Nuclear magnetic resonance spectroscopic studies of pyridine methyl derivatives binding to cytochrome c

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The binding of pyridine methyl derivatives (2-, 3- and 4-methylpyridine) to horse heart ferricytochrome c (cyt c) by displacing methionine-80 was studied by 1H NMR spectroscopy to elucidate the effects of the different methyl substitution positions on the affinity and kinetics of binding to cytochrome c and the hyperfine-shifted NMR signals of the ligand–cytochrome c complex. Two-dimensional exchange spectroscopy (2D-EXSY) showed that except for 2-methylpyridine (2-mpy) these pyridine derivatives can form stable complexes with cytochrome c. The complexes 3-mpy–cyt c and 4-mpy–cyt c exhibit different hyperfine shift patterns compared to that of py–cyt c. The temperature dependence of the methyl resonances of 3-mpy–cyt c differs from those of 4-mpy– and py–cyt c. Kinetic and equilibrium data for the binding of 3- and 4-mpy to cyt c have been obtained by 2D-EXSY. Based on these data a comprehensive comparison between the binding properties of these pyridine derivatives and those of pyridine towards cyt c was made. The 1H NMR resonances of 3-mpy–cyt c have also been assigned including the heme peripheral protons and some aliphatic and aromatic side chain protons.