NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPIC STUDIES OF PYRIDINE METHYL-DERIVATIVES BINDING TO CYTOCHROME-C

The binding of pyridine methyl derivatives (2-, 3- and 4-methylpyridin e) to horse heart ferricytochrome c (cyt c) by displacing methionine-8 0 was studied by H-1 NMR spectroscopy to elucidate the effects of the different methyl substitution positions on the affinity and kinetics o f binding to cytochrome c and the hyperfine-shifted NMR signals of the ligand-cytochrome c complex. Two-dimensional exchange spectroscopy (2 D-EXSY) showed that except for 2-methylpyridine (2-mpy) these pyridine derivatives can form stable complexes with cytochrome c. The complexe s 3-mpy-cyt c and 4-mpy-cyt c exhibit different hyperfine shift patter ns compared to that of py-cyt c. The temperature dependence of the met hyl 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 derivative s and those of pyridine towards cyt c was made. The H-1 NMR resonances of 3-mpy-cyt c have also been assigned including the heme peripheral protons and some aliphatic and aromatic side chain protons.