Conversion of Human Neuroglobin into a Multifunctional Peroxidase by Rational Design

Article abstract of DOI:10.1021/acs.inorgchem.0c03777

Protein design has received much attention in the last decades. With an additional disulfide bond to enhance the protein stability, human A15C neuroglobin (Ngb) is an ideal protein scaffold for heme enzyme design. In this study, we rationally converted A15C Ngb into a multifunctional peroxidase by replacing the heme axial His64 with an Asp residue, where Asp64 and the native Lys67 at the heme distal site were proposed to act as an acid-base catalytic couple for H2O2 activation. Kinetic studies showed that the catalytic efficiency of A15C/H64D Ngb was much higher (～50-80-fold) than that of native dehaloperoxidase, which even exceeds (～3-fold) that of the most efficient native horseradish peroxidase. Moreover, the dye-decolorizing peroxidase activity was also comparable to that of some native enzymes. Electron paramagnetic resonance, molecular docking, and isothermal titration calorimetry studies provided valuable information for the substrate-protein interactions. Therefore, this study presents the rational design of an efficient multifunctional peroxidase based on Ngb with potential applications such as in bioremediation for environmental sustainability.

Full text of DOI:10.1021/acs.inorgchem.0c03777

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Article
Conversion of Human Neuroglobin into a Multifunctional
Peroxidase by Rational Design
Shun-Fa Chen, Xi-Chun Liu, Jia-Kun Xu, Lianzhi Li,* Jia-Jia Lang, Ge-Bo Wen, and Ying-Wu Lin*
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ABSTRACT: Protein design has received much attention in the last
decades. With an additional disulfide bond to enhance the protein
stability, human A15C neuroglobin (Ngb) is an ideal protein scaffold for
heme enzyme design. In this study, we rationally converted A15C Ngb
into a multifunctional peroxidase by replacing the heme axial His64 with
an Asp residue, where Asp64 and the native Lys67 at the heme distal
site were proposed to act as an acid−base catalytic couple for H2O2
activation. Kinetic studies showed that the catalytic efficiency of A15C/
H64D Ngb was much higher (∼50−80-fold) than that of native
dehaloperoxidase, which even exceeds (∼3-fold) that of the most
efficient native horseradish peroxidase. Moreover, the dye-decolorizing
peroxidase activity was also comparable to that of some native enzymes. Electron paramagnetic resonance, molecular docking, and
isothermal titration calorimetry studies provided valuable information for the substrate−protein interactions. Therefore, this study
presents the rational design of an efficient multifunctional peroxidase based on Ngb with potential applications such as in
bioremediation for environmental sustainability.
the last decades for the biodegradation of highly toxic
halophenols.28,29 By introducing a negatively charged residue
near the heme proximal ligand His89, the M86E Amphitrite
ornata DHP exhibited an ∼9-fold higher turnover number
(kcat) than that of the wild-type (WT) enzyme.30 Much
progress has also been made in the design of functional heme
enzymes using the protein scaffold of Mb.18,31−36 For example,
the Asp64 mutant of Mb mimicking the active site of
chloroperoxidase (CPO) can facilitate the formation of the
reactive intermediate, an oxoferryl heme π-cation radical
named compound I.37,38
Inspired by these findings, in a previous study, we designed a
double-mutant of F43Y/H64D Mb that exhibits a catalytic
efficiency of more than 1000-fold than that of native
dehaloperoxidase, which has potential applications in the
bioremediation of toxic halophenols.39 In-depth structural
analysis of the dye-decolorizing peroxidases (DyPs) further
revealed the importance of the acidic residue in the heme distal
site. The B-type DyPs adopt a highly conserved catalytic Asp−
Arg couple to enhance the rate of peroxide heterolysis in which
the acidic aspartate is crucial for H2O2 deprotonation.40−42
INTRODUCTION
■
Neuroglobin (Ngb) is a member of the globin superfamily with
a high O2 binding affinity, whereas it shares less than 25%
sequence identity with myoglobin (Mb) and hemoglobin
(Hb).1,2 As predominantly expressed in nervous systems such
as the brain and retina, it plays a critical role in neuron
protection.1,3−7 Ngb is a monomeric and hexacoordinated
heme protein with a bis-His (His64/His96) coordination and
has a typical three over three α-helical sandwich fold.8−10 In
addition, the human Ngb is endowed with an intramolecular
disulfide bond (Cys46−Cys55) between helixes C and D that
regulate the binding behavior of small ligands (O2, NO,
CO).11−13 Replacement of the heme axial ligand His64 can
change the protein function. For example, the H64Q variant is
a ligand-trap antidote that tightly binds CO, while the H64A
variant can serve as an efficient nitrite reductase.14,15 We also
showed that the H64M Ngb variant is capable of self-oxidation
to produce methionine sulfoxide and sulfone.16 Recently, we
found that an Ala-to-Cys mutation at position 15 in human
Ngb can introduce an additional intramolecular disulfide bond
with Cys120, which significantly improved the stability of the
protein and prevented protein oligomerization.17 These
features make the A15C Ngb mutant a promising model for
the design of heme enzymes.
Received: December 24, 2020
Published: February 4, 2021
Protein design such as the design of heme enzymes
including peroxidases has received much attention for potential
applications in industry, bioremediation, biobleaching, and so
on.18−27 Since the discovery of the first native dehaloperox-
idase (DHP) in 1996, significant advance has been achieved in
https://dx.doi.org/10.1021/acs.inorgchem.0c03777
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The X-ray structure of human Ngb shows that it has a
residue of Lys67 near the distal heme site (Figure 1A).
titration studies. The spectra showed that the Soret band of
A15C/H64D Ngb decreased slightly and the visible band
shifted from 630 nm to 615 nm upon the titration of TCP
(Figure 2A) or TBP (Figure S3). These results suggest that
Figure 1. Rational protein design based on the Ngb scaffold. (A) X-
ray crystal structure of WT Ngb (PDB code 4MPM).10 (B) Modeling
structure of A15C/H64D Ngb constructed by molecular dynamic
simulation. The residues Asp64 and Lys67 are shown as red and
marine sticks, respectively. The water molecule (w) bound to the
heme iron is depicted as a red sphere. The H-bond between Asp64
and w is indicated as a black dashed line. The designed disulfide bond
between Cys15 and Cys120 is colored violet.
Moreover, as aforementioned, the A15C Ngb mutant is an
ideal protein scaffold for heme enzyme design. Therefore, in
this study we introduced a His-to-Asp mutation at position 64
of A15C Mb. The residues Asp64 and Lys67 in the double
mutant of A15C/H64D Ngb were proposed to form a catalytic
acid−base couple, as shown by the modeling structure (Figure
1B). Thus, the double mutant was expected to exhibit
enhanced peroxidase activity.
Figure 2. (A) UV−Vis titration of 0−500 μM TCP to 10 μM A15C/
H64D Ngb in 100 mM potassium phosphate buffer (pH 7.0) at 25
°C. The spectral changes at 615 nm upon TCP titration are shown as
an inset. (B) EPR spectra of 0.5 mM ferric A15C/H64D Ngb in the
absence (black) and presence of 1 equivalent TCP (cyan) or TBP
(red).
RESULTS AND DISCUSSION
■
To test our idea, we expressed and purified A15C/H64D Ngb
as well as a serine mutant A15C/H64S Ngb for comparison.
The purified proteins were certificated by an electrospray
ionization mass spectrometer (Figure S1). It showed a mass of
16939.0 Da for A15C/H64D Ngb and 16911.5 Da for A15C/
H64S Ngb, respectively, which was in agreement with the
calculated mass of the apo-protein with two disulfide bonds
(A15C/H64D, 16939.4 Da; A15C/H64S, 16911.4 Da). These
double mutants exhibited similar UV−Vis spectra, which differ
from that of A15C Ngb (Figure S2). In the ferric form, the
Soret band was blue-shifted to 406 and 405 nm in A15C/
H64D Ngb and A15C/H64S Ngb, respectively. The major
visible bands were at 500 and 630 nm, which were similar to
those of H64M Ngb (500, 630 nm) and met Mb (532, 630
nm).16,43 These spectral features support our design in which
the residue Asp64 or Ser64 is located in the heme distal site
with a water molecule coordinated to the ferric heme ion.
To test whether the substrate of dehaloperoxidase, such as
2,4,6-trichlorophenol (TCP) and 2,4,6-tribromophenol (TBP),
can bind to the designed enzyme, we first performed UV−Vis
both TCP and TBP bind to the heme distal site and affect the
heme micro-environment, especially for the distal water
coordination.
Moreover, we applied electron paramagnetic resonance
(EPR) spectrometry to probe the heme coordination state in
the absence or presence of TCP or TBP (Figure 2B). The
ferric A15C/H64D Ngb exhibited typical 6-coordinated high-
spin (6cHS) heme signals with g tensor calculated to g⊥ = 5.53
and g|| = 1.99, which were similar to other globins with H2O/
His coordination.39,44 A small fraction of low-spin species was
also detected with gz = 2.82 and gy = 2.15. In the presence of
substrates, the signal of g⊥ shifted to 5.44 and the intensity was
enhanced, which indicates an enhancement of the 6cHS
component of A15C/H65D Ngb, especially for the binding of
TBP. gz shifted to 2.64 in the presence of TCP and TCB,
whereas gy exhibited a different behavior. gy was unchanged
upon TCP loading, while it shifted to 2.27 upon TBP loading
likely due to a larger structural alteration by TBP binding to
the heme center. These observations showed that the TCP/
TBP binding alters the heme micro-environment of A15C/
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H65D Ngb, as also shown by the UV−Vis titration (Figure
2A).
To confirm that the designed A15C/H64D variant actually
exhibits dehaloperoxidase activity, we first collected the time-
dependent UV−Vis spectra in the presence of substrate TCP
(Figure 3A) or TBP (Figure 3B) using H2O2 as an oxidant. As
Table 1. Kinetic Parameters for H2O2-Dependent
Dehalogenation of TCP and TBP Catalyzed by WT, A15C,
A15C/H64S, and A15C/H64D Ngb, with DHP A and HRP
Shown for Comparison
protein
kcat (s−1
)
Km (mM)
k
cat/Km (M−1·s−1
)
TCP
WT Ngb
A15C Ngb
A15C/H64S Ngb
A15C/H64D Ngb
DHP A46
HRP46
0.03 0.01
0.02 0.01
13.95 0.51
43.85 0.62
13.67
0.34 0.08
0.21 0.06
0.58 0.04
0.13 0.01
2.07
88
95
24,000
340,000
6600
571.3
5.40
106,000
TBP
WT Ngb
A15C Ngb
A15C/H64S Ngb
A15C/H64D Ngb
DHP A46
HRP46
0.03 0.01
0.03 0.01
2.90 0.08
8.40 0.08
1.83
2.12 0.64
1.39 0.36
0.20 0.02
0.06 0.01
1.02
14
22
14,500
140,000
1790
223.4
4.53
49,300
of A15C/H64S Ngb and the native enzyme DHP for TCP
dehalogenation. Moreover, A15C/H64D Ngb exhibited ∼4.5-
fold, ∼16-fold, and ∼42-fold smaller Km values as compared to
those of A15C/H64S Ngb, DHP, and the native enzyme,
horseradish peroxidase (HRP), respectively.46 As a result, the
overall catalytic efficiencies (kcat/Km) were ∼14-fold, ∼51-fold,
and ∼3.2-fold higher than those of A15C/H64S Ngb, DHP,
and HRP, respectively. In addition, A15C/H64D Ngb also
exhibited ∼10-fold, ∼78-fold, and ∼2.8-fold higher catalytic
efficiencies for TBP dehalogenation as compared to A15C/
H64S Ngb, DHP, and HRP, respectively. Taken together, the
rationally designed A15C/H64D Ngb showed excellent
dehalogenation activities, which are more effective than those
of the native DHP A and HRP (Table 1).
Besides the dehalogenation activity, we also evaluated the
dye-decolorizing peroxidase (DyP) activity using the reactive
blue 19 (RB19) as a typical substrate. The control studies
showed that WT and A15C Ngb hardly exhibited dye-
decolorizing activities (Figure S6) due to the lack of an open
heme active site by bis-His coordination. Remarkably, the
A15C/H64D Ngb mutant can oxidize RB19 to a colorless
product in several seconds (Figure 4). The initial rate was
calculated by monitoring the decay of the absorption at 595
nm. The optimal concentration of H2O2 was found to be 1
mM (Figure S7). The steady-state reactions by varying the
RB19 concentration were performed to measure the kinetic
parameters (Figure 4C). As listed in Table 2, A15C/H64D
Ngb exhibited an ∼4-fold higher kcat value and an ∼2.9-fold
lower Km value than those of A15C/H64S Ngb. Therefore, the
overall catalytic efficiency of A15C/H64D Ngb is ∼12-fold and
∼32-fold higher than that of A15C/H64S Ngb and the native
VcDyP, respectively.47 Moreover, it is double that of TfuDyP48
and exceeds that of TvDyP1,49 although it is still lower than
that of TcDyP.50
Figure 3. UV−Vis spectral changes of 0.1 mM TCP (A) and 0.1 mM
TBP (B) dehalogenation catalyzed by 2.5 μM A15C/H64D Ngb
upon the addition of 0.5 mM H2O2 in 50 mM potassium phosphate
buffer (pH 7.0) at 25 °C. Steady-state rates of oxidation as a function
of TCP (C) and TBP (D) concentration catalyzed by WT (black),
A15C (cyan), A15C/H64S (yellow), and A15C/H64D (red) Ngb.
The experiments were carried out in triplicate.
shown in the spectra, the absorptions of TCP (244, 312 nm)
and TBP (249, 316 nm) decreased rapidly. A characteristic
peak at 272 or 290 nm increased, which corresponds to the
oxidation product of 2,6-dichloroquinone (DCQ) and 2,6-
dibromoquinone (DBQ), respectively. The formation of the
oxidation products was also confirmed by ESI-MS studies
(Figure S4). The time-dependent absorbance changes at 272
and 290 nm showed that TCP or TBP was converted to DCQ
or DBQ within 40 s as catalyzed by A15C/H64D Ngb in the
presence of H2O2. These results suggest that the rationally
designed A15C/H64D Ngb is indeed an efficient dehaloper-
oxidase.
Since TCP/TBP was rapidly oxidized by A15C/H64D Ngb,
we then measured the kinetic parameters for the steady-state
reactions at various substrate concentrations using stopped-
flow spectroscopy. The optimal concentration of H2O2 was
found to be 20 mM for TCP dehalogenation and 5 mM for
TBP dehalogenation, respectively (Figure S5). Control experi-
ments were performed for A15C and WT Ngb under the same
conditions. The kinetic parameters were obtained by fitting the
data to a Michaelis−Menten equation (Figure 3C,D). Note
that no substrate inhibition was observed for both substrates,
which was different from that observed for the dehalogenation
catalyzed by DHP or F43Y/H64D Mb.39,45
To explore the oxidation mechanism for the peroxidase
activity, we inspected the H2O2 activation ability of these Ngb
variants by stopped-flow spectroscopy studies. The results
showed that A15C/H64D Ngb reacted with H2O2 rapidly and
two peaks at ∼539 nm and ∼575 nm appeared in 1 s (Figure
S8), indicating the formation of the reactive oxoferryl heme
intermediate, named compound II.51 The observed rate
constant (kobs) was measured by fitting the decay curve of
As listed in Table 1, the kcat value was increased by 3 orders
of magnitude for A15C/H64D Ngb-catalyzed TCP dehaloge-
nation as compared to the control reactions catalyzed by WT
or A15C Ngb. The kcat value was also ∼3-fold higher than that
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Figure 5. Activation of H2O2 by A15C/H64D Ngb. (A) Stopped-flow
spectra upon mixing 5 μM A15C/H64D Ngb in 100 mM potassium
phosphate buffer (pH 7.0) and 0.5 mM H2O2 for 10 s. The decay of
the Soret band at 406 nm is shown as an inset. (B) Plots of the
observed rate constants (kobs) versus H2O2 concentrations. The linear
fit of data yielded the apparent rate constant (k1) of compound II
formation as the slope of the line.
protein complexes were obtained by using Autodock with the
modeling structure of A15C/H64D Ngb as a receptor. Out of
10 predicted models for each complex, the three structures
with the lowest energy were visualized. In the cases of TXP (X
= Cl, Br), the docking structures revealed that these small
substrates bound to the protein distal pocket close to the
protein surface (Figures 6A and S9). The phenolic hydroxyl
Figure 4. Dye-decolorizing peroxidase activity of the Ngb mutants.
(A) Stopped-flow spectra upon mixing 2.5 μM A15C/H64D Ngb in
the presence of 50 μM RB19 with 1 mM H2O2 in 100 mM potassium
phosphate buffer (pH 7.0) at 25 °C. (B) Time-dependent absorbance
change at 595 nm for A15C/H64D Ngb-catalyzed RB19 decolorizing
in A. The solution before and after H2O2 oxidation is shown as insets.
(C) Steady-state rate of oxidation as a function of RB19 concentration
catalyzed by A15C/H64D (red) and A15C/H64S (orange) Ngb.
Table 2. Kinetic Parameters for H2O2-Dependent Oxidation
of RB19 Catalyzed by A15C/H64S and A15C/H64D Ngb,
with Some Native DyPs Shown for Comparison
protein
kcat (s−1
)
Km (μM)
k
cat/Km (M−1·s−1
)
A15C/H64S Ngb
A15C/H64D Ngb
VcDyP47
2.06 0.11
8.38 0.62
1.3 0.3
10
28.64 3.00
10.03 2.26
50 20
71,900
836,000
26,000
345,000
629,600
2 300 000
TfuDyP48
29
TvDyP149
TcDyP50
23.8 0.62
37.8 3.34
96
2
42
1
the Soret band to a pseudo-first-order equation (Figure 5A).
The formation rate of compound II (k1) was obtained by a
linear fitting of kobs versus H2O2 concentration (Figure 5B). It
showed that A15C/H64D Ngb can activate H2O2 with a rate
constant (k1 = 1.47 0.02 mM−1·s−1) ∼7-fold faster than that
of A15C/H64S Ngb (k1 = 0.22
0.03 mM−1·s−1), which
strongly supports our design based on the acid−base catalytic
Figure 6. Docking structures of TCP- (A) and RB19- (B) A15C/
H64D Ngb complexes with three lowest energies. The substrates are
shown as sticks. The heme moiety is colored orange. The protein
matrix is depicted as a gray cartoon or surface. H-bonds between
substrates and protein are indicated as a black dashed line.
mechanism as demonstrated by the native DyPs.41
We further performed molecular docking studies to gain
more insight into the substrate binding mechanism of A15C/
H64D Ngb. The TXP−protein (X = Cl, Br) and RB19−
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group of TXP formed a hydrogen bond (H-bond) with the
distal Asp64, thus stabilizing the binding of TXP to the protein.
The substrate was further stabilized by the H-bond with Lys67.
Moreover, the binding was enhanced by the π−π stacking
between the benzene rings of TXP and Phe49. As shown in
Tables S2 and S3, the lower binding energy of TBP indicates a
stronger binding compared to that of TCP, which agrees with
the lower Km value for TBP binding to A15C/H64D Ngb
(Table 1). These observations may because TBP has a large
van der Waals force compared to that of TCP binding to the
protein. In addition, the complex structure showed that the
binding of TXP affects the H-bond network around the heme-
coordinated water molecule and the catalytic Asp64-Lys67
pair, which provides structural information for the TXP-
induced UV−Vis spectral change at 630 nm (Figures 2A and
S3).
over native DHP in catalytic efficiency but also eliminates the
substrate inhibition effect caused by the internal binding
model.53 It should be noted that although the catalytic
efficiency cannot match that of our previously designed
artificial dehaloperoxidase, F43Y/H64D Mb,39 the kcat of
A15C/H64D Ngb was increased by 60% without an inhibition
effect of TXP. Because the wastewater could be contaminated
by both halophenols and industrial dyes, the study suggests
that A15C/H64D Ngb is an efficient multifunctional
peroxidase, which has potential applications in bioremediation
for environmental sustainability.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c03777.
■
sı
In the case of RB19 binding, it showed that the dye bound
to the heme edge on the protein surface where a series of
interactions occurred (Figures 6B and 7A). The terminal
Experimental details, UV−Vis spectra, mass spectra,
kinetic studies, and docking results (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Lianzhi Li − School of Chemistry and Chemical Engineering,
Liaocheng University, Liaocheng 252059, China;
orcid.org/0000-0002-8447-3803; Email: lilianzhi1963@
163.com
Ying-Wu Lin − School of Chemistry and Chemical
Engineering, University of South China, Hengyang 421001,
China; Laboratory of Protein Structure and Function,
University of South China Medical School, Hengyang
421001, China; orcid.org/0000-0002-2457-0871;
Email: ywlin@usc.edu.cn
Authors
Shun-Fa Chen − School of Chemistry and Chemical
Engineering, University of South China, Hengyang 421001,
China
Figure 7. Binding of RB19 to A15C/H64D Ngb. (A) 2D interaction
diagram between RB19 and protein in the docking model with the
lowest energy. (B) ITC data for titration of 10 μM A15C/H64D Ngb
with 500 μM RB19 in 100 mM potassium phosphate buffer (pH 7.0)
at 25 °C. The top panel shows the raw data, and the bottom panel
shows the integrated heat versus substrate/enzyme ratio. The data are
fitted to a one-site binding model.
Xi-Chun Liu − School of Chemistry and Chemical Engineering,
University of South China, Hengyang 421001, China
Jia-Kun Xu − Key Lab of Sustainable Development of Polar
Fisheries, Ministry of Agriculture and Rural Affairs, Lab for
Marine Drugs and Byproducts of Pilot National Lab for
Marine Science and Technology, Yellow Sea Fisheries
Research Institute, Chinese Academy of Fishery Sciences,
Qingdao 266071, China
Jia-Jia Lang − Laboratory of Protein Structure and Function,
University of South China Medical School, Hengyang
421001, China
Ge-Bo Wen − Laboratory of Protein Structure and Function,
University of South China Medical School, Hengyang
421001, China
sulfate group of RB19 formed multiple H-bonds with Asn45
and Lys67 near the distal heme site, and the 2-sulfate group on
the anthracene ring formed an H-bond with Lys95. Major van
der Waals interactions include those between the anthracene
ring and Leu70, the benzene ring, and the side chain of Lys67.
These protein−substrate contacts resulted in a moderate
binding affinity of RB19 to A15C/H64D Ngb. This was further
supported by the isothermal titration calorimetry (ITC) study,
which showed a one-site binding model, with an association
constant of 1.32 0.04 × 104 M−1 (Figure 7B).
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.0c03777
CONCLUSIONS
■
Author Contributions
In summary, we have successfully converted human Ngb into
an efficient peroxidase, A15C/H64D Ngb, with both
dehalogenation and dye-decolorizing activities even exceeding
some of native enzymes. The rationally designed acidic Asp64
together with native Lys67 at the heme distal site enhanced the
activation rate of H2O2, presumably through the acid−base
catalytic mechanism.41,52 The Asp64 substitution also facili-
tated the substrate binding as compared to the Ser64
substitution. A15C/H64D Ngb not only has an advantage
S.-F.X., X.-C.L., and J.-K.X. contributed equally. The manu-
script was written through contributions of all authors. All
authors have given approval to the final version of the
manuscript.
Funding
This work was supported by the National Natural Science
Foundation of China (no. 21977042), Special Project of Major
Scientific and Technological Innovation in Shandong Province
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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