Time-Resolved Fluorescence of Flavin Adenine Dinucleotide in Wild-Type and Mutant NADH Peroxidase. Elucidation of Quenching Sites and Discovery of a New Fluorescence Depolarization Mechanism

Abstract

Time-resolved polarized fluorescence experiments have been carried out on the FAD of tetrameric NADH peroxidase from Enterococcus faecalis and three mutant enzymes, C42A, C42S, and Y159A, respectively. In particular Tyr159 and, in part, Cys42 turned out to be the amino acids which are responsible for the strong dynamic quenching of flavin fluorescence, because two picosecond 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 structure of the wild-type enzyme. Steady-state fluorescence spectroscopy indicated that the Tyr159 mutant has the same fluorescence yield as both Cys42 mutants suggesting that static fluorescence quenching prevails in the tyrosine mutant. Cys42 is the amino acid which is probably responsible for the static quenching in the wild-type enzyme and Y15...