On the ligands in charge-transfer complexes of porcine kidney flavoenzyme D-amino acid oxidase in three redox states: A resonance Raman study

To investigate the structural modulation of ligands and their interaction i n the active-site nanospace when they form charge-transfer (CT) complexes w ith D-amino acid oxidase (DAO) in three redox states, we compared Raman ban ds of the ligands in complex with DAO with those of ligands free in solutio n. Isotope-labeled ligands were synthesized for assignments of observed ban ds. The COO- stretching of ligands observed around, 1,370 cm(-1) downshifte d by about 17 cm(-1) upon complexation with oxidized, semiquinoid and reduc ed DAO, except for the case of reduced DAO-N-methylisonicotinate complex (8 cm(-1) downward shift); the interaction mode of the carboxylate group with the guanidino group of Arg283 and the hydroxy moiety of Tyr228 of DAO is s imilar in the three redox states. The C=N stretching mode (1,704 cm(-1)) of Delta (1)-piperideine-2-carboxylate (D1PC) downshifted to 1,675 and 1,681 cm(-1) upon complexation with reduced and semiquinoid DAO, respectively. Th e downward shifts indicate that the C=N bond is weakened upon the complexat ion. This is probably due mainly to charge-transfer (CT) interaction betwee n D1PC and semiquinoid or reduced flavin, i.e., the partial electron donati on from the highest occupied molecular orbital (HOMO) of reduced flavin or a singly occupied molecular orbital (SOMO) of semiquinoid flavin to the low est unoccupied molecular orbital (LUMO), an antibonding orbital, of D1PC. T his speculation was supported by the finding that the magnitude of the shif t is smaller by 5 cm(-1) (observed at 1,680 cm(-1)) in the case of reduced DAO reconstituted with 7,8-Cl-2-FAD, whose reduced form has lower electron- donating ability than natural reduced FAD. The amount of electron flow was estimated by applying the theory of Friedrich and Person [(1966) J. Chem. P hys. 44, 2166-2170] to these complexes; the amounts of charge transfer from reduced FAD and reduced 7,8-Cl-2-FAD to D1PC were estimated to be about 10 and 8% of one electron, respectively, in the CT complexes of reduced DAO w ith D1PC.