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
نویسندگان
چکیده
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
منابع مشابه
Flavin fluorescence dynamics and photoinduced electron transfer in Escherichia coli glutathione reductase.
Time-resolved polarized flavin fluorescence was used to study the active site dynamics of Escherichia coli glutathione reductase (GR). Special consideration was given to the role of Tyr177, which blocks the access to the NADPH binding-site in the crystal structure of the enzyme. By comparing wild-type GR with the mutant enzymes Y177F and Y177G, a fluorescence lifetime of 7 ps that accounts for ...
متن کاملEvidence for a novel mechanism of time-resolved flavin fluorescence depolarization in glutathione reductase.
Time-resolved flavin fluorescence anisotropy studies on glutathione reductase (GR) have revealed a remarkable new phenomenon: wild-type GR displays a rapid process of fluorescence depolarization, that is absent in mutant enzymes lacking a nearby tyrosine residue that blocks the NADPH-binding cleft. Fluorescence lifetime data, however, have shown a more rigid active-site structure for wild-type ...
متن کاملSensing cell metabolism by time-resolved autofluorescence.
We built a time-resolved confocal fluorescence spectroscopy system equipped with the multichannel time-correlated single-photon-counting technique. The instrument provides a unique approach to study the fluorescence sensing of cell metabolism via analysis of the wavelength- and time-resolved intracellular autofluorescence. The experiments on monolayered cell cultures show that with UV excitatio...
متن کاملTime-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence.
A time-resolved spectroscopic imaging system is built to study the fluorescence characteristics of nicotinamide adenine dinucleotide (NADH), an important metabolic coenzyme and endogenous fluorophore in cells. The system provides a unique approach to measure fluorescence signals in different cellular organelles and cytoplasm. The ratios of free over protein-bound NADH signals in cytosol and nuc...
متن کاملMulti-NMR and fluorescence spectra study the effects of aluminum(III) on coenzyme NADH in aqueous solutions.
The interactions of dihydronicotinamide adenine dinucleotide (NADH) with Al(III) in near neutral aqueous solutions were studied by means of multinuclear (31P, 27Al, 1H and 13C)-NMR and fluorescence spectra techniques. The results suggested that Al(III) interacts with NADH by occupying the binding sites of pyrophosphate oxygen atoms and locks the adenine moiety of coenzyme in an anti folded conf...
متن کامل