Measurement of the kinetic isotope effect for the oxidation of NADH at a poly(aniline)-modified electrode

Article abstract of DOI:10.1021/ja028943e

Kinetic isotope measurements using [4,4-²H₂]NADH and [4-¹H, 4-²H]NADH have been used to investigate the mechanism of the electrochemical oxidation of NADH at poly(aniline)-poly(vinyl sulfonate)-modified electrodes. The experiments show a primary kinetic isotope effect for the reaction of 4.2. This is consistent with literature values for the corresponding isotope effect for the oxidation of NADH by two-electron oxidants in homogeneous solution. The result demonstrates that transfer of H from NADH to the modified electrode occurs in the rate-limiting step within the reaction complex. Copyright

Full text of DOI:10.1021/ja028943e

Published on Web 03/13/2003
Measurement of the Kinetic Isotope Effect for the Oxidation of NADH at a
Poly(aniline)-Modified Electrode
Philip N. Bartlett* and Evelyne Simon
Department of Chemistry, UniVersity of Southampton, Southampton, SO17 1BJ, UK
Received October 15, 2002; E-mail: p.n.bartlett@soton.ac.uk
The kinetic isotope effect has been widely used to investigate
the mechanisms of reactions in homogeneous solution. This
Communication extends the use of the technique to study the
mechanism of a reaction at a chemically modified electrode. At a
chemically modified electrode, redox molecules immobilized at the
electrode surface are used to mediate between successive one-
electron-transfer reactions at the electrode and oxidation or reduction
of the species in solution. The electrochemical oxidation of NADH
to NAD+ provides an attractive model system on which to test ideas
Figure 1. Suggested mechanism for the mediated oxidation of NADH at
a poly(aniline)-coated electrode. Reaction of the oxidized, emeraldine form
about the design of modified electrodes.1 The oxidation of NADH
of the polymer with NADH produces NAD+ and the reduced, leucoemer-
to NAD+ involves the transfer of two electrons and one proton
aldine form of the polymer which is then reoxidized at the electrode in
and cleavage of a C-H bond. Direct electrochemical oxidation of
NADH only occurs at high overpotentials and is accompanied by
rapid poisoning of the reaction because the electrode surface is
fouled by the polymeric products of side reactions which occur
because the one-electron oxidation intermediates formed in the
reaction are unstable and highly reactive. The design of chemically
modified electrodes for the oxidation of NADH is also of great
practical interest because the oxidation of NADH at low potentials
is an essential requirement in the development of amperometric
biosensors based on a wide range of NADH-dependent dehydro-
genases.
two, sequential one-electron steps to complete the catalytic cycle.
In earlier work we have shown that electropolymerized poly-
(aniline) films grown in the presence of large polymeric counter-
anions are excellent modified electrodes for NADH oxidation in
neutral solution at low potential.2-4 Using a kinetic model in which
a reactive complex first forms between the NADH and polymer
and then oxidation of NADH occurs within this reactive complex,2-4
we were able to fit the electrochemical data from an extensive set
of experiments with films of different thickness, and for poly-
(aniline) films with different counteranions. According to this model
the oxidation of NADH can be represented by
Figure 2. Plot of the oxidation current as a function of the NADH
concentration recorded at 0.10 V vs SCE at a poly(aniline)-poly(vinyl
sulfonate)-modified electrode (electrode area ) 0.38 cm2, film thickness
) 5 µm) rotated at 9 Hz in 0.1 M citrate/phosphate buffer pH 7.0 at 25 °C.
The NADH concentrations have been corrected to allow for concentration
polarization in the diffusion layer of the rotating disk. Results for two
experiments are shown. In the first experiment aliquots of [4,4-2H2]NADH
were added (2) first up to a total concentration of 3.5 mM followed by
aliquots of [4,4-1H2]NADH (b). In the second experiment aliquots of [4,4-
1H2]NADH were added (9). The solid lines show the best fit of the data to
the theory.
NADH + site T NADHsite
NADHsite f NAD+site + H+ + 2e
NAD+site T NAD+ + site
KM
kcat
(1)
(2)
(3)
of the poly(aniline)-poly(vinyl sulfonate) films used in these
experiments were carefully chosen so that we were in a regime
where the reaction of NADH occurs throughout the whole of the
film since this ensures the maximum sensitivity of the current on
kcat.2,3 Figure 2 shows plots of the oxidation current for [4,4-1H2]-
NADH and the doubly deuterated compound, [4,4-2H2]NADH as
a function of concentration. In the experiment aliquots of [4,4-2H2]-
NADH were added to the cell first and the current recorded. As
shown in the figure, the current initially increases and then reaches
at plateau at around 3.5 mM [4,4-2H2]NADH. To be sure that any
differences in the currents for the two isotopomers arise from a
kinetic isotope effect and are not caused by changes in the activity
of the poly(aniline) film between measurements, we then added
aliquots of [4,4-1H2]NADH to the cell containing 3.5 mM [4,4-
2H2]NADH. The current now increases further with increasing [4,4-
1H2]NADH concentration clearly indicating that the [4,4-1H2]NADH
reacts more rapidly at the modified electrode. The electrode was
where site represents a reaction site on the polymer, KM is the
dissociation constant for the reaction complex, and kcat is the first-
order rate constant for oxidation of NADH within the reaction
complex. We also suggested that the reason the poly(aniline)
composite films are such good mediators for NADH oxidation is
that the oxidation can occur by the transfer of a hydride from NADH
to one of the nitrogens in the poly(aniline) chain followed by two
rapid, successive one-electron oxidations of the poly(aniline) to
regenerate the catalytic site, see Figure 1.
To investigate this model in greater detail, we prepared mono-
and dideuterated samples of NADH by following standard literature
procedures5 and studied the kinetics of their reaction at the poly-
(aniline)-poly(vinyl sulfonate)-modified electrode. The thicknesses
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J. AM. CHEM. SOC. 2003, 125, 4014-4015
10.1021/ja028943e CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
are a combination of the primary kinetic isotope effect and the
secondary kinetic isotope effect.8 Combining our results from the
experiments for [4,4-2H2]NADH and [4-1H, 4-2H]NADH, we obtain
estimates for the primary and secondary kinetic isotope effects of
4.2 and 0.91.9 This value for the primary kinetic isotope effect is
within the range of those reported in the literature for the reactions
of NADH with a variety of two-electron oxidants in homogeneous
solution8,11-13 and confirms that transfer of H from the NADH to
the polymer occurs in the rate-limiting step within the reaction
complex. Our results are inconsistent with rate-limiting electron
transfer followed by proton and electron transfer and indicate that
the reaction proceeds either by hydride transfer or by hydrogen
atom transfer followed by rapid electron transfer.14 Our results
demonstrate that kinetic isotope measurements can be usefully
applied to study the mechanisms of reactions at chemically modified
electrodes.
Figure 3. Plot of the oxidation current as a function of the NADH
concentration under the same conditions as Figure 2. Results for two
experiments are shown. In the first experiment aliquots of [4-1H,4-2H]NADH
were added (2) first up to a total concentration of 3.5 mM followed by
aliquots of [4-1H2]NADH (b). In the second experiment aliquots of [4,4-
1H2]NADH were added (9). The solid lines show the best fit of the data to
the theory.
Acknowledgment. This work was supported by ONR.
Supporting Information Available: Details of preparation of
deuterated NADH samples, polymerization of poly(aniline) films, and
measurement conditions. This material is available free of charge via
the Internet at http://pubs.acs.org.
Table 1. Rate Constants for the Reaction of [4,4-1H2]NADH and
[4,4-2H2]NADH at a Poly(aniline)-Poly(vinyl sulfonate)-Modified
Electrode Derived from the Analysis of the Data in Figure 2
k
cat/s-1
KM/K , mM
kcat,HH/kcat,DD
s
[4,4-1H2]NADH
[4,4-2H2]NADH
0.100 ( 0.001
0.026 ( 0.001
3.4 ( 0.1
2.2 ( 0.2
4.0
References
(1) (a) Bartlett, P. N.; Simon, E.; Toh, C. S. Bioelectrochemistry 2002, 56,
177-122. (b) Gorton, L.; Dom´ınguez, E. Mol. Biotechnol. 2002, 82, 371-
392.
Table 2. Rate Constants for the Reaction of [4,4-1H2]NADH and
[4-1H, 4-2H]NADH at a Poly(aniline)-Poly(vinyl sulfonate)-Modified
Electrode Derived from the Analysis of the Data in Figure 3
(2) Bartlett, P. N.; Wallace, E. N. K. J. Electroanal. Chem. 2000, 486, 23-
31.
(3) Bartlett, P. N.; Birkin, P. R.; Wallace, E. N. K. J. Chem. Soc., Faraday
Trans. 1997, 93, 1951-1960.
k
cat/s-1
KM/K , mM
kcat HH/kcat,HD
,
s
(4) Bartlett, P. N.; Simon, E. Phys. Chem. Chem. Phys. 2000, 2, 2599-2606.
(5) Brown, A.; Fisher, H. F. J. Am. Chem. Soc. 1976, 96, 5682-5688.
(6) Values of kcat were calculated by assuming that each imine unit in the
emeraldine form of the polymer acts as a site for reaction and calculating
the number of imine units from the voltammetry of the film. This probably
under estimates kcat because the large size of the NADH molecule will
mean that adjacent sites will be blocked when the NADH is adsorbed.
(7) Our deuteration scheme selectively adds the first deuterium to the A-face
of the NADH molecule. See Supporting Information for details.
(8) Powell, M. F.; Bruice, T. C. J. Am. Chem. Soc. 1983, 105, 7139-7149,
(9) The values for the primary and secondary kinetic isotope effects were
calculated from the values of kcat,HH/kcat,DD and kcat,HH/kcat,HD using the
equations given by Powell and Bruice8 which assume no difference in
the reaction rate for the two faces of the molecule. For a change from sp3
to sp2 a secondary kinetic isotope effect greater than 1 is expected. The
calculated value for the apparent secondary kinetic isotope effect of 0.91
could be explained by a difference in either KM or kcat by a factor of 1.2
to 1.3 for the two faces of NADH. Such an effect has been reported for
the reaction of NADH with 4-cyano-2,6-dinitrobenzenesulfonate10 where
a factor of 1.2 difference in reaction rate was found.
[4,4-1H2]NADH
0.100 ( 0.004
0.067 ( 0.003
3.5 ( 0.3
2.4 ( 0.1
1.5
[4-1H, 4-2H]NADH
then washed and the experiment repeated to record the response to
[4,4-1H2]NADH on its own. As shown in the figure, the response
for [4,4-1H2]NADH is significantly larger than that for [4,4-2H2]-
NADH. Analysis of the data in Figure 2 using the model given in
6
our earlier paper3 gives the values for kcat and KM/KS in Table 1,
where KM/KS is the ratio of the dissociation constant for the reaction
complex to the partition coefficient for NADH within the polymer
film. From the table we see that deuteration of the NADH has little
effect on the ratio KM/KS but significantly alters the values of kcat.
This is consistent with our model and gives a kinetic isotope effect,
(10) Kurz, L. C.; Frieden, C. J. Am. Chem. Soc. 1980, 102, 4198-4203.
(11) Lee, I.-S. H.; Jeoung, E. H.; Kreevoy, M. M. J. Am. Chem. Soc. 2001,
123, 7492-7496.
k
cat,HH/kcat,DD, of 4.0. We also carried out the same experiment using
the monodeuterated compound, A-side [4-1H, 4-2H]NADH,7 Figure
3. Again there is a kinetic isotope effect, but in this case it is
considerably smaller. Table 2 gives the rate constants derived from
(12) Ohno, A.; Yasui, S.; Yamamoto, H.; Oka, S.; Ohnishini, Y. Bull. Chem.
Soc. Jpn. 1978, 51, 294-296.
(13) Ostovic, D.; Roberts, R. M. G.; Kreevoy, M. M. J. Am. Chem. Soc. 1983,
105, 7629-7631.
(14) Kitani, A.; Miller, L. L. J. Am. Chem. Soc. 1981, 103, 3595-3597.
these data, and now we find a kinetic isotope effect, kcat,HH/kcat,HD
of 1.5.8 The kinetic isotope effects measured in these experiments
,
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