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,Antonie J.W.G Visser

doi:10.1074/jbc.m600609200

Adrie H Westphal, Andrey Matorin + Show 4 more

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https://doi.org/10.1074/jbc.m600609200

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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 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 micros) 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.

Highlights

A spectacular breakthrough in single-molecule fluorescence detection of flavoenzymes was reported for cholesterol oxidase, in which single enzymatic turnovers were observed in real-time by monitoring the redox state via FAD fluorescence [3, 4]
We have equipped p-hydroxybenzoate hydroxylase (PHBH) with a fluorescent Alexa488 dye that can act as sensor for the flavin redox state
The sensing is based on modulation of resonance energy transfer from the Alexa488 dye by the two redox states of the flavin

Summary

Introduction

A spectacular breakthrough in single-molecule fluorescence detection of flavoenzymes was reported for cholesterol oxidase, in which single enzymatic turnovers were observed in real-time by monitoring the redox state via FAD (flavin) fluorescence [3, 4]. The flavin molecule bound in flavoenzymes has unfavorable photophysical properties: an intrinsic high triplet yield and in most cases low molecular brightness due to severe quenching of fluorescence [16,17,18] Another approach to overcome these limitations is to use a second, strongly fluorescent donor label that can communicate with the flavin via spFRET. Single-molecule fluorescence detection can provide insight in the conformational dynamics of single flavoenzymes in relation to catalysis We illustrate this approach using the dimeric flavoprotein p-hydroxybenzoate hydroxylase (PHBH, EC 1.14.13.2), a prototype for enzymes involved in the monooxygenation of an aromatic substrate [19]. The distance between Alexa488 and the isoalloxazine ring of the FAD within the same subunit amounts to 39 Å

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