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J. Biol. Chem., Vol. 281, Issue 16, 11074-11081, April 21, 2006

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

Adrie H. Westphal, Andrey Matorin, Mark A. Hink, Jan Willem Borst, Willem J. H. van Berkel, and Antonie J. W. G. Visser^¶1

From the Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, the MicroSpectroscopy Centre, P. O. Box 8128, 6700 ET Wageningen, and the ^¶Department of Structural Biology, Faculty of Earth and Life Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands

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 Förster 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 µs) 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.

Received for publication, January 20, 2006

^* This work was supported by a VLAG postdoctoral fellowship (to A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 31-317-482-862; Fax: 31-317-484-801; E-mail: ton.visser{at}wur.nl.

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