Hybrid Amyloid-Based Redox Hydrogel for Bioelectrocatalytic H2 Oxidation

Article abstract of DOI:10.1002/anie.202101700

An artificial amyloid-based redox hydrogel was designed for mediating electron transfer between a [NiFeSe] hydrogenase and an electrode. Starting from a mutated prion-forming domain of fungal protein HET-s, a hybrid redox protein containing a single benzyl methyl viologen moiety was synthesized. This protein was able to self-assemble into structurally homogenous nanofibrils. Molecular modeling confirmed that the redox groups are aligned along the fibril axis and are tethered to its core by a long, flexible polypeptide chain that allows close encounters between the fibril-bound oxidized or reduced redox groups. Redox hydrogel films capable of immobilizing the hydrogenase under mild conditions at the surface of carbon electrodes were obtained by a simple pH jump. In this way, bioelectrodes for the electrocatalytic oxidation of H₂ were fabricated that afforded catalytic current densities of up to 270 μA cm^?2, with an overpotential of 0.33 V, under quiescent conditions at 45 °C.

Full text of DOI:10.1002/anie.202101700

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Hybrid amyloid-based redox hydrogel for bioelectrocatalytic H2
oxidation
Authors: Nicolas Duraffourg, Maxime Leprince, Serge Crouzy, Olivier
Hamelin, Yves Usson, Luca Signor, Christine Cavazza,
Vincent Forge, and Luca Albertin
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
Hybrid amyloid-based redox hydrogel for bioelectrocatalytic H
oxidation
2
[
a]
[a]
[a]
[a]
[b]
Nicolas Duraffourg, Maxime Leprince, Serge Crouzy, Olivier Hamelin, Yves Usson, Luca
[
c]
[a]
[a]
[a]
Signor, Christine Cavazza, Vincent Forge and Luca Albertin*
Dedicated to the memory of Dr. Serge Crouzy
[
a]
Dr N. Duraffourg, M. Leprince, Dr. S. Crouzy, Dr O. Hamelin, Dr C. Cavazza, Dr V. Forge, and Dr L. Albertin
Univ. Grenoble Alpes, CNRS, CEA, IRIG, Laboratoire de Chimie et Biologie des Métaux, 38000 Grenoble, France
E-mail: luca.albertin@cea.fr
Dr. Y. Usson
Univ. Grenoble Alpes, CNRS, CHU Grenoble Alpes, Grenoble INP , TIMC-IMAG, 38000 Grenoble, France
[
[
b]
c]
*
Dr. L. Signor
Univ. Grenoble Alpes, CEA, CNRS, IRIG, IBS, 38000 Grenoble, France
Supporting information for this article is given via a link at the end of the document.
Abstract: An artificial amyloid-based redox hydrogel was
designed for mediating electron transfer between a [NiFeSe]
hydrogenase and an electrode. Starting from a mutated prion-
forming domain of fungal protein HET-s, a hybrid redox protein
(TOF); iii) excellent product selectivity, decreasing the need for
downstream purification; and iv) greatly decreased reliance on
non-aqueous solvents and precious metals.
A major challenge in bioelectrocatalysis is to establish electrical
communication between enzymes and electrode surfaces: [
most enzymes have an active centre buried deep beneath their
surface and surrounded by an insulating protein shell, which
prevents direct electron transfer (DET) with the electrode. [
Although DET is easier for enzymes with built-in electron transfer
pathways, such as hydrogenases, [4] the enzyme still needs to be
adsorbed on the surface of the electrode with an appropriate
orientation, so that the most accessible electron relay is close
enough to the electrode to allow tunnelling. [5]
1a]
containing
a single benzyl methyl viologen moiety was
synthesized. This protein was able to self-assemble into
structurally homogenous nanofibrils. Molecular modelling
confirmed that the redox groups are aligned along the fibril’s axis,
and are tethered to its core via a long, flexible polypeptide chain
that allows close encounters between the fibril-bound oxidized or
reduced redox groups. Redox hydrogel films capable of
immobilizing the hydrogenase under mild conditions at the
surface of carbon electrodes, were obtained by a simple pH jump.
In this way, novel bioelectrodes for the electrocatalytic oxidation
3]
Redox hydrogels – i.e. cross-linked polymer networks featuring
reversible redox moieties tethered to their backbone chains - are
the only electron-conducting phase in which water-soluble
2
of H were fabricated that afforded catalytic current densities of up
-2
to 270 µA cm , with an overpotential of 0.33 V, under quiescent
conditions at 45°C.
[
6]
chemicals and biochemicals can dissolve and diffuse.
Accordingly, they are of much interest in bioelectrocatalysis, since
they can immobilize a wide range of embedded enzyme
molecules, irrespective of their spatial orientation and distance,
while allowing the diffusion of reactants, products and ions to and
from the bulk of the solution. Moreover, self-standing redox
hydrogel films immobilize the biocatalyst within a hydrophilic and
solvated matrix that protects it from denaturation. [7]
Introduction
Efficient electrocatalysts are needed in technological applications
ranging from energy conversion and storage, to biofuel cells, and
the production of electrofuels, commodity chemicals and
materials. [ In nature, all biological systems extract energy and
nutrients from their environment and carry out the metabolic
processes associated with life with high molecular and energetic
efficiency. Redox enzymes - a particular class of biocatalysts –
catalyse many of the reactions involved in these processes, and
the design, fabrication, and use of enzyme-modified electrodes is
an active field of research. [2]
Amyloid-based hydrogels (AbHs) [8] are water-swollen, three-
dimensional networks formed by cross-linking of protein fibrils
with a structure rich in cross-β-sheets. [ The exact mechanism of
gelation is complex and varies from one system to another, but
involves a combination of non-covalent interactions (i.e. hydrogen
1]
9]
bonding, hydrophobic and electrostatic interactions) and
entanglement of the fibrils. [
8, 9c, 10]
The ability of AbHs to hold large
amounts of water is due to the large number of hydrophilic and
charged functional groups present on the surface of protein fibrils.
Potential advantages of enzymatic bioelectrocatalysis over non-
biological systems are as follows: i) higher energy efficiency, due
to lower applied overpotentials; ii) very high catalytic activities
*
Institute of Engineering Univ. Grenoble Alpes
1
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RESEARCH ARTICLE
Scheme 1. Synthesis of redox nanofibrils from a self-assembling hybrid protein and preparation of amyloid-based redox hydrogel films.
Together with viscoelasticity, these physical properties make
AbHs good mimics of the cytosol, extracellular matrix, and
biofilms in which many enzymes and bacteria have evolved
and/or normally function. Hence, the development of electron-
conducting AbHs would enable their use as biomimetic,
immobilizing matrices for bioelectrocatalysis.
into redox nanofibrils and gels. Thus, a single protein could serve
as platform for the design of AbRxH possessing varying
electrochemical and catalytic properties. Interestingly, nanofibrils
of HETPFD are highly stable in aqueous solution thanks to the
numerous intra- and inter-molecular hydrogen bonds and salt-
bridges between β-strands. The structure of the fibrils is known at
atomic resolution, [18] and is not influenced by the mutation of a
few amino acids outside the hydrophobic core of the solenoid or
the C-terminal semiflexible loop. [19] Also, their morphology can be
controlled by tuning the electrostatic interaction of individual fibrils
via pH and ionic strength of the solution. [20] [21]
Baldwin et al. [11] first proposed the use of amyloid fibril scaffolds
to display redox proteins in a linear array in order to favour inter-
protein electron transfer via conformational sampling at the
protein–protein interface and, ultimately, electron transfer over
long distances. [12] Rengaraj et al. [13] and Altamura et al. [14] then
applied this concept to the preparation of protein nanowires
featuring a contiguous alignment of metalloproteins on their
surface. To this end, they exploited the ability of the prion-forming
domain (PFD; residues 218 to 289) of the heterokaryon
incompatibility protein HET-s from the fungus P. anserina
We therefore postulate that HETPFD could serve as self-
associating building block for the rational design of AbRxHs in
which the position of artificial prosthetic group(s) along the fibril
can be finely controlled by choosing the appropriate anchoring
point in the polypeptide chain. In this manner, gel matrices could
be engineered for mediated electron transfer (MET) from an
electrode to an enzyme or for more complex electrocatalytic
reactions involving multiple redox and catalytic sites. Here, we
report the first use of such a strategy, in which the [NiFeSe]-
hydrogenase from Desulfomicrobium baculatum (DbH-[NiFeSe])
(
HETPFD) [15] to self-organize into amyloid fibrils with high structural
homogeneity, and to do so in a reproducible way even when fused
to another protein domain of comparable size.[16] In this way, they
constructed a fusion protein comprising a small rubredoxin from
Methanococcus voltae connected to the C-terminus of HETPFD
.
[
22]
Following a pH jump, these nanowires self-assembled into redox
hydrogel films capable to mediate electron-transfer between an
electrode and an imbedded laccase [14] or [NiFe] hydrogenase. [13]
This work demonstrated the possibility of developing amyloid-
based redox hydrogels (AbRxHs) and using them in
bioelectrocatalysis. In theory, by changing the nature of the
metalloprotein displayed on the fibrils’ surface, one could design
AbRxH spanning the whole potential window of redox proteins,
i.e. -700 to +700 mV vs SHE, [17] thus allowing their application in
a wide range of electrochemical and photoelectrochemical
devices. [ In practice, however, the need to purify the PFD-
containing fusion protein under denaturing conditions limits its
scope to redox domains that are either extremely stable or that
can be easily refolded in their holo form once the chaotropic agent
removed. Furthermore, (i) the active centre of the redox domain
has to be close enough to the surface as to allow effective
interprotein electron transfer; (ii) its longitudinal size along the
fibril axis should not lead to steric hindrance and prevent self-
association of the fusion protein; and (iii) a new fusion protein has
to be designed, expressed, and refolded whenever the potential
of the redox matrix has to be changed.
was wired to carbon electrodes via a self-assembled AbRxH
prepared from a hybrid prion forming domain. DbH-[NiFeSe] was
selected because it has one of the highest catalytic activities
toward H
2
oxidation among the NiFe hydrogenases (TOF = 4000
−
1
[22]
s ),
and because – like other [NiFeSe]-hydrogenases – it is
inactivated under oxidizing electrochemical conditions. The latter
aspect allows a comparison with previously reported Nernst
buffers for this class of enzymes. [5, 23]
.
7]
Results and Discussion
Synthesis and structure of redox nanofibrils
A mutant of HETPFD possessing a single cysteine residue at the
N-terminus and a His6 tag at the C-terminus (CysHETPFD) was
constructed as self-associating building block that can be
selectively functionalized via sulfhydryl chemistry.[24] The protein
does not contain Met residues and, at pH values near neutrality,
Cys is alkylated by water-soluble benzyl bromide derivatives
considerably faster than any other amino acid.[
25]
-1
Recombinant CysHETPFD (M
r
= 9037.0 g mol , theoretical pI =
A more versatile approach to designing AbRxHs (Scheme 1)
would involve: (i) production of a self-associating protein carrying
one or more functional group(s) amenable to orthogonal chemical
functionalization; (ii) site-selective conjugation of artificial
prosthetic group(s) (e.g. metal complex or organic molecule)
possessing redox or catalytic properties matching the target
application; (iii) self-association of the resulting hybrid construct
7.24) was produced by heterologous overexpression in E. Coli
and 80–140 mg of purified protein were obtained per litre of shake
culture (i.e. 40-70 mg of per gram of wet cells; see Figure S1 and
S2). Unfolded (monomeric) CysHETPFD was conjugated to
bromobenzyl methyl viologen (40 eq.) by nucleophilic substitution
under denaturing conditions (GuHCl 5M, pH 7.3, 20°C; Scheme
2
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RESEARCH ARTICLE
S2). Benzyl methyl viologen (BMV) was chosen as redox relay
since viologen moieties undergo efficient electron transfer with
Self-association of the hybrid protein (0.13 mM in AcOH 1%) was
induced by consecutive buffer exchange (citrate 20 mM pH 3.6,
then HCl ~0.1 mM), and progress of fibrillation was monitored by
circular dichroism though the appearance of a positive peak at
195 nm and a negative peak at 214 nm (Figure 1), both
characteristic of the β-strand conformation predominant in
[26]
hydrogenases either in freely diffusing
forms. [27] Also, they display fast reaction kinetics with O
enabling the design of redox hydrogels that protect (bio)catalysts
or polymer-bound
, [28] thus
2
against oxygen damage (although protection from O
explored in this study). [
2
was not
23, 27d, 29]
[31]
amyloid structures.
After purification by affinity chromatography, MADI-TOF-MS
analysis confirmed that the isolated protein (BMV-HETPFD
Fibrillation was complete after 23 hours and the length and
morphology of the fibrils was observed by low voltage TEM
without staining (Figure 2, Figure S6). Single-stranded fibrils of
BMV-HETPFD - hereafter indicated as p(BMV-HETPFD) - were
predominant but coexisted with double- and triple-stranded fibres
resulting from lateral association. Analogous findings were
reported for fibrils of wild type HETPFD prepared under similar
conditions,[ confirming the limited impact of the small artificial
prosthetic group on fibrils morphology.
)
featured a single benzyl methyl viologen group (Figure S3),
whose absorbance band at 260 nm is clearly visible in the UV-Vis
absorbance spectrum (Figure S4a). When a deoxygenated
solution of hybrid protein was reduced with sodium dithionite
under Ar, the solution turned blue-violet in color and the
characteristic absorbance bands of a monomeric (398, 604 and
32]
7
32 nm) and dimeric (370, 560 and 880 nm) bipyridinium radical
cation appeared in its absorption spectrum (Figure S4b).[30]
Measurement of a small sample of individual fibrils (n=100) in
TEM images indicates that number average contour length was
L
n
 1.4 µm (which corresponds to an average length / width
aspect ratio of 350) and that fibrils were heterogeneous in length
0.2 µm  L  3.7 µm,  = 0.9 µm). The L value obtained in this
(
n
manner is certainly underestimated, since long individual fibrils
are more difficult to identify due to their tendency to intertwine and
overlap with other fibrils; and because they often extend beyond
the visualization area of a TEM image.
Starting from a 5-repeat structure of fibrils of HET-s(218-289)
resolved by ssNMR (PDB 2RNM), [
18b]
a 12-repeat model for
p(BMV-HETPFD) fibrils was built by Molecular Dynamics
simulation and energy minimization. As shown in Figure 3, benzyl-
methyl-viologen groups are loosely aligned along the fibril axis
and anchored at 9.4 Å intervals to the central β-solenoid via a
flexible polypeptide chain 10 amino acids long. By exploring its
conformational space in solution, the latter enables close
encounters between redox relays of proteins that are up to four
positions apart along the fibril axis, i.e. overcoming up to four
consecutive functionalization defects in the fibril. This encounters
support self-exchange reactions among the nanofibril-bound
oxidized/reduced redox groups and, ultimately, MET between the
hydrogenase and the electrode.
Figure 1. CD spectrum of p(BMV-HETPFD) redox nanofibrils (0.1 mM HCl pH
3.5) and of the unfolded prion-forming domain (AcOH 1% v/v pH 2.6) in the UV
region. Upon polymerization, a transition occurs from a disordered conformation
to one rich in β-strands, with a pronounced minimum at 214 nm. [31]
Figure 3. Model of p(BMV-HETPFD) fibril. Different colours indicate different
polypeptide chains; benzyl methyl viologen groups are shown in purple.
Amyloid-based redox hydrogels - AbRxHs
Nanofibrils of p(BMV-HETPFD) expose a majority of acidic, basic,
hydrophilic and positively charged residues on their surface
(
Figure S7), among which hydrophobic patches are interspersed.
Figure 2. Low voltage TEM image of p(BMV-HETPFD) in 0.1 mM HCl pH 3.5: the
sample mostly consists of single-stranded fibrils with some double- and triple-
stranded fibres, a few examples of which are indicated by arrows.
3
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RESEARCH ARTICLE
Figure 4. (a) SEM image of a dried single-layer p(BMV-HETPFD) gel film formed on a glassy carbon electrode. b) Topography of an analogous film drop-cast on a
borosilicate glass slide (drop volume 2.5 µL) as recorded by DHM. Films of circular shape ~2 mm in diameter were typically otained, and only quarter discs could
be imaged by DHM. The scale of the z-axis is nm, whereas x and y axis are in µm.
At pH < 4 and low ionic strength, lateral association is
unfavourable due to electrostatic repulsion between positively
charged fibrils and, at low concentration, a majority of single-
stranded filaments are observed under these conditions (Figure 2,
Figure S6). At higher pH values though, net surface charge will
be much diminished and electrostatic attraction between
oppositely charged patches, as well as hydrophobic interaction
between nonpolar patches, will lead to later association of
nanofibrils into larger fibres and bundles.
branched and dendritic viologen-polyethylenimine solutions drop-
cast under similar non-gelling conditions. [34]
To gain a better insight into the morphology of swollen
electroactive and electrocatalytic films, the topography of various
AbRxHs was investigated by Digital Holographic Microscopy
(DHM) in transmission mode. [35] DHM is a quantitative phase
microscopy technique that enables full-field imaging of the
structure and dynamics of transparent samples from a single
recorded hologram. Vertical accuracy as low as a few tens of
We hypothesised that, above a critical fibril overlap concentration,
a sudden decrease in net surface charge would result in a 3D
supramolecular network of entangled nanofibres of varying
morphology, cross-linked by physical junction zones.Accordingly,
the initial suspension of positively charged nanofibrils in 0.1 mM
nanometers is achievable under adequate conditions. [35b]
.
Circular films 2 mm 0.1 mm in diameter were obtained by drop-
casting 2.0 µL of nanofibrils’s suspension onto borosilicate glass
slides (as measured by optical microscopy). Quarter films were
imaged by DHM both in the dry and hydrogel state (Figure S11).
Although surface energy and roughness of the glass slides used
for DHM differ from that of GC, the radial profile obtained for a dry
single-layer p(BMV-HET) film (Figure 4 b and Figure S9) is similar
to that observed by stylus profilometry with a peak-to-valley ratio
of ~1.7. The fact that absolute thickness is smaller in the DHM
experiment compared to stylus profilometry is probably the result
of the smaller volume applied (2.0 µL vs 3.0 µL). Rehydration and
cross-linking in the supporting electrolyte swelled the film without
any variation in diameter, while the overall profile was maintained.
This property is important for ensuring reproducibility of the
coating process.
-1
HCl was concentrated 5 ½ fold by ultrafiltration (cfinal  3.8 g L ),
and films were prepared by drop casting 2.0 µL to 4.0 µL onto
planar surfaces, followed by drying of the sessile droplet in the
open atmosphere. Simple rehydration in the supporting
electrolyte used for electrochemical experiments (TrisHCl 20-50
mM / NaCl 50 mM, pH 7.6) effected both later association of the
fibrils into larger fibres and their physical cross-linking, yielding
transparent hydrogel films.
Figure 4 a shows a SEM image of the dried gel coating a glassy
carbon electrode: as expected, it is a dense and intricate network
of fibres and bundles with lateral dimension of 100-150 nm and
4
00-700 nm, respectively (see Figure S8a for more SEM images).
Electrochemistry and bioelectrocatalysis with AbRxHs
The profile of the dried film before physical cross-linking was
recorded by stylus profilometry and indicates a thickness varying
from 1.7 µm to 3.0 µm with some accumulation of material both at
the centre and at the outer rim of the circular deposit (drop volume
Cyclic voltammograms of p(BMV-HETPFD) hydrogel films on
Glassy Carbon (GC) and Edge Plane Pyrolytic Graphite (EPPG)
electrodes (Figure 5 a, c) display a reversible redox couple with
half wave potential E1/2 of -0.29 V and -0.28 V, respectively, at all
tested scan rates; i.e. a value 0.12 V higher than the formal
3.0 µL, film diameter 2.5 mm; Figure S9). Departure from a flat
film morphology is often observed in films obtained by drop-
casting methods and it is the result of a complex interplay
between mass and heat transport processes taking place within
the drying droplet. [33] In our case though, film inhomogeneity is
much less pronounced than the “coffee-ring effect” reported for
+
+
+•
potential of the Vio /Vio redox couple of free BMV in solution
(E1/2= -0.408 V). [
36]
4
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RESEARCH ARTICLE
Figure 5. Cyclic voltammograms (a, b) and dependency of the cathodic and anodic currents on scan rate (c, d) for p(BMV-HETPFD) hydrogel films on GC and EPPG
electrodes. Background capacitive current was subtracted and the intercept of linear regression functions was forced through the origin; current densities at scanning
-
1
-1
rates of 0.35 V s and 0.70 V s where excluded from linear regression calculations in plot (d). (See Figure S13 for more voltammograms). Conditions: 2 µL drop
coating, supporting electrolyte TrisHCl 20 mM (GC) or 50 mM (EPPG) / NaCl 50 mM, pH 7.6.
This shift to higher redox potentials was anticipated, having been
consistently observed for redox polymers in which a viologen
where C is concentration of redox species, DCT is the apparent
charge-transport diffusion coefficient, and T is temperature. The
+
•
derivative is anchored to a positively charged macromolecular
slope measured for the oxidation of Vio moieties in p(BMV-
matrix. [
27a, 27d, 34, 37] [38]
It is due to the polyelectrolyte nature of the
HETPFD) (3.010 A cm
-5
-2
V
-1/2 1/2
s ) is 5.1 times higher than what
[
14]
redox hydrogel, in which the electrostatic, excluded volume, and
van der Waals interaction fields couple in a complex way and
determine the free energy of the system.[39] As a result, the formal
potential of viologen moieties in p(BMV-HETPFD) hydrogels is 50
mV more positive than that of [4Fe-4S] clusters of DbH-[NiFeSe]
observed for rubredoxin in RdHET-based AbRxHs on GC.
Since equilibrium concentration of protein units is expected to be
similar in the two hydrogels, DCT must be at least one order of
magnitude higher in the biohybrid matrix. This result was
expected in virtue of the smaller size - hence higher Dphys - of
methylviologen as compared to rubredoxin.[43] Nonetheless, the
implied DCT is still 2 to 3 orders of magnitude lower than what
(
Figure S13), [22, 40] thus providing a sufficient driving force for ET
between the hydrogenase and the redox matrix.
For modified GC electrodes, peak-to-peak separation (E
p
) at low
reported for a methyl viologen-polyethylenimine hydrogel (DCT
=
4.7 x10-9 cm s ).
2
-1 [29a]
A possible explanation involve inter-wire
scanning rates was ~50 mV, close to the 57 mV expected for a
reversible monoelectronic diffusion-controlled redox system. [41]
At increasing scan rates, background-subtracted peak current
densities J increased linearly with the square root of scan rate v1/2
charge transfer being hindered by limited surface contact and/or
high distance; and the limited Brownian motion of large rigid
[43-44]
nanofibers diminishing the collision frequency of redox sites.
(
Figure 5 b), indicating that electron transfer within the hydrogel
A different behaviour was observed for modified EPPG electrodes
(Figure 5 c, d): compared to analogous GC electrodes, higher
current densities were recorded at all scanning rates and the
amount of faradaic charge exchanged during the reduction of
Vio+• moieties was 8 times higher (v = 100 mV s-1).
film is governed by semi-infinite linear diffusion. [42] According to
the Randles–Sevcik equation, the slope of the linear dependency
scales as:
1
퐷CT
2
slope ∝ 퐶 ( 푇
)
(1)
5
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Figure 6. Topography of (A) single-layer electroactive p(BMV-HETPFD), and (B) single-layer and (C) double layer p(BMV-HETPFD) / DbH-[NiFeSe] bioelectrocatalytic
hydrogel films drop-cast on a glass slide. The vertical scale is the same for all graphs, but it is in arbitrary units, whereas x- and y-axis are in µm.
Also, linear regression between peak current density J and v½ is
less convincing (Figure S14b): instead J scales tightly with v up
negatively charged enzyme (pI = 5.7) would penetrate the
nanofibers mesh and be entrapped by electrostatic interaction
with the multiple positive charges on the nanofibers.[49] Close
inspection of the swelling process, however, revealed that most
of the enzyme solution was not absorbed by the film at equilibrium,
and was effectively wasted.
1
00 mV s-1 as in a surface-confined electron transfer process
(
Figure 5 d), but the relationship breaks down at higher scanning
rates. Lastly, E is 27 mV at a low scanning rate ─ i.e. lower
p
than the 57 mV expected for a reversible monoelectronic
diffusion-controlled redox system ─ and increases almost linearly
In an attempt to increase the efficiency of immobilization, a
second protocol was tested in which the enzyme was pre-mixed
with an equal volume of nanofibril suspension before being
applied to electrode. Hence, double-layer electrocatalytic films
were prepared by drop coating a first layer of p(BMV-HETPFD) as
describe above, followed by drop casting 2µL or 4 µL of a 1:1
mixture of p(BMV-HETPFD) / DbH-[NiFeSe] on the top of it, drying
in the open atmosphere and immersion in the working electrolyte
to complete electrostatic cross-linking.
Topography of the electroactive and bioelectrocatalytic hydrogel
films obtained in this manner is shown in Figure 6. Compared to
a single-layer p(BMV-HETPFD) film (a), the addition of enzyme
resulted in an accumulation of material at the centre of the film
disk (b and c), as indicated by the increased thickness, while the
footprint of the film remained unchanged. The effect was
particularly pronounced for double-layer electrocatalytic films due
to the further addition of p(BMV-HETPFD) fibres.
with v½ to reach 51 mV at 100 mV s and 89 mV at 700 mV s
-1
-1
(
Figure S14d).
Detailed mechanistic investigation of these phenomena is beyond
the scope of this study, but the role played by the EPPG surface
as well as the nature of nanostructured polyelectrolyte
electroactive layer shall be taken into account. In particular, the
chemical heterogeneity on the edge plane (with aromatic,
hydrophilic, and carboxylate functionalities),[45] the topological
roughness of the EPPG surface and the presence of narrow basal
plane terraces a few micrometres long [46] make it particularly
suitable for the adsorption of p(BMV-HETPFD) nanowires.
Furthermore, as reported for simpler redox polyelectrolyte
films,[47] the electrochemistry of AbRxHs is the result of a number
of molecular processes (i.e. counter-ion diffusion, ion pairing,
water transport, nanofibers rearrangement) that are linked to
electron current density and are complex to model. [48]
Concerning the stability of the electroactive layer on EPPG, after
Figure 7 depicts cyclic voltammograms obtained under Ar (solid
3
days in the supporting electrolyte the intensity of the anodic
2
line) and H atmosphere (dashed line) for GC (a) and EPPG
wave due to viologen moieties decreased by 45%, suggesting
desorption and/or disassembly of the redox hydrogel film (Figure
S15).
electrodes (b) modified with p(BMV-HETPFD) / DbH-[NiFeSe]
hydrogel coatings. Experiments were realized under quiescence
conditions with a supporting electrolyte solution saturated either
Bioelectrodes for H
[
[
different protocols were implemented, which involved the same
amount of enzyme being applied onto the electrode.
In the first protocol, single-layer bioelectrocatalytic films were
2
oxidation were fabricated by imbedding
NiFeSe]-hydrogenase from Desulfomicrobium baculatum (DbH-
NiFeSe]) into the AbRxH films coating carbon electrodes. Two
2
with Ar or with H ; while gentle flushing of the cell head-space with
the appropriate gas was maintained throughout the experiment.
Under Ar atmosphere, the redox couple for the first viologen
reduction was clearly observed for double-layer electrodes
(Figure 7 a, b and Figure S16), but the anodic wave was
systematically more intense than the cathodic one. This effect is
probably due to slow hydrogen evolution taking place at low
obtained by drop casting
3 µL of p(BMV-HETPFD) fibrils
suspension on the electrode followed by drying in the open
atmosphere. The dry film was then swollen with 2 µL of buffered
DbH-[NiFeSe] solution (pH 7.6), which also effected physical
cross-linking by changing the surface charge of the nanofibrils.
We postulated that, during the swelling process, part of the
2
potentials, followed by rapid H oxidation at higher ones, DbH-
[NiFeSe] being a poor catalyst for HER but very active toward H
2
oxidation.[22] In the case of single-layer GC electrodes, a small
HER catalytic wave was indeed visible (Figure S16a).
6
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Angewandte Chemie International Edition
10.1002/anie.202101700
RESEARCH ARTICLE
-
1
Figure 7. Cyclic voltammograms under quiescent conditions of modified GC (a) and EPPG (b) electrodes (scan rate 10 mV s ). Curves for single-layer (blue series)
and double–layer (red series) p(BMV-HETPFD) / DbH-[NiFeSe] hydrogel coatings under Ar (solid line) or H atmosphere (dashed line) are shown. (c)
Chronoamperometry at E = +0.20 V (GC) and +0.00 V (EPPG) under H atmosphere. Coating volumes: GC single-layer, 3µL fibrils + 2µL enzyme; GC double-layer,
µL fibrils + 4µL mix; EPPG double-layer, 3µL fibrils + 2µL mix. Supporting electrolyte TrisHCl 20 mM (50 mM for chronoamperometry GC) / NaCl 50 mM, pH 7.6.
2
2
4
Under
H
2
atmosphere, pronounced catalytic waves were
Single- and double-layer coatings of GC electrodes lead to similar
catalytic current profiles (Figure 7 a, series at 25°C), with a
monotonic increase of current density with applied potential that
slows down above -0.15 V but never reaches a plateau. This
behaviour is independent of operating temperature and suggests
that H diffusion is not limiting catalytic current. In terms of
2
electrocatalytic efficiency, then, the two coating protocols seem to
be equivalent.
observed that are centred at the formal potential of the viologen
electron relays. Since the latter is 150 to 170 mV more positive
than the 2H / H
+
2
couple (E°`H+/H2 = -0.449 V at pH 7.6), the MET
chain involving BMV moieties is limiting the turnover of the
enzyme and DET can be excluded. According to the model of
Léger, [50] the active site of a multicenter redox enzyme feels the
potential of a ET relay under the condition that the ET chain limits
the enzymatic turnover. In this case, the ET relay acts as a Nernst
buffer and protects the enzyme from high potential deactivation.
However, if enzymatic turnover is limiting, fast ET chains involving
artificial electron relays will display the same properties as the
direct ET of enzymes directly adsorbed to electrodes, i.e. a
catalytic wave centred on the redox potential of the enzyme’s
active site or of the substrate. In this case, protection from high
potential would not be achieved.
Two control experiments were conducted with the enzyme either
directly adsorbed on a EPPG electrode, or immobilized within an
hydrogel of electroinactive p(HETPFD) fibrils at the surface of the
electrode. Similar results were obtained with the two coating
protocols (Figure S12): under Ar atmosphere, the [4Fe-4S]
electron-transfer signals of the enzyme are clearly visible after
subtraction of the capacitive current (E1/2 = -0.28 and -0.35 V,
2
It is worth noting that current density due to H oxidation at +0.40
V and 45 °C was 6 times higher than previously obtained at 60°C
with [NiFe] hydrogenase from Aquifex aeolicus embedded in
RdHET-based AbRxH (a GC electrode was used in both cases),
[
13]
thus confirming the relevance of the new bioelectrode design.
Under continuous turnover conditions at +0.20 V, a slow but
steady decline in catalytic current was observed, and after 80 min.
only ~65 % of the initial activity remained (Figure 7 c). A
complementary experiment (Figure S17) showed that the loss in
catalytic current matches the reduction in charge exchanged
during cyclic voltammetry for the reduction/oxidation of viologen
moieties in the matrix. Excluding hydrolysis or degradation of
BMV groups, it follows that gradual exfoliation and/or disassembly
of the hydrogel is the main cause for the limited stability of the
electrode over time. Enzyme inactivation and/or diffusion away
from the electrode surface cannot be exclude though.
respectively). [22, 40]. In the presence of H
at -0.40 V (E°`H+/H2 = -0.449 V at pH 7.6) but levels off between -
.20 V (co-immobilization) and 0.0 V (direct adsorption) due to
, an anodic wave starts
2
Results with modified EPPG electrodes were rather different
(Figure 7 b). For applied potentials higher than -0.15 V, the
monotonic increase of current density stopped and J started
0
oxidative inactivation of the enzyme.[5] Reactivation takes place
when the potential is scanned to negative values and a new,
slightly less intense, anodic wave is observed in the following
cycle.
-2
oscillating around a plateau of ~270 µA cm . This value is over
three-times higher than the current density recorded for modified
GC electrodes at the same potential (E = -0.15 V) and
temperature (45 °C), the only other difference being the coating
protocol ‒ which was previously found to have little influence on
catalytic currents (see Figure 7 a, series at 25°C).
For comparison, with p(BMV-HETPFD)-modified electrodes no
oxidative inactivation of the enzyme took place at applied
potentials as high as +0.40 V. Also, when DbH-[NiFeSe] was
immobilized within an hydrogel of electroinactive p(HETPFD) fibrils,
the catalytic current due to DET (E = - 0.20 V, T = 25 °C) was 0.21
µA; whereas the same experiment carried out using p(BMV-
HETPFD) fibrils afforded a catalytic current of 1.3 µA (single-layer
Current density fluctuations of  7% with a period of ~1 min were
also recorded in chronoamperometric experiments under
continuous H oxydation conditions at +0.00 V, but average
2
current did not decline over an 80 min period, thus proving the
robustness of the bioelectrode on this time scale (Figure 7 c). The
fluctuations in J at high potential might be due to local fluctuations
in substrate transport, pH and/or nanofibres morphology that do
not take place at the slower reaction rates achieved with GC
electrodes.
2
trace in Figure 7 a for a film area of 0.049 cm ). Hence, the AbRxH
effectively mediates electron transfer between the electrode and
the hydrogenase and acts as a Nernst buffer protecting it from
high potential deactivation.
7
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Angewandte Chemie International Edition
10.1002/anie.202101700
RESEARCH ARTICLE
Conclusion
thank Dr. J. Tan for the purification of Cys-HETPFD samples, Dr. S.
Ménage for useful discussions and suggestions, and Dr. J.
Willison for proofreading the manuscript.
An artificial amyloid-based redox hydrogel was designed for
mediating electron transfer from a [NiFeSe] hydrogenase to an
electrode. Starting from a mutated prion-forming domain of fungal
protein HET-s, a hybrid redox protein was synthesized that is
capable of self-assembling into structurally homogenous
nanofibrils in a predictable way. Molecular modelling confirmed
that redox groups are loosely aligned along the fibril’s axis and
tethered to the central solenoid via a long flexible polypeptide
chain. By exploring its conformational space in solution, the latter
enables self-exchange reactions among the fibril-bound
oxidized/reduced redox groups and, ultimately, mediates electron
transport between the hydrogenase and the electrode.
At high concentration, a sudden change in surface charge of the
fibrils leads to their lateral association into protein nanofibres and
physical cross-linking through non-covalent interactions. This
property was exploited to prepare redox hydrogel films that are
capable of immobilizing a hydrogenase at the surface of carbon
electrodes under mild conditions. Topography investigations
revealed some accumulation of material both at the centre and at
the outer rim of the circular deposit, but no “coffee-ring effect”.
Based on these properties, new bioelectrodes for the
Keywords: hybrid prion forming domain • supramolecular
polymer • protein nanowire • redox hydrogel• bioelectrocatalysis
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RESEARCH ARTICLE
Entry for the Table of Contents
A biohybrid prion-forming domain was synthesized that is capable to undergo hierarchical self-assembly into single-stranded protein
nanowires, nanofibres and supramolecular redox hydrogels. This property enabled us to immobilize a [NiFeSe] hydrogenase within a
water-swollen network of conductive nanofibres via a gentle non-covalent cross linking process, while ensuring its wiring to carbon
electrodes for electrocatalysis.
1
0
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