Tài liệu Báo cáo khoa học: Structural and mechanistic aspects of flavoproteins: probes of hydrogen

MINIREVIEW

Structural and mechanistic aspects of flavoproteins:

probes of hydrogen tunnelling

Sam Hay, Christopher R. Pudney and Nigel S. Scrutton

Manchester Interdisciplinary Biocentre and Faculty of Life Science, University of Manchester, UK

Introduction

There is now fairly widespread recognition that

enzyme-catalysed C–H bond cleavage reactions can

occur by quantum mechanical tunnelling [1–5]. The

role of protein dynamics in these reactions is still hotly

debated and it has been proposed that promoting

vibrations, nonequilibrated fast (sub-ps) dynamics,

could modify the reaction barrier and profoundly

influence the reaction rate [4,6–12]. In recent years, we

have investigated H-transfer reactions in a number of

enzymes, primarily quinoprotein [4,13,14] and flavo￾protein [8,15–20] systems. Using a combination of

experimental and computational approaches, we have

shown that H-transfer reactions can occur by ‘deep’

tunnelling and the reaction can be enhanced by local￾ized dynamics in the enzyme active site – putative pro￾moting vibrations. Although it is fairly well established

that enzymatic H transfers often involve tunnelling,

the role of promoting vibrations remains contentious

[21]. In this minireview, we describe experimental

methods we have recently employed and developed to

probe the role of environmental coupling ⁄ promoting

vibrations in H-transfer reactions in the Old Yellow

Enzyme (OYE) family of flavoproteins.

Hydrogen tunnelling

Because of wave ⁄ particle duality, electrons and light

atoms have appreciable (de Broglie) wavelengths. The

Keywords

high pressure; H-tunneling; kinetic isotope

effect; kinetic isotope fractionation; multiple

reactive conformations; Old Yellow Enzyme;

promoting vibration; protein dynamics;

quantum mechanics; stopped-flow kinetics

Correspondence

N. S. Scrutton, Manchester Interdisciplinary

Biocentre and Faculty of Life Science,

University of Manchester, 131 Princess

Street, Manchester M1 7ND, UK

Fax: +44 161 306 8918

Tel: +44 161 306 5152

E-mail: [email protected]

(Received 23 December 2008, revised 28

April 2009, accepted 1 May 2009)

doi:10.1111/j.1742-4658.2009.07121.x

At least half of all enzyme-catalysed reactions are thought to involve a

hydrogen transfer. In the last 10 years, it has become apparent that many

of these reactions will occur, in part, or in full, by quantum mechanical

tunnelling. We are particularly interested in the role of promoting vibra￾tions on H transfer, and the Old Yellow Enzyme family of flavoproteins

has proven to be an excellent model system with which to examine such

reactions. In this minireview, we describe new and established experimental

methods used to study H-tunnelling in these enzymes and we consider

some practical issues important to such studies. The application of these

methods has provided strong evidence linking protein dynamics and H-tun￾nelling in biological systems.

Abbreviations

DHFR, dihydrofolate reductase; EIE, equilibrium isotope effect; ET, electron transfer; GO, glucose oxidase; KIE, kinetic isotope effect; MR,

morphinone reductase; OYE, Old Yellow Enzyme; PETNR, pentaerythritol tetranitrate reductase; RHR, reductive half-reaction.

3930 FEBS Journal 276 (2009) 3930–3941 ª 2009 The Authors Journal compilation ª 2009 FEBS