An Artificial Hemoprotein with Inducible Peroxidase- and Monooxygenase-Like Activities

Article abstract of DOI:10.1002/chem.202002434

A novel inducible artificial metalloenzyme obtained by covalent attachment of a manganese(III)-tetraphenylporphyrin (MnTPP) to the artificial bidomain repeat protein, (A3A3′)Y26C, is reported. The protein is part of the αRep family. The biohybrid was fully characterized by MALDI-ToF mass spectrometry, circular dichroism and UV/Vis spectroscopies. The peroxidase and monooxygenase activities were evaluated on the original and modified scaffolds including those that have a) an additional imidazole, b) a specific αRep bA3-2 that is known to induce the opening of the (A3A3′) interdomain region and c) a derivative of the αRep bA3-2 inducer extended with a His₆-Tag (His₆-bA3-2). Catalytic profiles are highly dependent on the presence of co-catalysts with the best activity obtained with His₆-bA3-2. The entire mechanism was rationalized by an integrative molecular modeling study that includes protein–ligand docking and large-scale molecular dynamics. This constitutes the first example of an entirely artificial metalloenzyme with inducible peroxidase and monooxygenase activities, reminiscent of allosteric regulation of natural enzymatic pathways.

Full text of DOI:10.1002/chem.202002434

Accepted Article
Accepted Article
Title: An artificial hemoprotein with inducible peroxidase- and
monooxygenase-like activities
Authors: Jean-Pierre Mahy, Kalani Kariyawasam, Thibault Di Meo,
Fabien Hammerer, Marie Valerio-Lepiniec, Giuseppe
Sciortino, Jean-Didier Maréchal, Philippe Minard, Agathe
Urvoas, and Rémy Ricoux
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202002434
Link to VoR: https://doi.org/10.1002/chem.202002434
01/2020

10.1002/chem.202002434
Chemistry - A European Journal
RESEARCH ARTICLE
An artificial hemoprotein with inducible peroxidase- and
monooxygenase-like activities
Kalani Kariyawasam,[a] Thibault Di Meo,[a,b] Fabien Hammerer,[a] Marie Valerio-Lepiniec,[b] Giuseppe
Sciortino,[c] Jean-Didier Maréchal,[c] Philippe Minard,[b] Jean-Pierre Mahy,*[a] Agathe Urvoas,*[b] and
Rémy Ricoux*[a]
[a]
Dr. K. Kariyawasam, Dr. T. Di Meo, Dr. F. Hammerer, Prof. J-P. Mahy, Dr. R. Ricoux
Institut de Chimie Moléculaire et des Matériaux d’Orsay (ICMMO), UMR 8182 CNRS,
Laboratoire de Chimie Bioorganique et Bioinorganique,
Bât. 420, Université Paris-sud, Université Paris‐Saclay, 91405 Orsay cedex, France
E-mail: remy.ricoux@u-psud.fr, jean-pierre.mahy@u-psud.fr
[b]
[c]
Dr. T. Di Meo, Dr. M. Valerio-Lepiniec, Prof. P. Minard, Dr. A. Urvoas
Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif‐sur‐Yvette cedex, France
E-mail: agathe.urvoas@i2bc.paris-saclay.fr
Dr. G. Sciortino, Prof. J-D. Maréchal
Departament de Química, Universitat Autònoma de Barcelona,
Edifici C.n., 08193 Cerdanyola del Vallés, Barcelona, Spain
Supporting information for this article is given via a link at the end of the document.
Abstract: We here report
metalloenzyme obtained by covalent attachment of
a
novel inducible artificial
The quest for efficient catalysts able to perform selective
oxidations in water under atmospheric pressure and at room
temperature is a field of research of upmost importance in modern
chemistry. Amongst the strategies that have caught the eye of
chemists for decades are those inspired on naturally occurring
oxidases.
Through evolution, Nature has built a panoply of enzymes for
catalytic oxidations. Prototypical families are cytochromes P450
that are able to catalyze the oxidation by O2 of numerous organic
a
manganese(III)-tetraphenylporphyrin (MnTPP) to the artificial
bidomain repeat protein, (A3A3’)Y26C. The protein is part of the
αRep family developed in our laboratory. The biohybrid was fully
characterized by MALDI-ToF mass spectrometry, circular
dichroism and UV-Vis spectroscopies. Its peroxidase activity was
tested against several typical peroxidase cosubstrates and its
monooxygenase activity against oxidation of thioanisole into
sulfoxide and using H2O2 as an oxidant. The catalytic tests were
performed on the original scaffold as well as including a) an
additional imidazole, b) a specific αRep bA3-2 that is known to
induce the opening of the (A3A3’) interdomain region and c) a
derivative of the αRep bA3-2 inducer extended with a His6-Tag
(His6-bA3-2). Catalytic profiles are highly dependent on the
presence of co-catalysts with the best activity obtained with His6-
bA3-2. The entire mechanism was rationalized by an integrative
molecular modeling study that includes protein-ligand docking
and large-scale molecular dynamics. From the overall study, it
appears that: i) the hydrophobic porphyrin cofactor fits well into
the (A3A3’)Y26C cavity, becomes protected by protein
environment and fully soluble in an aqueous buffer; ii) the
biohybrid alone does not display any catalytic activity, even in the
presence of an imidazole co-catalyst because of a closed
conformation of the system; iii) the presence of the bA3-2 inducer
leads to noticeable activity by allowing the opening of the binding
site, and iv) the best activity is obtained with the additional
presence of the His6-Tag with one or two of the histidines able to
coordinate to the metal acting as co-catalyst in the reactions. To
the best of our knowledge, this constitutes the first example of an
entirely artificial metalloenzyme with inducible peroxidase and
monooxygenase activities, reminiscent of allosteric regulation of
natural enzymatic pathways.
compounds, including poorly reactive alkanes. As
a
consequence, man-made P450s have become for decades the
focus of intensive research. Two lines have been undertaken: i)
the protein engineering of natural occurring P450s [1][2] and ii) the
identification of artificial biomimetic de novo constructs.[3]
Artificial metalloenzymes (ArMs) mimicking cytochromes P450
are mostly based on synthetic metalloporphyrins that have long
proven to be efficient catalysts for oxidation reactions in organic
solvents. Adapting these compounds to biocatalytic conditions is
a tremendous tour de force with many pieces to put together.
Among key questions to solve are i) the identification of non-
natural substitutive electron transfer partners (i.e., reductases) ii)
the solubilization of metalloporphyrins in aqueous medium and iii)
the transition to cellular media that include the control over the
reaction to occur in the correct location in the cell.
Over years of investigation, satisfying solutions to the first and
second aspects have been provided by using H2O2, that
constitutes a water-soluble equivalent of O2 + 2e- + 2 H+ and
releases H2O as side product, and by providing metalloporphyrin
microenvironments that promote their solubility in water as well as
promote asymmetric environment. Those microenvironments
were first achieved by substituting metalloporphyrins with water-
soluble derivatives (i.e. carboxylates or sulfonate). However,
attention has shifted in the last decades to the development of
artificial metalloenzymes (ArMs) where porphyrin derivatives are
included into biological hydrophilic receptors [4][5][6][7][8] and that
ease water solubilization as well as a protective and asymmetric
environment in the vicinity of the first coordination sphere of the
metal. Today, following the pioneering work by Hayashi and
Introduction
1
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10.1002/chem.202002434
Chemistry - A European Journal
RESEARCH ARTICLE
collaborators, most P450 ArM systems are obtained by direct
supramolecular anchoring [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24]
of the artificial metalloporphy-rinoid cofactor into protein hosts.
Despite promising results, such incorporation allows limited
stability of the cofactor into the protein receptor and this, in its turn,
challenges the biocatalytic viability (i.e. inclusion in cellular
environments). Covalent attachment of the cofactor to the host,
another common strategy used in ArM design, is an interesting
alternative to improve the stability of the biohybrids. However, the
size of porphyrinic macrocycle makes it difficult to identify
convenient hosts and linkers. In fact, to date, only Watanabe and
coworkers [25] reached a covalent attachment of hemes to a
heteroprotein assembly isolated from bacteriophage T4 by a
maleimido group linker.
Despite the vivid field of research that man-made P450s
represents, their incorporation into cellular environments still
remains a major step ahead. Because oxidases could be
extremely reactive, hence hazardous for the cell, it is fundamental
to previously secure the identification of stable porphyrin-host
constructs and look for molecular mechanisms to control their
activity. Looking for allosteric effectors could be an interesting way
to move forward along this line. Allosteric proteins are
omnipresent in natural regulatory processes like genetic control,
epigenetic modulators, and molecular pathways,[26] but to date,
there is only very few examples of allosterically controlled ArMs.
[27][28][29][30]
(A3A3’)Y26C.[33][34] The protein has shown to adopt a closed
bivalve shell-like conformation. Molecular modeling identified that
this cavity was wide enough for the anchoring of even larger metal
complexes such as meso-tetraphenylporphyrin. Moreover, in a
posterior study, we also observed that large conformational
changes occur when in presence of a specific protein effector
bA3-2 and that the interdomain region can largely open and close
in an inducible manner. [35] Thus, from an enzyme designer point
of view, the artificial αRep proteins gather the required properties
for the development of ArMs dedicated to the catalysis of
oxidation reactions. They appear convenient to fully
accommodate a Mn-tetraphenylporphyrin, are compatible to
covalent anchoring and also display conformational changes at
the large scale that could be controlled by an adequate molecular
inducer.
The work reported in this manuscript is a major proof-of-concept
of artificial P450 induced systems. It is the first example of an
artificial hemoprotein in which the synthetic metallo-tetraaryl-
porphyrin cofactor is covalently anchored to a cysteine residue in
its protein binding pocket and which activity is controlled by the
addition of the specific αRep protein binder, bA3-2. [35] This
species triggers not only a peroxidase activity, which allows the
efficient oxidation of several typical peroxidase cosubstrates, but
also of a monooxygenase activity that allows the selective
oxidation of thioanisole into sulfoxide. The best activity was
obtained with His6-bA3-2, the protein bearing a His6-Tag that also
acted as a co-catalyst in these reactions. An integrative molecular
modeling study allowed proposing a model for this activation
mechanism.
We have recently shown that a new class of thermostable artificial
proteins named αReps, [31][32] developed in our laboratory from a
library of artificial proteins, could be converted into artificial
metalloenzymes by covalent grafting of a synthetic metal
complex. These αRep proteins are easily produced in E. coli,
highly stable under various pH ranges, and present a wide
hydrophobic core. We already tested the concept by creating
As far as we know, this is the first occurrence of an artificial
metalloenzyme with protein-cofactor induced peroxidase and
monooxygenase activities, reminiscent of the activation of
catalytic activities of enzymes by chaperone proteins or allosteric
regulators.
artificial
Diels-Alderases
by
covalent
attachment
of
phenanthroline- or terpyridine-copper(II) complexes into an
specific αRep single chain asymmetric bidomain variant named
Figure 1. A) Model for the (A3A3’)Y26C-NHMal-TPP biohybrid based on the crystallographic structure of A3 homodimer (3LTM), B) Covalent attachment of Mn(III)-
NHMal-TPP into (A3A3’)Y26C by nucleophilic addition of the sulfide function of Cys 26 on its maleimide group.
reaction
of
meso-mono-4-aminophenyl-triphenylporphyrin
Results and Discussion
NH2-TPP [36] with 3-maleimidopropionic acid in the presence of
DCC [37] followed by a reaction with manganese chloride [38] to
affordthe Mn(III) porphyrin that was insoluble in water (see SI, Fig.
S1). The biohybrid was finally assembled by incubation of the
protein (A3A3’)Y26C with 10 equiv. of Mn(III)-NHMal-TPP for 3
days at 4°C, with 25% DMSO in the coupling buffer (50 mM Na-
phosphate pH 7.75, 150 mM NaCl).
Biohybrid preparation
The (A3A3’)Y26C-NHMal-TPP-Mn(III) biohybrid was prepared in
three steps (See SI). The single-chain bidomain protein in which
a cysteine-free domain was linked with a domain bearing the
Y26C mutation ((A3A3’)Y26C) was first produced and purified as
recently described.[34] NHMal-TPP was then synthetized by
2
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10.1002/chem.202002434
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RESEARCH ARTICLE
Figure 2. Spectral properties of the (A3A3’)Y26C-NHMal-TPP-Mn(III) biohybrid : A) MALDI -ToF spectrum of (A3A3’)Y26C (blue) and BH-MnTPP (green), B) CD
spectrum of a 3 M BH-MnTPP (green) and 4 M A3A3 WT (blue) solution in 10 mM HEPES pH 8 at 20°C, C) UV-visible spectrum of a 2.3 M BH-MnTPP solution
in 10 mM HEPES 150 mM NaCl pH 8 at 20°C.
After removal of excess Mn(III)-NHMal-TPP by extraction with
butan-2-one, the (A3A3’)Y26-NHMal-TPP-Mn(III) (BH-MnTPP)
biohybrid was recovered in the aqueous phase. MALDI -ToF MS
characterization showed a major peak at 43178 Da, confirming
the covalent binding of Mn(III)-NHMal-TPP (M = 833 Da) on
(A3A3’)Y26C (M = 42331 Da) (Fig. 2A). Most proteins would not
remain folded under such rude conditions; yet the circular
dichroism spectrum of the biohybrid was almost superimposable
to that of the wild type A3A3 symmetric bidomain protein (Fig. 2B),
indicating that its 3D structure was not altered after covalent
attachment of the porphyrin cofactor in the hydrophobic pocket
even in the presence of 25% DMSO and the subsequent
treatment with butan-2-one. Finally, UV-visible spectroscopy in 10
mM HEPES 150 mM NaCl pH 8 at 20°C that displayed main
bands at 471 nm ( = 65340 M-1cm-1), 570 nm ( = 8160 M-1cm-1)
and 604 nm ( = 6835 M-1cm-1) (For  calculations see SI, Fig. S2),
characteristic of the Mn(III)-porphyrin cofactor (Fig. 2C).
the presence of 40 M bA3-2, and effectively, a peroxidase
activity was observed in the presence of 4 mM imidazole with an
initial rate of 278 M DAP/µmol BH-MnTPP /min, and 14.5 M
DAP was produced after 4 h. (Fig. 3A, trace d). Noteworthy, when
imidazole was deleted no activity was observed (Fig. 3A, trace c),
indicating that the oxidation of OPD only occurred once access to
the cavity was open to allow both imidazole to coordinate the Mn
ion and OPD to transfer electrons to the intermediate Mn-H2O2
derived oxidizing intermediates. More amazing, when bA3-2 was
replaced by 40 M of its precursor-tagged protein, His6-bA3-2, a
more efficient oxidation of OPD characterized by an initial rate of
800 M DAP /µmol BH-MnTPP/min was observed and 28.5 M
DAP was produced after 4 h (Fig. 3A, trace e). Strikingly the initial
rate value was 2.9-fold higher than that obtained in the presence
of 40 M bA3-2 and 4 mM imidazole. Replacing His6-bA3-2 by
another His6-Tag labelled αRep protein not selected against αRep
A3 (His6-αRep-ns), that does not bind the bidomain A3A3’
proteins, did not lead to any peroxidase activity (Fig. 3A, trace f).
Since, in addition, no peroxidase activity was observed with either
bA3-2, or His6-αRep-ns alone, it is clear that the necessary
conditions to trigger an optimal peroxidase activity, required both
the presence of the bA3-2 protein to open the access of the
reducing cosubstrate to the Mn-porphyrin cofactor substrate and
of imidazole or, much better, of its His6-Tag label as a co-catalyst.
Two further experiments confirmed that the His6-Tag did indeed
act as a co-catalyst in the peroxidase reaction. First, the formation
of DAP was performed in the presence of 2 M BH-MnTPP and
increasing concentrations of either bA3-2 or His6-bA3-2 (0-160
M). A 72 nM.min-1 initial rate of DAP formation was observed in
the presence of 160 M bA3-2, whereas an optimal rate of 320
nM.min-1 was reached with only 40 M of His6-bA3-2 (Fig. 3B).
Second, when this initial rate was monitored as a function of pH
(pH 4 – 8.5), in the presence of either 40 M bA3-2 without (b)
and with (c) 4 mM imidazole or (d) 40 M His6-bA3-2 alone, an
increase in the rate of DAP formation as a function of pH was
obtained in both (c) and (d) cases (Fig. 3C), with respective
optimal rates of 158 nM.min-1 and 422 nM.min-1 at pH 8.5, which
is in agreement with a contribution of the imidazole side chain of
a histidine to catalysis. It should be noted that at pH 4, the different
proteins partners precipitate in solution before the catalysis.
Catalysis of oxidation reactions
The catalytic activity in oxidation of BH-MnTPP was then assayed
using H2O2 as oxidant in the presence of imidazole as co-catalyst,
and a typical peroxidase cosubstrate, ortho-phenylenediamine
(OPD). The two electrons oxidation of OPD in the presence of
H2O2 leads to the 2,3-diaminophenazine (DAP) absorbing at 418
nm ( = 16700 M-1cm-1) (see SI, Fig. S3).[39]
When 500 M OPD was allowed to react with 125 M H2O2 in the
presence of 2 M BH-MnTPP, no product was detected after 4 h
(Fig. 3A, trace a) and, in the presence of 4 mM imidazole, a small
amount of DAP was formed (Fig. 3A, trace b). This lack in
peroxidase activity suggested that the Mn(III)-NHMal-TPP
cofactor was buried too deeply in the cavity of the bidomain
protein, thus preventing the OPD to approach the porphyrin
periphery and deliver electrons to the H2O2 derived oxidative
species. To facilitate the access to the metalloporphyrin cofactor,
we then thought about widening the opening of the cavity using
another protein from the αRep library, named bA3-2, that was
previously selected by phage display[32] and was recently found to
be able to bind tightly to the bidomain A3A3’ proteins and to open
their bivalve shell-like conformation.[35] The reaction was then
performed under the same conditions as described above but in
3
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10.1002/chem.202002434
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RESEARCH ARTICLE
Figure 3. Oxidation of 500 M OPD by 125 M H2O2 catalyzed by 2 M BH-MnTPP in 10 mM HEPES buffer pH 8 at 20°C: A) Time course of the oxidation with
BH-MnTPP a) alone (a, black) and in the presence of b) 4 mM imidazole (b, orange), c) 40 M bA3-2 (c, green), d) 40 M bA3-2 and 4 mM imidazole (d, blue), e)
40 M His6-bA3-2 (e, red) f) 40 M His6-αRep-ns (f, purple), B) Rate of oxidation in the presence of increasing concentrations of bA3-2 (green), bA3-2 and 4 mM
imidazole (blue), His6-bA3-2 (red) and His6-αRep-ns (purple), C) Rate of oxidation as a function of pH in 10 mM citrate/MES/HEPES/CHES buffers for BH-MnTPP
alone (a, black) and in the presence of 40 M bA3-2 (b, green), 40 M bA3-2 and 4 mM imidazole (c, blue), 40 M His6-bA3-2 (d, red) and 40 M His6-Resp-ns
(e, purple).
A concerted activation mechanism
to the manganese (III) ion. However, this value remained about
20-fold higher than that found in the case of His6-bA3-2 (KS = 49
µM) suggesting that the binding of bA3-2 to A3A3’ positioned the
imidazole ring of one of the histidines of the His6-Tag in a very
favorable position for its coordination on the manganese ion.
Overall, the aforementioned results suggest that the His6-Tag of
His6-bA3-2 plays a key role in triggering the peroxidase activity of
the BH-MnTPP biohybrid: histidine coordinates to the Mn(III) ion,
possibly modulating its redox potential. In that regard it could play
the same role as the proximal histidine in the catalytic pocket of
heme peroxidases which participate in their well-known push-pull
mechanism (Fig. 6a).[21]
Complementary absorbance spectrophotometry studies were
performed to clarify the interaction between the His6-Tag label of
His6-bA3-2 and MnTPP. The addition of 100 M His6-bA3-2 led to
the modification of the MnTPP spectrum, with a hypochromic of
the Soret band of the MnTPP together with a bathochromic shift
of  and  bands from 572 to 575 nm and 606 to 611 nm
respectively indicating a possible coordination of the metal by
His6-bA3-2 (Fig. 4, red).
Under the same conditions, the addition of 4 mM imidazole to a
10 M solution of BH-MnTPP resulted in an intensity decrease of
the Soret band together with no significant shift of the  and 
bands (Fig. 4, orange). The same reaction in the presence of 100
M non-labeled bA3-2 resulted in a new spectrum similar to that
obtained in the presence of 100 M His6-bA3-2, with  and 
bands at 575 nm and 611 nm respectively, suggesting that Mn
coordination was facilitated by the presence of bA3-2 (Fig. 4, blue).
In contrast, the addition of 100 M bA3-2 to a 10 M solution BH-
MnTPP led to an intensity decrease of the Soret band without
making an impact on  and  bands (Fig. 4, green). It suggests
that the binding of bA3-2 to the bidomain led to widen the opening
of the cavity and thus a high exposure of the MnTPP cofactor to
aqueous media. Taken together, these results indicate that
spectroscopic changes could be due to the coordination of one
imidazole side chain of one of the His6-Tag histidines to the metal
center of the porphyrin upon His6-bA3-2 binding. Accordingly,
these spectral evolutions are similar to the ones reported by
Mahmoudi et al. for the coordination of imidazole to
Mn(TPP)OAc.[40] Indeed, upon addition of 1 mM imidazole to a 10
mM solution of Mn(TPP)OAc in CH2Cl2 saturated with water, a
decrease of the intensity of the Soret band at 477 nm and slight
shifts of the bands at 577 and 616 nm respectively towards 580
and 618 nm could be estimated from their reported spectra.
Figure 4. UV-visible spectrum of 10 M BH-MnTPP in 10 mM HEPES buffer
pH 8 (black) alone or in the presence of (orange) 4 mM ImH, (red) 100 M
His6-bA3-2, (green) 100 M bA3-2, or (blue) 100 M bA3-2 and 4 mM ImH.
The inset panel shows a focus of the same spectra between 550 and 560 nm.
The binding constants (KS) for the formation of Mn(III)-NHMal-
TPP-ImH were determined by differential spectroscopy (see SI,
Fig. S5). A binding constant KS = 98 mM was found for imidazole
in the case of the biohybrid alone, which decreased to 1 mM in
the presence of 40 M bA3-2, in line with the opening of the cavity
of A3A3’ by bA3-2 leading to an easier coordination of imidazole
To examine the scope of the reaction, two other peroxidase
cosusbtrates, 3,3’,5,5’-tetramethylbenzidine (TMB) and
ortho-dianisidine (ODA) (see SI, Fig. S3) were oxidized by H2O2
under the same conditions as previously described for OPD
(Table 1). A poor activity was found in the presence of the
biohybrid and 4 mM ImH with initial rates of oxidized product (Vi)
4
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10.1002/chem.202002434
Chemistry - A European Journal
RESEARCH ARTICLE
of 0.4 nM.s-1 for ODA and 0.6 nM.s-1 for OPD and an optimal
amount of product obtained after 4h of 4.3 M for ODA and 4.5
M for OPD. The addition of 40 M bA3-2 triggered the
peroxidase activity in the case of OPD by a factor of 3, and a factor
of about 5 in the case of ODA which is a larger substrate than
OPD.
Table 1. Oxidation of typical peroxidase cosubstrates by H2O2 in the presence of the BH-MnTPP and various co-catalysts.
Catalyst[a]
BH-MnTPP + 4 mM ImH
BH-MnTPP + 40 µM bA3-2
+ 4 mM ImH
BH-MnTPP
+ 40 µM His6-bA3-2
Vi (nM.s-1)
Product (µM)
Vi (nM.s-1)
Product (µM)
Vi (nM.s-1)
Product (µM)
Substrate
OPD (at 418 nm)
ODA (at 450 nm)
TMB (at 650 nm)
TMB (at 450 nm)
0.6
0.4
-
4.5
4.3
-
1.8
14.5
5.5
8.9
28.5
10
2
-
4
-
12.7
1.9
9.2
1.8
-
-
-
-
[a] Conditions: 500 µM substrate, 125 µM H2O2 in 10 mM HEPES buffer pH 8 at 20°C for 4 h in the presence of 2 µM BH-MnTPP and: a) 4 mM imidazole, b) 4 mM
imidazole and 40 µM bA3-2 c) 40 µM His6-bA3-2. The concentration of product was measured after 4h in all experiments.
Finally, the best activity was obtained for the 3 substrates in the
presence of 40 M His6-bA3-2, with Vi ranging from 5.5 nM.s-1 for
OPD to 12.7 nM.s-1 for TMB and an optimal amount of product
ranging from 1.8 M for TMB to 28.5 M for OPD.
higher with system 2 than with system 1 by a factor 2.2 in the case
of OPD and 3.7 with ODA (Table S1). Overall, the best catalytic
efficiencies were observed when ODA was used as substrate with
both systems.
In the case of TMB, two products can be detected by UV-visible
spectrophotometry, that are the charge transfer complex
(detected at 652 nm) and the diimine derivative (detected at 450
nm) (see SI, Fig. S3). Overall TMB appeared to be a poor
substrate, and only the system associating BH-MnTPP with His6-
bA3-2 allowed the appearance of small amounts of both products.
The kinetic parameters for the oxidation of OPD and ODA were
determined in order to compare the two systems associating 2 M
BH-MnTPP with i) 40 M bA3-2 and 4 mM ImH (system 1) or ii)
40 M His6-bA3-2 (system 2). As the peroxidase activity involves
two substrates, apparent kinetic parameters were determined by
using one of the substrates in large excess (2 mM) and by varying
the concentration of the second substrate, and then conversely
(Fig. 5). The data were fitted using a hyperbolic function
corresponding to the Michaelis-Menten model and the resulting
apparent kinetics parameters are summarized in Table S1. In all
cases, system 2 resulted in higher catalytic constants than system
1, suggesting the His6-Tag takes part in the catalytic process. kcat
values increased respectively by a factor 1.7, from 2.7 to 4.5 min-
1 for OPD and, by a factor 8.2, from 3.1 to 25.5 min-1 for ODA
(Table S1). The same effect was observed for the cosubstrate, as
kcat values for H2O2 increased respectively by a factor 3.4 from 1.5
to 5.1 min-1, for OPD oxidation, and by a factor 5.4, from 0.5 to 2.7
min-1, for ODA oxidation (Table S1). These higher kcat values
highlight the much better efficiency of His6-bA3-2 to facilitate the
transformation of the Mn-H2O2 adduct into an highly oxidizing Mn-
Oxo species.[5][6]
Figure 5. Initial rates of OPD and ODA oxidation with H2O2 by BH-MnTPP with
His6-bA3-2 (blue) or by BH-MnTPP with bA3-2 and 4 mM ImH (red). (A) H2O2
variation in the presence of 2 mM ODP; (B) H2O2 variation in the presence of 2
mM ODA; (C) OPD variation in the presence of 2 mM H2O2; (D) ODA variation
in the presence of 2 mM H2O2. For all the series of experiments, the data were
fitted using a hyperbolic model to determine the apparent kinetics parameters
(see SI table S1)
When compared to other metalloporphyrin catalysts, it appears
the peroxidase reactions catalyzed by the BH-MnTPP biohybrids
(best kcat value for BH-MnTPP in the presence His6bA3-2 = 0.5 s-
1, Table S1) are slower than those reported for HRP (kcat = 5 s-1),
Due to its reactivity towards proteins, the concentration H2O2 was
not raised above 2 mM, thus allowing only the determination of
apparent Michaelis constants and efficiency catalytic values for
H2O2, OPD and ODA substrates. The Michaelis constants, KM for
H2O2 were higher than 0.5 mM in all cases (Table S1), indicating
that the systems were not saturated with the H2O2 cosubstrate.
These values were very similar with both catalysts, indicating that
the binding of the H2O2 on the manganese ion was similar in the
presence of bA3-2 and ImH and in the presence of His6-bA3-2.
However, the catalytic efficiencies with respect to H2O2 were
Microperoxidase
8
(kcat
= 10 s-1), Fe-porphyrin-antibody
complexes (kcat = 1-15 s-1) but are about as fast than with Fe-
porphyrin complexes (0.35-2.8 s-1),[19] and Mn-MP8 (3.4 s-1) [41].
However, owing to rather low KM values for H2O2 (KMapp for H2O2
= 400-900 M, Table S1), the catalytic efficiency of the BH-
MnTPP biohybrids for H2O2 (Best value for BH-MnTPP in the
5
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10.1002/chem.202002434
Chemistry - A European Journal
RESEARCH ARTICLE
presence of His6bA3-2, with OPD as substrate kcat/KMapp = 130 M-
1s-1, Table S1) was in the range of those reported for Fe-
porphyrins and Fe-porphyrin-antibody complexes (3-4.8 x 103 M-
1s-1) [19] and Mn-HRP (1.7 x 103 M-1s-1),[42] but stays lower than that
observed with HRP (1.8 x 107 M-1s-1) [42] and Fe-MP8 (2.4 x 104 M-
1s-1).[19] As for the catalytic efficiency for the peroxidase
cosubstrates, that of the BH-MnTPP biohybrids stays below the
highest values observed for the oxidation of ABTS by H2O2
catalyzed by HRP (5.8 x 106 M-1s-1 at pH 4.6) and E2L-(TD)-
Fe(III)-Mimochrome VI (5.8 x 106 M-1s-1).[43]. However, concerning
the two cosubstrates used in this study; first, the best catalytic
efficiency obtained for ODA, kcat/KM app = 936 M-1s-1 with BH-
MnTPP in the presence of bA3-2 and ImH as catalyst (Table S1),
is in the range of those reported with Xln10A-Fe(TpCPP) (1.1 x
103 M-1s-1) and MP8-3A3 (3 x 104 M-1s-1) [43] as catalysts. Second,
the best catalytic efficiency obtained for OPD, kcat/KM app = 253 M-
1s-1 with BH-MnTPP in the presence of His6-bA3-2 as catalyst
(Table S1), is lower than that reported with HRP (3.95 x 106 M-1s-
1) as catalyst but much higher than that reported with PCN-222-
Fe (13.6) as catalyst [43].
In order to examine whether the BH-MnTPP-based systems were
also able to perform oxygen atom transfer reactions, the oxidation
of thioanisole by H2O2 was then performed in the presence of BH-
MnTPP under different conditions. In the case of BH-MnTPP
alone, no product was detected (Fig. 6 column A), but when 4 mM
imidazole was added, 18 M of sulfoxide was formed after 3 hours
(Fig. 6 column B). Further addition of bA3-2 led to an increase of
the concentration of sulfoxide to 33 M (Fig. 6 column D),
whereas finally the highest concentration of sulfoxide, 64 M (Fig.
6 column C), was obtained after 3 hours in the presence of His6-
bA3-2.
It is noteworthy that when the same reaction was performed in the
presence of 18O-labeled hydrogen peroxide, H218O2, mass
spectrometry analysis of the obtained product confirmed the
formation of 18O labeled sulfoxide (see SI, Fig. S4) in line with an
oxygen atom transfer from H2O2 catalyzed by the Mn-TPP moiety.
However, chiral HPLC analysis of the reaction mixture showed
the formation of racemic sulfoxide.
The best catalyst, BH-MnTPP in the presence of His6bA3-2 as
co-catalyst, then leads to a TON of 6.5 in 3 hours. This value can
be compared to those already reported for the sulfoxidation of
thioanisole catalyzed by artificial metalloenzymes that have been
reported in the literature [44]. This value is in the range of those
reported for Mn-horseradish peroxidase (HRP) (4 [44]) and Mn-
salen-M(T39C/L72C) (7 [45]) but it is far from the best values
reported for Mn-Corrole-BSA (150 [18] and the best one to date
Mn-mimochrome 6a (MnMC6*a, 870 [44]). It is also far from the
values observed for the best Fe-artificial enzymes (1500 for
FeMC6*a, [44], and 145 for Fe(TpCPP-Xln 10A, [46] that are
generally the best catalysts for the monooxygenase.
These results are in agreement with the above mentioned results
for the oxidation of typical peroxidase substrates, and confirm that
the association of the BH-MnTPP with the His6-bA3-2 protein
leads to the best catalyst for the monooxygenation of thioanisole,
due to the combination of the opening of the (A3A3’) bidomain by
the bA3-2 protein and of the possible involvement of its His6-Tag
in the formation of highly oxidative manganese-oxo species (Fig.
6a).
Molecular Modelling
With the aim to shed light on the molecular events leading to the
switchable catalysis, a molecular modelling study was performed
as it has now become a key player in sustaining ArM design with
or without X-ray structures available. [47][48][49][50] First, a series of
large Molecular Dynamics (MD) was carried on: ii) BH-MnTPP; ii)
BH-MnTPP in presence of the triggering His6-bA3-2 protein, and
iii) His6-bA3-2 included BH-MnTPP with different possible
coordination between the Mn(III) ion and histidine residues from
the His6-Tag of bA3-2 (His5, His7, His8 of the His6-Tag from the
N-terminal protein sequence M1R2G3S4H5H6H7H8H9H10). The
MDs served to evaluate the flexibility of the bio-hybrid, the viability
of substrate binding cavities for the reaction to take place, and
finally the best possible axial coordination for the metal throughout
a N-His donor.
Posterior to the molecular dynamics simulations, further insights
on catalysis was pursued via protein-ligand docking of the
substrate to convenient cavities.
Figure 6. Sulfoxidation of thioanisole by H2O2 catalyzed by BH-MnTPP in the
presence of various co-catalysts: a) Mechanism, b) Sulfoxidation of 500 M
thioanisole by 1 mM H2O2 : A) in the presence of 10 M BH-MnTPP alone and
in the presence of, B) 4 mM imidazole, C) 100 M His6-bA3-2, D) 100 M bA3-
2 with 4 mM imidazole, in 10 mM HEPES 150 mM NaCl buffer pH 8 at 20°C for
3 hours.
6
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10.1002/chem.202002434
Chemistry - A European Journal
RESEARCH ARTICLE
Figure 7. Schematic representation of the conformational behavior of the
systems: a) BH-MnTPP closed conformation; b) His6-bA3-2-BH-MnTPP in open
conformation; c) His6-bA3-2-BH-MnTPP after coordination of His5; d and e)
dynamic equilibria between most sampled conformations after coordination of
His5 or His7 and H2O, and f) considering the coordination of His8. Each system
was studied along 200 ns of MD for total explorations of 1 s. Atomistic
representations of such conformations are shown in Figure S7 and trajectories
of the dynamics in movies S1-5.
Figure 8. A global view of conformation IV and V of the enzyme including the
triggering His6-bA3-2 coordinating the metal center by His5 is depicted in panels
a) and b) respectively. The best thioanisole docking solution for conformation IV
(GoldScore 37.48), and V (GoldScore 37.61) are reported respectively in panels
c) and d). Amino acid side chains within 5 Å from the ligand are also shown.
The analysis of the 200 ns MD of BH-MnTPP clearly shows a
stable closed conformation, displaying the catalytic cofactor
buried into the cavity between the two monomers and particularly
close to its A3 subunit (Fig. 7a). Little room exists in this pocket
so that substrate could bind and the catalysis could occur. The
MD simulation with inserted His6-bA3-2 presents a substantially
different behavior that consists in first, the separation of the two
subunits opening the dimer´s shell (ca. 50ns), then an inter-
domain rotation (ca. ~50 ns) that finally leads to an open
conformation stable along the remaining 100 ns of the simulation
(Fig. 7b). In this calculation, the MnTPP cofactor stands close to
its A3 interface as in the closed form, but the wide opening of the
dimeric BH allows histidine residues from the bA3-2 His6-Tag to
approach the Mn(III) ion in proximal axial position. The residue
that presents the longest residency in such position is the His5
sidechain. Therefore, a subsequent MD was carried coordinating
N-His5, in the apical vacancy, of the metal. Along the 200 ns, the
relative conformation of the two monomers is almost conserved
while the MnTPP cofactor attains to a slight rotation enlarging the
cavity at the A3 interface and enabling a H2O axial coordination
(Fig. 7c). This simulation shows a dynamic equilibrium between
the MnTPP in contact with A3 or slightly rotated with respect to it
(Figure 7d and e). Interestingly, along the MD trajectories, some
negatively charged amino acids side chains (Glu48’ and Asp57)
are found close to the axial ligand suggesting that they could
assist the H2O2 deprotonation and heterolytic cleavage of the O-
O bond leading to Mn-oxo species responsible for the oxidation
reaction (Fig. 6a). For the sake of the study, two similar MDs were
carried out considering the coordination by N-His7 and N-His8 of
His6-bA3-2 to the metal since a certain amount of conformations
of the previous simulation lead to close distances between their
imidazole nitrogen atoms and manganese. Both MDs show an
increase of conformations where the MnTPP is rotated and
separated with respect to A3 (Fig. 7e). In particular, with the
coordination by His8 the rotation of the MnTPP cofactor leads to
its exposition to the solvent (Fig. 7f).
Overall, MD simulations show that the biohybrid construct
provides with the necessary dynamical behavior to open the
system, allowing histidine coordination and finally providing stable
geometries adequate for substrate binding.
Docking calculations for the thioanisole binding were further
carried out on the most sampled conformations of each trajectory
considering the sixth axial position occupied by water and oxo
ligand after Mn(III) oxidation. The results, summarized in Table
S2 in SI, suggest that: i), the BH-TPP closed form presents an
adequate cavity for the substrate binding and also to host an
imidazole (Fig. S6a); ii) for open conformations obtained in the
MDs, the substrate presents good affinities, particularly those with
coordination by His5 and iii) coordination of His7 and His8 leads
to a wide MnTPP-A3 cavity, making these the worst candidates to
bind the thioanisole substrate since no clear pocket is defined in
these structures (See SI, Table S2).
Considering the pre-catalytic Mn-oxo complex, the better bindings
are reached for conformations IV and V (models with coordinated
His5, Fig. 8a and 8b) displaying a high affine binding site.
Conformation IV is characterized by the polar oxo-thiol and Q57-
thiol, the CH- Ph-TPP, and VdW D57, V88, S85 (Fig. 8c).
Conformation V shows a slight open cavity enabling a − Ph-
TPP stacking, oxo-thiol VdW Q92, V88, and F119 (Fig. 8d). It can
be highlighted that for each docking assay, both pro-S and pro-R
configurations are observed with similar affinities suggesting that
the cavity not display enantio-selective features (see SI, Table S2).
Conclusion
The possibility to build new-to-Nature enzymes compatible with
cellular media is crucial in the green chemistry agenda. Major
breakthroughs in this field are coming from the development of
artificial metalloenzymes although some designs look like far from
reaching objectives. One of those are de novo cytochromes P450
7
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RESEARCH ARTICLE
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particular care has to be given to the protection of the metallic
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Supporting Information
The Supporting Information includes: Additional figures, (Figures S1 –
S12), experimental procedures, and characterization data
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Acknowledgements
We thank the “Initiative Doctorale Interdisciplinaire IDEX”
program of University Paris-Saclay for the financial support of
author T.D.M. G.S. and J.-D.M. gratefully acknowledge the
financial support from the Spanish MINECO (CTQ2017-87889-P).
G.S. thanks Regione Autonoma della Sardegna (grant
RASSR79857) for a post-doctoral fellowship.
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Keywords: artificial metalloenzyme • inducible activity •
hemoprotein • porphyrin biohybrid • Oxidation • artificial repeat
proteins
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Entry for the Table of Contents
The first example of an artificial metalloenzyme with triggerable oxidation activities, reminiscent of the activation or allosteric
regulation of the catalytic activity of enzymes by specific regulatory proteins.
10
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