NMR studies of 4-methylimidazole binding to cytochrome c: effects of methyl substituent on the binding affinity, the orientation of the ligand plane, and the electronic structure of the heme

The binding of 4-methylimidazole (mim) to cytochrome c (cyt c) has been studied by H-1 NMR spectroscopy. The kinetic and thermodynamic parameters for the reaction were calculated. The assignment of a number of signals has led to the determination of the magnetic susceptibility tensor of mim-cyt c. It turned out that the orientation of the imidazole ring of mim was different from that of Him in Him-cyt c. This difference was due to the steric interaction between the 4-methyl and the surrounding peptides in the heme cavity. The pseudocontact and contact shifts of the four heme methyl groups in mim-cyt c were calculated. The hyperfine shift pattern and heme electron structure of mim-cyt c were compared with those of native cyt c and Him-cyt c.