Understanding the role of oxo and peroxido species in the glutathione peroxidase (GPx)-like activity of metal based nanozymes

Article abstract of DOI:10.1016/j.ica.2018.09.045

Nanomaterials having enzyme-like activities (nanozymes) attract significant attention. Particularly, metal-based nanozymes that exhibit superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities find applications in controlling the redox state of the cells. It has been shown that V₂O₅ functionally mimic the GPx enzyme by catalytically reducing H₂O₂ in the presence of glutathione (GSH). In this paper, we discuss the role of oxo and peroxido species in the GPx activity of V₂O₅ and MoO₃. A detailed mechanistic study reveals that the GPx activity of these materials depends on the reactivity of oxo moiety on the surface of the material with H₂O₂ to form the peroxido linkages.

Full text of DOI:10.1016/j.ica.2018.09.045

Accepted Manuscript
Research paper
Understanding the role of oxo and peroxido species in the glutathione peroxidase
(
GPx)-like activity of metal based nanozymes
Sourav Ghosh, Sanjay Prasad, Govindasamy Mugesh
PII:
S0020-1693(18)31296-9
https://doi.org/10.1016/j.ica.2018.09.045
ICA 18503
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Accepted Date:
21 August 2018
17 September 2018
Please cite this article as: S. Ghosh, S. Prasad, G. Mugesh, Understanding the role of oxo and peroxido species in
the glutathione peroxidase (GPx)-like activity of metal based nanozymes, Inorganica Chimica Acta (2018), doi:
https://doi.org/10.1016/j.ica.2018.09.045
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Understanding the role of oxo and peroxido species in
the glutathione peroxidase (GPx)-like activity of metal
based nanozymes
Sourav Ghosh, Sanjay Prasad and Govindasamy Mugesh*
Department of Inorganic & Physical Chemistry, Indian Institute of Science,
Bangalore 560 012; Email: mugesh@iisc.ac.in
Abstract
Nanomaterials having enzyme-like activities (nanozymes) attract significant attention.
Particularly, metal-based nanozymes that exhibit superoxide dismutase (SOD), catalase
(
CAT) and glutathione peroxidase (GPx) activities find applications in controlling the redox
state of the cells. It has been shown that V functionally mimic the GPx enzyme by
catalytically reducing H in the presence of glutathione (GSH). In this paper, we discuss
the role of oxo and peroxido species in the GPx activity of V and MoO . A detailed
mechanistic study reveals that the GPx activity of these materials depends on the reactivity of
oxo moiety on the surface of the material with H to form the peroxido linkages.
2
O
5
2
O
2
2
O
5
3
2
O
2
1
. Introduction
Excessive generation of reactive oxygen species (ROS) such as hydrogen peroxide (H
2
O
2
),
-
hydroxyl radical (˙OH) and superoxide (O
2
) induce oxidative stress resulting in damage of
biomolecules such as DNA, proteins, and lipids [1-2]. In the long term, these effects can lead
to various pathophysiological conditions like neurodegeneration, HIV activation, diabetes,
cardiovascular diseases, cancer, and aging [3-4]. The biological paradox is that ROS at
moderate concentration act as signalling molecules in various biochemical reaction pathways,
which are important for immune response in cells [5]. However, a very low concentration of
ROS will slow down metabolism and cause immunosuppression. Therefore, to conserve
proper cellular function, a fine balance between the level of ROS and the antioxidant
machinery is important. In this context, major cellular antioxidant enzymes such as
superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and several
small molecules like glutathione (GSH), lipoic acid, vitamin C etc can protect the cells from
oxidative damage and maintain the level of ROS bellow toxic threshold to maintain the redox
homeostasis [6-7].
Nanomaterial-based artificial enzymes (nanozymes) have attracted significant interest due to
their high catalytic efficiency, robustness, and non-toxicity compared to enzyme mimetic
organic molecules, metal complexes, and metal-organic frameworks [8-12, 30]. Recently, we
have demonstrated the potent GPx mimetic activity of V
role in redox regulation in vitro [13]. Among three major redox enzymes, glutathione
peroxidase (GPx) plays an important role in the regulation the level of H by converting it
to H O using GSH as a cofactor. As developing new potential GPx-mimetic nanozymes is a
2
O
5
based nano-isozymes and their
2
O
2
2
challenging task, detailed mechanistic understanding of the activity of nanomaterials is
crucial to design suitable materials for biomedical research. In this paper, we report the
synthesis and GPx-like activity of V
2
O
5
and MoO nanowires. We have described the
3

plausible mechanism of GPx-like activity for metal oxide based nanozymes. The intermediate
species (M-peroxido) involved in the enzymatic cycle was characterized by in-situ FT-IR and
FT-Raman spectroscopy.
2
. Experimental
2
.1. Reagents and materials
Vanadyl sulfate monohydrate (VOSO
.H
4 2
O), sodium molybdate dihydrate (Na
2
MoO
4
.2H O)
2
potassium bromate (KBrO ), Conc. HCl, Conc. HNO
3
3
, absolute ethanol, acetone was
obtained from S. D. Fine Chemicals Pvt. Ltd. Reduced β-nicotinamide adenine dinucleotide
phosphate (β-NADPH) was obtained from Sisco Research Laboratories Pvt. Ltd. (SRL)
India. Hydrogen peroxide 30% (H
2
O
2
), Sodium dihydrogen phosphate (NaH
2
PO ), disodium
4
hydrogen phosphate (Na HPO ), reduced glutathione (GSH), glutathione reductase (GR),
2
4
t
cumene hydroperoxide (CuOOH)), tert-butyl hydroperoxide ( BuOOH) were purchased from
Sigma Aldrich and were used as received. The molecular structure of some of the compounds
are shown in supplementary information (Fig. S1). Double distilled (dd) water and ultrapure
water were used during all the chemical synthesis and experiments respectively.
2
.2. Instrumental
Powder X-ray diffraction (PXRD) was recorded to check phase purity and crystallinity of the
materials, on Phillips PANalytical diffractometer using a CuKα (λ = 1.5406 Å) radiation. The
emission current 30 mA and accelerating voltage 40 kV were used during PXRD
measurement. To visualize the morphology of the nanomaterials, the samples were placed on
carbon tape, and then imaged using scanning electron microscopy (SEM) on FEI Sirion UHR
SEM. Transmission electron microscopy (TEM), High resolution transmission electron
microscopy (HRTEM), X-ray mapping images and Energy dispersive spectroscopy (EDS)
were recorded on JEOL transmission electron microscope operating at 200 kV after casting a
drop of nanoparticle dispersion in isopropyl alcohol, over Cu grid. FT-Raman spectra and
FT-IR spectra were recorded using Renishaw in-Via Raman Microscope (Renishaw Inc, UK),
with excitation wavelength 514 nm and PerkinElmer FT-IR spectrometer respectively.
SHIMADAZU UV-2600 spectrophotometer was used to acquire all the UV-Vis absorption
spectra of the enzyme mimetic activity assays. X-ray photoelectron spectroscopy (XPS) was
performed using AXIS Ultra, KRATOS ANALYTICAL, SHIMADAZU.
2
2
.3. Experimental methods
.3.1. Preparation of V O nanowires (VNw)
2
5
VNw was synthesized by previously reported procedure [14]. Briefly, 1.5 g of VOSO
and 0.835 g of KBrO was mixed thoroughly in 30 ml ultrapure water. The colour of the
solution changed from blue to green to finally orange-yellow. The solution was stirred for 45
min to get orange-yellow coloured precipitate followed by addition of 4.5 ml conc. HNO and
the solution was further stirred to get yellow transparent solution which was transferred to a
4
.H O
2
3
3
ο
5
0 ml Teflon- lined stainless-steel autoclave and kept in the oven at 190 C for 24 h and then
cooled naturally. The resultant bright coloured precipitate was filtered by 0.22 µm membrane
filter paper and washed thoroughly with double distilled water and acetone. Finally, the
ο
compound was dried at 70 C for 12 h.

2
.3.2. Preparation of MoO
3
nanowires (MNw)
MNw was synthesized by previously reported procedure [15]. First, 2.42 g of
Na MoO .2H O was dissolved in 32.75 ml ultrapure water in a 100 ml round bottom. After
that, 7.25 ml of conc. HNO was added slowly into the solution and stirred for 30 min at 25
2
4
2
3
ο
C. This precursor solution was transferred to a 50 ml Teflon-lined stainless-steel autoclave
ο
and kept in the oven at 190 C for 24 h. The white precipitate was obtained after the resultant
solution was filtered through 0.22 µm membrane filter paper and washed thoroughly with
double distilled water until the pH of the filtrate became 7. Finally, 10 ml absolute ethanol
ο
was used to wash the precipitate after which the compound was dried at 70 C for 12 h.
2
.3.3. Glutathione peroxidase (GPx) like activity assay
GPx-like activity of the synthesized V and MoO nanowires (VNw and MNw) was
2
O
5
3
followed spectrophotometrically by using the standard GR-coupled GPx assay (Fig. 1) [11].
Fig. 1. Schematic of the glutathione reductase (GR) coupled GPx assay used to measure the
activity of the nanozymes.
The rate of the reaction was quantified by following the decrease in the absorbance of
−
1
NADPH at 340 nm. 1 mL reaction mixture consisted of catalyst (20 µg mL ), GSH (2 mM),
NADPH (0.2 mM), GR (∼ 1.7 U) and H (200 µM) in pH 7.4 phosphate buffer (100 mM).
All the assays along with respective controls were carried out at 25°C. Using Beer-Lambert’s
2
O
2
−
1
−1
law, rate of decrease in the absorbance of NAPDH (ε = 6220 M cm ) was converted to rate
+
of conversion of NADPH to NADP which is equal to the rate of conversion of H
Equation 1, 2).
2
O
2
to H O
2
(
…
..(1)
…
..(2)

Sigma plots were obtained by plotting V vs [S] (Equation 3) and kinetic parameters like
substrate binding affinity (K ) and maximum reaction velocity (Vmax) were obtained from the
Lineweaver-Burk plots (Equation 4), where V is the rate of reaction, [S] is concentration of
substrate (in our case, H and GSH).
M
2
O
2
…
..(3)
..(4)
…
2
.3.4. In-situ FT-Raman and FT-IR Spectroscopy
Changes in the intensity of the peaks associated with metal oxo bond (M=O) vibration and
metal peroxido bond, during the reaction were studied by in-situ FT-Raman spectroscopy
using excitation wavelength of 514 nm, 50xL objective lens, 10 sec exposure and 3
accumulations (power <10 m|W|). Peak for metal peroxido bond were observed using FT-IR
spectroscopy, spectra were recorded using pellets made by grinding KBr with materials
together.
3
. Results and discussion
3
3
.1. Characterizations of V
2
O
5
(VNw) and MoO (MNw) nanowires
3
.1.1. Crystal properties and morphology
Crystalline nature of the VNw and MNw was confirmed by XRD patterns (Fig. 2) which
were indexed to the standard V orthorhombic phase (a = 11.5160 Å, b = 3.5656 Å, c =
.3727 Å, JCPDS = 41-1426, Space group Pmmn) [14] and the standard orthorhombic phase
2
O
5
4
of MoO (a = 3.9620 Å, b = 13.8580 Å, c = 3.6970 Å, JCPDS = 05-0508, Space group Pbnm)
3
[
15]. The wire like morphology of VNw and MNw were confirmed by SEM and transmission
electron microscopy TEM images (Fig. 3). HRTEM images of VNw and MNw along with
their selected area electron diffraction pattern (SAED) are shown in Fig. 4. The lattice fringes
observed for VNw are (200) and (110) with d-spacing of 0.58 nm and 0.34 nm respectively,
with the interfacial angle of 72.8º. Similarly, in case of MNw, two perpendicular crossing
lattice fringes (100) and (001) with the d-spacing of 0.39 nm and 0.36 nm respectively were
observed. In both the cases, the SAED pattern were well indexed along [001] and [010] zone
axis for VNw and MNw respectively. This result is in accordance with the PXRD patterns
where the most intense peaks for VNw and MNw correspond to (001) and (040) planes
respectively.

Fig. 2. XRD pattern of V
2
O
5
nanowires (VNw) and MoO nanowires (MNw).
3
Fig. 3. Scanning electron microscopy (SEM) images of (a) VNw, (c) MNw. Both the samples
are imaged in powder form after casting on carbon tape. Transmission electron microscopy
(
TEM) images of (b) VNw, (d) MNw.

Fig. 4. High resolution transmission electron microscopy (HRTEM) images of (a) VNw, (c)
MNw. SAED patterns of (b) VNw, (d) MNw. The zone axes were found to be [001] and
[
010] for VNw and MNw respectively.
3
.1.2. Optical properties
FT-Raman (Fig. 5a) and FT-IR spectra (Fig. 5b) show peaks corresponding to the pure
-
1
orthorhombic phase of VNw and MNw materials. In Raman spectra, the peak at 993 cm
-1
corresponds to the terminal (V=O) and peak at 996 cm corresponds to (Mo=O) resulting
from unshared oxygen of V and MoO crystal respectively [16-21]. Similarly, in FT-IR
the peak at 1020 cm and 996 cm are due to V-oxo (V=O) and Mo-oxo (Mo=O)
respectively. The weak peak observed at 1643 cm in the FT-IR spectra of both VNw and
MNw is due to adsorbed water molecule on the surface of nanomaterials (M-OH ). The
is due to the metal bound OH vibration
case [24]. The detailed analysis of all the peaks present
2
O
5
3
-1
-1
-
1
2
-1
intense peak at 1384 cm in the spectra of MoO
Mo-OH) unlike in the case of V
3
(
2
O
5
in the FT-Raman and FT-IR spectra of VNw and MNw is given in the supplementary
information (Table S1 and S2).

-
1
Fig. 5. (a) FT-Raman spectra from 50 to 1200 cm and (b) FT-IR spectra from 400 to 4000
-1
-1
cm of VNw and MNw respectively. The peaks observed in the range of 400 to 1100 cm in
FT-IR spectra are due to the M-O stretching vibrations.
3
.1.3. Energy-dispersive X-ray spectroscopy (EDS) and X-ray mapping
EDS spectra of VNw and MNw (Fig. S2) showed signal for Vanadium (V), Molybdenum
(
Mo) and Oxygen (O) confirming the elemental purity. The small signal of silicon (Si) in the
EDS spectrum of MNw was from the silicon wafer substrate. The signal of copper (Cu) in the
EDS spectrum of VNw was due to copper grid. Selected area bright field (SABF) images of
and X-ray mapping images of VNw and MNw further confirmed the elemental purity of the
materials and indicated the homogenous distribution of vanadium (V), oxygen (O) in the
nanowires of V
2
O
5
and of molydenum (Mo), oxygen (O) in MoO nanowires (Fig. S3).
3
3
.1.4. X-ray photoelectron spectroscopy (XPS)
XPS spectra of VNw and MNw are shown in supplementary information (Fig. S4, Fig. S5).
For both the samples, the wide spectrum was recorded in the range of 0 to 1200 eV. In case
of VNw vanadium (V) and oxygen (O) peaks were present in the spectra. Similarly,
molybdenum (Mo) and oxygen (O) peaks were observed for MNw, reassuring the elemental
purity of the synthesized nanomaterials. The binding energies and FWHM of V2p3/2 (517.63
eV) and V2p1/2 (524.95 eV) peaks correspond to the 5+ state of vanadium (V) in VNw (Fig.
S4d). O1s spectrum for VNw was deconvoluted to get three components O1s_a, O1s_b and
O1s_c, corresponding to terminal oxygen (V=O), the linkage oxygen (O-V-O or V3-O) and
the oxygen from adsorbed water molecules or hydroxyl groups on the V
2
5
O surfaces (Fig.
S4d) respectively. As according to Mendialdua et al., the difference in binding energies
between the O1s (VOx) core level and the V2p3/2 level {Δ= BE (O1s) – BE (V2p3/2)} can
be used as an important parameter in determination of the oxidation state of V
2
5
O materials.
Experimentally, this difference (Δ) was found to be 12.9 eV, which further confirms the
oxidation state of vanadium in VNw to be +5 [22]. Similarly, in case of molybdenum the
binding energies are 232.86 eV and 235.99 eV for 3d5/2 and 3d3/2 levels respectively (Fig.
S5d). The binding energies and FWHM of the peaks confirm the +6 state of molybdenum
(
Mo). The difference between the binding energy of the orbitals {Δ = BE (3d5/2) – (3d3/2)}
was observed to be 3.13 eV which confirms the +6 state of molybdenum in the synthesized
MNw [23]. The peak originating from oxygen in MNw was deconvoluted into two

components. The binding energy and FWHM of O1s_a corresponds to the metal bound
oxygen (Mo=O, O-Mo-O or Mo O) in the crystal lattice and O1s_b peak can be ascribed to
3
hydroxo species (Mo--OH) attached on the surface of MNw (Fig. S5d) . The presence of Mo-
OH on MNw was further confirmed from FT-IR spectra of MNw, where the strong peak at
-1
1
384 cm was observed which corresponds to Mo—OH vibrations. Such peaks were not
observed in the case of VNw [24].
3
.2. Glutathione peroxidase (GPx) like activity of VNw and MNw
GPx-like activity of VNw and MNw were checked using glutathione reductase (GR)-coupled
assay by monitoring the decrease of absorbance of NADPH (at 340 nm) using UV-vis
spectrophotometer. As shown in Fig. 6a, VNw was more efficient in catalysing reduction of
H
2
O
2
using GSH when compared to MNw but at higher concentration of H
efficient GPx-like activity. In the absence of any one of the required constituents of the assay,
no GPx-like activity was observed for both the materials, i.e., no reduction of H takes
, organic peroxides such as BuOOH and CuOOH were also
used for the activity assay but none of the materials were able to reduce the organic peroxides
significantly, indicating their selectivity in reducing H
2
O , MNw shows
2
2
O
2
t
place (Fig. S6). Other than H
2
O
2
2
2
O .
Fig. 6. (a) Comparison of the GPx-like activity in the presence of three different peroxides,
t
H
2
O , tert-butyl hydroperoxide ( BuOOH) and cumene hydroperoxide (CuOOH). Assay
2
-
1
conditions: nanozymes (20 ng μl ), NADPH (0.2 mM), GSH (2 mM), GR (~1.7 units),
peroxide in phosphate buffer (100 mM, pH 7.4) at 25 °C. (b) Monitoring the decrease in the
absorbance of NADPH at 340 nm, during the reaction, under assay conditions as well as

various control conditions. (c) Comparison of initial rates for GPx-like activity observed
MNw and in case of the control conditions. Assay conditions were same as mentioned earlier
except the concentration of H
2
O2, which was 1.0 mM in this case. The initial rate was shown
in condition 1 after subtracting the rate observed at condition 9.
Glutathione peroxidases requires both H and GSH, simultaneously to reduce H
2
O
2
2
O
2
to H O
2
and thereby converting GSH to GSSG. To understand the effect of the concentration of
individual substrates on the rate of the reaction and their binding on the surface of our
nanozymes, we performed the activity assay at various concentrations of H
GSH (0-4 mM) and determined the kinetic parameters, Michaelis constant (K
2
O
2
(0-4 mM) and
) and
M
maximum velocity (Vmax). A typical Michaelis-Menten kinetics was observed for both the
both the substrates (Fig. S6). The kinetic parameters were obtained from the corresponding
Lineweaver-Burk plots (Fig. S6, Table 1) indicate the significant differences in the substrate
binding affinity between VNw and MNw.
Table 1
The kinetic parameters of MNw and VNw obtained from the Lineweaver-Burk plots.
-
1
Nanozyme Substrate
H O
K
M
(mM)
.61 ± 0.18
3.06 ± 0.03
.04 ± 0.00
1.28 ± 0.06
Vmax (µM min )
5
840.34 ± 26.48
323.62 ± 3.09
192.31 ± 6.58
279.3 ± 18.2
2
2
MNw
GSH
H O
0
2
2
[
13]
VNw
GSH
The K
M
(mM) values obtained for VNw and MNw indicate that H
2
2
O molecule binds ~140
times strongly on the surface of VNw in comparison with MNw, whereas the binding affinity
of GSH is 2.4 times stronger for VNw than that of MNw. This indicates that the reaction with
H
2
O2, on the surface of nanozymes, plays the key role in determining the rate of the reaction
and therefor the overall GPx-like activity.
3
.3. Mechanistic understanding for the GPx-like activity of nanozymes by in-situ
spectroscopy
To understand the mechanism of action and to identify the intermediate(s) involved during
the reaction, in-situ FT-Raman spectra were recorded after sequential addition of various
components used the assay mixture during GPx-like catalytic cycle (Fig. 7a). There were
observable changes in the spectra after upon treatment of the material with GSH, NADPH,
and/or GR, suggesting that the material is unaltered in such reducing conditions. In contrast, a
-
1
-1
rapid generation of new peak at 570 cm and its overtone 1150 cm along with a significant
-1
decrease in the intensity of peroxide (O—O at 877 cm from H
was added [25-29]. These observations indicate that the reaction of H
nanozyme is the first step in the GPx-like catalytic cycle.
2
O
2
) peak was observed once
H
2
O
2
2
O
2
with the

Fig. 7. (a) Probing the intermediate species by in-situ Raman spectroscopy during GPx-like
-
1
-1
catalytic cycle of VNw. * Peaks for the V-peroxido species (570 cm and 1150 cm ) (b)
-1
-1
Time-dependent in-situ FT-IR spectra (830 to 1050 cm ) of MNw (400 to 1500 cm in Fig.
S8a) after addition of 1 M H . There was a simultaneous increase in the intensity of the
peak of Mo-peroxido (Mo(O)
2
O
2
-1
2
) at 885 cm and decrease in the intensity of Mo-oxo (Mo=O)
peak at 995 cm was observed with time. (c) Time-dependent in-situ FT-IR spectra (830 to
-1
-1
1
050 cm ) of MNw upon treating with 1 M CuOOH. In this case, no significant changes
-1
were observed in the spectra unlike the case with H
2
O . (450 to 2200 cm in Fig. S8b).
2
In the time-dependent FT-IR spectra, recorded during the reaction for both VNw and MNw
nanozymes, we could observe the decrease in the intensity of metal-oxo (M=O) peaks and
simultaneous increase of metal peroxido peaks upon reaction with H
of organic peroxides ( BuOOH, CuOOH), we could not observe the formation of metal-
2
O specifically. In case
2
t
peroxido species in either of MNw and VNw (Figs. 7c and 8c,d) which is in agreement with
our observations from GPx-like activity assays that our nanozymes specifically reduce H
2
O
2
but not the organic peroxides (Fig. 6a). In Fig. 7b peak for molybdenum-oxo (Mo=O) at 995
-
1
-1
cm decreases and peak for Mo-peroxo (Mo(O
unlike the reaction with cumene hydroperoxide and tert-butyl hydroperoxide (Figs. 7c
and S8c,d) [26-27]. Interestingly, we could see that the rate of conversion of V-peroxido (935
2
)) at 885 cm increases upon addition of
H
2
O
2
-1
-1
cm ) species from V-oxo (1020 cm ) is faster for VNw than MNw (Figs. 8a and 7b) [29].
These results explain the difference in the initial rate in the GPx-like activity of VNw and

MNw. To understand the reactivity of peroxido intermediate and the role of GSH in the GPx-
like cycle, GSH was added in the mixture after 20 mins of addition of H to the nanozyme
2
O
2
-1
which resulted in the decrease of intensity of the peak at 935 cm indicating the reaction of
GSH with the peroxido intermediate (Fig. 8b)
Fig. 8. Time-dependent in-situ FT-IR spectroscopy of VNw in presence of different
-
1
-1
peroxides (1 M). (a) FT-IR spectra of VNw (900 to 1050 cm ) (400 to 1500 cm in Fig. S7a)
-
1
in presence of H
2
O . The intensity of the peak at 1020 cm (V=O) decreased whereas the
2
-1
intensity of the peak at 935 cm (V-peroxido) increased over time. (b) Upon addition of GSH
-1
we could see the decrease of the peak intensity at 935 cm indicates the reaction of GSH with
the peroxido intermediate. FT-IR spectra of VNw in the presence of (c) cumene
hydroperoxide and (d) tert-butyl hydroperoxide. It is clear from the absence of the peak for
V-peroxido at 935 cm and the unaltered intensity of the peak of V-oxo at 1020 cm , that
there is no formation of V-peroxido intermediate on the surface of VNw upon treatment with
the organic peroxides.
-
1
-1
3
.4. Understanding the mechanism of catalyst regeneration cycle by in-situ FT-Raman
As H reacts with the native VNw/MNw to generate the active species of catalytic cycle
2
O
2
(
M-peroxido), we thought of investigating the fate of M-peroxido intermediate in the absence
of GSH by time dependent FT-Raman spectroscopy. It was observed that while the intensity
of the V-oxo peak decreases drastically after addition of H , the peak reappears after some
time in both VNw and MNw (Figs. 9a-d). On the other hand, the intensity of peak for
2
O
2

-1
peroxide (O—O at 877 cm from H
2
O
2
) decreases with time indicating the reaction between
H
2
O
2
and nanozymes. In case of VNw we could see the formation of peroxido species (at 570
-
1
-1
cm and 1150 cm ) (Fig. 9b and S7b) initially and later it vanishes completely [25,28].
These results indicate the peroxido species formed on the surface of nanozymes are quite
unstable and it can revert to the native nanozyme in the absence of GSH where further
reaction with the peroxido intermediate does not take place.
Fig. 9. Time-dependent FT-Raman spectroscopy of (a, b) VNw and (c, d) MNw in presence
-1
of 1 M H
2
O
2
. For VNw we could monitor the peak at 877 cm (O—O vibration from H
2
O
2
)
up to 10 min but in case of MNw it was observable till 20 min. In case of VNw, intensity of
-1
the peak at 993 cm (V-oxo) is decreasing initially (up to 10 min) with time and later
-1
-1
increases whereas the intensity of the additional peaks at 570 cm (Fig. S7b) and 1150 cm
V-peroxido) increases initially but later decreases. This indicates the unstable nature of
(
peroxido intermediate formed on the surface of VNw and regeneration of catalyst over time.
Similar results could be seen in case of MNw as well where the intensity of the peak at 995
-1
cm (Mo-oxo) decreases initially up to 20 min and later increases.
3
.5. Mechanism of GPx-like catalytic cycle and catalyst regeneration cycle
Based on the observations from extensive in-situ spectroscopic studies, we could propose a
plausible mechanism (Fig. 10) for metal oxide based nanozymes and their GPx-like activity.
In the first step, the metal-oxo bond (A) on the surface of nanomaterials interacts with the
H
2
O
2
molecule, one of the substrates of GPx enzyme, and gets converted to metal peroxido
species (C) which was confirmed by FT-IR as well as FT-Raman spectroscopy in both (VNw

and MNw) nanozymes. The peroxido intermediate (C) is the active species which gets into
the catalytic cycle of GPx enzymes. Intermediate C reacts with one molecule of GSH to form
an unstable species D which further reacts with another molecule of GSH to form a relatively
-1
stable intermediate metal oxo-hydroxo (E). The peak at 1384 cm in FT-IR (Fig. S7a) was
detected for VNw upon reaction with H and could be assigned as V-OH which was absent
before the reaction in the native nanozyme [24]. Intermediate E further reacts with H and
2
O
2
2
O
2
regenerates the peroxido species C. As the peroxido intermediate is unstable and it can revert
to the native material after catalysis by eliminating water molecule, in the absence of GSH. In
case of organic peroxides, we could not detect the intermediate C. It may be the bulkiness of
the organic moiety and other electronic factors preventing the formation of intermediate C
which render the organic peroxides unsuitable as substrates for our nanozymes (Fig. S9).
Fig. 10. Glutathione peroxidase (GPx)-like catalytic cycle and catalyst regeneration cycle.
4
. Conclusions
Metal-based redox active nanozymes can regulate the cellular redox state in mammalian cells
by functionally mimicking the antioxidant enzymes. In this regard, nanozymes having
glutathione peroxidase (GPx) activity is important as such materials can catalytical reduce the
potentially harmful hydrogen peroxide to water in the presence of cellular reducing agents
such as glutathione (GSH). The metal-based nanozymes, V
2
O
5
and MoO mediate the
3
reduction of hydrogen peroxide with GSH and functionally mimics the GPx activity. The
catalytic mechanism for this activity involves the formation of a reactive peroxido bond,
which acts as a catalytically active species in the reaction. The cleavage of the peroxido bond
with the nucleophilic GSH, followed by the formation of a hydroperoxido species are
responsible for the observed catalytic activity. Several in situ Raman spectroscopic
experiments reveal that the reactivity of the oxo moiety with hydrogen peroxide depends the

nature of the metal and morphology of the materials. The change in the reactivity is
responsible for the differences in the GPx activity of metal oxide-based nanozymes.
Acknowledgements:
This study was supported by the Science and Engineering Research Board (EMR/IISc-
0
1/2016) and DST Nanomission (SR/NM/NS-1380/2014), New Delhi. G.M. acknowledges
the SERB/DST for the award of J. C. Bose National Fellowship. S. G. thanks the Indian
Institute Science, Bangalore, for a fellowship. The authors thank Prof. S. Umapathy for
providing Raman spectroscopy facility. The authors thank Mr. Punarbasu Roy for his help in
preparing some of the figures. We also thank the MNCF Facility, CeNSE, IISc for the
spectroscopic and microscopic facilities.
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