J. Phys. Chem. B 1998, 102, 10431-10439
10431
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 Antonie J. W. G. Visser,*,† Petra A. W. van den Berg,† Nina V. Visser,† Arie van Hoek,† Harrold A. van den Burg,‡ Derek Parsonage,‡ and Al Claiborne‡ MicroSpectroscopy Centre, Department of Biomolecular Sciences, Wageningen Agricultural UniVersity, P.O. Box 8128, NL-6700 ET Wageningen, The Netherlands, and Department of Biochemistry, Wake Forest UniVersity Medical Center, Winston-Salem, North Carolina 27157 ReceiVed: May 6, 1998; In Final Form: September 1, 1998
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
