Crystal-facet-dependent denitrosylation: Modulation of NO release from S-nitrosothiols by Cu2O polymorphs

Article abstract of DOI:10.1039/c9sc01374a

Nitric oxide (NO), a gaseous small molecule generated by the nitric oxide synthase (NOS) enzymes, plays key roles in signal transduction. The thiol groups present in many proteins and small molecules undergo nitrosylation to form the corresponding S-nitrosothiols. The release of NO from S-nitrosothiols is a key strategy to maintain the NO levels in biological systems. However, the controlled release of NO from the nitrosylated compounds at physiological pH remains a challenge. In this paper, we describe the synthesis and NO releasing ability of Cu₂O nanomaterials and provide the first experimental evidence that the nanocrystals having different crystal facets within the same crystal system exhibit different activities toward S-nitrosothiols. We used various imaging techniques and time-dependent spectroscopic measurements to understand the nature of catalytically active species involved in the surface reactions. The denitrosylation reactions by Cu₂O can be carried out multiple times without affecting the catalytic activity.

Full text of DOI:10.1039/c9sc01374a

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Crystal-Facet-Dependent Denitrosylation: Modulation of NO
Release from S-nitrosothiols by Cu2O Polymorphs
Sourav Ghosh, Punarbasu Roy, Sanjay Prasad and Govindasamy Mugesh*
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560012,
India
*Corresponding author, Email: mugesh@iisc.ac.in
Keywords: copper oxide • crystal facets • nanozymes • nitric oxide • s-nitrosothiols
Abstract: Nitric oxide (NO), a gaseous small molecule generated by the nitric oxide synthase
(NOS) enzymes, plays key roles in signal transduction. The thiol groups present in many proteins
and small molecules undergo nitrosylation to form the corresponding S-nitrosothiols. The release
of NO from S-nitrosothiols is a key strategy to maintain the NO levels in biological systems.
However, the controlled release of NO from the nitrosylated compounds at physiological pH
remains a challenge. In this paper, we describe the synthesis and NO releasing ability of Cu2O
nanomaterials and provide the first experimental evidence that the nanocrystals having different
crystal facets within the same crystal system exhibit different activities toward S-nitrosothiols. We
used various imaging techniques and time-dependent spectroscopic measurements to
understand the nature of catalytically active species involved in the surface reactions. The
denitrosylation reactions by Cu2O can be carried out multiple times without affecting the catalytic
activity.
Introduction
In 1998, the Nobel Prize was awarded to Robert F. Furchgott, Louis J. Ignarro and Ferid Murad
“for their discoveries concerning nitric oxide as a signalling molecule in the cardiovascular
system.”1 Their research had shown that nitric oxide (NO), a transient free radical with a half-life
of ∼5s, is involved in transmitting cellular signals in mammals. Now, it is well known that NO is
involved in a variety of important biological processes such as a signalling molecule in the nervous
system, vasodilator in the cardiovascular system, and it is used as a key molecule by the immune
system to fight against infections.2 However, the imbalance between the nitrosylation and
denitrosylation activities of enzymes affect the NO concentration (Fig. 1), which ultimately results
in the formation of nitrosylated proteins via a covalent binding of NO to the cysteine residues of
1

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proteins.3 The nitrosylation of proteins lead to various deleterious effects, which include the
monomerization of functionally active endothelial nitric oxide synthase (eNOS), leading to the
production of superoxide species that can react with NO to generate the highly toxic peroxynitrite
(ONOO-) and elevation of oxidative stress in the cells.4 It has been shown that nitrosylated Hsp90
inhibits the ATPase activity, which leads to the decrease in the eNOS activity (Fig. 1).5 As NO is
synthesized in endothelial cells by eNOS, the low level of NO due to impairment of the enzyme
activity affects the endothelial function. On the other hand, elevated levels of NO cause nitrosative
stress related signalling, leading to post-translational modification of many proteins, neurotoxicity
and apoptosis.6 NO concentration above 1.0 µM results in nitrosative stress and necrotic cell
death by generating a large amount of reactive nitrogen species (RNS). Therefore, the biological
effects of NO are highly dependent on its concentration.6
Fig. 1 Chemical biology of nitric oxide (NO). The nitrosylation/denitrosylation reactions play important role
in the maintenance of NO levels in the mammalian cells. An excessive nitrosylation can lead to protein
misfolding, oxidative stress and impairment of NO synthesis by the nitric oxide synthase (NOS) enzymes.
Synthetic compounds such as organic nitrates (RONO2) and nitrites (RONO), metal
nitrosyl complexes, N-nitrosamines (RN(NO)R′), S-nitrosothiols (RSNO) have been used to
2

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generate NO for biological applications.7 S-nitrosothiols (RSNOs) such as S-nitrosoalbumin, S-
nitroso-N-acetyl-penicillamine (SNAP), and S-nitrosoglutathione (GSNO) have been used
extensively for storage and release of NO.8 As several diseases such as asthma, cystic fibrosis,
Parkinson’s disease, heart failure, and stroke are associated with dysregulated S-nitrosylation,
regulating the NO levels is crucial for the maintenance of normal physiology.6 In biological
systems, the removal of NO from nitrosylated proteins and small molecules is mediated by
denitrosylases enzymes, which maintain the NO homeostasis and dynamically regulate the
cellular signal transduction.6 To date, two physiologically relevant enzymatic denitrosylating
systems have been demonstrated, which include the thioredoxin (Trx)/thioredoxin reductase
(TrxR) and the glutathione (GSH)/GSNO reductase (GSNOR) systems.6g,h A few selenium-based
compounds, transition metal (Cu and Fe) complexes, metal organic frameworks, polymers and
nanomaterials have been reported to mediate the release of NO from S-nitrosothiols (RSNO).9
However, the controlled release of NO from S-nitrosylated compounds at physiological pH
remains a challenge. Recently, Schoenfisch and co-workers developed polyurethanes and
cyclodextrins-based compounds for NO release.7c,d On the other hand, Meyerhoff and co-workers
used a polymer doped with SNAP for storage and release of NO.7e,f In this paper, we report the
synthesis and NO releasing activity of Cu2O nanocrystals10a,b and show, for the first time, that
nanocrystal polymorphs having different crystal facets10 within the same crystal system exhibit
different activity toward S-nitrosothiols. We also describe the nature of catalytically active species
involved in the surface reactions using various time-dependent spectroscopic studies.
Results and discussion
Synthesis and Characterization of Cu2O Nanocrystals: Three different morphologies of Cu2O
nanomaterials – rhombic dodecahedra (RDh), cube, and octahedra (Oh) were synthesized using
either copper (II) chloride (cube and Oh) or copper (II) sulfate (RDh) under reducing conditions.11
All the morphologies were thoroughly characterized by scanning electron microscopy (SEM) and
transmission electron microscopy (TEM), which confirmed the distinguishable differences
between the nanomaterials (Fig. 2a-c). To understand the crystalline nature of the materials,
powder X-ray diffraction (PXRD) measurements were carried out and the diffraction peaks were
well indexed to the standard cubic fcc Cu2O structure (JCPDS = 78-2076, a=b=c= 4.267 Å and
ɑ=β=γ= 90º) (Fig. 2d).11 The two strong peaks (110) and (220) in RDh indicate the presence of
{110} facets in this material. Similarly, the strong (200) and (111) peaks confirm the presence of
{100} and {111} facets in Cube and Oh, respectively. The EDS spectrum of all the morphologies
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and X-ray mapping of Oh confirmed the presence and distribution of copper (Cu) and oxygen (O)
atoms in all materials (Fig. S1 and S2). X-ray photoelectron spectroscopy (XPS) analysis was
performed to determine the oxidation state of the metal ions in the materials. The binding energies
(BE) and the difference in the BE between the Cu2p3/2 and Cu2p1/2 peaks (~19.8eV) confirm that
copper exists in +1 oxidation state in all the morphologies (Fig. S5, Table S4).10b,17 To gain insight
into the bonding connectivity between the copper and oxygen atoms in the fcc Cu2O
nanomaterials, FT-IR and FT-Raman spectra were recorded (Fig. S3, S4 and Table S3). In the
Raman spectra, an intense peak observed at 219 cm-1 can be assigned to the 2nd order overtone
of Cu2O; corresponding to 2Γ-(12) mode.17c,d The peak observed at 635 cm-1 in the FT-IR is ascribed
to the stretching vibration of Cu2-O.17c The well-defined sharp peaks at 219 cm-1 (FT-Raman) and
635 cm-1 (FT-IR) demonstrate the high structural quality of the synthesized materials, which
corelates well with the X-ray diffraction results. (Fig. 2d).
Denitrosylation of S-nitrosothiols by Cu2O nanocrystals: The denitrosylation of S-nitrosothiols
(RSNO) by Cu2O nanocrystals was monitored spectrophotometrically by following the decrease
in the absorbance of RSNO at 335 nm over time. (Fig. 2f).15 As S-nitrosothiols are sensitive to
light and heat, it is important to store all the samples in dark at low temperatures (-20 °C) to avoid
any decomposition. As shown in Fig. 2g, all three materials were found to be efficient in the
denitrosylation of SNAP. The rate of the reactions was determined by using the molar extinction
coefficient of respective RSNO at 335 nm as shown in the Supplimentary Information (Fig. 2e and
Table S1). A comparison of the activities of the nanomaterials with SNAP indicates that Oh
exhibited the highest activity in the series and the rate of denitrosylation observed for this material
was found to be almost five- and two-fold higher than that of RDh and cube, respectively (Fig.
3c). A similar trend was observed with other S-nitrosothiols - GSNO, SNAC, CYSNO and
HCYSNO (Fig. 3a-b, S6 and Table S2). These observations clearly indicate that the
denitrosylation of RSNO by Cu2O nanomaterials is morphology-dependent. Interestingly, the rate
of denitrosylation of RSNO is not directly proportional to the surface area of the materials (Fig.
3c, S8). The surface area, pore diameter and pore volume measured by the Brunauer-Emmett-
Teller (BET) method (Fig. S7) indicate that cube with the higher surface area (5.4 m2g-1) than that
of Oh (3.2 m2g-1) exhibits an almost 3-fold lower activity after normalization with respect to surface
area as compared to that of the Oh nanocrystals.
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Fig. 2 (a-c) SEM and TEM (inset) images of RDh, cube and Oh, respectively. (d) PXRD pattern of fcc cubic
Cu2O materials. (e) Chemical structure of S-nirosothiols used as substrates for the release of nitric oxide.
(f) Denitrosylation of SNAP by RDh. The decrease in the absorbance of SNAP (500 µM) was monitored
upto 90 min with time interval of 2 min by UV-Vis spectrophotometer in presence of RDh (2 ng µL-1). (g)
Denitrosylation of SNAP by three different Cu2O nanomaterials. The decrease in the absorbance of SNAP
was monitored at 335 nm by UV-Vis spectrophotometer. Assay condition: SNAP (500 µM), Cu2O (2 ng µL-1
), 0.1 M sodium phosphate buffer (pH 7.4), 25 °C.
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Confirmation of the NO release at physiological pH: To confirm the release of NO from RSNO by
Cu2O nanomaterials, various spectroscopic (UV-Vis, fluorescence and EPR) methods were
developed. For the fluorescence-based detection of NO, 2,3-diaminonaphthalene (DAN) assay
was used.18 Briefly, a smaller vial containing Cu2O nanomaterials (50 ng.µL-1 in 0.1 M phosphate
buffer pH 7.4) was placed inside a larger vial containing DAN (1.0 mM in DMSO) solution such
that the two solutions do not mix with each other. The larger tube was then sealed (Fig. S9) and
a solution of GSNO (20 mM) was injected into the smaller vial having the Cu2O nanomaterials.
The in-situ generated NO from the smaller vial diffuses into the larger vial and reacts with DAN to
produce 2,3-naphthotriazole (NATH), which is weakly fluorescent, but it exhibits strong
fluorescence in basic medium due to the formation of 2,3-naphthotriazole anion (NAT).18 The
excitation and emission wavelengths used for the experiments were 362 and 405 nm, respectively.
When the denitrosylation was carried out without catalysts, no significant fluorescence was
observed, confirming that the release of NO is mediated by the Cu2O nanomaterials (Fig. 3d and
S10). Further, the amount of NO released during the denitrosylation was also quantified using the
DAN-based fluorescence assay. These experiments confirmed that the amount of NO produced
in the Cu2O-mediated denitrosylation corresponds well with the decrease in the concentration of
S-nitrosothiols as shown for GSNO (Fig. S11).
The release of NO was also confirmed by electron paramagnetic resonance (EPR) spectroscopy
using a similar experimental set-up. For this purpose, (dtc)2Fe complex was used for trapping the
NO released from GSNO to produce the EPR active (dtc)2FeNO (Fig. 3e). The room temperature
EPR spectrum shows three lines (S = 1/2) at g = 2.01 with A (14N) = 13.0 G, which is characteristic
of (dtc)2FeNO complex.16b The very weak EPR signal observed for GSNO in the absence of
nanomaterials indicate that the release of NO from GSNO is very slow and it leads to the formation
of only minor amount of (dtc)2FeNO complex under identical experimental conditions. (Fig. 3f and
Table S5). We also used an UV-Vis spectroscopy technique in which the formation of a nitrosyl
complex (TPP)CoNO from (TPP)CoII was monitored. The red shift of Q-band from 527 nm to 535
nm indicated the formation the nitrosyl complex and no shift of Q-band was observed in the
absence of Cu2O nanomaterials.16b The presence of Co-bound nitric oxide (TPP)CoNO complex
was further confirmed by FT-IR spectroscopy. The peak at 1696 cm-1 corresponds to N=O
stretching vibration of (TPP)CoNO,19 which is absent in the native metal complex i.e. (TPP)CoII
(Fig. S12). In addition to the specroscopic techniques mentioned above, we also used the
ferrocene-encapsulated (2-hydroxypropyl)-β-cyclodextrin (β-HCD-Fc) to observe the release of
NO by a visual colour change (yellow to blue) of the solution due to the formation of β-HCD-Fc+
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(reaction with NO and β-HCD-Fc).14 The characteristic band at 618 nm observed in the UV-Vis
spectrum confirms the formation of β-HCD-Fc+ (Fig. S13).
Fig. 3 (a-b) A comparison of the rate of denitrosylation by three different Cu2O nanomaterials. (c) The actual
rates of denitrosylation of SNAP (500 µM) in the presence 2 ng µL-1 of Cu2O and a comparison with the
rates obtained by normalizing the concentration with respect to 10 mm2 of the surface area. Assay
conditions: 0.1 M sodium phosphate buffer (pH 7.4) at 25 °C. (d) Detection of NO by a fluorescence assay
using DAN (5 µM). (e) The reaction of (dtc)2Fe with NO generated from the reaction of GSNO with Cu2O.
(f) Room temperature electron paramagnetic resonance (EPR) spectra of [(dtc)2FeNO] complex.
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Formation of glutathione disulfide (GSSG) in the denitrosylation of GSNO: Earlier reports
suggested that copper-mediated denitrosylation of RSNO results in the formation of disulfide
(RSSR) along with the release of NO.9f,15 To understand whether the release of NO from GSNO
is accompanied with the formation of glutathione disulfide (GSSG), we established a coupled
assay in which the formation of GSSG can be confirmed by using the enzyme glutathione
reductase (GR) (Fig. 4a). In mammalian cells, GR catalyzes the reduction of the disulfide bond in
GSSG using NADPH as co-factor to generate GSH, which is important to maintain the cellular
redox homeostasis.20 After incubating GSNO and Cu2O for 45 min, the activity of GR was
monitored by measuring the decrease in the absorbance of NADPH at 340 nm. A rapid decrease
in the absorption of NADPH was observed, indicating that the reaction of GSNO with Cu2O indeed
generates GSSG in addition to NO. Although the generation of NO from GSNO by Cu2O is
accompanied by the oxidation of Cu(I) to Cu(II) and the formation of GS, the highly reactive GS
undergoes oxidation by Cu(II) to produce GSSG. When the experiments were carried out in the
absence of Cu2O, no significant decrease in the absorbance of NADPH was observed. Other
control experiments revealed that NADPH or GR in the presence of NADPH do not mediate any
significant denitrosylation of GSNO (Fig. 4b, S14).
Fig. 4 (a) A coupled assay system involving glutathione reductase (GR) and NADPH to confirm the
formation of GSSG. (b) Monitoring the decrease in the absorbance of NADPH at 340 nm under various
conditions.
Mechanistic investigations of the denitrosylation reactions
Analysis of the exposed crystal-facets on the Cu2O surface: The difference in the initial rates
observed for the denitrosylation of RSNO by Cu2O having different morphologies indicate that the
denitrosylase activity is morphology-dependent and activity follows the order RDh < Cube < Oh.
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Surprisingly, no correlation was observed between the activity and the surface area of the
materials (Fig. 4a), indicating that the arrangement or orientation of the atoms on exposed facets
of the material surface may be responsible for the remarkable difference in the denitrosylation
activity of Cu2O nanomaterials having various morphologies. To understand the growth direction
and exposed facets in the materials, we analyzed the selected area electron diffraction (SAED)
patterns, high resolution TEM (HRTEM) images and their FFT (Fast-Fourier-Transform) patterns
of the materials.10 The inter-planer spacing and the angles between the planes observed in the
HRTEM images and the spots in SAED and FFT patterns were well indexed in accordance with
the crystal structure of fcc cubic Cu2O . The analysis revealed that the surface of the three
morphologies, RDh, cube and Oh, consists of {011}, {001} and {111} facets, respectively (Fig. 5,
S15 and S16).
Fig. 5 (a-c) The selected area electron diffraction (SAED) pattern of RDh, Cube and Oh respectively. (d-e)
The HRTEM of RDh, Cube and Oh, respectively. The Fast-Fourier-Transform patterns for the Cube and
Oh are shown as insets. (g-i) Zoomed HRTEM images of RDh, Cube and Oh, respectively. The
arrangement corresponding to the crystal facets {011} for RDh, {001} for Cube and {111} for Oh are given
as insets.
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The role of surface properties in the denitrosylation activity: To understand the effect of catalyst
concentration in the denitrosylation of RSNO, we have monitored the reaction of SNAP using the
Oh polymorph at various concentrations. It was observed that the rate of denitrosylation increased
linearly with an increase in the concentration of nanomaterial, indicating that the number of copper
atoms plays an important role in the denitrosylation activity (Fig. 6a). As discussed in the previous
sections, the activity of Oh was found to be almost three times higher than that of RDh or cube
when the catalyst concentrations were normalized with respect to the surface area (Fig. 3c). Even
when the concentration of the catalysts was normalized with respect to the number of copper
atoms present on the surface of the nanomaterials (Table 1 and Table S6-S7), the activity of Oh
was found to be almost 1.5- and 1.2-fold higher than that of RDh and cube, respectively (Fig. 6b).
These observations indicate that the {111} crystal facet present in Oh is more reactive than the
{011} and {001} facets present in RDh and cube, respectively. To understand the remarkably high
reactivity of the {111} surface in Oh, we carried out isothermal titration calorimetry (ITC)
experiment using GSH as a model subtrate to find out the thermodynamic parameters for the
surface of the nanomaterial (Fig. S22). These experiments revealed that the dissociation constant
of GSH (Kd) for the {111} surface is much lower than that of the {011} facet, indicating a stronger
binding of GSH to the surface with {111} facets as compared to that with the {011} facets. The
difference in the surface charge (zeta-potential, ζ) of the nanomaterials possibly leads to the
change in the binding affinity of RSNO towards Cu2O surfaces (Table 1 and S21).21
Table 1 The surface area, total number of copper atoms, surface charge (zeta-potential) and the
dissociation constants (Kd) for GSH obtained for the three nanomaterials.
Number of
Crystal-Facet on
Nanomaterials
Surface
Dissociation
Constant Kd
(µM)*
Surface
Area#
Copper Atoms
on Surface
(x1013)
Surface
Charge (mV)
{011} - RDh
{001} - Cube
{111} - Oh
8√2d2
6d2
2√3d2
1.00
1.89
4.83
-10.9
6.6
24.0
69.3
37.4
19.2
d = edge length of single unit. #for a single unit. *from isothermal titration calorimetry.
Probing the intermediates in the catalysis by spectroscopy: Several spectroscopic studies were
performed to understand the nature of intermediates formed on the surface during the catalysis.
The time-dependent FT-IR spectroscopic experiments for the reaction of SNAP with Cu2O Oh
indicated that the peak at 635 cm-1 corresponding to the Cu(I)-O vibration became
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Fig. 6 (a) Effect of Cu2O Oh concentration on the denitrosylation of SNAP. (b) A comparison of the
denitrosylation activity of RDh, cube and Oh after normalizing with the number of copper atoms on the
surface. (c) Mechanism of Cu2O-mediated NO release from RSNO, involving a redox cycle between Cu(I)
and Cu(II) species. (d) Time-dependent FT-IR spectra and (e) Time-dependent FT-Raman spectra of the
nanomaterials during the denitrosylation of SNAP by Cu2O Oh. (f, g) Short-range XPS spectra of Cu 2p
and O 1s, respectively, recorded on the materials recovered after the catalysis.
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broader and the characteristic peaks for Cu(II)-O vibrations appeared at 518, 539 and 565 cm-1
after 10 min of reaction time (Fig. 6d), which indicated the existence of mixed phases i.e. Cu2O
and CuO during the catalysis.17c,22 The peaks below 600 cm-1 started disappearing with time and
after 60 min, only a sharp peak at 635 cm-1 along with an additional peak for CuO at 565 cm-1
were observed (Fig. 6d). Similar experimental results were also observed by time-dependent FT-
Raman spectroscopy. The peak around 630 cm-1 corresponding to the Cu(I)-O stretching vibration
became broader after 15 min, indicating the presence of both Cu(I) and Cu(II) oxide. The
characteristic Raman spectral peak for CuII(OH)2 was detected at 470 cm-1 after 15 min along with
two other broad peaks at 291 cm-1 and 630 cm-1.17c,d The formation of CuIIO was identified after
30 min of catalysis by observing the peak at 265 cm-1, which disappeared over the time. After 60
min, the peaks mainly corresponding to the vibrations of Cu(I)-O were observed (Fig. 6e).17c,d To
further understand the nature of chemical species formed on the surface of the nanomaterials,
XPS of copper (Cu 2p) and oxygen (O 1s) were recorded after the catalysis. The shake-up satellite
peaks observed for copper confirms the presence of Cu(II) species along with Cu(I) on the surface
of the nanomaterials.17,23 Similarly, the deconvoluted O 1s spectra showed peaks corresponding
to Cu(II)- and Cu(I)-oxide along with a peak for copper hydroxide (Cu-OH).17,23 The small peak at
162.9 eV observed in the XPS of sulphur can be ascribed to the presence of trace amount of
copper-bound thiolate (Cu(SR)x) as an intermediate (Fig. 6f-g).16b,24 The weak signals for sulfur
bound to the material was also observed in the X-ray mapping images obtained after the catalysis
(Fig. S20).25
Analysis of the surface after catalysis by PXRD, SEM and TEM: The intermediate phases of
catalyst on the surface of nanomaterials were identified by studying the PXRD of the
nanomaterials recovered after the catalysis (Fig. 7a). In addition to the most intense peaks of
Cu2O, a few smaller peaks corresponding to CuII(OH)2.H2O, CuII(OH)2 and CuIIO species were
identified on the surface of the catalyst (Fig. 7a). The minor peaks for the Cu(II) species
disapperead after treatment of the catalyst with 6.0 mM ascorbic acid (D in Fig. 7a). When the
concentration of ascorbic acid was increased further to 0.6 M, a complete reduction of the
nanomaterials to elemental copper (Cu) was observed (JCPDS: 01-1241 as shown in E in Fig.
7a). These results correlate well with the proposed mechanism of NO release from RSNO
involving the initial oxidation of Cu(I) to Cu(II),15b,c which can be reduced back to Cu(I) by the
thiolates (RS-) produced in the first step of the denitrosylation reaction (Fig. 6c-g). Warren and co-
workers studied the reactivity of copper(II) thiolates towards S-nitrosothiols and proposed a
pathway for the generation of NO within the coordination sphere of copper model complexes.24b,c
However, the facile redox changes between the Cu(I) and Cu(II) in Cu2O is further confirmed by
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carrying out the denitrosylation reactions using the materials recovered from the first cycle. In fact,
the nanomaterials can mediate multiple cycles of the denitrosylation reactions without loss of the
catalytic activity (Fig. 7b and S19). The SEM and TEM images for Oh on materials recovered from
the reaction mixture after the catalysis indicated that their morphology and surface remain
unaltered, suggesting that the nanomaterials are quite stable under the reaction conditions (Fig.
7c). The HRTEM images of the materials recorded after multiple reactions cycles indicate that the
exposed facets present on the surfaces of material were also unaffected (Fig. 7d).
Fig. 7 (a) PXRD study of Cu2O Oh after the catalysis. A: parent Cu2O Oh, B: catalyst recovered after 60
min, C: catalyst recovered after 60 min and heated at 60 °C for 12h, D: treated with 6.0 mM of ascorbic
acid after heating, E: treated with 0.6 M ascorbic acid after heating. (b) Experiments demonstrating the
recyclability of Cu2O Oh (0.2 ng µl-1) using five cycles of 1.0 mM CYSNO in 0.1 M phosphate buffer (pH
7.4) at 25 °C. (c) SEM and TEM (inset) images of Oh after the catalysis. (d) HRTEM and corresponding
FFT (inset) of Oh after the catalysis.
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Conclusions
We demonstrated in this paper that Cu2O-based nanomaterials can release NO from S-
nitrosothiols and the catalytic activity is morphology-dependent. This study provides the first
experimental evidence that the denitrosylating activity of Cu2O nanocrystals can be altered by
changing the crystal facets within the same crystal system. Surprisingly, no direct correlation
between the activity and the surface area of the materials was observed. The different
arrangement of the atoms on the surface is found to be responsible for the remarkable change in
the denitrosylation activity of Cu2O nanomaterials having various morphologies. A detailed
mechanistic investigation revealed that the higher activity of the Cu2O nanomaterial having Oh
morphology as compared to that of RDh and cube is due to the presence of the {111} crystal facet
in Oh, which is more efficient than the {011} and {001} facets present in RDh and cube,
respectively, in the denitrosylation of S-nitrosothiols. Further, the Cu2O Oh nanocrystals having
{111} facets may be employed for a quick release of NO, whereas the RDh morphology having
the {011} facets will be useful for a slow and sustained release of NO. Therefore, the facet-
dependent denitrosylation activity described in this paper may find potential applications in
modulating the NO bioavailability under disease conditions where the physiological NO signaling
is severely impaired. For example, the decrease in the NO levels in endothelial cells is known to
cause endothelial dysfunction, leading to several vascular diseases. The materials that can
improve the levels of NO in these cells are likely to be potential candidates for the treatment of
vascular disorders.
Experimental section
Synthesis of Cu2O cubes and Cu2O octahedra
CuCl2.2H2O (341 mg) and PVP (for cubes: 0 g, for octahedra: 17.8 g) were dissolved in 200 mL
of ultrapure water.11 The solution was heated to 55 ºC and 20 mL of 2M NaOH solution was added
to the solution under constant stirring. After 30 min, 20 mL of 0.6 M ascorbic acid solution was
added to the reaction mixture dropwise and the resultant solution was stirred at 55 ºC for 5 h (for
cubes) or 3 h (for octahedra). After cooling the reaction mixture to 25 ºC, brick coloured
nanomaterials were obtained by centrifugation of the reaction mixture at 9000 rpm for 10 min,
followed by washing with ultrapure water and absolute ethanol. Finally, the nanomaterials were
dried in oven at 50 ºC for 24 h.
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Synthesis of Cu2O rhombic dodecahedra
CuSO4.10H2O (1 g), dissolved in 160 mL of ultrapure water, was treated with 16 mL of oleic acid
and 80 mL of absolute ethanol under vigorous stirring.11 After the reaction mixture was heated to
100 ºC under constant stirring, 40 mL of 0.8 M NaOH solution was added dropwise. After stirring
the mixture for 5 min, D-glucose solution (13.68 g dissolved in 120 mL of ultrapure water) was
added and stirring was continued for 2 h. The nanomaterials were obtained by centrifugation of
the reaction mixture at 9000 rpm for 10 min, followed by washing with ultrapure water and absolute
ethanol. Finally, the nanomaterials were dried in oven at 50 ºC for 24 h.
Synthesis of S-nitrosoglutathione (GSNO) and other RSNO
S-nitrosthiols were synthesised by following the literature method.12 Glutathione (GSH, 100 mg,
0.325 mmol), dissolved in 383 µL of ultrapure water, was treated with 38 µL of concentrated
hydrochloric acid. The solution was stirred in an ice bath (0-4 °C). The pH of the solution was
maintained between 1 and 2. After the complete dissolution of GSH, 23 mg (0.325 mmol) of
sodium nitrite was added in dark. The colour of the solution turned red immediately. After stirring
the reaction for additional 10 min, the red coloured solution was aliquoted in Eppendorf tubes and
stored at -80 °C.
Other S-nitrosothiols, CYSNO, HCYSNO and SNAC were synthesised by following a similar
procedure using L-cysteine, L-homocysteine and L-N-acetylcysteine (NAC), respectively.
Synthesis of bis(N,N-diethyldithiocarbamato)iron(II) ((dtc)2FeII) complex
The title compound was synthesized following a reported procedure with minor modifications.13
Briefly, FeSO4.7H2O (217 mg, 0.78 mmol) and diethylammonium diethyldithiocarbamate (347 mg,
1.56 mmol) were added in dichloromethane (10 mL) and the reaction mixture was stirred for 8 h.
The resultant brown solution was filtered, and the brown filtrate was dried under reduced pressure
to obtain (dtc)2FeII as a brown solid, which was then recrystallized in diethyl ether to get pure
(dtc)2FeII.
Synthesis of β-HCD-Fc
To a 10 mM solution of ferrocene (Fc) (50 mL) in dichloromethane, 10 mM aqueous solution of
(2-hydroxypropyl)-β-cyclodextrin (β-HCD) (50 mL) was added and the mixture was stirred at 45
°C under reflux for 24 h. The aqueous fraction from the reaction mixture containing ferrocene
entrapped in β-HCD complex (β-HCD-Fc) was collected.14 The synthesis was repeated several
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times, the aqueous fractions were collected, and the combined solution was freeze-dried to get a
concentrated solution (yellow) of β-HCD-Fc, which was used for the detection of nitric oxide.
Measurement of denitrosylation of RSNO by nanomaterials
The denitrosylation of S-nitrosothiols (RSNO) by three different Cu2O nanomaterials was
monitored by UV-Vis spectrophotometer.15b The decrease in the absorbance of RSNO (at 335
nm) was monitored under time-drive mode to quantify the rate of catalysis by the nanozymes. The
assay conditions for different RSNO is given in the Supplementary Information (Table S2).
Trapping of nitric oxide by (TPP)CoII complex
An experimental set-up was designed for the detection of NO using (TPP)CoII complex.16 A small
vial (0.75 mL) containing GSNO (20 mM in 0.1 M phosphate buffer, pH 7.4) and Cu2O
nanomaterials (50 ng µL-1) were placed inside a large vial (5 mL) containing 1.0 mM (TPP)CoII in
dichloromethane (DCM). The solutions were kept undisturbed for 45 min. It was observed that
the nitric oxide gas to generated from GSNO diffused into the dichloromethane solution and
reacted with (TPP)CoII to form (TPP)CoNO complex. The UV-Vis spectrum of the DCM solution
confirmed the formation of (TPP)CoNO complex from the red shift of the Q-band from 527 nm to
535 nm. Two control experiments were performed. In one case, the experiment was performed in
the absence of Cu2O nanomaterials in the assay buffer and in another case, the experiment was
carried out in the absence of both Cu2O nanomaterials and GSNO. The UV-Vis spectra of the
DCM solution from the control experiments showed no shift in the Q-band. Similar experiments
were carried out using other S-nitrosothiols (HCYSNO and SNAC) as nitric oxide source and the
formation of (TPP)CoNO complex was confirmed by UV-Vis and FT-IR spectroscopic experiments.
Time-dependent FT-Raman and FT-IR spectroscopy
The catalysis was performed using Cu2O Oh as the model catalyst and SNAP as substrate in 0.1
M sodium phosphate buffer (pH 7.4) at 25 °C. After each time point, the catalyst was recovered
from the reaction mixture by centrifugation and washed with acetone several times. The recovered
catalyst was used for the spectroscopic studies. FT-Raman spectroscopy was performed on
Renishaw in-Via Raman Microscope (Renishaw Inc, UK) by using an excitation wavelength of
514 nm, 50 x L objective lens,10 sec exposure and 3 accumulations (power <10 m|W|). The FT-
16

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IR spectra were recorded on PerkinElmer instrument using KBr pellet made with the nanomaterial
recovered from the reaction mixture.
Isothermal titration calorimetry (ITC)
The stock solution of the catalyst (2 mg mL-1) was prepared by dispersing it in isopropyl alcohol
(IPA) and the stock solution of GSH was prepared in 0.1 M sodium phosphate buffer pH 7.4. ITC
experiments were performed using Microcal VP-ITC at 25 °C. The catalyst (10 ng µL-1) were
dispersed in ultrapure water and loaded onto the sample cell. The ligand solution (GSH, 1.0 mM),
loaded in the injection syringe, was titrated into the sample cell as a series of injections (aliquots
of 10 μL) separated by 4 min interval with constant stirring at 307 RPM. Control titrations with the
ligand into water in the sample cell and water titrated with catalyst in the sample cell were carried
out to confirm that the heat of dilution was negligible. The data were fitted with Microcal Origin 5
software. After subtraction of the respective controls from the main titration profile, the binding
constant (Ka) and enthalpy (ΔH) were obtained by fitting the titration curves to a one-site binding
model. The dissociation constant (Kd) values were calculated from 1/Ka.
Conflicts of interest
The authors declare no competing financial interest.
Acknowledgements
This study was supported by the Science and Engineering Research Board (SERB, EMR/IISc-
01/2016) and Nano Mission (SR/NM/NS-1380/2014), Department of Science and Technology
(DST), New Delhi. S. G. and P. R. acknowledge the Indian Institute of Science, Bangalore, for a
research fellowship. S. G. thanks Mr. Estak Ahmed and Arnab for helping in EPR spectroscopy
with X-band JEOL (JES FA200) instrument. The authors thank Prof. S. Umapathy and Prof. P.
Thilagar for providing Raman spectroscopy and UV-Vis spectroscopy facilities, respectively. We
also thank the AFMM Facility and CeNSE, IISc for the spectroscopic and microscopic facilities.
S.G. thanks Aparna for helping in ITC experiment. G. M. acknowledges the SERB/DST for the
award of J. C. Bose National Fellowship (SB/S2/JCB-067/2015).
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Table of Contents
Crystal Facet-dependent Cu2O-mediated denitrosylation of S-nitrosothiols and release of nitric oxide (NO)
are described.
1