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Journal of Chemical Sciences

, Volume 119, Issue 2, pp 123–133 | Cite as

Time-resolved fluorescence analysis of the mobile flavin cofactor in p-hydroxybenzoate hydroxylase

  • Petra A W Van Den Berg
  • Koert Grever
  • Arie Van Hoek
  • Willem J H Van Berkel
  • Antonie J W G Visser
Open Access
Article

Abstract

Conformational heterogeneity of the FAD cofactor in p-hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations; the ‘in’ conformation with the isoalloxazine ring located in the active site, and the ‘out’ conformation with the isoalloxazine ring disposed towards the protein surface. Fluorescence-lifetime analysis of these complexes revealed similar lifetime distributions for the ‘in’ and ‘out’ conformations. The reason for this is twofold. First, the active site of PHBH contains various potential fluorescence-quenching sites close to the flavin. Fluorescence analysis of uncomplexed PHBH Y222V and Y222A showed that Tyr222 is responsible for picosecond fluorescence quenching free enzyme. In addition, other potential quenching sites, including a tryptophan and two tyrosines involved in substrate binding, are located nearby. Since the shortest distance between these quenching sites and the isoalloxazine ring differs only little on average, these aromatic residues are likely to contribute to fluorescence quenching. Second, the effect of flavin conformation on the fluorescence lifetime distribution is blurred by binding of the aromatic substrates: saturation with aromatic substrates induces highly efficient fluorescence quenching. The flavin conformation is therefore only reflected in the small relative contributions of the longer lifetimes.

Keywords

Time-resolved fluorescence flavin fluorescence lifetime distribution quenching by electron transfer enzyme conformations maximum entropy method 

Abbreviations

GR

glutathione reductase

MEM

maximum entropy method

PHBH

p-hydroxybenzoate hydroxylase

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Copyright information

© Indian Academy of Sciences 2007

Authors and Affiliations

  • Petra A W Van Den Berg
    • 1
    • 2
  • Koert Grever
    • 1
  • Arie Van Hoek
    • 2
  • Willem J H Van Berkel
    • 1
  • Antonie J W G Visser
    • 1
    • 2
    • 3
  1. 1.Laboratory of BiochemistryWageningen UniversityWageningenThe Netherlands
  2. 2.Microspectroscopy CentreWageningenThe Netherlands
  3. 3.Department of Structural Biology, Faculty of Earth and Life SciencesVrije Universiteit AmsterdamAmsterdamThe Netherlands

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