Evidence for a novel mechanism of time-resolved flavin fluorescence depolarization in glutathione reductase.

P.A.W. van den Berg, A. van Hoek, A.J.W.G. Visser

Research output: Contribution to journalArticleAcademicpeer-review

24 Citations (Scopus)

Abstract

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 GR than for the tyrosine mutants. These results suggest that the rapid depolarization in wild-type GR originates from an interaction with the flavin-shielding tyrosine, and not from restricted reorientational motion of the flavin. A novel mechanism of fluorescence depolarization is proposed that involves a transient charge-transfer complex between the tyrosine and the light-excited flavin, with a concomitant change in the direction of the emission dipole moment of the flavin. This interaction is likely to result from side-chain relaxation of the tyrosine in the minor fraction of enzyme molecules in which this residue is in an unsuitable position for immediate fluorescence quenching at the moment of excitation. Support for this mechanism is provided by binding studies with NADP+ and 2'P-5'ADP-ribose that can intercalate between the flavin and tyrosine and/or block the latter. Fluorescence depolarization analyses as a function of temperature and viscosity confirm the dynamic nature of the process. A comparison with fluorescence depolarization effects in a related flavoenzyme indicates that this mechanism of flavin fluorescence depolarization is more generally applicable.
Original languageEnglish
Pages (from-to)2577-2586
Number of pages10
JournalBiophysical Journal
Volume87
Issue number4
DOIs
Publication statusPublished - 2004

Keywords

  • escherichia-coli
  • lipoamide dehydrogenase
  • nadh peroxidase
  • binding-site
  • polarized fluorescence
  • azotobacter-vinelandii
  • adenine-dinucleotide
  • angstrom resolution
  • crystal-structure
  • energy-transfer

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