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

摘要

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.