Electrochemistry of the [4Fe4S] cluster in base excision repair
proteins: tuning the redox potential with DNA
Phillip L. Bartels
†
,
Andy Zhou
†
,
Anna R. Arnold
†,3
,
Nicole N. Nuñez
‡
,
Frank N. Crespilho
§
,
Sheila S. David
‡
, and
Jacqueline K. Barton
†,*
†
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
CA 91125
‡
Department of Chemistry, University of California Davis, Davis, CA 95616
§
Instituto de Química de São Carlos, University of São Paulo, Brazil
Abstract
Escherichia coli
Endonuclease III (EndoIII) and MutY are DNA glycosylases that contain [4Fe4S]
clusters and that serve to maintain the integrity of the genome after oxidative stress.
Electrochemical studies on highly-oriented pyrolytic graphite (HOPG) revealed that DNA binding
by EndoIII leads to a large negative shift in midpoint potential of the cluster, consistent with
stabilization of the oxidized [4Fe4S]
3+
form. However, the smooth, hydrophobic HOPG surface is
non-ideal for working with proteins in the absence of DNA. In this work, we use thin film
voltammetry on a pyrolytic graphite edge electrode to overcome these limitations. Improved
adsorption leads to substantial signals for both EndoIII and MutY in the absence of DNA, and a
large negative potential shift is retained with DNA present. In contrast, the EndoIII mutants
E200K, Y205H, and K208E, which provide electrostatic perturbations in the vicinity of the
cluster, all show DNA-free potentials within error of wild type; similarly, the presence of
negatively charged poly-L glutamate does not lead to a significant potential shift. Overall, binding
to the DNA polyanion is the dominant effect in tuning the redox potential of the [4Fe4S] cluster,
helping to explain why all DNA-binding proteins with [4Fe4S] clusters studied to date have
similar DNA-bound potentials.
Graphical abstract
*
Corresponding Author: to whom correspondence should be addressed at jkbarton@caltech.edu.
3
Present address: Intel Corporation, Hillsboro, OR 97124
Author Contributions
All authors have given approval to the final version of the manuscript.
Supporting Information. This material is available free of charge via the Internet at
http://pubs.acs.org
: Background CV scans, scan
rate dependence of protein signals on PGE, [Ru(NH
3
)
6
]
3+
CVs on PGE.
HHS Public Access
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Published in final edited form as:
Langmuir
. 2017 March 14; 33(10): 2523–2530. doi:10.1021/acs.langmuir.6b04581.
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INTRODUCTION
E. coli
endonuclease III (EndoIII) is a DNA glycosylase that excises oxidized pyrimidines
from DNA, functioning as part of the base excision repair (BER) pathway in order to
maintain the integrity of the genome (
1
). EndoIII contains a [4Fe4S]
2+
cluster that is
relatively insensitive to reduction and oxidation in solution (
2
); as a result, it was initially
proposed that the cluster served only a structural role within the protein. MutY is another
E.
coli
BER glycosylase, homologous to EndoIII, that also contains a [4Fe4S]
2+
cluster (
3
).
MutY, found in organisms from bacteria to man, is involved in the repair of oxoG:A
mismatches (
4
); in humans, inherited defects in MUTYH are associated with a familial form
of colon cancer known as MUTYH-associated polyposis (MAP) and many MAP-associated
variants are localized near the [4Fe4S] cluster (
4
). Furthermore, in the case of MutY, it has
been shown that the cluster is not required for folding or stability (
3
), or direct participation
in the intrinsic glycosidic bond hydrolysis catalysis (
5
), making the widespread presence of
conserved, non-catalytic [4Fe4S] clusters difficult to explain.
Notably, the earliest studies with EndoIII and MutY looked only at free protein in solution,
neglecting the effect of DNA binding on redox potential. Experiments carried out on DNA-
modified electrodes have demonstrated that, in both EndoIII and MutY, the cluster
undergoes a negative shift in potential associated with binding to the DNA polyanion and is
activated toward reversible redox activity (
6
). In these experiments, DNA monolayers were
formed on gold electrodes, and, upon addition of EndoIII or MutY, a reversible signal with a
midpoint potential ranging from 60–95 mV versus NHE was observed. Importantly, the
introduction of just a single mismatch or abasic site into DNA led to signal attenuation,
showing that electron transfer between the protein and the electrode was through the
π
-
stacked base pairs in a process known as DNA-mediated charge transport (DNA CT) (
7
). In
this process, charge is funneled from the electrode surface through the
π
-stack of the DNA
bases to reach the redox probe (a protein in this case); the only requirement is that the probe
must be electronically coupled to the DNA
π
-stack. Remarkably, the sensitivity to base
stacking observed with EndoIII and MutY was comparable to that obtained using small
molecules such as Nile blue or methylene blue that intercalate directly into the base stack.
The expanded potential window of highly-oriented pyrolytic graphite (HOPG) and the
ability to form pyrene-modified DNA films on the surface made it possible to directly
compare the potential of proteins in the presence and absence of DNA (
8
). Experiments with
EndoIII revealed that DNA binding shifts the reduction potential of the [4Fe4S]
3+/2+
couple
by −200 mV to favor oxidation. Thermodynamically, this shift corresponded to a large (~3
orders of magnitude) increase in the DNA binding affinity of the oxidized form of the
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protein. Crystal structures of EndoIII and MutY with and without DNA do not show any
significant structural change upon DNA binding (
9
–
12
), so this dramatic result was
attributed to a combination of electrostatic effects resulting from the negatively charged
DNA backbone and decreased solvent accessibility of the cluster in DNA-bound protein,
which is in agreement with the known sensitivity of [4Fe4S] clusters to their local
environment (
13
). By demonstrating that DNA binding brought the redox potential of
EndoIII into a biologically relevant window, this result served to explain the previously
observed redox insensitivity of free EndoIII and provided evidence in favor of a redox role
for the DNA-bound protein cluster.
Since these experiments were carried out, a wide range of DNA processing enzymes have
been revealed to contain [4Fe4S] clusters with properties similar to EndoIII and MutY.
These include the
Archaeoglobus fulgidus
uracil DNA glycosylase (UDG), archaeal and
eukaryotic versions of the nucleotide excision repair helicase XPD and the
E. coli
R-loop
maturation helicase DinG (
14
), all of which were found to have similar DNA-bound
potentials (~80 mV versus NHE) as measured on DNA-modified gold electrodes (
6
,
7
). The
similar DNA-bound midpoint potentials and picosecond kinetics of DNA CT together
suggested that DNA CT could provide a means for these enzymes to localize efficiently to
the vicinity of their target lesions (
15
). Indeed, experiments carried out both
in vitro
and
in
vivo
have led to the development of a model for DNA repair in which two [4Fe4S] cluster
proteins use DNA CT to communicate with each other over long molecular distances via
electron transfer self-exchange reactions (
7
,
15
). As evidenced through the potential shift,
DNA binding activates the proteins toward oxidation to the [4Fe4S]
3+
state (
8
). When the
DNA intervening between the two proteins is undamaged, the self-exchange reaction can
proceed efficiently, with the result that one of the DNA-bound proteins is reduced and its
affinity for DNA lowered. This protein is then free to diffuse to another region of the
genome. However, in the case of an intervening mismatch or lesion that impairs CT by
disrupting
π
-stacking, this self-exchange reaction is inhibited. Both proteins then remain
bound to the DNA in the vicinity of the lesion, significantly reducing the range over which
the slower processes of diffusion must occur and facilitating repair of a relatively large
genome on a biologically relevant time scale (
15
).
While DNA binding is clearly of critical importance to the redox activity of these enzymes,
it is not clear that it represents the only way to modulate the potential. It was recently
reported that carboxylic acid monolayers had a similar activating effect as DNA, although, in
contrast with the above model, they identified the relevant couple as the [4Fe4S]
2+/+
rather
than the [4Fe4S]
3+/2+
couple (
16
). With respect to the latter point, the high potential of the
reversible DNA-bound signal on both gold and HOPG, EPR spectroscopy of oxidized DNA
bound EndoIII and MutY, and the observation of both couples in the expected potential
regimes on HOPG support the original [4Fe4S]
3+/2+
assignment (
6
,
8
,
15
). Furthermore, this
assignment is in agreement with the known potential ranges accessed by the [4Fe4S]
3+/2+
couple of HiPIPs (
17
). Regardless of redox couple assignment, the possibility of activation
by other molecules remains an interesting point deserving further investigation.
In addition to other molecules, charged amino acid residues near the cluster might also be
expected to affect the potential. This was explored in a recent study in which several EndoIII
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mutants, E200K, Y205H, and K208E, were prepared and extensively characterized on DNA-
modified gold electrodes; although these residues are located within 5 Å of the cluster, all of
the mutants had indistinguishable DNA-bound midpoint potentials (
18
). Overall, these
observations suggested that DNA binding was the dominant environmental effect in
modulating potential, but the narrow accessible potential window on gold prevented further
investigation.
In this work, we used direct electrochemistry on carbon electrodes to address the capacity of
DNA and other polyanions to activate [4Fe4S] proteins for redox activity and to assess the
ability of local electrostatics to shift the potential of EndoIII in the absence of DNA.
Because the hydrophobic surface of HOPG is unsuitable for protein adsorption and difficult
to prepare (
8
,
19
–
20
), we turned to the rougher, more hydrophilic pyrolytic graphite edge
(PGE) electrode for these experiments, using the technique of thin film voltammetry to
immobilize proteins in a stable layer on the electrode surface (
21
–
24
). To enhance signal
sizes, we also included single-walled carbon nanotubes (CNT) when possible, taking
advantage of their high conductivity and the additional 3-dimensional surface area they can
provide for protein binding (
21
). In summary, this platform provided an ideal and reliable
way to improve our understanding of the factors important to tuning the potential of DNA
processing enzymes containing [4Fe4S] clusters.
MATERIALS and METHODS
EndoIII Overexpression and Purification
WT
E. coli
EndoIII was overexpressed in BL21star-(DE3)pLysS cells containing a pET11-
ubiquitin-His
6
-
nth
construct and purified as detailed previously (
18
), with the exception that
the final buffer contained 10% rather than 20% glycerol (20 mM sodium phosphate, pH 7.5,
0.5 mM EDTA, 150 mM NaCl, 10% glycerol). For electrochemical experiments, glycerol
was removed from the protein solution using a HiPrep 26/10 desalting column (GE
Healthcare) equilibrated with a buffer consisting of 20 mM sodium phosphate, pH 7.5, 0.5
mM EDTA, 150 mM NaCl. Following buffer exchange, the protein was concentrated in two
steps. First, 10,000 molecular weight cutoff (MWCO) Amicon Ultra 15 mL centrifugation
filter units (Millipore) were used to concentrate each protein solution to a total volume of 1
mL or less. Samples were then transferred to 10,000 MWCO Amicon Ultra 0.5 mL
centrifugation filter units (Millipore) and concentrated until the initially yellow protein
solutions were very dark in color (approximately 300 μL final volume from 6 L of bacterial
culture). Protein purity was confirmed by SDS-PAGE. Immediately following concentration
of the sample, the [4Fe4S] cluster loading ratio was calculated by dividing the total [4Fe4S]
cluster concentration as determined from the UV-visible absorbance spectrum using
ε
410
=
17,000 M
−1
cm
−1
by the total protein concentration as measured in a Bradford assay; typical
cluster loading ratios for WT EndoIII were 70–75%.
MutY Overexpression and Purification
MBP (Maltose Binding Protein)-MutY fusion protein was expressed and purified using a
slightly modified version of a previously reported protocol (
25
). Modifications to the
protocol included changes in “buffer A” to a resuspension buffer (20 mM sodium phosphate,
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pH 7.5, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 10% glycerol) and use
of an amylose column to eliminate the necessity of a streptomycin sulfate and ammonium
sulfate precipitation. During the amylose preparation, the sample was washed with amylose
wash buffer (20 mM sodium phosphate, pH 7.5, 200 mM NaCl, 1mM EDTA pH 8) and
eluted in amylose elutant buffer (20 mM sodium phosphate, pH 7.5, 200 mM NaCl, 1 mM
EDTA pH 8, 10 mM maltose). The resultant fractions were concentrated using an
ultrafiltration cell with a 10,000 MWCO filter with stirring at 4°C. Protein was then diluted
10-fold in heparin buffer A (20 mM sodium phosphate, pH 7.5, 1 mM EDTA, 5% glycerol in
water), applied to a Pharmacia Hi-trap heparin column on an AKTApurifier FPLC system,
and eluted using a 10% linear gradient in heparin buffer A to 100% heparin buffer B (20mM
sodium phosphate, pH 7.5, 1mM EDTA, 5% glycerol, and 1 M NaCl in water). MBP-MutY
eluted at 450 mM NaCl (45% heparin buffer B). Purity of protein samples was confirmed via
12% SDS page stained with SYPRO orange. The [4Fe4S] cluster loading was determined
using the UV-visible absorbance at 410 nm (
ε
410
= 17,000 M
−1
cm
−1
) and at 280 nm (
ε
280
=
143,240 M
−1
cm
−1
); samples were typically 65–75% loaded.
DNA preparation
DNA strands for EndoIII experiments were purchased from Integrated DNA Technologies,
with sequences as follows:
20-mer:
5
′
-GTG AGC TAA CGT GTC AGT AC-3
′
Complement:
5
′
-GTA CTG ACA CGT TAG CTC AC-3
′
Single-stranded DNA oligomers (5 μmol) were resuspended in MilliQ water and purified by
ethanol precipitation. Briefly, 1000 μL of cold 200 proof ethanol and 50 μL of 3 M NaCl
were added to 100 μL single-stranded DNA in water and vortexed; DNA solutions were then
frozen in liquid nitrogen for rapid precipitation and spun at 16,000 RCF (25 minutes) to
form a pellet which was then re-dissolved in EndoIII storage buffer (20 mM sodium
phosphate, pH 7.5, 0.5 mM EDTA, 150 mM NaCl). Single-stranded DNA was quantified by
UV-vis using
ε
260
values calculated using the Integrated DNA Technologies oligo analyzer
tool; these were 197,800 M
−1
cm
−1
for the 20-mer strand and 190,200 M
−1
cm
−1
for its
complement. Equimolar amounts of each strand were then annealed by incubation at 90°C
for 5 minutes followed by slow cooling to ambient temperature.
For MutY experiments, DNA substrates containing oxoG (8-oxo-guanine) or FA (2
′
-fluoro-
adenine) were synthesized at the University of Utah DNA and Peptide Synthesis Core
Facility and unmodified strands were ordered from Integrated DNA Technologies. The
following DNA duplexes were used:
15-mer:
5
′
-GGA GCC A
X
G AGC TCC-3
15-mer Complement:
3
′
-CCT CGG T
Y
C TCG AGG-5
′
30-mer:
5
′
-CGA TCA TGG AGC CAC
X
AG CTC CCG TTA CAG-3
′
30-mer Complement:
3
′
-GCT AGT ACC TCG GTG
Y
TC GAG GGC AAT
GTC-5
′
X
= G or oxoG and
Y
= C, FA, or A
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Oligonucleotides containing the central oxoG or FA were deprotected and cleaved from the
column by incubation in NH
4
OH; 2-mercaptoethanol was added into oxoG samples to
prevent oxidation. The cleaved DNA substrates were dissolved in H
2
O, filtered with a 0.2
μm filter, and HPLC purified using a Beckman Gold Nouveau system with a Waters
AP1DEAE 8HR column; a 10–100% gradient of 90:10 H
2
O/acetonitrile with 2 M NH
4
Ac
was used in purification. Isolated fractions were dried down and de-salted using SEP-PAK
cartridges, and DNA integrity was confirmed using MALDI-MS. All DNA substrates were
stored dried in the −20°C freezer prior to annealing.
Electrochemistry
All electrochemical experiments were performed on an edge-plane pyrolytic graphite
electrode (Pine Research Instrumentation) with a geometric surface area of 0.196 cm
2
. To
generate a rough surface suitable for protein binding, the electrode was abraded with 400
grit sandpaper and cleaned by sonication for 1 minute each in ethanol and water. After
sonication, the absence of electroactive impurities was verified by scanning in EndoIII
storage buffer (20 mM sodium phosphate, 0.5 mM EDTA, 150 mM NaCl, pH 7.5) or MutY
storage buffer (20 mM sodium phosphate, 1 mM EDTA, 150 mM NaCl, pH 7.6) as
appropriate.
Single-walled carbon nanotubes (CNTs) were found to enhance greatly the signal size of
adsorbed protein, so they were included in the formation of all thin films unless otherwise
noted. Protein thin films were formed from several (typically 3–6) alternate layers of 10 μL
single-walled carbon nanotubes (CNT) in water (0.25 mg/mL) and 10 μL EndoIII (150 μM
in storage buffer) or MutY (50 μM) in a 1:1 mix with aqueous CNTs. Each layer was gently
dried under an argon gun, and the process was repeated until the surface was coated by a
viscous film, which was then secured with 5% Nafion in water (diluted from 10% in water
as purchased) to prevent dispersal (
21
). For experiments including DNA, CNTs generally
hindered electrochemical signals, so these films were formed in their absence. Poly-L
glutamate (MW 50–100 kDa) was used to assess the effects of a negatively charged non-
substrate on potential. Although Nafion also carries a negative charge at the pH values used,
it was applied only to the top of a multilayer film to form a binding layer, minimizing
interactions with the electroactive protein; in contrast, poly-L glutamate and DNA were
incorporated directly into the thin film with protein to maximize any possible interactions.
After thin film formation, 50 μL of EndoIII or MutY storage buffer was pipetted on top of
the film and an Ag/AgCl reference in 3 M NaCl and Pt auxiliary electrode were submerged
in the resulting droplet. Reduction potential, current, and charge measurements were then
taken by cyclic voltammetry (CV), square wave voltammetry (SQWV) and differential pulse
voltammetry (DPV); all experiments were conducted at ambient temperature (20 °C).
Electroactive area was determined by plotting the scan rate dependence of the CV current
generated by 1 mM [Ru(NH
3
)
6
]Cl
3
in storage buffer and applying the Randles-Sevcik
equation (
26
),
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(1)
I
p
is the peak current in amperes, F is Faraday’s constant (96485 C·mol
−1
), R is the universal
gas constant (8.314 J·(mol·K)
−1
), T is temperature in K, n is the number of electrons
transferred per CV peak, A is electrode area in cm
2
, D is the diffusion coefficient in cm
2
·s
−1
(9.0 × 10
−6
for [Ru(NH
3
)
6
]
3+
;
27
), C° is bulk protein concentration in mol·cm
−3
, and
ν
is
the scan rate in V·s
−1
. Potentials were converted to NHE by adding 0.212 V to the value
measured by Ag/AgCl, using the value of 0.209 mV value at 25 C given by the supplier,
BASi®, and applying a temperature correction (
28
). To prevent leakage of NaCl into the
buffer and subsequent wandering of the reference potential, the glass frit of the electrode
was immersed in a gel loading pipet tip containing 3 M NaCl with 4% dissolved agarose,
and dried in this mix overnight. CNTs, 10% aqueous Nafion, poly-L glutamate, and
[Ru(NH
3
)
6
]Cl
3
were purchased from Sigma-Aldrich, while the Ag/AgCl reference electrode
in 3 M NaCl was purchased from BASi®.
RESULTS
Direct Electrochemistry of WT EndoIII and MutY
To examine variations in EndoIII potentials with various substitutions or binding partners,
EndoIII thin films anchored to the surface by Nafion were prepared on a PGE electrode. In
the absence of DNA, a quasi-reversible signal was observed by CV (Figure 1). The signal
under these conditions was relatively small (3 ± 1 μC reductive peak, −4 ± 1 μC oxidative
peak), and the reductive peak partially overlapped the much larger wave of oxygen
reduction, making it more challenging to quantify. By adding 0.25 mg/mL CNTs to form a
protein/CNT/Nafion thin film, the peak areas increased by an order of magnitude to reach 16
± 3 μC and −18 ± 5 μC reductive and oxidative peaks, respectively, while the peak potentials
remained unaltered from those without the CNTs at 74 ± 20 mV and 162 ± 18 mV (all
potentials vs NHE).
The addition of EndoIII and CNTs was associated with a large increase in the capacitance;
much of this increase was due to CNTs, as seen in CNT/Nafion thin films, but the protein
itself certainly contributed (Figure S1). Notably, the high conductivity of CNTs amplifies
redox events at the surface; a CNT/Nafion film shows reversible peaks around 200 mV vs
NHE and −80 mV vs NHE, both of which show no splitting and are likely attributable to the
reversible reduction of surface oxides on the edge plane and even on the CNTs themselves
(Figure S1;
29
–
31
). Indeed, the 200 mV peaks were invariably present, although smaller, in
buffer alone and the −80 mV peaks varied in size based upon the freshness of the CNT
suspension applied, consistent with this assertion. The presence of protein on the surface
markedly suppressed both of these peaks, and the EndoIII signal differed from the
background both by its potential, which was essentially identical to that measured in the
absence of CNTs, and in the occurrence of peak splitting; the latter suggested a slower
process, in agreement with reports of other proteins adsorbed on carbon (
21
).
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WT MutY thin films were prepared just as with EndoIII, although the stock concentrations
were somewhat lower (~50 μM for MutY compared to ~150 μM for EndoIII). MutY
displayed a quasi-reversible signal similar to EndoIII on CNT/Nafion thin films, with CV
peak potentials centered at 100 ± 9 mV for the reductive peak and 162 ± 3 mV for the
oxidative peak (Figure 1). Notably, the potentials were within error of the values obtained
for EndoIII. The respective peak areas were 2.3 ± 0.3 μC and −3.4 ± 0.1 μC, about an order
of magnitude smaller than EndoIII and indicative of lower surface coverage.
For both EndoIII and MutY, the current exhibited a linear dependence on the scan rate
(Figure S2), confirming that the protein was adsorbed to the electrode surface rather than
diffusing in from solution; this relationship was present whether or not CNTs were included.
Surface coverage was initially determined simply by converting the total CV peak charge at
a scan rate of 100 mV/s into pmol using Faraday’s constant and dividing by the geometric
surface area of the electrode. Because the PGE surface is uneven, the geometric surface area
can underestimate the electroactive area by a factor as large as 10
4
(
32
). Indeed, using the
geometric area of the electrode (0.196 cm
2
) gave a surface coverage of 550 ± 300 pmol/cm
2
for 75 μM EndoIII stock, over 10 times larger than reported for ferredoxin thin films on PGE
(40 pmol/cm
2
;
32
) and over 100 times larger than CNT/Nafion/protein thin films on glassy
carbon (2–6 pmol/cm
2
;
21
).
By taking the scan rate dependence of the current for [Ru(NH
3
)
6
]
3+
in EndoIII storage
buffer and applying the Randles-Sevcik equation (Equation 1), the electroactive surface area
was determined to be 1.0 cm
2
, about 5 times larger than the simple geometric area. When
this correction was applied to a thin film formed from 75 μM EndoIII stock, a value of 108
± 60 pmol/cm
2
was obtained, which is still high but much closer to previously published
results on PGE (
32
). Applying the same correction to films formed from 25 μM MutY stock
gave a coverage of 29 ± 6 pmol/cm
2
, around 25% of that measured for 75 μM EndoIII. To
facilitate a more direct comparison, surface coverage on thin films formed with 25 μM
EndoIII was measured to be 51 ± 8 pmol/cm
2
, indicating that MutY adsorption was
absolutely less extensive than EndoIII. This result is not surprising, given that unmodified
MutY is 39 kDa while EndoIII is only 24 kDa; the 42 kDa N-terminal MBP tag on MutY
would only enhance this issue.
Adsorption of proteins to the electrode surface made it possible to estimate electron transfer
rate (k
ET
) and transfer coefficient (
α
) values using the Laviron method for diffusionless
systems (Figure S2;
33
), where
α
is a measure of transition state symmetry, taking on values
between 0 and 1. The MutY signal was too small to measure the currents at high scan rates,
but this analysis could be carried out for EndoIII. In the case of EndoIII, we obtained a k
ET
of 3 ± 0.6 s
−1
and
α
values of 0.4 and 0.6 for the reductive and oxidative peaks, respectively.
Assuming that electron transfer is the only reaction taking place on the electrode, the values
of
α
imply a quasi-reversible system. Importantly, electron transfer rates are similar to those
reported for other redox-active enzymes/proteins adsorbed to carbon electrodes in the
presence of CNTs and Nafion (
21
).
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