycera dibranchiata monomeric hemoglobin; HSQC, heteronuclear single quantum coherence; HMBC, heteronuclear multiple-bond correlation; SE, sensitivity enhancement; INEPT, insensitive nuclear enhancement by polarization transfer; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy. Introduction The ground rules for the mechanism by which hemoglobin (Hb) and myoglobin (Mb) control ligand affinity at the heme prosthetic group have been es- tablished from detailed comparisons of structures of ligated and unligated forms of these proteins (Bald- win and Chothia, 1979; Shaanan, 1983; Perutz et al., ∗ To whom correspondence should be addressed. 1987; Quillin et al., 1993). Several possible ligand trajectories that involve the movement of flexible re- gions within the protein have been proposed on the basis of molecular dynamics simulations (Case and Karplus, 1979; Kottalam and Case, 1988). Recent time-resolved X-ray crystallography of photolyzed Mb-CO provides structural snapshots of the amino acid and heme reorganizations that are important in CO recombination and rebinding (Srajer et al., 1996). Amino acids that play a role in these proposed mech-