Real-time Enzyme Dynamics Illustrated with Fluorescence Spectroscopy of p-Hydroxybenzoate Hydroxylase

A.H. Westphal, A. Matorin, M.A. Hink, J.W. Borst, W.J.H. van Berkel, A.J.W.G. Visser

Research output: Contribution to journalArticleAcademicpeer-review

19 Citations (Scopus)

Abstract

We have used the flavoenzyme p-hydroxybenzoate hydroxylase (PHBH) to illustrate that a strongly fluorescent donor label can communicate with the flavin via single-pair Forster resonance energy transfer (spFRET). The accessible Cys-116 of PHBH was labeled with two different fluorescent maleimides with full preservation of enzymatic activity. One of these labels shows overlap between its fluorescence spectrum and the absorption spectrum of the FAD prosthetic group in the oxidized state, while the other fluorescent probe does not have this spectral overlap. The spectral overlap strongly diminished when the flavin becomes reduced during catalysis. The donor fluorescence properties can then be used as a sensitive antenna for the flavin redox state. Time-resolved fluorescence experiments on ensembles of labeled PHBH molecules were carried out in the absence and presence of enzymatic turnover. Distinct changes in fluorescence decays of spFRET-active PHBH can be observed when the enzyme is performing catalysis using both substrates p-hydroxybenzoate and NADPH. Single-molecule fluorescence correlation spectroscopy on spFRET-active PHBH showed the presence of a relaxation process (relaxation time of 23 mus) that is related to catalysis. In addition, in both labeled PHBH preparations the number of enzyme molecules reversibly increased during enzymatic turnover indicating that the dimer-monomer equilibrium is affected.
Original languageEnglish
Pages (from-to)11074-11081
JournalJournal of Biological Chemistry
Volume281
Issue number16
DOIs
Publication statusPublished - 2006

Keywords

  • single-molecule kinetics
  • pseudomonas-fluorescens
  • conformational dynamics
  • crystal-structure
  • energy-transfer
  • wild-type
  • catalysis
  • protein
  • binding
  • resolution

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