The structures of sperm whale myoglobin (Mb) and human hemoglobin (Hb) complexed with methyl, ethyl, n-propyl and n-butyl isocyanide (MNC, ENC, nPNC and nBNC, respectively) were determined by X-ray crystallography. The polar isocyano head groups of the alkyl isocyanides (RNC's) have similar affinities for heme iron, whereas the size and stereochemistry of the alkyl groups cause the different RNC ligands to cross varied steric barriers when entering or exiting the protein. Four Mb structures were determined at a resolution $$ 100$\sp\circ$ rotation about its C$\sb\alpha$-C$\sb\beta$ bond. Relatively immobile side chains of amino acids in the heme pocket allowed the alkyl groups of MNC and ENC to lie in the hydrophobic interior of the heme pocket. The larger alkyl groups of nPNC and nBNC projected into the hydrophilic exterior entrance to the heme pocket.
Data for the structures of Hb complexed with MNC, ENC, nPNC and nBNC (pH 6.7) were collected to $\sim$2.2A. In $\alpha$ subunits, the alkyl moieties of the ligands lay approximately parallel to the heme plane. In $\beta$ subunits, the alkyl groups lay at about a 45$\sp\circ$ angle from the heme plane. The alkyl groups of the ligands occupied the hydrophobic interior of the heme pocket in both subunits. Little disorder in His-E7 was observed in either subunit.
Ligand binding causes greater tertiary structure changes in the $\alpha$ and $\beta$ subunits than in myoglobin. These changes in tertiary structure in response to isocyanide binding appear to cause the $\alpha$ and $\beta$ subunits to have more of a T-state tertiary conformation than does the oxy complex, and may explain the lesser cooperative effect seen in isocyanide binding compared to oxygen binding.