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

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
wavelength of H (used here to denote H
+
,H
•
and H
)
)
is $1A
˚
and thus similar to a typical bond length,
whereas the wavelength of deuterium is shorter by a
factor of $
ffiffiffi
2
p
. As a consequence, the position of H
(and to a lesser extent, D) is somewhat diffuse, and
H transfer may involve an appreciable degree of quan-
tum mechanical tunneling, in which H or D transfer
occurs by ‘tunnelling’ through part of the reaction
barrier rather than by passing over the barrier as is the
case in a classical transition-state reaction [22]. It is
accepted that long-range electron transfer (ET) reac-
tions occur by tunnelling [23,24] and we now have
nearly 20 years of both experimental and computa-
tional evidence demonstrating that H-tunnelling reac-
tions can also occur during enzyme-catalysed reactions

F:C:ðÞexp À
DG
z
k
B
T
!
ð1Þ
where V is the electronic coupling, F.C. is the Frank–
Condon nuclear wave function overlap (related to the
de Broglie wavelength of the H or D) and DG
à
is the
Marcus activation energy. The activation energy is
described by the driving force, DG
0
, and reorganization
energy in the standard way:
DG
z
¼ DG
0
þ k
ÀÁ
2
=4k ð2Þ
The driving force dependence of H transfer in the
flavoprotein glucose oxidase (GO) was investigated by
Brinkley & Roth [29]. The endogenous FAD was
substituted with other chemically modified flavins with

i
Dr
2
=2h
ÀÁÂÃ
exp ÀE
X
=k
B
TðÞdX
ð3Þ
where l and x are the mass and frequency of the
transferred H or D and E
x
is the environmental energy
or promoting vibration, which reduces the H-transfer
distance from an equilibrium distance, r
0
,byDr =
(r
0
) r
X
) [9,31,32]. The kinetic isotope effect (KIE =
k
H
⁄ k
D
) arises because of differences in the mass,
frequency and consequently the transfer distance of H

modest (< 10) but the temperature dependencies of
these KIEs are quite varied (Table 1).
S. Hay et al. Hydrogen tunnelling in biological systems
FEBS Journal 276 (2009) 3930–3941 ª 2009 The Authors Journal compilation ª 2009 FEBS 3931
The reductive half-reaction (RHR) of MR and PET-
NR occurs in three steps:
E
ox
þ NADðPÞH À!
binding
½E
ox
Á NADðPÞH
CT
À!
reduction=
HÀtransfer
E
red
Á NADðPÞ
þ
À!
product
release
E
red
þ NADðPÞ
þ
ð4Þ
MR reacts only with NADH, whereas PETNR has a

strates – one generic substrate being cylcohexen-1-one.
With many of the oxidative substrates tested, the oxi-
dative half-reactions of MR and PETNR are fully rate
limiting during steady-state turnover. Consequently,
the steady-state KIE on the RHR hydride transfer is
unity and steady-state analysis is clearly not appropri-
ate to study these reactions. However, in MR, we have
measured a KIE of 3.5 ± 0.2 on k
cat
for the hydride
transfer from the reduced FMN to cylcohexen-1-one
Table 1. Kinetic isotope effects observed in selected flavoproteins. CO, choline oxidase; DD, human class 2 dihydroorotate dehydrogenase;
FDTS, flavin dependent thymidylate synthase; GO, glucose oxidase; MAO A ⁄ B, monoamine oxidase A ⁄ B; MR, morphinone reductase; PAO,
L-phenylalanine oxidase; PETNR, pentaerythritol tetranitrate reductase; TMADH, trimethylamine dehydrogenase; TSOX, heterotetrameric sar-
cosine oxidase; nd, not determined or reported. The KIEs are for pre-steady-state flavin reduction by the denoted substrate unless otherwise
stated. Isotope effects are H ⁄ D unless otherwise stated.
Enzyme Substrate 1° KIE DDH
à
(kJÆmol
)1
) A
àH
: A
àD
Ref.
PETNR b-NADPH 7.0 ± 0.04
a
6.5 ± 2.76
a
0.51 ± 0.04 [15,18]

n.d. n.d. [77]
DD dihydroorotate 3.77 ± 0.08 n.d. n.d. [78]
TMADH trimethylamine 4.6 ± 0.4 0.5 ± 5.2
f
7.8 ± 1.2 [16]
PAO
L-phenylalanine 5.4 ± 0.3 0.2 ± 0.03 5.2 ± 0.2 [72]
TSOX sarcosine 7.3
e
0.6 ± 2.1 5.4 ± 0.4 [17]
a
Revised from previously reported, manuscript in preparation.
b
Data from k
cat
⁄ K
m
measurements.
c
Data not corrected for the calculated
commitment to catalysis.
d
Data from H ⁄ T isotope effect.
e
No error given.
f
Data for the H172Q mutant.
g
Calculated from the KIE and
DDH

and in the case of the oxidative half-reaction of MR,
the observed double KIE should be: 3.5 · 2.3 = 8.0,
which is in agreement with the observed value of
8.2 ± 1.4 [15]. Although it has been argued that vio-
lation of the rule of geometric mean may be used as
evidence for H-tunnelling [36–38], the oxidative KIEs
in MR are not measurably temperature dependent – a
diagnostic of ground state H-tunnelling [15].
Using a stopped-flow spectrometer, it is possible to
determine most of the rate constants for the steps in the
OYE RHR reaction (Eqn 4) above. However, care must
be made to keep the samples anaerobic by either using
an anaerobic glove box or by adding glucose ⁄ GO. The
binary complex has a characteristic p-p charge transfer
(CT) absorbance and NAD(P)H binding and dissocia-
tion can be measured by following the formation of this
CT absorbance at, for example, 555 nm while perform-
ing a concentration dependence [35,39]:
k
obs
¼ k
off
þ k
on
½NADðPÞHð6Þ
Similarly, the rate of hydride transfer can be deter-
mined because H transfer is concomitant with FMN
reduction. By following the bleaching of FMN absor-
bance at $465 nm while performing a concentration
dependence [15,18,35,39], it is possible to characterize

and the apparent rate of H transfer, k
red
, is 56 and
33 s
)1
in MR and PETNR, respectively [18,35,39].
Product (NAD(P)
+
) inhibition of MR and PETNR is
very weak suggesting that NAD(P)
+
rapidly dissoci-
ates from the active site once FMN reduction occurs.
We have been unable to measure the reverse rate of
hydride transfer, k
ox
, in either enzyme and it appears
to be close to zero [18,40]. We have also determined
the driving force for hydride transfer during the RHR
of MR with NADH to be $60 kJÆmol
)1
[40], which
is also consistent with an effectively irreversible
H transfer.
We have mutated various amino acid residues within
the active site of MR and PETNR [19,41,42]. In the
wild-type enzymes, FMN reduction occurs as a mono-
exponential process (Fig. 2) – greatly simplifying the
stopped-flow analysis. In the H186A and N189A
active-site mutants in MR (Fig. 1A), FMN reduction

coenzyme binding (no KIE on k
off
⁄ k
on
), the observed
KIE is essentially the intrinsic KIE. Using stopped-
flow methods, we have measured the temperature
dependence of the rate of H transfer in both MR and
PETNR. For convenience, we tend to measure k
obs
(in
0.20
0.15
0.10
0.05
0.00
0.01
0.0
0.1
0.00
0.05
0.10
0.15
0.20
Absorbance
Absorbance
20 40
0.1
wt
N189A

perature dependent [15,19,35]. We typically analyse
these data in terms of Eyring (transition state) theory:
ln
k
obs
T

¼ ln
k
B
h

þ
DS
z
T
À
DH
z
RT
ð8Þ
with KIE
obs
¼ k
H
obs
=k
D
obs
and the temperature dependence

and H
s
are deuterated
[18]. We have been able to use stopped-flow methods to
measure quite accurately both the magnitude and tem-
perature dependence of a-2° KIEs during the RHR in
MR and PETNR [18,40], and for hydride transfer in the
thermophilic dihydrofolate reductase (DHFR) from
Thermotoga maritima [43]. The equilibrium isotope
effect (EIE) on NAD(P)H oxidation was measured by
Cook & Cleland to be 1.13 [44]. The observation of a-2°
KIEs values larger than the EIE was rationalized by
Huskey & Schowen [36] because of coupling of the
motion between the 2° hydrogen (labelled in Fig. 1A)
and the 1° (transferred) hydrogen during an H-tunnel-
ling reaction. We have measured identical a-2° KIE val-
ues of $1.2 in MR and PETNR, which are significantly
larger than the EIE [18,40]. We have also measured the
double KIE in MR [18] and shown that in this reaction,
the rule of geometric mean (Eqn 5) is most likely vio-
lated [39]. We have shown computationally that the
H transfer in MR occurs by deep tunnelling [28] so Hus-
key & Schowen’s [36] interpretation of inflated 2° KIEs
would seem plausible. However, we have measured a
normal (KIE £ EIE) and temperature-independent a-2°
KIE in TmDHFR, yet this reaction proceeds by 50-80%
tunneling, depending on the temperature [25,43]. A simi-
lar observation has been observed in the Escherichia coli
DHFR [45]. Consequently, it would appear that inflated
a-2° KIEs may be indicative of a tunnelling contribution

95% isotopologue purity (based on
1
H NMR spectra,
see Pudney et al. [18] for examples), with the corre-
sponding impurity being the protiated coenzyme.
Kohen [49] recently developed syntheses for extremely
high-purity NADPH isotopologues and the method of
McCracken et al. [49] has been reported to yield >
99% isotopologue purity – a purity necessary when
performing competitive isotope experiments. We also
describe our synthesis of 1,4,5,6-tetrahydroNAD(P)H.
We typically prepare (R)-[4-
2
H]-NADH by the ste-
reospecific reduction of NAD
+
(500 mg) with 1-[
2
H
6
]-
ethanol (1 g) using yeast alcohol dehydrogenase
(200 U) and aldehyde dehydrogenase (100 U) in
20 mm Taps pH 8.5 (20 mL) at room temperature.
This method is a slight modification of the procedure
reported in Viola et al. [48]. (R)-[4-
2
H]-NADPH is pre-
pared through a stereospecific reduction of NADP
+

H]-glucose (150 mg) using glucose
Hydrogen tunnelling in biological systems S. Hay et al.
3934 FEBS Journal 276 (2009) 3930–3941 ª 2009 The Authors Journal compilation ª 2009 FEBS
dehydrogenase (150 U) in Taps pH 8.5 (10 mL) at
room temperature. This method is a slight modifica-
tion of the procedures reported in Ottolina et al. [50]
and the reactions typically take 3 h. (R,S)-[4,4-
2
H
2
]-
NADH is prepared by stereospecific oxidation of
(S)-[4-
2
H]-NADH (300 mg) with 100 mm cyclohexen-
1-one catalysed by 10 lm MR in Taps pH 8.5
(10 mL). The deuterated NAD
+
is purified in the same
manner as for (R)-[4-
2
H]-NADH. (R,S)-[4,4-
2
H
2
]-
NADH is then prepared through a further stereospe-
cific reduction of [4-
2
H]-NAD

$1.06 are obtained.
We purify the coenzymes using anion-exchange (Q-
Sepharose) chromatography, eluting NADH and
NADPH isotopologues (including the tetrahydro forms)
in $200 and $500 mm ammonium bicarbonate, respec-
tively [18]. All of the enzymes used in these syntheses
(excluding MR) are available from Sigma-Aldrich (St.
Louis, MO, USA) and the coenzymes are available
from Melford Laboratories (Chelsworth, UK). We use
extinction coefficients of 6.22 mm
)1
Æcm
)1
at 340 nm for
NAD(P)H isotopologues and 16.8 mm
)1
Æcm
)1
at 289
nm for NAD(P)H
4
[34]. Usually the enzymatic synthesis
of (R)-[4-
2
H]-NAD(P)H does not proceed to comple-
tion. We have observed that freezing or freeze-drying
the reaction volume before purification usually leads to
the formation of a significant impurity of undeuterated
NAD(P)H. Consequently, on this scale, it is important
to purify the reaction volume as quickly as possible after

ðÞþk
D
f
D
ð10Þ
where f
D
is the fraction of substrate deuteration, which
can usually be determined quite accurately by
1
H NMR [18,48,52] or possibly MS [18,53]. In Fig. 3,
we use Eqn (10) to model the effect of partial deutera-
tion on the observed rate and KIE of an H transfer
reaction. If k
D
is underestimated then so too will be
DDH
à
and the effect of f
D
on the apparent temperature
dependence of the KIE is also shown in Fig. 3. We
determined Eqn (10) empirically and this relationship
is quite approximate. Nevertheless, we have been able
to correct the RHRs of MR and PETNR and also the
RHR of aromatic amine dehydrogenase with benzyl-
amine using Eqn (10) [39]. However, further studies
are required to confirm the general validity of this
correction method. That fractionation can occur
emphasizes the need for: (a) care in preparing high-

0
0.0 0.2 0.4 0.6 0.8 1.0
Fr
ac
ti
o
n
deu
t
e
r
a
ti
o
n
ΔΔH
‡
/ kJ·mol
–1
Fig. 3. The effect of substrate isotopic purity on the observed rate
of deuterium transfer (filled squares) and the corresponding KIE
(open circles) (A), and on the temperature dependence (B) of a
modelled H-transfer reaction. The data are modelled using Eqn (10)
with k
H
=5s
)1
, a KIE of 5 and various values of DDH
à
.

using Northrop’s model [58], nor with a simple non-
adiabatic H-tunnelling model (e.g. Eqns 1–3) when
pressure simply causes a compression of the reaction
barrier [60]. However, we found that we could qualita-
tively model the data by invoking a promoting vibra-
tion that changes frequency with pressure [8]. We have
since refined this analysis and recently described a sim-
ple nonadiabatic H-tunnelling model which explicitly
includes pressure as a variable [61]:
k
H
=k
D
$ exp l
D
x
D
À l
H
x
H
½r
0
þ Dr:p
ÀÁ
2
=2h
no
 exp À l
D

B
C
8
6
KIE
KIE
4
2
10
8
6
4
2
10
KIE
obs
8
6
4
2
2.0
1.5
1.0
0.5
0.0
3.2
3.3
3.4
3.5
3.6

ure/kbar
1.0
0.5
0.0
Fig. 4. A variable pressure H-tunnelling model (Eqn 3) [61]. The KIE
pressure dependence is modeled when (A) the frequency of the
promoting motion or (B) the H-transfer distance changes with pres-
sure. Positive values of Dj and Dr reflect increases in frequency
and distance with pressure, respectively. The data are modeled
with j =5JÆm
)2
, r
0
= 0.52 A
˚
(KIE
0
= 5) and only one parameter in
each plot is varied. It is possible for both Dj and Dr to vary with
pressure (as we have modelled in MR) [61], causing curvature in
the KIE versus pressure plots. We have also plotted (C) the com-
bined pressure and temperature dependence of the observed KIE
on hydride transfer during the reductive half reaction of morphinone
reductase. The data are taken from Hay et al. [8]. We have not plot-
ted error bars for clarity but the average error in the KIE for this
data set is ±5% and the minimum and maximum error is 1% and
18%, respectively.
Hydrogen tunnelling in biological systems S. Hay et al.
3936 FEBS Journal 276 (2009) 3930–3941 ª 2009 The Authors Journal compilation ª 2009 FEBS
should only be used qualitatively, but is useful to esti-

active site. Further studies are required to determine
whether this is a general phenomenon.
Other experimental probes
In addition to temperature and hydrostatic pressure, it
is possible to experimentally probe enzymatic H-tun-
nelling reactions using additional experimental parame-
ters and we briefly discuss the use of varying the
solvent composition to probe the effect of solvent
dielectric and viscosity on H transfer chemistry.
It is predicted from von Smoluchowski’s theory [63]
that the rate of a diffusion-controlled (bimolecular)
reaction will be inversely proportional to the bulk
solution viscosity. The effect of viscosity on a unimo-
lecular reaction is more complicated but can be
described in combination with the Eyring equation
according to Ansari et al. [64]:
k
obs
¼
k
B
T
h
1 þr
g þr

exp
DS
z
R

enzyme. Fitness was defined as a reduction (away from
unity) in the Arrhenius pre-exponential ratio (A
D
: A
T
)
[70,71]. In a more conventional study, we found that
the magnitude and temperature dependence of the pre-
steady-state rate and KIE for proton tunnelling during
the RHR of the quinoprotein methylamine dehydroge-
nase are unchanged following the addition of 30%
glycerol – an increase in solvent viscosity of approxi-
mately two- to threefold [13]. Conversely, a decrease in
KIE and increase in apparent enthalpy for the RHR
of l-phenylalanine oxidase upon the addition of 30%
glycerol has been reported [72]. In a more systematic
study, we recently showed that the rate of coenzyme
capture decreases, whereas the rate and KIE of
hydride transfer during the RHR in MR are invariant
over a 10-fold increase in solution viscosity [20]. We
found it was possible to use a conventional stopped-
flow to make these measurements by varying the
viscosity between $0.9 and 9 cP at 25 °C with the
addition of 0–60% w ⁄ w glycerol. Addition of > 60%
glycerol leads to mixing artefacts that precluded
further measurements. The addition of glycerol to the
solvent will also reduce the solvent dielectric. We inde-
pendently probed the role of solvent dielectric on the
RHR of MR by measuring the temperature depen-
dence in this reaction in the presence of ethanol.

Acknowledgement
This work was funded by the UK Biotechnology and
Biological Sciences Research Council. NSS is a
BBSRC Professorial Fellow.
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