A RAMAN-STUDY ON THE C(4)=O STRETCHING MODE OF FLAVINS IN FLAVOENZYMES - HYDROGEN-BONDING AT THE C(4)=O MOIETY

Raman spectroscopy was used to investigate the hydrogen bonding at the C(4)=O moiety of the isoalloxazine nucleus in a series of flavins and flavoproteins. Isotope effects of Raman bands confirmed that the band observed around 1,710 cm(-1) is mainly derived from C(4)=O stretching vibrational mode, A linear correlation was observed between the frequ ency of C(4)=O stretching and the chemical shift of C-13(4), suggestin g that the data from both Raman and NMR spectroscopies reflect a commo n perturbation, i.e., hydrogen bonding, The maximum difference of C(4) =O frequency among flavins and flavoproteins examined is 36 cm(-1) [1, 723 cm(-1) for riboflavin-binding protein (Kim, M. and Carey, P.C. (19 93) J. Am. Chem. Sec. 115, 7015-7016) and 1,687 cm(-1) for the complex of medium-chain acyl-CoA dehydrogenase with acetoacetyl-CoA]; the max imum difference of 40-70 kJ/mol in the hydrogen bonding strength at th e C(4)=O exists among flavoproteins. By use of an empirical linear cor relation between the frequency of C=O stretching and the bond length o f the C=O, it is estimated that the maximum difference in the bond len gth among flavoproteins treated here is ca, 0.017 Angstrom. The hydrog en bonding at the C(4)=O in medium-chain and short-chain acyl-CoA dehy drogenases becomes stronger upon complexation with substrate analogs, Since the hydrogen bonding at the C(4)=O is expected to enhance the el ectron-accepting capacity of the N(5) position, substrate-binding itse lf probably raises the reactivity of flavin, through enhancing the hyd rogen bonding.