13C nuclear magnetic resonance studies of the binding of alkyl isocyanides to soybean leghemoglobin; comparison with animal myoglobins.

13C NMR of labelled alkyl isocyanide ligands has been used with a view to probe the protein environment around the heme site of Soybean leghemoglobin, and comparatively, those of sperm whale myoglobin and monomeric Glycera hemoglobin. The terminal carbon of the isocyanide, which is known to be highly sensitive to change in hybridization of the nitrogen, could be expected to reflect the movement of the alkyl group through steric interactions. Three alkyl isocyanides (alkyl = methyl, ethyl & n-butyl) have therefore been used and the 13C0 chemical shift values were measured for each ligand bound to the various proteins studied. In all cases, the 13C0 resonance of the bound ligand were shifted considerably down-field with respect to those of the free unbound species, but the pattern of these displacements revealed more pronounced steric hindrance in the case of some proteins compared to others. The modifications of the chemical shift values of binding delta delta = delta bound -- delta free) were least in the case of leghemoglobin; moreover, the delta delta values were insensitive to the length of the alkyl chain (methyl to n-butyl) when bound to leghemoglobin, in contrast to the other proteins examined. The results are interpreted as arising from a diminished steric hindrance to isocyanide binding with leghemoglobin, in conformity with the recently published X-ray structure which reports the existence of a large heme pocket on the distal side.