Proton nuclear magnetic resonance studies of isonitrile-heme protein complexes

Steric interactions between bound ligand molecules and the valine E11 methyl groups of human hemoglobin and sperm whale myoglobin have been examined directly by high resolution NMR techniques. The methyl proton resonances of this amino acid are shifted markedly upfield and away from the bulk of the protein resonances by the shielding effect of circulating pi electrons in the porphyrin ring. We have monitored the valine resonance in the presence of CO and a series of isonitriles and found considerable shifts in its position, both between the various protein complexes and among the different liganded states. The ring current shifts of the gamma 1-methyl group of Val E11 in the CO forms of isolated alpha and beta chains and myoglobin are -2.70, -2.91, and -3.30 ppm, respectively. In all the proteins, these positions show little change in going from bound CO to bound methyl and ethyl isocyanide. In alpha subunits and myoglobin, n-propyl and n-butyl isocyanide binding produces marked decreases in the magnitude of these shifts, indicating that the valine residue has been forced away from the center of ring by the presence of these large ligand molecules. In the case of beta subunits, however, only tert-butyl isocyanide produces a marked decrease (from -2.91 to -1.99 ppm) in the ring current shift of the valine methyl protons. New peaks were observed in the isonitrile-protein spectra and identified as ligand proton resonances by comparing the spectra of normal and totally deuterated isonitrile complexes. The magnitudes of the ring current shifts for the terminal methyl protons of ethyl isocyanide suggest a linear geometry for the Fe equal to C equal to N - C bonds in beta chains and a bent geometry for alpha chains. The bent geometry in alpha subunits appears to be dictated by the position of the Val E11 methyl group which is located further up from the heme plane but closer to the heme center than the corresponding position of the beta subunit residue. The free energy changes for ethyl isocyanide binding to the two chains are nearly identical, suggesting that the linear and bent geometries are energetically equivalent. Myoglobin ethyl isocyanide complexes exhibit ligand ring current shifts intermediate to those observed for the hemoglobin subunits. Assignment of resonances and positions to the alkyl protons of the longer isonitriles is more difficult.