Reversible redox of NADH and NAD+ at a hybrid lipid bilayer membrane using ubiquinone

Article abstract of DOI:10.1021/ja204014s

Here, we report the reversible interconversion between NADH and NAD ⁺ at a low overpotential, which is in part mediated by ubiquinone embedded in a biomimetic membrane to mimic the initial stages of respiration. This system can be used as a platform to examine biologically relevant electroactive molecules embedded in a natural membrane environment and provide new insights into the mechanism of biological redox cycling.

Full text of DOI:10.1021/ja204014s

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Reversible Redox of NADH and NAD+ at a Hybrid Lipid Bilayer
Membrane Using Ubiquinone
Wei Ma,† Da-Wei Li,† Todd C. Sutherland,‡ Yang Li,† Yi-Tao Long,*,† and Hong-Yuan Chen§
†Shanghai Key Laboratory of Functional Materials Chemistry and Department of Chemistry, East China University of Science and
Technology, 130 Meilong Road, Shanghai 200237, P.R. China
‡Department of Chemistry, University of Calgary, 2500 University Drive NW, Calgary, Canada T2N1N4
§Key Laboratory of Analytical Chemistry for Life Science and Department of Chemistry, Nanjing University, 22 Hankou Road, Nanjing
210093, P.R. China
S Supporting Information
b
hydrophobic core of the phospholipid bilayer of the inner
membrane of mitochondria.14 As an essential cofactor, ubiqui-
ABSTRACT: Here, we report the reversible interconver-
sion between NADH and NAD+ at a low overpotential,
none also serves as a mobile carrier, transferring electrons and
protons in the respiratory chain,15 generating interest as a system
which is in part mediated by ubiquinone embedded in a
biomimetic membrane to mimic the initial stages of respira-
tion. This system can be used as a platform to examine
biologically relevant electroactive molecules embedded in a
natural membrane environment and provide new insights
into the mechanism of biological redox cycling.
to clarify electron-/proton-transfer processes. On the other hand,
the NADH/NAD+ coenzyme couple is one of the most im-
portant redox mediators and functions as biology’s reducing
agent.16 The electrochemical oxidation of NADH to enzymati-
cally active NAD+ has attracted attention because NADH
participates in a variety of enzymatic reactions, including more
than 300 dehydrogenases.17 Interestingly, the direct electroche-
mical oxidation of NADH at conventional electrodes is both
electrochemically irreversible and requires high overpotentials.16
This overpotential was overcome by using redox mediators, such
as quinones.19À21 The interaction between the mediator and
NADH oxidation has been explained in terms of a hydride
transfer mechanism in which the mediator accepts a hydride.22
To our knowledge, despite extensive efforts to overcome the
NADH overpotential with redox mediators, reversible electro-
chemical interconversion between NADH and NAD+ to mimic the
reactions present in the respiratory chain has not been reported. The
only reported study that shows the reversible interconversion
between NADH and NAD+ without application of an overpotential
is mediated by complex I.23 Furthermore, the redox wave for the
NADH/NAD+ complex I-mediated redox couple is sigmoidal, has a
large peak-to-peak separation, and was carried out in an aqueous
solution, not in a biomimetic membrane environment.
Herein, we report a biomimetic membrane model in which
ubiquinone is embedded in a HBM that contains the NADH/
NAD+ redox couple (QnS-HBM-NADH/NAD+) as shown in
Scheme 1. The findings represent the first report of reversible
NADH/NAD+ interconversion caused by a ubiquinone media-
tor in a biomimetic membrane model. The first step to form the
membrane model is the modification of a gold electrode surface
with a disulfide derivative of ubiquinone using SAM techniques.
Three ubiquinone-terminated disulfides with different alkyl
spacers (QnS, n = 1, 5, and 10, respectively)24 were synthesized
and used to modify the gold electrodes (Supporting Information
[SI]), assess monolayer stability, and modulate the kinetics of
charge transfer. After SAM formation, the ubiquinone-modified
gold electrode was then incubated in a vesicle solution, which is
uring the initial stages of respiration, nicotinamide adenine
D
dinucleotide (NADH):ubiquinone oxidoreductase (complex I)
catalyzes a two-electron transfer from NADH to ubiquinone,
coupled to a transmembrane four-proton translocation, helping
to provide the proton-motive force required for the synthesis of
adenosine triphosphate.1 The well-known ‘Q cycle’ model in
mitochondria is based on the tight coupling of both NADH and
ubiquinone.2
Although the interactions between NADH and ubiquinone
have been studied extensively in biological systems, a biomimetic
model system has been elusive because of the inherent complexity.
Recent insight into the structures of electrode-supported hybrid
bilayer membranes (HBMs) has provided a platform to investi-
gate biological applications.3À6 A typical HBM system consists of
a phospholipid layer physisorbed to a self-assembled monolayer
(SAM) of alkanethiols that are covalently attached to a gold substrate.
The polar head groups of the phospholipids are orientated away
from the gold surface to the aqueous solution, and the hydrophobic
tails orient toward the hydrophobic SAM.7À10 The alkylthiols
typically form a complete hydrophobic layer at the gold substrate
and provide the driving force for the formation of HBM.8
Although HBM strategies are diverse and well-established, there
are very few examples of embedding redox-active molecules in a
HBM system.11À13 When redox molecules self-assemble onto
the surface of a gold substrate, a HBM is easily formed by the
adsorption of lipid vesicles to produce a supported hybrid lipid
bilayer, as illustrated in Scheme 1. The HBM environment re-
sembles the biological membrane, which can dramatically affect
the properties of the embedded redox molecule.13
Unlike most redox molecules, ubiquinone, also known as
coenzyme Q, is composed of the redox active quinoid moiety
possessing a tail of long isoprenoid units and is found at the
Received: May 2, 2011
Published: July 20, 2011
r
2011 American Chemical Society
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Scheme 1. Structures of Three Ubiquinone-Terminated
Disulfides (QnS) and Schematic of Biomimetic Membrane
Containing Ubiquinones and NADH/NAD+ Redox Systems
(QnS-HBM-NADH/NAD+)
consistent with the formation of a HBM and indicates that
NADH/NAD+ do not induce large defects in the HBM. The
high-resolution XPS spectra (Figure S23, SI) and TM-AFM
images (Figure S24, SI) also confirmed the formation of HBM
layers and the inclusion of NADH/NAD+. Panels aÀc of Figure 1
show cyclic voltammetry (CV) redox waves associated with the
2eÀ, 2H+ redox reaction of ubiquinones (Q1S, Q5S, and Q10S)
that are embedded in the HBM without NADH/NAD+ coen-
zyme in 0.1 M phosphate buffer solution (PBS, pH 7.0) at a scan
rate of 100 mV sÀ1. Note the resulting CV waves are consistent
3
with water being present in the HBM. The formal redox potentials
of Q1S, Q5S, and Q10S experience slight changes of À0.11,
À0.10, and À0.08 V, respectively, perhaps attributed to the
bilayer depth penetration. The formal redox potentials of QnS in
the HBMs are in agreement with reported values of ubiquinone
at À0.13 V vs SCE of pH 7.0 (Table S2, SI). The three ubi-
quinones, QnS-HBM, of Figure 1aÀc qualitatively show an increase
in peak separation, indicating a slowing of charge transfer rate
with increasing alkyl chain lengths. The slower charge transfer
kinetics associated with longer alkyl chains is commonly observed in
SAMs of redox-active molecules27 and is consistent with an
electron transfer process that is forced to proceed at a larger
distances between ubiquinone and electrode surface. A quanti-
tative kinetic analysis, using Laviron’s formalism, is carried out in
the SI (Figures S25ÀS26), and the results are consistent with a
two-electron, two-proton transfer process28 for surface-confined
quinone monolayers. On the basis of the peak-to-peak separation
between the oxidation and reduction waves, the voltammetric
responses range from quasi-reversible for Q1S-HBM to irrever-
sible for Q5S-HBM and Q10S-HBM.
Panels dÀf in Figure 1 show the same QnS-HBMs that now
contain the lipid-embedded NADH/NAD+. The ubiquinone in
the HBM still undergoes similar redox chemistry as described
above; however, a second redox event is evident after NADH/
NAD+ has been immobilized in the HBMs (Q1S-HBM-NADH/
NAD+, Q5S-HBM-NADH/NAD+, and Q10S-HBM-NADH/NAD+,
respectively). Importantly, the new redox couple is ascribed to
the reversible NADH/NAD+ redox reaction at a low overpotential.
The role of the lipid bilayer in the reversible NADH/NAD+
redox reaction was confirmed by forming a QnS-HBMs system
that does not contain NADH/NAD+ in the lipid membrane, and
instead NADH/NAD+ was added to the electrolyte. With NADH/
NAD+ in solution (not embedded in the bilayer membrane) CV
experiments were conducted to probe the reversible nature of the
NADH/NAD+ redox reaction; reversible redox peaks were not
observed, and NADH was only irreversibly oxidized at a potential
of ∼0.60 V vs SCE (Figure S27, SI). Lipid confinement of
NADH/NAD+ plays two critical roles: (1) higher localized
concentration, thus more collisional probability between ubiqui-
none and NADH/NAD+; (2) the hydrophobic environment of
lipid favors communication between ubiquinone and NADH/
NAD+. To confirm that reversible NADH/NAD+ interconver-
sion was mediated by surface-confined ubiquinone rather than
lipid confinement only, a control experiment in which QnS was
replaced by an alkylthiol monolayer (Figure S28, SI) in a NADH/
NAD+ embedded HBM was conducted and resulted in irrever-
sible oxidation of NADH at a potential (∼0.60 V vs SCE). We
propose that the surface-attached ubiquinone acts as an efficient
mediator to enhance the heterogeneous proton-coupled electron
transfer reaction in the oxidation reaction of NADH to NAD+.
The reverse reduction reaction of NAD+ to NADH conversion
is unlikely mediated by ubiquinone due to the difference in
Figure 1. CVs at 100 mV sÀ1 of the ubiquinone HBM system in
3
the absence (aÀc) and presence (dÀf) of embedded NADH/NAD+.
Q1S-embedded HBM system (a and d), Q5S-embedded HBM system
(b and e), and Q10S-embedded HBM system (c and f) .
known to form suspended lipid bilayers by fusion with the surface
of SAM-modified gold electrode.7À10 A subset of vesicles also
contained embedded NADH/NAD+ following established pro-
cedures of Fainstein25 and Bourdillon.26 This approach to sup-
ported bilayer formation allows for control of the ubiquinone
electron transfer rate (by tether length modification) and inves-
tigations into communication between ubiquinone and the NADH/
NAD+ redox couple in a biologically relevant medium.
HBMs were characterized with electrochemical impedance
spectroscopy (EIS), high-resolution X-ray photoelectron spectro-
scopy (XPS), and tapping-mode atomic force microscopy (TM-
AFM). The observed capacitance changes in EIS (Figure S22, SI)
of the electrode after exposure to the lipid vesicle solution is
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is observed at 1071 cmÀ1. The intense peak position is con-
sistent with the ring breathing mode of the oxidized nicotinamide
ring for NAD+ at 1032 cmÀ1 32,33 considering the influence of the
,
lipid membrane and ubiquinone molecules at the gold mesh
electrode. Interestingly, the SERS bands changed significantly
with variation in the applied voltage to À0.50 V, as shown in
Figure 2, curve b. Two main features can be highlighted from the
spectrum curve b. First, the strong diagnostic peaks at 1180 cmÀ1
for NADH,33,34 which must have a significant contribution from
a reduced nicotinamide ring mode in NADH. Second, though the
overall shape of the Raman spectra is conserved, some subtle
changes occur, suggesting the formation of different electroche-
mical-dependent NADH/NAD+ species. The new peaks indicate
that the NAD+ molecules are transformed to NADH at suffi-
ciently negative potentials. After applying an oxidizing potential
of +0.50 V, the diagnostic NADH peak at 1180 cmÀ1 disappears
and the characteristic band of 1071 cmÀ1 of the oxidized nico-
tinamide ring is observed, as shown in Figure 2, curve c. Clearly,
Figure 2 shows that changes in the Raman peaks are consistent
with NAD+ molecules being electrochemically transformed to
NADH species reversibly as a function of applied potential.
Practical methods for the redox cycling of NADH and NAD+
are of significant interest in biocatalysis.35,36 More importantly,
enzymatically active NADH/NAD+ should be regenerated to
allow enzymes to continue their turnover because hundreds of
dehydrogenase enzymes rely on the NADH/NAD+ coenzyme
couple in biological systems.17 However, direct electrochemical
NADH oxidation at a bare electrode surface does not permit
regeneration of the biologically active NAD+. Here, we performed
enzymatic experiments on the Q5S-HBM on a larger-scale gold
mesh electrode in a solution containing NADH dispersed in PBS,
such that absorbance changes in solution could be monitored by
UVÀvis spectroscopy. As shown in Figure S29a (SI), the UVÀvis
characteristic absorption peak of NADH at 340 nm disappears slowly
at an applied potential of ∼0.50 V vs Ag/AgCl. Alcohol dehydro-
genase (ADH) catalyzes the oxidation of ethanol to acetaldehyde in
the presence of NAD+, which is reduced to NADH. To confirm that
the NAD+ produced electrochemically was active in a biological
system, ADH and ethanol were added to the solution. As shown in
Figure S29b (SI), the absorption peak at 340 nm gradually increases,
demonstrating that the electrochemically produced NAD+ can be
used enzymatically to drive the conversion of ethanol to acetaldehyde.
In conclusion, we synthesized three ubiquinone-terminated
disulfides with different alkyl spacers to form SAMs on gold
electrodes. Biomimetic lipid bilayer membranes were then formed
on the SAMs that contained embedded NAD+ and NADH.
Importantly, we have shown that reversible interconversion
between NADH and NAD+ could occur at a low overpotential
when both ubiquinone, as a redox mediator, and NADH/NAD+
were embedded in a lipid bilayer. Further evidence for the
reversible interconversion NADH/NAD+ was obtained by
in situ SERS, and spectroelectrochemical UVÀvis experiments
confirmed that the electrochemical NADH oxidation at the
ubiquinone HBM allows for the regeneration of biologically
active NAD+. Furthermore, this biomimetic membrane system
could be useful as a platform to examine several biologically
relevant electroactive molecules in lipid bilayer membranes.
Figure 2. SERS spectra of Q5S-HBM with embedded NAD+ on SERS-
active rough gold mesh substrates (Q5S-HBM-NAD+) as a function of
applied potential measured using a 785-nm laser at 10 mW with 20 s of
acquisition time. (a) Open circuit potential (OCP); (b) applied potential of
À0.50 V vs Ag/AgCl; (c) applied potential of +0.50 V vs Ag/AgCl.
reduction peak potentials in CVs dÀf in Figure 1. Further, the
observed NADH/NAD+ formal potential in the HBMs is shifted
compared those in the literature at À0.56 V vs SCE of pH 7.0
(Table S2, SI) and may reflect a higher proton concentration in
the HBM than in the bulk solution. Cumulatively, these experi-
ments suggest that reversible interconversion between NADH
and NAD+ occurs at a low overpotential when both ubiquinone
and NADH/NAD+ are immobilized in lipid bilayers.
Interestingly, the CV experiments of the reversible NADH
and NAD+ redox reaction show a kinetic difference, as assessed
by the peak separation that parallels the kinetics of the ubiqui-
none charge transfer reaction. The change in the rate of NADH/
NAD+ reaction could result from a specific interaction between
ubiquinone and either nicotinamide or adenine. Alternatively,
the NADH/NAD+ may not have easy access to the gold surface
with the more tightly packing, longer alkyl chains and the greater
distance in the Q10S monolayer. The kinetics of the NADH/
NAD+ redox reaction further highlights the important role that
the ubiquinone and lipid membrane must play in mediating the
charge transfer reaction.
Surface enhanced Raman scattering (SERS) is a tool to monitor
electrochemical phenomena even down to single molecule
levels29 and optical modulation30,31 was used here to monitor the
reversible electrochemical interconversion between NADH and
NAD+ in the biomimetic membrane model.
In situ SERS spectroelectrochemical experiments were used to
monitor NAD+ and NADH by electrochemical modulation. To
clearly elucidate the interconversion between NAD+ and NADH,
only NAD+ was immobilized in the lipid bilayer for SERS study,
and Figure 2 shows the SERS spectra obtained under different
potentials. The spectra were acquired as a function of the applied
potential in 0.1 M PBS (pH 7.0) on a Q5S-HBM-NAD+ SERS-
active gold mesh substrate. The potential was scanned from open
circuit potential to À0.5 V then to +0.50 V, such that NAD+ evolves
from its oxidized state (NAD+), to being fully reduced (NADH)
and returns to its oxidized form (NAD+). The nicotinamide ring
structures of NAD+ and NADH have unique spectroscopic signa-
tures and yield Raman spectral changes in intensity and peak position.
An initial spectrum of NAD+ acquired before an applied voltage
is shown as Figure 2, curve a. Under open circuit conditions, a sig-
nificant signal from the intense SERS band of Q5S-HBM-NAD+
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental details. This material
b
is available free of charge via the Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION
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R. P. J. Am. Chem. Soc. 2007, 129, 16249–16256.
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O.; Woo, H. Y.; Loꢀpez Navarrete, J. T. J. Am. Chem. Soc. 2004, 126,
13363–13376.
Corresponding Author
ytlong@ecust.edu.cn
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’ ACKNOWLEDGMENT
This research was supported by the Natural Science Foundation of
China (Grant No. 91027035, 20933007), the Fundamental Research
Funds for the Central Universities (Grant No. WK1013002) and the
open research fund from Key Laboratory of Analytical Chemistry for
Life Science of Nanjing University. Y.T.L. is supported by The
Program for Professor of Special Appointment (Eastern Scholar) at
Shanghai Institutions of Higher Learning.
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