Catalase, catecholase and phenoxazinone synthase-like activities of homodinuclear co(II), ni(II), cu(II) and zn(II) complexes including oxime group

Article abstract of DOI:10.4067/s0717-97072018000304072

Homodinuclear (1) Co(II), (2) Ni(II), (3) Cu(II) and (4) Zn(II) metal complexes containing oxime group were tested for their catalase, catecholase and phenoxazinone synthase-like activity by volumetric and spectrophotometric procedures. Catalase-like acti

Full text of DOI:10.4067/s0717-97072018000304072

J. Chil. Chem. Soc., 63, Nº 3 (2018)
CATALASE, CATECHOLASE AND PHENOXAZINONE SYNTHASE-LIKE ACTIVITIES OF HOMODINUCLEAR
Co(II), Ni(II), Cu(II) and Zn(II) COMPLEXES INCLUDING OXIME GROUP
GÜVENÇ GÖRGÜLÜa, BÜLENT DEDEb*
aDepartment of Science Education, Faculty of Education, Mehmet Akif Ersoy University, Burdur, Turkey
b*Department of Chemistry, Faculty of Science & Art, Süleyman Demirel University, Isparta, Turkey
ABSTRACT
Homodinuclear (1) Co(II), (2) Ni(II), (3) Cu(II) and (4) Zn(II) metal complexes containing oxime group were tested for their catalase, catecholase and
phenoxazinone synthase-like activity by volumetric and spectrophotometric procedures. Catalase-like activity of the complexes was studied by measuring
the evolved dioxygen resulted from the disproportionation reaction of potentially harmful hydrogen peroxide. Catecholase and phenoxazinone synthase-like
enzyme activities were followed by the increase in absorbance at 400 and 433 nm resulted from the oxidation reaction of 3,5-di-tert-butylcatechol to 3,5-di-tert-
butylquinone and 2-aminophenol to 2-aminophenoxazine-3-one, respectively. Among the studied homodinuclear complexes, complex (3) showed the highest
catalytic efficiency for three enzymes tested. Catalytic efficiency of the complexes was found 3>1>4=2 for catalase-like, 3>1>2>4 for catecholase-like and
3>1>4>2 for phenoxazinone synthase-like activity. Relatively higher catalytic activity of the Cu(II) complex is thought to be related to the lower redox potential
of Cu(II) ion and better proximity of the chosen substrates with the complex (3).
Keywords: Homodinuclear metal complexes, oxidoreductases, catalase, catecholase, phenoxazinone synthase
1.
INTRODUCTION
2.
MATERIALS AND METHODS
Metal ions have been found to play a crucial role in many biological
systems. At least one-third of all proteins appear to contain metal ions and
even the nucleozymes such as ribozymes (RNA enzymes) appear to be
metalloenzymes.1,2 These metal ions can modify electron flow in a substrate
or enzyme, thus effectively controlling an enzyme-catalyzed reaction. Without
the appropriate metal ion, a biochemical reaction catalyzed by a particular
metalloenzyme would proceed very slowly. Bio-inorganic chemistry comes
into the play at this point which encompasses a variety of disciplines, ranging
from inorganic chemistry and biochemistry to spectroscopy, molecular
biology, and medicine.3 It is not possible to use the natural enzyme as a drug
due to its delivery problems and instability in solution. Therefore, synthetic
compounds able to mimic natural enzymes have been designed and studied by
the researchers. 4-8
Organic chelating ligands containing the –C=N–OH group have been
named as oxime compounds.9 Oxime ligands have a growing interest due to
their physical, chemical and biological properties, besides their reactivity
patterns. They find a broadening application field in biological systems,10,11
medicinal chemistry,12,13 electrochemical and electrooptical sensors.14
Several types of ligand systems, which can bind two metal ions in close
proximity, have been used as biomimetic studies of binuclear metalloenzyme
and metalloproteins due to their interesting catalytic properties, their ability
to stabilize unusual oxidation states and possibilities for magnetic interaction
between two metal ions.15,16 These complexes have found many applications
as catalysts for specific purposes, as mimics for metallobiomolecules, and in
investigations concerning the mutual influence of two or more metal centers on
the electronic, magnetic and electrochemical properties of such closely spaced
metal centers.17,18
2.1. Physical Measurements
All the reagents and solvents were of reagent-grade quality and purchased
from commercial suppliers. The UV-Vis measurements were done on a PG
T80+ spectrophotometer.
2.2. Catalase-like Activity
Volumetric measurements of evolved dioxygen during the
disproportionation reactions of the H2O2 with homodinuclear complexes were
modified and applied from Kaizer et al. as follows: A 50 cm3 three-necked
round-bottom flask containing a solution of the complexes (0.005 mmol solid
sample) in DMF (10 cm3) was placed in a water bath (25°C). One of the necks
was connected to a burette and the others were stoppered by a rubber septum.
While the solution was being stirred, H2O (1.33 mmol, 0.150 cm3) was injected
into it through the rubber septum using 2a microsyringe. Volumes of evolved
dioxygen were measured at 1 min intervals by volumetry17 Pyridine (50 mg)
was introduced into the reaction vessel as an accelerator, before the addition of
H2O2. Observed initial rates were expressed as moldm-3s-1 by taking the volume
of the solution (10 cm3) into account and calculated from the maximum slope
of the curve describing the evolution of O2 versus time.
2.3. Catecholase-like Activity
The catalytic oxidation of the model substrate 3,5-DTBC (3,5-di-tert-
butylcatechol) was evaluated by homodinuclear Co(II), Ni(II), Cu(II) and
Zn(II) complexes spectrophotometrically in dioxygen-saturated methanol by
monitoring the increase in absorbance at 400 nm, corresponding to the formation
of the quinone product 3,5-DTBQ (3,5-di-tert-butyl-o-benzoquinone).23 The
observed rate constant (kobs) values of the metal complexes for the 3,5-DTBQ
formation were obtained from eq (1).
Oxidoreductases are
a broad group of enzymes that catalyze the
oxidation and reduction reactions. They involve various life-sustaining
reactions ranging from the simplest organism to human beings. Catalase (EC
1.11.1.6), catecholase (catechol oxidase: EC 1.10.3.1) and phenoxazinone
synthase (o-aminophenol oxidase: EC 1.10.3.4) are the members of his group.
Catecholase and phenoxazinone synthase contain Cu(II), while catalase contain
Fe(II) in their active sites.
ln(A∞/A∞-At)=(kobs)1t
eq (1)
A
and A are the absorbance of the formed 3,5-DTBQ at time = ∞ and
time =∞t, respetctively.
In this study, we investigated previously synthesized and characterized19
homodinuclear (1) Co(II), (2) Ni(II), (3) Cu(II), (4) Zn(II) metal complexes’
three different enzymatic activities namely; catalase, catecholase and
phenoxazinone synthase. Enzymes are picked up due to their biochemical
importance and the mimicking capacity of our complexes. Catalase is
responsible for disproportionation reaction of potentially harmful hydrogen
peroxide to oxygen and water.20 Likewise, catecholase enzyme catalyzes the
conversion of 3,5-di-tert-butylcatechol to 3,5-di-tert-butylquinone21. Finally
phenoxazinone synthase catalyzes the formation of 2-aminophenoxazine-3-one
(APX) from 2-aminophenol (OAPH).22 All three enzymes are metabolically
important as well as the substrates and the products.
2.4. Phenoxazinone Synthase-like Activity
The reaction between 2-aminophenol (OAPH) and dioxygen in the
presence of catalytic amount of homodinuclear Co(II), Ni(II), Cu(II) and
Zn(II) complexes was performed. Complexes (1.67 x 10-4 M each) and
correspondingly the substrate; 2-aminophenol (OAPH) (12.5 x 10-3 M) were
dissolved and completed to 25 mL of DMF as the final volume. Spectrum
scan was carried out between 300-600 nm with 30 sec intervals for 25 repeats.
The oxidation reaction of OAPH was monitored by following the increase in
absorbance at 433 nm by UV–Vis spectrophotometer, which is a typical band
for 2-aminophenoxazine-3-one (APX).22
The observed rate constant (kobs) values of the metal complexes for the
e-mail: bulentdede@sdu.edu.tr
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J. Chil. Chem. Soc., 63, Nº 3 (2018)
APX formation were obtained from eq (2).
3.
RESULTS AND DISCUSSION
ln(A∞/A∞-At)=(kobs)2t
eq (2)
Complexes, investigated for their catalytic activity, were synthesized as
described before.19 They were also verified for their physical and spectroscopic
properties which explained before by our research group. All complexes were
formed in the mole ratio of 2:2 metal:ligand (Figure 1). Results obtained were
compatible with the previous data.19
A∞ and At are the absorbance of the formed APX at time = ∞ and time =
t, respectively.
Figure 1. General structure of homodinuclear metal complexes.
[M:(1) Co(II), (2) Ni(II), (3) Cu(II), (4) Zn(II)]
3.1. Catalase-like Activity
The disproportion reaction of peroxide is completed up to 13 minutes. For
one molecule of complexes, corresponding number of H2O2 disproportionated
has been found 28, 13, 36, 13 for Co(II), Ni(II), Cu(II) and Zn(II) at the 1st
minute of the reaction and Co(II), Ni(II), Cu(II) and Zn(II) reached the highest
number of disproportionated H2O2 as 71, 20, 154, 28 at 10th, 3rd, 13th and 6th
minutes, respectively.
For all complexes, the reaction rate of the first minute is faster than the
succeeding minutes. The rapid dioxygen evolution rate reached as soon as the
reaction was initiated and slowed down as the reaction proceeded which can
be attributed partially to occupation of the complex molecules. Initial rates of
the reactions have been calculated and showed in Table 1. Cu(II) homodimer
complex showed the highest catalytic activity among all complexes. The
catalytic efficiency of the complexes at first minute was obtained as 3>1>4=2.
These complexes were tested for their mimicking capacity of catalytic
activity of catalase, catecholase and phenoxazinone synthase enzymes.
Homodinuclear Cu(II) and Mn(II) complexes are used rather than the
heterodinuclear ones for the catalase-like activity studies by researchers.
Synthesized homodinuclear Co(II), Ni(II), Cu(II) and Zn(II) complexes have
been studied for catalytic activity in decomposing H2O2 at 25oC by volumetric
measurements. The catalytic activity of the complex towards hydrogen
peroxide was investigated in N,N-dimethylformamide (DMF). In the absence
of pyridine as a heterocyclic base, the complex disproportionated hydrogen
peroxide slowly. The time course of the O2 evolution can be seen from Figure 2.
Table 1. Initial reaction rates of complexes 1-4 for the disproportion of
H2O2
Complexes
(1) Co(II)
(2) Ni(II)
(3) Cu(II)
(4) Zn(II)
Rate (moldm-3s-1)
1.18 x 10-5
0.53 x 10-5
1.51 x 10-5
0.53 x 10-5
Catalase-like activity results of our complexes are comparable with the
previous results. Gao et al. studied 3 different dinuclear Cu(II) complexes and
expressed lower rate values ranging from 1.35x10-5 to 0.2x10-5 in moldm-3s-1.24
Similarly, Pires et al. studied mononuclear Co(III), Cu(II) and Ni(II) complexes
and obtained the best activity from Ni(II) complex. They found Ni(II) complex’s
initial rate value as (2.0x10-7 moldm-3s-1) which was 1.7 times higher than their
previous study containing tetranuclear Fe(III) complex.25 When compared to
previous studies performed in similar experimental conditions and expressed
in similar units, our complexes showed higher and, regarding some studies,
significantly higher catalase-like activity. On the other hand, our complexes
have far more lower catalytic efficiency compared to natural catalase enzyme.26
The proposed reaction mechanism for disproprotionation of hydrogen
peroxide can be seen from Scheme 1. At the first step of the reaction, hydrogen
Figure 2. Increase in O2 yield resulted from H2O2 disproportion
Catalase mimicking studies have been repeated several times to ensure
consistency of the results.
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J. Chil. Chem. Soc., 63, Nº 3 (2018)
peroxide attacks the complex resulting in the removal of two molecules of
coordinated water. In the next step, while Cu(II) is oxidized to Cu(III), O-O
bond of hydrogen peroxide goes through a homolytic breakdown forming
hydroxyl radical-Cu(III) moieties. Cu(III)-OH intermediate goes back to its
original conformation by taking another hydrogen peroxide yielding a molecule
of oxygen and coordination waters bonded back to Cu(II) atom.
Figure 4. Spectrum scan of catecholase catalyzed reaction taken by 30
sec. intervals. The increase in absorbance at 400 nm is characteristic for 3,5-di-
tert-butylquinone
Scheme 1. A proposed mechanism for catalase-like enzyme activity of
the complexes
3.2. Catecholase-like Activity
Figure 3. Oxidation reaction of the 3,5-di-tert-butylcatechol by catecholase
enzyme to the 3,5-di-tert-butylquinone in the presence of O2
All complexes were also tested for their catecholase-like activity
spectrophotometrically by following the increase in absorbance at 400 nm
which is characteristic for 3,5-di-tert-butylquinone. Overall reaction can be
seen in Figure 3. Methanol is used as a solvent due to good solubility of the
substrate and product as well as the complexes. In Figure 4, dinuclear Cu(II)
complex spectrum scan was chosen to exhibit since it exerted the highest
activity among four synthesized complexes. A gradual increase in absorbance
value at 400 nm has been shown by the measurements taken 30 sec. intervals.
Other complexes showed the same pattern of spectrum scan with lower
absorbance values.
Figure 5 shows the relative observed rate constants (k ) of the complexes
which are equal to the slope of the best lines. Accorodbisng to the results,
homodinuclear Cu(II) complex exerted the highest activity revealed by the
highest slope (kobs=0.143 min-1). The relative rates of the complexes may be
shown like 3>1>2>4 which correspond to; Cu(II), Co(II), Ni(II) and Zn(II),
respectively.
Figure 5. Comparison of relative observed rates of complexes for
catecholase-like activity
[(1) Co(II), (2) Ni(II), (3) Cu(II), (4) Zn(II)]
Compared to previous studies, our complexes showed higher and lower
catecholase-like catalytic efficiency. Chen et al. studied mononuclear Cu(II)
complex and compared to previously synthesized dinuclear Cu(II) complex’s
catecholase-like activity. They found kobs(min-1) values ranging from 0.0418
to 0.0545 min-1 in different temperature, pH and complex concentration for
mononuclear and from 0.0396 to 0.0541 min-1 for dinuclear Cu(II) complex.27
Neves et al. studied dinuclear mixed valence Fe(III) and Fe(II) complex and
obtained higher kobs values (0.696 min-1) compared to our results (0.143 min-1
for Cu(II)).28
The reaction mechanism for catecholase described below in three steps
starts with the bonding of 3,5-DTBC with dinuclear complex releasing two
hydrogens. In this step, Cu(II) ions of the complex are reduced to Cu(I). In
the second step which resembles a redox cycle, Cu(I) reacts with O forming
the Cu(II)(O2)2- intermediate. Then, this intermediate reacts with two2 protons
to form a complex with original conformation and a molecule of hydrogen
peroxide. This reaction mechanism is experimentally evidenced by Chyn and
Urbach. 29
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J. Chil. Chem. Soc., 63, Nº 3 (2018)
2Cu(II) + 3,5-DTBC
2Cu(I) + O2
2Cu(I) + 3,5-DTBQ + 2H+
2Cu(II)(O2)2-
(1)
(2)
(3)
2Cu(II)(O2)2- + 2H+
2Cu(II) + H2O2
3.3. Phenoxazinone synthase-like activity
Figure 6. Formation of 2-aminophenoxazine-3-one (APX) from
2-aminophenol (OAPH)
Figure 8. Comparison of relative observed rate of complexes for catalytic
oxidation of OAPH to APX
The oxidation of OAPH to APX in the presence of O2 by complexes (1),
(2), (3) and (4) in DMF as a solvent at room temperature was shown in Figure
6. The proportion of OAPH to O2 was indicated as 2:1,5.
[(1) Co(II), (2) Ni(II), (3) Cu(II), (4) Zn(II)]
The time evolution of Uv-Vis spectra is shown in Figure 7. Spectral
data reveals the time course formation of the product APX. As the other
enzyme-like activities dinuclear Cu(II) complex showed the highest activity
for phenoxazinone synthase. Generally, a range of wavelength (410-440) is
given for APX,30 we preferred to take the highest absorbance value for rate
calculations which is 433 nm. To eliminate the autooxidation of OAPH,
we previously tested the same experiment without the complex which gave
insignificant increase at 433 nm.
Complexes (1), (2), (3), (4) showed comparable results. Recently, Mitra
et al. studied mononuclear Co(III), tetranuclear Cu(II) complex’s phenoxazine
synthase-like activity. They obtained kobs value as 0.03787 min-1 and 0.0188
min-1 which are approximately 5 times lower than our Co(II) and 11 times
lower than Cu(II) complex, respectively.31,32
The mechanism of OAPH oxidation catalyzed by the metal complexes
contains steps (1)-(4) and begins with the binding of the OAPH to the metallic
center. This intermediate decomposes subsequently and produces OAP• free
radical intermediate which is the rate-controlling step (2). 2-aminophenoxy
radical was detected30,33 and unable to detect34 due to fast disproportionation
by the other researchers using ESR spectroscopy. Fast disproportionation of
OAP• radical results in the formation of 2-benzoquinone monoimine (BQMI)
intermediate. The catalytic reaction sequence is completed by reaction of
2-benzoquinone monoimine with another mole of OAPH which gives the final
product APX.35
OAPH-Cu(II)....
OAPH + Cu(II)....Cu(II)
OAPH-Cu(II)....Cu(II)
.OAP
(1)
(2)
(3)
(4)
Cu(II)
.OAP (radical)
BQMI
Phenoxazinone
(APX)
BQMI+ OAPH
[O2]
4. CONCLUSION
Our study revealed that four different homodinuclear complexes showed
relatively high catalytic activities mimicking catalase, catecholase and
phenoxazinone synthase compared to the previous studies. Higher activity
of the complexes, especially Cu(II), may be due to their dinuclear nature
confirming that the dinuclear complexes show higher activity compared
to the mononuclear ones of the same metal ions. Other factors for obtained
results may be the proximity and right orientation of our complexes with the
substrates which are obvious in coherence of catechol and dinuclear complex.
Among the studied four, Cu(II) pair containing complex showed the highest
activity for three oxidoreductase enzymes. Compared to other metal ions,
copper has relatively lower redox potential which may be responsible for the
higher activity. Among enzymes that catalyze oxidoreductase reactions, copper
is one of the most abundant metal ion acting as a cofactor. Besides, two of
the subjected enzymes of this study namely; catecholase and phenoxazinone
synthase bear copper ion. Considering all these properties, oxime group
containing dinuclear Cu(II) complexes have the potential to be a model for
enzyme-like activity studies.
Figure 7. Time evolution UV-Vis spectra of APX formation from OAPH
catalyzed by dinuclear Cu(II) complex in the presence of O2. Spectra were
taken 30 sec intervals
The observed rate constant (kobs) values of the metal complexes for the
APX formation were obtained from eq (2). Rate constant values obtained from
the slope of the best line in Figure 8. The highest activity is shown by complex
(3) (kobs=0.214) as in catalase and catecholase-like reactions. Different from
catecholase, complex (4) showed insignificantly higher activity compared to
complex (2). The catalytic efficiencies of the complexes were as 3>1>4>2.
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