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

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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 10 ns) which can be attributed to energy transfer among the flavins in the tetramer. The rapid ns depolarization is due to excited-state charge transfer between Tyr159 and flavin, which leads to a change of transition moment out of the plane of the isoalloxazine ring. The latter process contributes to a major extent to the observed fluorescence anisotropy decay and can be considered as an unusual source of fluorescence depolarization.

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