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 approximately 90% of the fluorescence decay could be attributed to quenching by Y177. Based on the temperature invariance for this lifetime, and the very high quenching rate, electron transfer from Y177 to the light-excited isoalloxazine part of flavin adenine dinucleotide (FAD) is proposed as the mechanism of flavin fluorescence quenching. Contrary to the mutant enzymes, wild-type GR shows a rapid fluorescence depolarization. This depolarization process is likely to originate from a transient charge transfer interaction between Y177 and the light-excited FAD, and not from internal mobility of the flavin, as has previously been proposed. Based on the fluorescence lifetime distributions, the mutants Y177F and Y177G have a more flexible protein structure than wild-type GR: in the range of 223 K to 277 K in 80% glycerol, both tyrosine mutants mimic the closely related enzyme dihydrolipoyl dehydrogenase. The fluorescence intensity decays of the GR enzymes can only be explained by the existence of multiple quenching sites in the protein. Although structural fluctuations are likely to contribute to the nonexponential decay and the probability of quenching by a specific site, the concept of conformational substates need not be invoked to explain the heterogeneous fluorescence dynamics.
van den Berg
P A
PA
MicroSpectroscopy Centre, Department of Biomolecular Sciences, Wageningen Agricultural University, The Netherlands.
van Hoek
A
A
Walentas
C D
CD
Perham
R N
RN
Visser
A J
AJ
D001665:Binding Sites / Q000235:genetics; D055592:Biophysical Phenomena; D001703:Biophysics; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005415:Flavins / Q000737:chemistry; D005454:Fluorescence Polarization; D005980:Glutathione Reductase / Q000737:chemistry* / Q000235:genetics; D010777:Photochemistry; D017354:Point Mutation; D011487:Protein Conformation; D013816:Thermodynamics
D005415:Flavins; D005980:Glutathione Reductase