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.

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-C-13, O-18 = C(2), or 4,10a-C-13(2) 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 1,630cm(-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 23cm(-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.