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

Abstract

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.

References

1. P. Georgs, S. C. Glauser and A. Schjter, J. Biol. Chem., 1967, 242, 1690.
2. N. Sutin and J. K. Yandell, J. Biol. Chem., 1972, 247, 6932.
3. P. D. Baker and A. G. Mauk, J. Am. Chem. Soc., 1992, 114, 3619.
4. W. Shao, H. Sun, Y. Yao and W. Tang, Inorg. Chem., 1995, 34, 680.
5. X. Hong and D. W. Dixon, FEBS Lett., 1989, 246, 105.
6. M. Smith and G. McLendon, J. Am. Chem. Soc., 1981, 103, 4912.
7. I. Morishima, S. Ogawa, T. Yonezawa and T. Iizuka, Biochim. Biophys. Acta, 1978, 532, 48.
8. W. Shao, Y. Yao, G. Liu and W. Tang, Inorg. Chem., 1993, 32, 6112.
9. G. Liu, Y. Chen and W. Tang, J. Chem. Soc., Dalton Trans., 1997, 795.
10. D. L. Bautigan, S. Ferguson-miller and E. Margolish, Methods Enzymol., 1978, 53D, 128.
11. J. G. Heap, W. J. Jones and J. B. Speakman, J. Am. Chem. Soc., 1921, 43, 1936.
12. G. Bodenhausen, H. Kogler and R. R. Ernst, J. Magn. Reson., 1984, 58, 370.
13. W. Shao and W. Tang, Spectrosc. Lett., 1994, 27, 763.
14. R. R. Ernst, G. Bodenhausen and A. Wokau, Principles of Nuclear Magnetic Resonance in One and Two Dimensions, Oxford University Press, 1983.
15. E. R. Johnston, M. J. Dellwo and J. Hendrix, J. Magn. Reson., 1986, 66, 399.
16. E. W. Abel, T. P. J. Coston, K. G. Orrell, V. Sik and D. Stephenson, J. Magn. Reson., 1986, 70, 34.
17. J. Lu, G. Liu, C. Yu, D. Zhu and W. Tang, Inorg. Chim. Acta, in the press.
18. Critical Stability Constants, R. M. Smith and A. E. Martell, Plenum Press, London, 1975, vol. 2, pp. 165–169.
19. G. Liu, W. Shao, X. Huang, H. Wu and W. Tang, Biochim. Biophys. Acta, 1996, 1277, 61.
20. INSIGHT II, version 97.0, MSI Corp., San Diego, CA, 1997.
21. D. C. Blumenthal and R. J. Kassner, J. Biol. Chem., 1980, 255, 5859.
22. M. M. M. Saleem and M. T. Wilson, Inorg. Chim. Acta, 1988, 153, 105.
23. H. Santos and D. L. Turner, FEBS Lett., 1986, 194, 73.
24. G. Williams, G. R. Moore, R. Porteus, M. N. Robinson, N. Soffe and R. J. P. Williams, J. Mol. Biol., 1985, 183, 409.
25. K. L. Bren, H. B. Gray, L. Banci, I. Bertini and P. Turano, J. Am. Chem. Soc., 1995, 117, 8067.
26. H. Senn, H. Bōhme and K. Wüthrich, Biochim. Biophys. Acta, 1984, 789, 311.
27. D. L. Turner, Eur. J. Biochem., 1995, 227, 829.
28. H. Senn and K. Wüthrich, Biochim. Biophys. Acta, 1983, 743, 69.
29. D. L. Turner, Eur. J. Biochem., 1993, 211, 563.
30. A. J. Wand, D. L. Di Stefano, Y. Feng, H. Roder and S. W. Englander, Biochemistry, 1989, 28, 186.
31. Y. Feng, H. Roder, S. W. Englander, A. J. Ward and D. L. Di Stefano, Biochemistry, 1989, 28, 195.
32. J. D. Satterlee and S. Moensch, Biophys. J., 1987, 52, 107.