Heme bound amylin self-assembled monolayers on an Au electrode: An efficient bio-electrode for O2 reduction to H2O

Article abstract of DOI:10.1039/c3cc49571j

Self-assembled monolayers of the water soluble hydrophilic part of naturally occurring amylin and its Arg11 mutant have been assembled on an Au surface, which are found to efficiently catalyze selective 4e ^-/4H⁺ O₂ reduction reaction (ORR) upon binding heme with a k_cat of ～10⁷ M^-1 s^-1 under ambient conditions, where the Arg11 residue plays the key role of proton transfer in determining the rate of ORR. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3cc49571j

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Heme bound amylin self-assembled monolayers
on an Au electrode: an efficient bio-electrode for
O2 reduction to H2O†
Cite this: Chem. Commun., 2014,
50, 3806
Received 18th December 2013,
Accepted 14th February 2014
Kushal Sengupta, Sudipta Chatterjee, Soumya Mukherjee, Somdatta Ghosh Dey*
and Abhishek Dey*
DOI: 10.1039/c3cc49571j
www.rsc.org/chemcomm
Self-assembled monolayers of the water soluble hydrophilic part His18 residue.24 Arg11, though not directly bound to heme, plays a
of naturally occurring amylin and its Arg11 mutant have been key role by providing hydrogen bonding interactions with the
assembled on an Au surface, which are found to efficiently catalyze propionate side chain of heme.24 Two cysteine residues (Cys2 and
selective 4eÀ/4H+ O2 reduction reaction (ORR) upon binding heme Cys7) present in the 1–19 domain do not bind heme24 and are thus
with a kcat of B107 MÀ1 sÀ1 under ambient conditions, where the available to bind to the Au(111) surface, as observed for other Cys
Arg11 residue plays the key role of proton transfer in determining appended peptides.25,26 Here, we report the formation of a self-
the rate of ORR.
assembled monolayer (SAM) of Ay (1–19) on the Au surface (AySAM
)
which when bound to heme acts as an excellent bio-electrode for O2
Electrochemical reduction of O2 to H2O with high catalytic rates reduction. Heme bound to AySAM (WT) and Arg11Asn mutant
and turnover numbers has significant implications in biofuel (AySAM-R11N) show 4eÀ/4H+ O2 reduction with minimal PROS
cells, bio-sensors, etc.1–3 While precious metals and metal-alloy formation having rates as high as 107 MÀ1 À1, which is several
s
based O2 reduction reaction (ORR) catalysts have been found to be orders of magnitude greater than most of the reported metallo-
promising candidates, their utility is limited by their high costs.3 porphyrin and metallo-enzyme based electrocatalysts.11,27
Metallo-porphyrins and metallo-corroles are reported to function
The cysteine residues present in Ay (1–19) bearing either a thiol
as good ORR electrocatalysts.4–9 In these cases the involvement of group or a disulfide bridge are used to form a monolayer on Au
partially reduced oxygen species (PROS) has proved to be detri- electrodes. Clean Au surfaces upon being immersed in a 0.1 mM
mental for the long term stability of these catalysts.4,10 Prior solution of Ay (1–19) show the presence of small and isolated
investigations have revealed that naturally occurring enzymes like features (Fig. 1A). These features have a height distribution of
laccases, immobilized directly on electrodes, reduce O2 at reason- 3–5 nm (Fig. 1B). Very few clusters ranging from 6 to 7 nm are also
able catalytic rates.11–13 The advantage of using a natural scaffold seen, which are randomly distributed. Unlike previous reports of
has been widely discussed and demonstrated in non-heme iron amyloid b peptides (associated with Alzheimer’s disease),26 these
proteins,14,15 electron transport proteins,16–18 oxidases19,20 and, AySAM surfaces do not show large structures resulting from
recently, in hydrogenases.21 Hence a naturally occurring protein/ peptide aggregation.28 Ay (1–19) has 19 residues and considering
peptides scaffold, which can bind heme and can be easily a 3.2 Å translation per residue, an upright Ay peptide should have
attached to electrodes, could form a potential biochemical scaf- a height of B60 Å (6 nm). This is more than the observed height
fold for heme based ORR catalysts.
of 3–5 nm. Alternatively, assuming the segments 1–7 runs parallel
Amylin (Ay) or the human islet amyloid polypeptide protein is
a principal constituent of fibrillar amyloid deposits observed in
patients suffering from type 2 diabetes mellitus. It comprises of
37 amino-acid residues (1–37) and is found in blood.22,23 Recent
studies establish that the non-amyloidogenic segments 1–19 is the
heme binding domain where a single heme is bound by the
Department of Inorganic Chemistry, Indian Association for the Cultivation of Science,
2A & 2B Raja S. C. Mullick Road, Jadavpur, Kolkata, 700032, India.
E-mail: icad@iacs.res.in, icsgd@iacs.res.in
† Electronic supplementary information (ESI) available: Experimental detials,
AFM, CV, SERRS, and BE data. See DOI: 10.1039/c3cc49571j
Fig. 1 (A) 3D topology AFM image of the amylin (1–19) modified Au surface
(AySAM). (B) Height distribution profile diagram of the corresponding surface.
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to the electrode, anchored by Cys2 and Cys7 at the ends, and
the remaining 12 residues (8–19) project out of the surface, an
approximate height of 4 nm (3.2 Â 12 Å) can be estimated for
such a construct consistent with the observed height of 4 Æ 1 nm
in the AFM data (Fig. 1). Note that similar results were observed in
the AFM images of the AySAM-R11N (Fig. S2, ESI†).29
AySAM formation on the Au electrode surfaces shields the
electrode and abolishes the electrocatalytic O2 reduction catalyzed
by bare Au electrodes (Fig. S3, ESI†). For further insulation of these
modified surfaces from the solution phase, the AySAM covered
surfaces were immersed in octanethiol solution which led to further
decrease in the capacitive current (Fig. S3, ESI†). Heme binding to
AySAM is characterized using Surface Enhanced Resonance Raman
Fig. 3 (A) CV data of heme–AySAM (red) and heme–AySAM-R11N mutant
(green) obtained in air saturated pH 7 buffer at a scan rate of 50 mV sÀ1
.
(B) Bar diagram representation of the amount of PROS produced by the
respective heme bound species.
Spectroscopy (SERRS).30,31 SERRS data of heme–AySAM show the n4, surface coverage suggests formation of a monolayer of site isolated
n3 and the n2 bands at 1370 cmÀ1, 1489 cmÀ1 and 1569 cmÀ1 heme–AySAM active sites. Multi-layers formed from physiadsorption
respectively, indicating the presence of a six coordinate (6C) FeIII have coverages 41014 molecules cmÀ2 34 In aerated buffer solutions,
.
high-spin (HS) species on the surface (Fig. 2A, red).32,33 Careful CV of heme–AySAM in the pH 7 buffer shows an electrocatalytic O2
fitting of the n2 band shows the presence of a 6C FeIII low-spin (LS) reduction current peaking at À150 mV (Fig. 3A, red). Similar results
component having the n2 band at 1586 cmÀ1 (Fig. S4, ESI†). Note are also observed in the R11N mutant. The potential for O2 reduction
that these values are in good agreement with those obtained for is B30 mV lower in the R11N mutant, consistent with the lower
heme–Ay complexes in solution.24 In the presence of imidazole the FeIII/II potential observed in this mutant relative to the WT. Rotating
n4, n3 and n2 bands of heme–AySAM shift to 1374 cmÀ1, 1505 cmÀ1 ring disc electrochemistry (RRDE) is used to estimate the amount of
and 1585 cmÀ1 respectively, indicating the conversion of the HS PROS, formed due to incomplete reduction of O2.35 In this technique
species to a pure 6C FeIII LS species (Fig. 2A, blue) and suggesting any O22À, i.e., a 2eÀ reduction product of O2, produced in the
the presence of an exchangeable ligand (likely to be H2O) in the modified Au disc electrode is radially diffused to the encircling Pt
heme–AySAM complex.32,33 Note that the structural organization of ring, which is held at a potential where the H2O2 is oxidized back
the heme bound AySAM-R11N SAM is similar to that of the WT to O2. The amount of PROS produced by heme–AySAM is about
(SERRS, absorption and capacitance data; Fig. S5–S7, ESI†).
9 Æ 0.5% while for the R11N mutant the amount of PROS produced
Cyclic voltammetry (CV) of heme–AySAM (WT) in degassed pH 7 is 6.6 Æ 1% (Fig. 3B and Fig. S9, ESI†).36 The decrease in PROS when
buffer under anaerobic conditions shows a well developed quasi- the Arg11 residue is mutated to Asn indicates that the acidic Arg11
reversible FeIII/FeII process at À108 mV vs. NHE (Fig. 2B, red; Fig. S8, residue, even if not directly involved in heme binding, is likely
ESI†). The AySAM-R11N mutant shows the FeIII/II redox couple at involved in 2nd sphere interaction with the active site, consistent
À120 mV (Fig. 2B, green). The surface coverages were measured with previous reports.26,37,38 Such interaction may provide efficient
from the area under the oxidation and reduction waves and were proton transfer pathways into the active site (vide infra).
found to be 9.8 Â 1012 molecules cmÀ2 and 2.0 Â 1013 molecules
Electrocatalytic O2 reductions of these species are further inves-
cmÀ2 for WT and R11N mutant, respectively (Table 1). Such low tigated using a rotating disc electrode (RDE). Linear Sweep Voltam-
metry (LSV) of heme–AySAM and the corresponding mutant show a
substrate diffusion limited catalytic O2 reduction current below
À200 mV (Fig. 4A and B). The O2 reduction current increases with
increasing rotation rates following the Koutecky–Levich equation,
iÀ1 = iK(E)À1 + iLÀ1, where iK(E) is the potential dependent kinetic
current expressed as nFA[O2]kcat
G
catalyst, and iL is the Levich current.
iL is expressed as 0.62nFA[O2](DO )2/3o1/2 À1/6
v
, where n is the number
2
of electrons transferred to the substrate, F is the faraday constant,
A is the macroscopic area of the disc (0.192 cm2), [O2] is the
concentration of O2 in an air saturated buffer (0.2 mM) at 25 1C,
kcat is the 2nd order rate of catalytic O2 reduction, Gcatalyst is the
Fig. 2 (A) SERRS data of heme–AySAM in pH 7 buffer (red) and in the pre-
sence of 100 mM imidazole in pH 7 (blue), collected under resting (oxidizing)
conditions. (B) CV data of heme–AySAM (red) and heme–AySAM-R11N mutant
catalyst concentration in moles cmÀ2, DO is the diffusion coefficient
2
of O2 (2.2 Â 10À5 cm2 sÀ1) at 25 1C, o is the angular velocity of the
disc and v is the kinematic viscosity of the solution (0.009 cm2 sÀ1) at
25 1C.35 The plot of iÀ1 at multiple rotation rates vs. the inverse
square root of the angular rotation rate (oÀ1/2) is linear. The number
of electrons (n) involved in the O2 reduction by a catalytic species
(green) in deoxygenated pH 7 buffer at a scan rate of 1 V sÀ1
.
Table 1 Electrochemical response of heme–AySAM
Gcatalyst
Species
(molcules cmÀ2
)
n
PROS (%) kcat (MÀ1 sÀ1
)
may be calculated from the slope and 2nd order rate of catalysis (kcat
)
B9.8 Â 1012
4
4
9 Æ 0.5 (1.8 Æ 0.6) Â 107
from the intercept of this linear plot. The slopes obtained from the
Heme–AySAM
Heme–AySAM-R11N B2 Â 1013
6.6 Æ 1.0 (2.7 Æ 0.4) Â 105 experimental data, at different potentials in the substrate diffusion
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thiol group of Cys2 and Cys7 residues. The AySAM assemblies on
Au electrodes bind heme and can be characterized using SERRS
and CV. The resultant heme–AySAM complex on the electrode is
very stable and can catalyze ORR at a rate of 107 MÀ1 sÀ1, which
is 100 times faster than any man-made metalloporphyrin
catalyst reported so far. The facile ORR by the WT heme–AySAM
complexes is due to the presence of Arg11 residue in the active
site, which likely provides a facile H+ transfer pathway. A facile
proton transfer pathway has been identified as a key factor for
an efficient ORR in both natural and artificial systems.9,44,45
We thank the SERC Fast Track Scheme SR/FT/CS-34/2010
(SGD) and SR/S1/IC-35/2009 (AD), Department of Science and
Technology, Govt. of India for funding this research. K.S., S.C.
and S.M. thank CSIR, India, for Senior (K.S. and S.C.) and
Junior (S.M.) Research Fellowships.
Notes and references
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Fig. 4 RDE plots of heme–AySAM (A) and heme–AySAM-R11N (B), in air
saturated pH 7 buffer at a scan rate of 50 mV sÀ1 at multiple rotations. (C) and
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