Resonance Raman study on reduced flavin in purple intermediate of flavoenzyme: use of [4-carbonyl-18O]-enriched flavin.

4-Carbonyl-18O]-enriched lumiflavin, riboflavin, and FMN were prepared by incubating each corresponding non-labeled flavin in 1 M Na18OH (H218O) at 25 degrees C. [4-Carbonyl-18O]FAD was prepared from the corresponding riboflavin by using FAD synthetase. Isotope effects by [4-carbonyl-18O]-labeling confirmed that the 1,709-cm-1 band in the IR spectrum of lumiflavin and the 1,711-cm-1 band in the Raman spectrum of FAD are mainly derived from C(4)=O stretching vibrational mode. The 1,605-cm-1 Raman band of the anionic reduced flavin in the purple intermediate of D-amino acid oxidase (DAO) with D-proline or D-alanine does not shift in DAO reconstituted with [4-carbonyl-18O]FAD, although it shifts with [4,10a-13C2]- or [4a-13C]FAD. Thus the band is mainly due to the C(4a)=C(10a) stretching vibrational mode and includes no contribution from C(4)=O stretching vibration. The band frequencies cover a fairly wide range (1,602-1,620 cm-1) depending on the enzymes. The frequencies of the reduced flavin in the purple intermediates of the dehydrogenases (medium-chain acyl-CoA, short-chain acyl-CoA, and isovaleryl-CoA dehydrogenases) are higher than those of the oxidases (DAO and L-phenylalanine oxidase). This indicates that the C(4a)=C(10a) bond order of reduced flavin in the dehydrogenases with the low reactivity for molecular oxygen is stronger than that in the oxidases with high reactivity. Therefore, the band frequency of C(4a)=C(10a) stretching may serve as an indicator of the reactivity of flavoprotein with molecular oxygen. Furthermore, strong hydrogen bonding of flavin at the N(1) moiety with the hydroxyl group of Thr136 in MCAD is probably responsible for the strong bond of the C(4a)=C(10a) of reduced flavin in the dehydrogenase.