© 2007 The Japanese Biochemical Society.
Intramolecular and Intermolecular Perturbation on Electronic State of FAD Free in Solution and Bound to Flavoproteins: FTIR Spectroscopic Study by Using the C = O Stretching Vibrations as Probes
1Department of Physiology, School of Health Sciences; Departments of 2Molecular Physiology and 3Molecular Enzymology, Graduate School of Medical Sciences, Kumamoto University, Honjo, Kumamoto 860-8556; and 4Department of Nursing, Kyushu University of Nursing and Social Welfare, Tomio, Tamana, Kumamoto 865-0062
*To whom correspondence should be addressed. Fax: +81-96-373-5490; E-mail: nishina{at}hs.kumamoto-u.ac.jp
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Abstract |
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The intramolecular and intermolecular perturbation on the electronic state of FAD was investigated by FTIR spectroscopy by using the C=O stretching vibrations as probes in D2O solution. Natural and artificial FADs, i.e. 8-CN-, 8-Cl-, 8-H-, 8-OCH3-, and 8-NH2-FAD labelled by 2-13C, 18O=C(2), or 4,10a-13C2 were used for band assignments. The C(2)=O and C(4)=O stretching vibrations of oxidized FAD were shifted systematically by the substitution at the 8-position, i.e. the stronger the electron-donating ability (NH2 > OCH3 > CH3 > H > Cl > CN) of the substituent, the lower the wavenumber region where both the C(2)=O and C(4)=O bands appear. In contrast, the C(4)=O band of anionic reduced FAD scarcely shifted. The 1,645-cm–1 band containing C(2)=O stretching vibration shifted to 1,630 cm–1 in the medium-chain acyl-CoA dehydrogenase (MCAD)-bound state, which can be explained by hydrogen bonds at C(2)=O of the flavin ring. The band was observed at 1,607 cm–1 in the complex of MCAD with 3-thiaoctanoyl-CoA. The 23 cm–1 shift was explained by the charge-transfer interaction between oxidized flavin and the anionic acyl-CoA. In the case of electron-transferring flavoprotein, two bands associated with the C(4)=O stretching vibration were obtained at 1,712 and 1,686 cm–1, providing evidence for the multiple conformations of the protein.
Key Words: artificial flavin, electron-transferring flavoprotein, FTIR spectroscopy, hydrogen bond, medium-chain acyl-CoA dehydrogenase
Abbreviations: CT, charge-transfer; ETF, electron-transferring flavoprotein; MCAD, medium-chain acyl-CoA dehydrogenase.
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