Time-resolved fluorescence of flavin adenine dinucleotide in wild-type andmutant NADH peroxidase. Elucidation of quenching sites and discovery of a new fluorescence depolarization mechanism

Time-resolved polarized fluorescence experiments have been carried out on t he FAD of tetrameric NADH peroxidase from Enterococcus faecalis and three m utant enzymes, C42A, C42S, and Y159A, respectively. In particular Tyr159 an d, in part, Cys42 turned out to be the amino acids which are responsible fo r the strong dynamic quenching of flavin fluorescence, because two picoseco nd fluorescence lifetime components <150 ps are clearly present in the wild -type enzyme and in the Cys42 mutants, while only one picosecond lifetime < 150 ps is present in the Tyr159 mutant. This observation is corroborated by the distance information obtainable from the known three-dimensional struc ture of the wild-type enzyme. Steady-state fluorescence spectroscopy indica ted that the Tyr159 mutant has the same fluorescence yield as both Cys42 mu tants suggesting that static fluorescence quenching prevails in the tyrosin e mutant. Cys42 is the amino acid which is probably responsible for the sta tic quenching in the wild-type enzyme and Y159A mutant. The time-resolved f luorescence anisotropy data showed a dependence on the emission wavelength. In case of proteins with Tyr159 present, less rapid depolarization is obse rved when the emission wavelength is at 526 nm, while depolarization of a f ew nanoseconds is more clearly visible at 568 nm. The rapid depolarization process was absent in the Y159A mutant irrespective of emission wavelength. The latter protein only showed a minor component of relatively long correl ation time (>10 ns) which can be attributed to energy transfer among the fl avins in the tetramer. The rapid ns depolarization is due to excited-state charge transfer between Tyr159 and flavin, which leads to a change of trans ition moment out of the plane of the isoalloxazine ring. The latter process contributes to a major extent to the observed fluorescence anisotropy deca y and can be considered as an unusual source of fluorescence depolarization .