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 †

Abstract

The binding of 4-methylimidazole (mim) to cytochrome c (cyt c) has been studied by 1H 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.