Hydrothermal synthesis of a paramagnetic alkali supermolecule, its effect on catalase inhibitory by spectroscopic and theoretical investigation

Article abstract of DOI:10.1016/j.ica.2020.119946

A sodium-based hydrogen maleate supercomplex formulated as [NaH(C₄H₂O₄). 3H₂O]_n; Na complex (C₄H₂O₄ = maleate) has been synthesized by a hydrothermal process at 120 °C for 48 h. The structure of Na complex has been investigated by elemental analysis, atomic absorption spectroscopy (AAS), Fourier-transform infrared (FT-IR) spectroscopy, thermal gravimetric analysis (TGA), differential thermal analysis (DTA), single-crystal X-ray diffraction (SC-XRD) and vibration sample magnetometer (VSM). The crystallography results revealed a triclinic structure of expected composition with space group P-1. Magnetic measurements in the applied field of ±15kOe revealed that Na complex has a paramagnetic behavior at room temperature. Furthermore, the thermal behavior of Na complex revealed a good stability for it. Catalase as an antioxidant enzyme plays a crucial role in the regulation of redox state. In this study, Na complex was used to investigate its effects on bovine liver catalase (BLC) and explore the underlying mechanism. Na complex interacts with BLC mainly via Van der Waals forces and hydrogen bonds. These interactions result in the decrease of BLC catalytic activity to 31percent. UV–Vis, synchronous and 3D fluorescence spectral results confirmed that the prepared complex induced some structural changes of BLC. Molecular docking also revealed the specific binding mode of Na complex with BLC.

Full text of DOI:10.1016/j.ica.2020.119946

InorganicaChimicaActa513(2020)119946
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Research paper
Hydrothermal synthesis of a paramagnetic alkali supermolecule, its effect on
catalase inhibitory by spectroscopic and theoretical investigation
Zohreh Razmara⁎, Fereshteh Shiri, Somaye Shahraki
Department of Chemistry, University of Zabol, P.O. Box 98613‐35856, Zabol, Iran
A R T I C L E I N F O
A B S T R A C T
A sodium-based hydrogen maleate supercomplex formulated as [NaH(C4H2O4). 3H2O]n; Na complex
Keywords:
Paramagnetic alkali supermolecule
Metal-based drug
Catalase
(C4H2O4 = maleate) has been synthesized by a hydrothermal process at 120 °C for 48 h. The structure of Na
complex has been investigated by elemental analysis, atomic absorption spectroscopy (AAS), Fourier-transform
infrared (FT-IR) spectroscopy, thermal gravimetric analysis (TGA), differential thermal analysis (DTA), single-
crystal X-ray diffraction (SC-XRD) and vibration sample magnetometer (VSM). The crystallography results re-
vealed a triclinic structure of expected composition with space group P-1. Magnetic measurements in the applied
field of 15kOe revealed that Na complex has a paramagnetic behavior at room temperature. Furthermore, the
thermal behavior of Na complex revealed a good stability for it. Catalase as an antioxidant enzyme plays a
crucial role in the regulation of redox state. In this study, Na complex was used to investigate its effects on
bovine liver catalase (BLC) and explore the underlying mechanism. Na complex interacts with BLC mainly via
Van der Waals forces and hydrogen bonds. These interactions result in the decrease of BLC catalytic activity to
31%. UV–Vis, synchronous and 3D fluorescence spectral results confirmed that the prepared complex induced
some structural changes of BLC. Molecular docking also revealed the specific binding mode of Na complex with
BLC.
Molecular docking
1. Introduction
movement of βPhe-280 out of the tunnel in the presence of alkali metal
cations. Crea et al. [10] examined the effects of alkali metal complexes
Supramolecules or supermolecules have generally been described as
structures that are formed by two or more molecules [1]. Both organic
and inorganic complexes can form supermolecules, through coordina-
tion bonds, hydrogen bonds, Van der Waals force or ionic bonds. The
supracomplex have many applications in magnetic materials, con-
ductors, sensors, vital biological processes, drugs such as antitumor
agents, antimalarial agents, anti-inflammatory, analgesic and anti-
bacterial agents [2–4]. The organic life depends on inorganic elements
for carrying out many vital processes [5]. The alkali metal ions such as
Na+ and K+ control essential biological processes of living cells [6],
and concentrated on some reiterative DNA sequences. These elements
have very much important role in nucleic acid folding, maintaining
blood pressure and balancing the fluids in and around the cells [7]. In
biological environments the alkali metal ions can interact with ligands
to form bi- (or poly-) nuclear complexes which may significantly affect
their activity [8]. Rhee et al. [9] reported a comprehensive study of the
effects of alkali metal cations on activity and inter subunit commu-
nication of tryptophan synthase. The results of their studies indicate the
opening of the tunnel connecting the α and β active sites by the
of 1, 2, 3, 4, 5, 6, myo-inositol hexakis-phosphate in the biological
systems. They found that the ligand of phosphate modify the bioa-
vailability of alkali cations and the resulted complex have antioxidant
and anticancer activity. Here, we aimed to (1) synthesize a complex
with a relatively high reactivity of sodium metal, and (2) investigate the
type of complex interactions with bio-macromolecules such as enzymes
and proteins. When providing a complex as a drug, there is some im-
portant points such as magnetic properties, solid-chiral structure, iso-
mers, phase transitions, and multiphase structures [11–16]. Para-
magnetic metal complexes have various medical applications. Although
the study of paramagnetic complexes has been reported on important
proteins such as enzymes [17], the study of paramagnetic super-
molecules on important bio macromolecules has not been reported.
Compared to many proteins, catalase has special importance; this pro-
tein is a key enzyme in the defense system. Any factor that can inhibit
catalase activity, can lead to ROS accumulation and cell damage.
Changes in the structure and activity of the catalase or damage to it
could have serious consequences for body. So, the effect of a new Na
complex on the function and structure of the bovine liver catalase was
⁎ Corresponding author.
E-mail address: razmara@uoz.ac.ir (Z. Razmara).
https://doi.org/10.1016/j.ica.2020.119946
Received 25 February 2020; Received in revised form 6 July 2020; Accepted 5 August 2020
Availableonline07August2020
0020-1693/©2020ElsevierB.V.Allrightsreserved.

Z. Razmara, et al.
InorganicaChimicaActa513(2020)119946
investigated by spectroscopic and molecular modeling methods. It is
hoped that this study will provide new insights into the toxicity effects
of relatively high reactivity metal complexes.
and H2O2, 1.0 × 10−2 M) and changes in hydrogen peroxide con-
centration were measured by the absorption spectrophotometer in the
presence of different concentrations of the Na complex (0–400 × 10−6
M) [20]. The following Eq. can be used to study catalase activity:
2. Experimental
(ΔA1/ΔA0) × 100%
Where, ΔA1 and ΔA0 are the adsorption changes of hydrogen peroxide
at λ = 240 nm in the presence of catalase, before and after the addition
of Na complex [21].
2.1. Reagents and chemicals
All reagents used in this work were prepared from highest-purity
available materials and used as received without further purification.
The water was distilled and deionized. Catalase from bovine liver was
purchased from Sigma-Aldrich Chemical Co. USA. The solution of Tris-
HCl buffer (0.05 M, containing 0.1 M NaCl) was used to maintain the
pH of the solution at 7.4. Stock solution of BLC (1 × 10−5 M) was
prepared in Tris-HCl buffer. The Na complex solution was prepared
with a concentration of 5 × 10−3 M in DI water.
2.6. Molecular docking study
The Smina software [22] run in Linux was used to find the forces
involved in the binding of Na complex and BLC. Also, the bioactive
conformer (pose) of Na complex was determined. The CIF file of X-ray
crystal structure of the Na complex was obtained from Cambridge
crystallographic data center (CCDC). While this complex was
a
polymer, we decided to use a timer as representative of the polymer.
The CIF of the timer complex file covert to SDF format by Mercury 4.3.0
software. The crystal structure of BLC with PDB ID 1TGU with resolu-
tion 2.8 Å was obtained from the RCSB Protein Data Bank (http://www.
pdb.org). In preparation for protein, we removed the three monomers
from four identical sequences of 1TGU and just kept a monomer file.
Because the active pocket is located inside the protein and it is far
enough from the boundaries of a single monomer. All water and non-
protein molecules were removed. In addition, Polar hydrogen were
added to catalase. The grid box of 32 × 32 × 36 Å3 was centered on Na
complex with coordinates of the complex in X, Y, Z planes at 23.58,
41.74 and 11.96, respectively.
Input files were prepared, and the Lamarckian genetic algorithm
was used for the blind docking in Smina. The highest magnitude energy
was selected as a best binding pose. To visualize and analyze the mo-
lecular structure, BIOVIA Discovery Studio client 2016 was also ap-
plied. In addition, the interacting energies between free amino acid
with the best binding pose of Na complex into the BLC binding site were
calculated by Molegro Molecular Viewer 2.5 (MMV) (http://www.
molegro.com/mmv-product.php).
2.2. Apparatus and methods
Elemental analyses of Na complex were carried out using a Perkin-
Elmer 2400 elemental analyzer. FT-IR spectrum of synthesized complex
was recorded in the range 4000–400 cm−1 using a Jasco FT-IR-430
spectrometer by KBr pellets. The weight and heating changes of syn-
thesized complex was simultaneously studied by TGA and DTA analysis,
respectively on a NETZSCH STA 409 PC/PG device in N2 atmosphere.
The thermal behavior was recorded at temperature ranging from RT to
700 °C at a constant heating rate of 10 °C/min in a quartz crucible.
Room temperature magnetization versus applied magnetic field was
measured using a vibrating sample magnetometer (VSM) by a super-
conducting quantum interference device (SQUID) in the applied mag-
netic fields up to 15,000 Oe. UV–Vis spectra were recorded on a JASCO
UV/Vis-7850 double-beam spectrophotometer using quartz cells with a
path length of 1.0 cm. Fluorescence spectra were recorded on the
Agilent Cary Eclipse Fluorescence Spectrophotometer. The excitation
and emission slit widths were set at 5.0 nm; λex = 280 nm and the
emission spectra range were used between 300 and 500 nm. To obtain
the appropriate fluorescence intensity, these spectra were modified for
the inner filter effect [18].
3. Results and discussion
2.3. Hydrothermal synthesis of Na complex
3.1. Characterization of synthesized complex
An aqueous solution (10 mL) of maleic acid (0.116 g, 1 mmol) was
added to an aqueous solution of Na2CO3 (0.105 g, 1 mmol). The re-
sulting solution was stirred for 30 min in air, then placed in a Parr
Teflonlined stainless steel vessel (50 cm3) at 120 °C for 2 days. After
cooling to room temperature, the solution was filtered off and then left
to evaporate in air at room temperature for 7 days. Crystals of Na
complex which is suitable for X-ray crystallography were collected in
73% yield. Anal. Calc. for C4 H9 Na O7: C, 24.98; H, 4.68 Found: C,
24.85; H, 4.64%.
3.1.1. Crystal structure
The Na complex crystallizes in a triclinic system with the space
group P −1 [23]. The asymmetric unit of Na complex comprises of one
Na+, one hydrogen maleate and three coordinated water molecules.
The Na+ is bonded to one hydrogen atom and five oxygen atoms
forming a distorted octahedral geometry. The polyhedral geometry
around centers of Na+ is shown in Fig. S1. A three-dimensional co-
ordinating supramolecular network is formed by strong OeH…O hy-
drogen bonds between the non-coordinated water molecules as donors
and the coordinated maleate anions (Fig. S2). A packing view of three-
dimensional network of Na complex along c-axis is shown in Fig. 1.
2.4. X-ray structure determination
Data collection of good quality crystals of synthesized complex was
performed on a four-circle KUMA KM4 diffractometer equipped with
graphite monochromatic Mo Ka radiation (0.71073 Å) at 100 K from
the sealed fine-focus X-ray tube. The crystal structure was solved by
direct methods using SHELXT-2014/7 to get all the positions of almost
all non-hydrogen atoms and refined by SHELXL-2018/3 program with
the anisotropic thermal displacement parameters [19].
3.1.2. FT-IR spectrum
The FT-IR spectrum of Na complex is shown in Fig. S3. A broad and
strong absorption band in the range of 3000–3600 cm−1 can be related
to the stretching vibrations of crystalized water molecules. Further-
more, the band appeared at about 1625 cm−1 can be assigned to
asymmetric stretching vibrations of carboxylate groups which overlaps
with the bending vibrations of crystallized water molecules [24–27].
Symmetric stretch of the COO−1 groups was appeared at about
1380 cm−1. Δυ= (υs (COO)-υas(COO)) = 245 cm−1 shows a unidentate
coordination mode for carboxylate groups [25]. The stretching vibra-
tion bands of CeH in-plane and out-of-plane bending modes appear at
1137, and 900 cm−1, respectively [28,29]. Moreover, frequency at
2.5. Activity evaluation of catalase
We used the reported method to investigate the effect of the Na
complex on catalase activity. Catalase and hydrogen peroxide con-
centrations were constant in this experiment (catalase, 1.0 × 10−8
M
2

Z. Razmara, et al.
InorganicaChimicaActa513(2020)119946
Fig. 1. Packing view of Na complex along c-axis.
1270 cm−1 is assigned to the CeO vibrations of maleate ligands. In
magnetization change is linear and the synthesized complex has a
paramagnetic behavior at room temperature, while there is no any
tendency to saturate even at 15,000 Oe. The magnetization value of Na
complex was found to be about 0.71 emu g−1. paramagnetic materials
have positive magnetic susceptibility while their atomic magnetic mo-
ments are uncoupled. Magnetic materials have many applications in
fluorescence techniques, environmental, drug delivery, therapeutic and
etc. Transportation of drugs using magnetic materials to a specific site
can eliminate side effects and also reduce the dosage required of the
drugs.
addition, the bond at 515 cm−1 corresponds to Na-O vibration.
3.1.3. Simultaneous thermal analysis
The simultaneous thermogravimetric analysis (TGA/DTA) curves of
synthesized complex in the temperature range of 25–700 °C is shown in
Fig. 2. This analysis was performed under N2 atmosphere, while the
temperature was linearly increased with a constant heating rate of
10 °C/min. As shown in the TAG curve, thermal analysis of Na complex
starts just above 110 °C. An initial weight loss in the region 110–140 °C
is due to the dehydration step and removal of the crystallized water
molecules. Two other weight losses over a wide temperature range of
220 to 500 °C correspond to the burning of all organic parts of the
complex and complete collapse of the structure, no weight loss was
observed after this temperature. Also, more information on the en-
thalpy changes of the synthesized complex was studied by differential
thermal analysis (DTA) and is shown in Fig. 2. One small endothermic
peak was observed in the temperature region of 110–140 °C, due to the
losses of uncoordinated water molecules. Three exothermic peaks in the
region 250–500 °C are probably due to the burning of the organic li-
gands.
3.2. Investigation of catalase activity
Influence of different concentrations of the Na complex (0–400 μM)
on the activity of BLC was shown in Fig. 4A. As Fig. 4A shows, the
addition of Na complex concentrations to the BLC solution reduces BLC
activity gradually. The inhibitory strength of this complex was in-
vestigated graphically as the IC50 value. This parameter refers to the
concentration of an inhibitor needed to reduce the activity of the en-
zyme to 50% [30]. Using Fig. 4A, IC50 value of 13 μM was calculated for
this complex. Similar behavior has been observed in some transition
metal complexes [30–33]. To study the inhibition mechanism of cata-
lase by studied complex, activity of a fixed concentration of BLC
(1.0 × 10−8 M) were evaluated in the presence of various concentra-
tions of H2O2 (0.01, 0.02, and 0.04 M) by different concentrations (0,
3.0, 6.00 and 12.00 × 10−7 M) of complex. According to the Line-
weaver–Burk plot (Fig. 4B and Table 1S), the Km values for inhibited
reactions were different from that of the control (free BLC), which re-
vealed mixed-type enzyme inhibition mode [34–36]. That means, the
Na complexes can bind to the free BLC as well as the enzyme-substrate
complex to reduce catalysis. The mixed-type inhibition is similar to
noncompetitive inhibition excluding binding of the substrate or in-
hibitor and can affect the enzyme binding affinity for the other. In this
type of inhibition, the inhibitor has both the competitive and non-
competitive inhibitory effects. Indeed, it can be said the studied com-
plex did not directly bind into the catalase activity site, but the binding
of it into the enzyme cavity influenced the microenvironment of the
catalase activity site which resulted in the reduced catalase activity
[17].
3.1.4. Magnetic properties
In order to study the magnetic behavior of synthesized complex, we
investigated its magnetic behavior at room temperature by VSM. A
typical plot of magnetization versus applied field in the range of 15
kOe is shown in Fig. 3. The hysteretic M−H curve shows that the
80
70
60
50
40
30
20
10
0
0
DTA (mW/mg)
TGA (mg)
-2
-4
-6
-8
-10
-12
-14
-16
3.3. Fluorescence quenching: Mechanism and binding parameters
0
100
200
300
400
500
600
700
Temperature/ ºC
Fluorescence emission of proteins is mediated by the aromatic
amino acids (Tyrosine, Tyr, Tryptophan, Trp, and Phenylalanine, phen).
Fig. 2. TGA and DTA plots of Na complex.
3

Z. Razmara, et al.
InorganicaChimicaActa513(2020)119946
0.8
0.6
0.4
0.2
0
-15000
-10000
-5000
0
5000
10000
15000
-0.2
-0.4
-0.6
-0.8
Applied Field (Oe)
Fig. 3. Magnetization as a function of applied field at room temperature of Na complex.
Fluorescence emission of these amino acids can change in the presence
of external reagents or change in the environment around them.
Monitoring fluorescence quenching of a protein under different che-
mical or physical conditions has been widely used for understanding
the protein–ligand interaction. The fluorescence spectra of BLC in the
presence of different concentrations of Na complex were shown in
Fig. 5A. As shown in this figure, free BLC shows a strong fluorescence
emission peak at 344 nm upon maximum excitation wavelength of
280 nm. The fluorescence intensity of BLC was quenched obviously
with the addition of Na complex, which indicated that an interaction
had occurred between BLC and Na complex. Furthermore, the fluores-
cence spectra of BLC has a blue shifted by 4 nm from 338 nm to 334 nm.
The blue shift in fluorescence spectra of BLC confirmed that the mi-
croenvironment of fluorophores became relatively more hydrophobic
or less exposed to the solvent as a result of the binding of the Na
complex and BLC.
where F0 and
F
are fluorescence intensities of BLC in two-state of ab-
kq 0, and[Q]are
sence and presence of Na complex, respectively.Ksv
,
,
τ
Stern–Volmer quenching constant, quenching rate constant, the average
lifetime of fluorescence in the absence of quencher (10−8 s), and the Na
complex (quencher) concentration, respectively. The KSV is obtained by
a linear regression plot of F0/F versus [Na complex] (Fig. 5B). The
linear Stern–Volmer plots obtained at two temperatures which con-
firmed the existence of only one kind of quenching mechanism (dy-
namic or static) [33]. Also, Ksv values are inversely related to the
temperature, confirming the static mechanism. Furthermore, the static
quenching mechanism and ground state complex formation were con-
firmed by the kq values that were greater than 2 × 1010
Bonding parameters such as binding constant (Kb) and the number
of binding sites (n) can be obtained from the following equation:
M .
−1s−1
F0 − F
log
= logKb + nlog [Q]
(2)
F
Fluorescence quenching mainly occurs by two mechanisms: dy-
namics (collisional encounters) and static (complex formation). To de-
termine the mechanism of fluorescence quenching of BLC in the pre-
sence of Na complex were measured at two temperatures 300 and 310 K
and the fluorescence data were analyzed according to the classical
Stern − Volmer equation [30]:
Fig. 5C displays log F0-F/F vs. log [Q] plot at 300 and 310 K.
Binding parameters are listed in Table 1. As the results presented in the
table show, an increase in the temperature is associated with a decrease
in the Kb values, indicating the formation of the ground state complex
between BLC and Na complex. This observation confirms the static
mechanism and is fully consistent with the KSV result. Furthermore,
given the values obtained for the n (approximately equal to one), it can
F0
F
= 1 + kqτ0 [Q] = 1 + Ksv [Q]
(1)
120
100
80
60
40
20
0
5
(A)
(B)
4
3
2
1
0
0
15 30 60 90 120 200 280 400
-0.1
-0.05
0
0.05
0.1
[Complex] μM
1/[H2O2] (mM-1)
Fig. 4. (A) Effect of different concentrations of Na complex on the BLC activity; [BLC] = 1.0 × 10−8 M, [Na complex] = 0–4.0 × 10−4 M. (B) Lineweaver–Burk plot
for BLC in the absence and presence of Na complex; [BLC] = 1.0 × 10−8 M, [Na complex] = (0, 3.0, 6.00 and 12.00 × 10−7 M).
4

Z. Razmara, et al.
InorganicaChimicaActa513(2020)119946
Table 1
Thermodynamic and binding parameters for interaction of BLC with Na complex.
T (K)
n
KSV (M−1
)
kq (M−1
)
Kb (M−1
)
ΔG° (kJ mol−1
)
ΔH° (kJ mol−1
−30.39
)
ΔS° (kJ mol−1 K−1
−0.050
)
300
310
0.99
0.95
0.10 × 104
0.85 × 103
0.10 × 1012
0.85 × 1011
0.44 × 103
0.30 × 103
−15.27
−14.76
(A)
338 nm
500
400
300
200
100
334 nm
0
300
350
400
Wavelength (nm)
450
500
2
1.6
1.2
0.8
0.4
0
0.5
0
(B)
(C)
-0.5
-1
-1.5
-5
-4
log[Na Complex]
-3
0
20
40
60
80
100
[Na Complex] × 105 M
Fig. 5. (A) Fluorescence emission spectra of BLC in the presence of different amounts of Na complex. [BLC] = 3 × 10−7 M, [Na complex] = 0–8.4 × 10−6 M. (B)
The Stern–Volmer curve of BLC quenching in the presence of various amounts of Na complex at two temperatures 300 K (○) and 310 K (●). (C) The plot of log (F0–F/
F) vs. log [Na complex] at two temperatures of 300 K (○) and 310 K (●).
be said that there is only one binding site for the Na complex at the BLC.
3.5. Synchronous fluorescence spectra
Synchronous fluorescence spectra at the fixed wavelength interval
(Δλ) of 15 nm and 60 nm were used to evaluate the alteration in the
environment around fluorophores that was induced by Na complex
interactions. The Δλ (the difference between excitation and emission
wavelengths) at 15 and 60 nm gives information about microenviron-
ment of Tyr and Trp, respectively. The effects of the Na complex on Tyr
and Trp residues in the synchronous fluorescence spectra of BLC are
shown in Fig. 6. In the presence of Na complex in the BLC solution with
Δλ = 60 nm, the intensity of BLC fluorescence decreased and no sig-
nificant shift was observed in the spectrum. So, this complex did not
significantly perturb the polarity around Trp residues. While, in the
presence of Na complex, the maximum emission of Tyr residue has an
obvious red shift from 290 nm to 249 nm, indicating that the Na
complex affected the conformation of BLC and made the micro-
environment of Tyr less hydrophobic.
3.4. Thermodynamic and binding forces
To determine thermodynamic parameters such as ΔH0, ΔS0, and ΔG0
the Van't Hoff Eq. (3) and Eq. (4): were used;
ΔH°
RT
ΔS°
R
Ln K = −
+
(3)
(4)
ΔG° = ΔH° − TΔS°
The binding forces between ligand and bio-macromolecules are
classified into hydrogen bonds, Van der Waals force, hydrophobic
forces and electrostatic interactions. The sign and magnitude of the
thermodynamic parameters can predict the type of acting forces in the
ligand–protein interactions. The hydrophobic interactions usually pre-
sent positive values of ΔH0 and ΔS0, whereas Van der Waals forces and
hydrogen bonding present negative values of ΔH0 and ΔS0; further-
more, negative values of ΔH0 along with positive values of ΔS0 confirm
that electrostatic interactions have main roles in the bio-macro-
molecules-ligand interaction process. The calculated thermodynamic
parameters for the interaction between Na complex and BLC were
presented in Table 1. Here, ΔS° < 0 and ΔH° < 0 showed that the main
binding forces in the Na complex-BLC system were Van der Waals and
hydrogen bonding. According to the negative values of ΔG°, the binding
interaction between Na complex and BLC is spontaneous.
3.6. UV–Vis absorption analysis
Protein conformation can also be evaluated by using UV–visible
spectrophotometry. The UV–Vis absorption spectra of BLC with and
without the Na complex were recorded (Fig. S4). Three absorption
bands at 210, 280, and 408 nm appear in the catalase spectrum. The
first peak around 210 nm is the characteristic peak of the framework
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Z. Razmara, et al.
InorganicaChimicaActa513(2020)119946
100
ꢊꢋ = 15 nm
290 nm
80
60
40
20
0
294 nm
270
280
290
Wavelength (nm)
300
310
ꢊꢋ = 60 nm
280 nm
280 nm
600
400
200
0
260
280
300
Wavelength (nm)
Fig. 6. Synchronous fluorescence spectra of the BLC-Na complex.
Fig. 7. 3D fluorescence spectra of BLC in the absence (I) and presence of (II) Na
complex.
conformation of the protein and two other peaks at 408 and 280 nm is
related to the heme molecule of catalase and aromatic amino acids (Trp,
Tyr, and Phe), respectively [37,38]. As it can be seen in the Fig. S4, the
absorbance peak of the BLC at 280 nm was gradually increased by the
addition of Na complex to BLC solution. This observation confirmed
that binding of the Na complex to BLC can lead to a change in the
structure of the enzyme’s active site [33]. Besides, the addition of Na
complex also caused a blue shift around the λmax = 280 nm (from
284 nm to 282 nm). This suggests that the hydrophobicity of the Trp,
Tyr and Phe environment may have decreased [39].
arm, heme moiety with a β-barrel, wrapping domain, and carboxy-
terminal portion. This structure was shown in Fig. 8A. In previous work,
we described the structure of BLC in detail [42]. Among the nine con-
formations of the complex that Smina based on the affinity score gen-
erated (Table 2), the conformer with −8.47 kcal mol−1 had best affi-
nity. The binding site for Na complex is located at the wrapping domain
(Fig. 8B). The docking results confirmed the driving forces for Na
complex binding to BLC are Van der Waals forces and hydrogen
bonding. When a distance between a non-hydrogen atom of a protein
and ligand is smaller than the sum of their VDW radii plus a threshold
of 0.5 Å, a VDW contact can be founded. Nitrogen and oxygen are
hydrogen bond acceptors while hydroxyl and amine groups are donor.
To form a hydrogen bond, the distance and angle between donor–-
acceptor should be ≤ 3.5 Å and ≤ 30, respectively [43]. The interac-
tion between the complex and BLC for the binding site is dominated by
hydrogen binding interactions with ASN141, ASP139, ASN337,
MET338 and ASN384 residues. These interactions with the length of
hydrogen bonds represented in Fig. 9. Also, several Van der Waals in-
teractions were observed with GLN386, SER336 and PRO344 residuals.
So, docking study results and data obtained from fluorescence
quenching of BLC emission in the presence of Na complex are the same,
thus confirming each other. Finally, the interaction energies of amino
acids in the docked complex are separately listed in Table 3.
3.7. 3D fluorescence measurements
The 3D fluorescence spectra of BLC and the BLC–Na complex are
presented at Fig. 7. In this figure, there are 4 distinct peaks namely A, at
λ
em = λex for first-order, B, at λem = 2λex for Rayleigh second order, 1,
at n → π∗ for Tyr/Trp and 2, at π → π∗ for polypeptide backbone. In the
presence of the Na complex, the intensity of these peaks decreases as
indicated polypeptide backbone and also, the Tyr/Trp microenviron-
ments of catalase are changed during the interaction between Na
complex and BLC.
3.8. Molecular docking analysis
Molecular docking study could be a useful technique to determine
the forces involved in the interaction between target proteins and small
molecules, and determine the exact binding sites [40,41]. Also, the
molecular docking approach is a complementary technique for experi-
mental data. The tertiary structure includes four domains; N-terminal
6

Z. Razmara, et al.
InorganicaChimicaActa513(2020)119946
Fig. 8. Molecular structure of subunit A of BLC by 1TGU(A) and schematic illustration of Na complex binding sites on BLC (B).
Table 2
Table 3
Smina score of BLC with different conformers of Na
complex.
Interaction energy between the Na complex and responsive amino acid
residues of BLC in molecular.
Pose
Affinity (kcal/mol)
docking.
Amino acid residues
Interaction energy kJ mol−1
1
2
3
4
5
6
7
8
9
−8.47
−8.44
−8.33
−8.29
−8.25
−8.06
−8.04
−7.97
−7.96
Asn141
Asn337
Asn384
Gln386
Gly140
Leu370
Met338
Pro377
Val382
−2.87518
−7.52342
−3.50131
−2.9627
−2.38952
−6.08408
−1.34118
−3.93209
−6.59939
4. Conclusion
body. In this work, a paramagnetic sodium coordination polymer based
on hydrogen maleate was successfully synthesized under hydrothermal
condition and characterized by various methods such as single crystal
X-ray diffraction, FT-IR, TGA/DTA and VSM. Interaction of above
Among the metal complexes presented as biological agents, most of
the transition metals complexes have been considered. The reason is the
high reactivity of these compounds which can lead to side effects on the
Fig. 9. 2D (right) and 3D (left) representation the residues of BLC active site around Na complex in the crystal structure of 1TGU.
7

Z. Razmara, et al.
InorganicaChimicaActa513(2020)119946
complex with BLC was evaluated using experimental and molecular
docking studies. The results revealed that Na complex could dramati-
cally inhibit the decomposition of hydrogen peroxide. The fluorescence
analysis showed that Na complex quenched BLC fluorescence by static
mechanism. Thermodynamic investigation and molecular docking
study also confirmed one binding site for Na complex on BLC.
Formation of Na complex-BLC system was a spontaneous processing
and Van der Waals forces and hydrogen bonds were the major forces
between BLC and Na complex. Synchronous fluorescence results con-
firm that Na complex has a more pronounced quenching effect on Tyr
residue than Trp residue. The molecular modeling results were in good
agreement with the experimental results and showed that the Na
complex binding site was located at the wrapping domain.
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Acknowledgements
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