Nitrite Reduction in Aqueous Solution Mediated by Amavadin Homologues: N2O Formation and Water Oxidation

Article abstract of DOI:10.1002/chem.201705385

Reactions of two vanadium(IV) complex anions that are homologues of amavadin, [V(HIDPA)₂]^2? and [V(HIDA)₂]^2? (HIDPA=N-oxyiminodipropionate, HIDA=N-oxyiminodiacetate), with the nitrite ion (NO₂^?) in aqueous solution were investigated by experimental (absorption spectroscopy in the visible range, through measurements of dioxygen formed in solution from water oxidation and identification of nitrogen oxide species of a gaseous atmosphere from nitrite reduction by using an IR analyser) and theoretical methods. Two reactions, mediated by the vanadium complexes, with environmental and biological significance, were observed in this system, namely, reduction of nitrite to N₂O and oxidation of water to molecular oxygen. The reduction of nitrite, as studied by DFT calculations, occurs through the formation of NO (ΔG^≠=14.3 kcal mol^?1), which is strongly dependent on pH and slightly endergonic, and is then easily converted into N₂O, with an overall activation barrier of ΔG^≠=11.8 kcal mol^?1. The later process includes dimerisation of NO assisted by one molecule of the V complex, protonation and oxidation of the formed ONNO^.? ligand by another amavadin molecule or by nitrite, and NO bond cleavage/proton transfer in the ONNOH^? ligand. The results indicate that amavadin exhibits an unusual nitrite reductase type activity that could be involved in nitrogen metabolism of Amanita muscaria and other fungi containing this vanadium complex.

Full text of DOI:10.1002/chem.201705385

A Journal of
Accepted Article
Title: Nitrite reduction in aqueous solution mediated by amavadin
homologues: N2O formation and water oxidation
Authors: José da Silva, Lúcia Dias, Nihel Bekhti, Maxim L. Kuznetsov,
José A. B. Ferreira, and Maria C. Bacariza
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Nitrite reduction in aqueous solution mediated by amavadin
homologues: N2O formation and water oxidation
Lúcia Dias,[a] Nihel Bekhti,[a] Maxim L. Kuznetsov*,[a] José A. B. Ferreira,[a] Maria C. Bacariza,[a] José
Armando L. da Silva*[a]
Abstract: Reactions of two vanadium(IV) complex anions –
homologues of amavadin, [V(HIDPA)2]2- and [V(HIDA)2]2- (HIDPA =
N-oxyiminodiproprionate, HIDA = N-oxyiminodiacetate) with nitrite-
–
ion (NO2 ) in aqueous solution were investigated by experimental
(absorption spectroscopy in the visible range, by measurements of
the dioxygen formed into solution from water oxidation and
identification of nitrogen oxide species of gaseous atmosphere from
nitrite reduction by an infrared analyser) and theoretical methods.
Two reactions, mediated by the V complexes, with environmental
and biological significance were observed in this system, i.e.
reduction of nitrite to N2O and water oxidation to molecular oxygen.
The reduction of nitrite, studied by DFT calculations, occurs via
formation of NO (G = 14.3 kcal/mol), which is strongly pH
dependent and slightly endergonic, and then it is easily converted
into N2O with the overall activation barrier G of 11.8 kcal/mol. This
process includes dimerization of NO assisted by one molecule of the
V-complex, protonation and oxidation of the formed ONNO•– ligand
Figure 1. Amavadin anion.
by another amavadin molecule or by nitrite and the NO bond
cleavage/proton transfer in the ONNOH– ligand. The results indicate
that amavadin exhibits an unusual nitrite reductase type activity that
could be involved in the nitrogen metabolism of Amanita muscaria
and other fungi bearing this V complex.
Introduction
Amavadin is a vanadium metallobiomolecule, isolated in
1972 by Bayer and Kneifel from the fungus Amanita muscaria.[1]
Later, it was also found in two other fungi of this genus that has
more than 600 species.[2].Natural amavadin is a vanadium(IV)
species bearing the [V(S,S-HIDPA)2]2– complex anion (S,S-
HIDPA = (S,S)-N-oxyimino-(2,2’)-diproprionate) with two ligands
derived from S,S’-N-hydroxyiminodiproprionic acid (Figure 1 and
Chart 1). Differently of the majority of vanadium(IV) complexes,
it does not bear the oxovanadium (V=O) group. Each ligand in
amavadin is tetradentate[3] and the coordination number of
vanadium is eight, which is rare for the first series of transition
metals.
Chart 1.
Amavadin may play an important biological role exhibiting
catalase- and peroxidase- type activities.[2,4,5] Its homologues
[V(HIDPA)2]2– (including diastereomers of the natural compound)
and [V(HIDA)2]2– (HIDA = N-hydroxyiminodiacetate, Chart 1) are
also known to mediate or catalyse a number of reactions, in
particular, various oxidation processes[2] such as hydroxylation,
oxygenation and peroxidative halogenation of alkanes and
benzene,[6] peroxidative oxygenation of benzene and
mesitylene,[7] carboxylation of linear and cycloalkanes,[8-13]
oxidation of alcohols[14] as well as the cyanide addition to
aldehydes.[14]
On the other hand, mechanistic details of reactions,[11,13,15]
NMR[16] and EPR[17] spectroscopic information, involving
amavadin and its homologues, have been obtained from DFT
calculations.
Recently, it was reported by some of us that amavadin in
acidic medium is an efficient mediator of water oxidation in the
presence of a strong oxidant (Ce4+).[15] This was the first example
of such a type of activity exhibited by a V complex under non-
photochemical conditions.[18] Experimental data and DFT
calculations suggested for this reaction a mononuclear Water
Nucleophilic Attack (WNA) mechanism (Scheme 1) which
includes (i) initial protonation of amavadin and its oxidation by
Ce4+ to the corresponding V(V) complex II, (ii) decoordination of
the protonated carboxylic group and ligation of a water molecule,
(iii) deprotonation of the highly acidic coordinated H2O molecule,
(iv) the ligand centred oxidation of the formed complex V by Ce4+
and (v) nucleophilic addition of another water molecule at the
OH– ligand in VI with simultaneous proton transfer to give the
hydrogen peroxide complex VII. The hydrogen peroxide is
further oxidized to O2 in a well-known metal-mediated process.
[a]
Centro de Química Estrutural, Instituto Superior Técnico,
Universidade de Lisboa
Av. Rovisco Pais, 1, 1049-001 Lisboa, Portugal
E-mail: max@mail.ist.utl.pt (MLK); pcd1950@tecnico.ulisboa.pt
(JALS)
Supporting information for this article is given via a link at the end of
the document.
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2–
Results and Discussion
O
O
H
O
+
H
HO
N
O
O
O
O
Reaction features and visible spectra of solutions
Addition of the oxidant NaNO2 in the aqueous solutions of
complexes [V(HIDA)2]2- or [V(HIDPA)2]2- (0.01 M) results in an
immediate colour change from the initial blue to red due to
oxidation of the vanadium(IV) complexes into the corresponding
vanadium(V) species. This behaviour is similar for samples
covered with aluminium foil and, hence, protected from the light
radiation and for the unprotected ones. Then, the colour of all
solutions returns to blue, although with different shades, within
several hours or days depending on the complex and conditions
(see below). Such a colour change was previously observed
O
IV
Ce
V
V
IV
O
N
O
N
O
N
N
O
N
H O
2
V
V
V
III
–Ce
O
O
O
O
O
O
O
II
O
III
OH2
OH
O
O
I
V
O
N
V
N
O
O
O
IV
H O
2
–
+
OH
–
OH
O2H2
–H O
OH
OH
3
OH
IV
III
O
O
N
Ce
O
O
N
O
O
2H O
2
IV
V
V
N
O
N
N
V
+
V
V
–H O
3
O
O
N
–Ce
O
O
O
O
O
O
[4]
with H2O2 or Ce(IV)[15] used as oxidants, and it was attributed
VI
VII
V
to the reduction of the V(V) complexes back to V(IV) as a result
of the water oxidation process. Formation of bubbles of gas was
detected visually in the solutions for both [V(HIDA)2]2- and
[V(HIDPA)2]2-.
Scheme 1. Proposed mechanism of water oxidation by Ce(IV)
mediated by the amavadin analogue.[15]
Stimulated by these exciting findings, we decided to
explore other oxidants milder than Ce4+ that mediated by
amavadin homologues could also oxidize water. We pointed our
The absorption spectra of [V(HIDA)2]2- and [V(HIDPA)2]2-,
with nitrite 0.005 M, are characterized by band with maxima at
570 nm and 565 nm, respectively (Figure 2 and 3). In the case
of [V(HIDA)2]2-, these spectra are similar for the light-protected
and unprotected samples. Colour stabilization is reached within
ca. 24 hours and a partial degradation of the complex occurs in
both cases, see Figure 2.
–
attention to nitrite (NO2 ) due to its great significance from the
environmental and biological viewpoints. To our knowledge, the
homogeneous chemical oxidation of water by nitrites was not yet
reported. Nitrite still exhibits a rather strong oxidizing activity in
acidic medium that is noticeably weaker than of Ce4+
[E°(Ce4+/Ce3+) = 1.62 V, E°(HNO2 + H+/NO + H2O) = 1.00 V,
E°(2HNO2 + 4H+/N2O + 3H2O) = 1.30 V].
Nitrite is a pollutant in drinking and waste water. Its
reduction to nitrogen oxides NO and N2O – which should be
captured – or preferentially to environmentally benign molecular
nitrogen underlies the water denitrification process and has a
tremendous ecological significance.[19]
Furthermore, the nitrite reduction is an important
biochemical process associated with e.g. neurotransmission,
immune response and apoptosis in various organisms.[20-26] Soil
bacteria metabolize nitrite to NO via nitrite reductases –
enzymes of the EC 1.7 group[27] with the iron,[28] copper[29] or
molybdenum[26] based active centres. Both NO and N2O –
Figure 2. Absorption spectrum of the aqueous solution of [V(HIDA)2 ]2– (0.01
M) (___) and spectra of the solution upon addition of NaNO2 (0.005 M) after
reaction completion for the light protected (· · ·) and unprotected (- - -)
samples.
another product of the biological nitrite reduction
– are
greenhouse gases that also participate in the destruction of the
ozone layer. Besides the anthropogenic way, the soil bacteria
are a principal cause of the NO emission. Various fungi including
A. muscaria also contribute to the nitrogen cycle with nitrite as
possible metabolic substrates.[30-32] Therefore, the understanding
of mechanistic details of the nitrite reduction is important in
environmental chemistry.[21]
Despite such
a
great importance, the chemical
homogeneous reduction of nitrites by inorganic transition and
internal transition metal compounds was investigated rather
scarcely. Most of the works deal iron[33-45] or copper[46-52]
complexes which mimic the nitrite reductases. However, the
–
NO2 reduction with Ru,[53] Mo,[54-56] Ti, Cr,[56] Co[57-59] and U[60]
was also reported. Vanadium species are unconventional for this
reaction, and there are only few publications about the nitrite
reduction by the V(III) species from an analytical approach[56,61]
and there is no one describing this reaction with the V(IV)
Figure 3. Absorption spectra of the aqueous solution of [V(HIDPA)2 ]2– (0.01
M) before (· · ·) and after addition of NaNO2 (0.005 M) and after the reaction
completion for the light protected (· · ·) and unprotected (- - -) samples.
complexes.
Additionally,
rational
design
based
on
metallobiomolecules[62-64] has been employed for similar purpose.
Herein, complexes related with a vanadiumbiomolecule with
metal in the oxidation state four are used for nitrite reduction,
suggesting a possible natural process which could occur in A.
muscaria and other fungi bearing this vanadium species. In the
first part of the article, experimental results of the reaction
between the homologues of amavadin and nitrite in aqueous
solution are presented. In the second part, a detailed theoretical
study of the reaction mechanism is discussed.
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associated with the V-mediated water oxidation – a process
whose mechanism was proposed and investigated previously.[15]
An eightfold increase of the dissolved oxygen (from 0.46 mg/L to
4.06 mg/L) was detected after 20 h. The amount of the released
oxygen is half of the initial amount of nitrite in solution (0.11
mmol/L vs. 0.22 mmol/L, respectively). This is in agreement with
the general reaction scheme (Scheme 2) involving the formation
of N2O but not NO as the final product of the nitrite reduction (in
the latter case, the amount of the oxygen released should be
four times lower than the amount of the nitrite consumed).
The reaction of nitrite with [V(HIDPA)2]2- depends on
exposure to the light radiation in contrast with [V(HIDA)2]2-. For
the light-protected samples the former complex returns to its
original blue colour after ca. 10 days, much slower than for
[V(HIDA)2]2-, and there is no sign of the complex degradation. A
typical example of [V(HIDPA)2]2- photodegradation at ambient
light conditions is shown in Figure 3. Upon irradiation (ca. 2h
with the xenon lamp) the return to blue colour of the solution
occurs much faster, exhibiting the complex degradation (see
Experimental Section).
2NO2– + 6H+
N2O + 3H2O
The oxidation of [V(HIDA)2]2- or [V(HIDPA)2]2- by nitrite
strongly depends on pH of the reaction mixture. In a strongly
acidic solution (pH = 1.72), the colour change from initial blue to
red occurs immediately upon addition of the oxidant. The
decrease of the reaction mixture acidity by addition of NaOH to
–
pH > 2.5 prevents the oxidation of the V complexes by NO2 ,
4{VV}
2H2O
4{VIV}
and no colour change is observed.
Gaseous reaction products
Gaseous species derived from the nitrite reduction for
three solutions, [V(HIDPA)2]2- (protected and unprotected from
light) and [V(HIDA)2]2- (protected from light), were qualitatively
identified by analysis of the gas phase above the solution using
the infrared analyser (see Supporting Information for details). In
all samples, N2O is the only detected reduction product in the
gaseous atmosphere, despite its high (comparing with other
gases) solubility in aqueous solution. The presence of the other
nitrogen oxides (NO and/or NO2) was not detected. The results
for [V(HIDPA)2]2- protected from light are shown in Figure 4.
Meanwhile, analysis of the literature on this topic[33,44,47,57,59,65]
suggests that the reaction may involve the formation of NO
(and/or hyponitrite) as an intermediate which is rapidly
converted into N2O mediated by complex. Theoretical DFT
calculations of the reaction mechanism confirmed this
hypothesis (see below).
O2 + 4H+
–
Scheme 2. Overall reaction of water oxidation with NO2 mediated by
amavadin or related complexes. {VIV} is amavadin or its homologues, {VV} is
the corresponding oxidized V(V) complex.
Theoretical mechanistic study of nitrite reduction
With aim to understand the mechanism of the nitrite
reduction with amavadin, theoretical calculations at the DFT
level of theory were undertaken. Taking into account the acidic
character of the reaction mixture, the monoprotonated form of
the amavadin model [V(HIDA)(HIDAH)] (1) was considered for
these calculations. The experimental observations suggest that
the nitrite reduction with amavadin consists of two main steps,
–
i.e. (i) reduction of NO2 to NO and (ii) conversion of NO into
N2O. Additionally, the possibility of the direct reduction of nitrite
to N2O is also considered. Each of these processes is discussed
in the following sections (and in Supporting Information).
Formation of NO. The reaction starts with the liberation of
one coordination place in 1 due to the cleavage of the V–O(H)
bond to give 2 which is slightly endergonic (by 2.9 kcal/mol).
The nitrite reduction strongly depends on the pH value of the
reaction mixture. Therefore, three main possible pathways were
considered, i.e. without further participation of a proton and with
additional involvement of one (for these two cases, see
Supporting Information for details) or two protons in the reaction
(Scheme 3).
Figure 4. Three sequential injections of the gas phase, above of [V(HIDPA)2]2-
after addition of nitrite and protected from light radiation.
Molecular oxygen is another gaseous product whose
formation was monitored with O2-measuring system (see
Supporting Information for details). The release of oxygen is
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3[VIV(HIDA)(HIDAH)](•NO)– (32•••NO) were obtained (Scheme 4).
In 37, NO is coordinated via the N atom forming a weak
coordination V–NO bond [l(V–NO) = 2.468 Å]. In 2•••NO, the
O
N
2–
O
2–
=
N
3
O
IV
O
V
O
V
2–
OH
OH
OH
O
O
O
V
O
N
O
N
O
O
N
O
O
V
V–ON distance is 3.145 Å and, therefore, NO is in the second
coordination sphere. No equilibrium structure corresponding to
the inner sphere complex with NO coordinated by the O atom
3[VIV(ON•)(HIDA)(HIDAH)]– was found. All attempts of its
N
N
O
N
O
–NO
O
O
O
O
3
5
TS1
(40.4)
(19.3)
O
(10.8)
–
NO
–
=
2
–
HNO2
OH
O
O
N
3
–
H
–
optimization led to the adduct 2•NO. The outer sphere complex
HO
N
O
–
HO
O
O
O
HO
N
O
V
O
IV
HO
N
HNO
IV
32•••NO is by 6.7 kcal/mol more stable than the inner-sphere
O
OH
VV
O
O
N
O
2
O
IV
N
O
N
V
V
V
O
N
O
N
O
O
N
O
N
O
O
3
O
O
–NO
complex 7. The low spin antiferromagnetic biradical structures
O
O
O
O
O
O
2
(2.9)
O
2•••HNO
of 2•••NO and 7 were also optimized but they are slightly less
stable (by 1.5 kcal/mol) than the corresponding high spin triplet
2
1
TS2
(31.1)
+
6
H
(14.3)
(0.0)
(15.2)
–
NO
2
+
H
O
3
structures.
O
O
O
H
O
2–
=
N
IV
OH
•
N
–
N
O
O
N
O
H
N
O
V
N
O
O
N
–
H
N
O
O
H
O
V
O
O
V
HO
HO
N
O
HO
N
O
O
HO
O
2–
4
O
IV
V
V
O
N
N
O
O
N
O
N
(13.3)
N
O
N
O
N
O
V
V
V
V
O
–2H
O
O
O
2
•
O
N
O
O
O
3
O
O
O
O
OH
0
N
2
•••NO
O
1
TS3
(13.5)
8
2•••NO
2
1/2
O
HO
N
O
(3.2)
(1.7)
HO
N
(33.9)
0
N
V
O
–2
O
Scheme 3. Mechanisms of the nitrite reduction to NO upon reaction with the
amavadin model 1 (relative Gibbs free energies are indicated in parentheses
in kcal/mol).
V
O
N
O
N
O
V
V
O
O
O
•
2
O
0
O
O
–
O
O
–
0
2 •••NO
–2
N
V
N
V
HO
IV
9
HO
O
(0.0)
(23.3)
O
N
N
O
O
N
N
O
Reaction with additional involvement of two protons. This
mechanism can operate in a sufficiently strong acidic medium
corresponding to the experimental conditions. The OH– anion
liberating as a result of the N–O bond cleavage in 2•••HNO2 is
captured by a proton (H3O+) that should be much more efficient
than the grab by a metal centre as in the pathway with additional
involvement of one proton (see Supporting Information). For
such a process, transition state TS3 was found. As a result of
the N–O bond cleavage and simultaneous proton transfer, NO
V
H
+
H
O
O
O
O
O
3
1
7
7
N
V
HO
N
O
O
(8.4)
(16.9)
O
N
O
V
O
10
(36.0)
Scheme 4. Initial steps of the NO-to-N2O conversion mechanism based on
the initial NO reduction (relative Gibbs free energies are indicated in
parentheses in kcal/mol).
associated with
a
V(V) complex ([V(HIDA)(HIDAH)](NO)
The reduction of NO by V(IV) in 32•NO results in the
20•••NO) and two water molecules are formed. No minimum
corresponding to the inner sphere V(V) or V(IV) complex with
coordinated NO or NO+ ligand was found. Thus, this process is
truly outer sphere including the electron transfer (ET) from V(IV)
to the NO moiety.
formation
of
the
closed
shell
V(V)
complex
1[VV(ON)(HIDA)(HIDAH)]– (18) where NO is coordinated to
vanadium by the oxygen atom (the V–ON bond length is 2.275
Å). However, the energy of such ET is 32.2 kcal/mol, i.e. too
high to permit the realization of this process. In contrast, the
intramolecular reduction of NO in 37 leads to the closed shell
V(V) complex 1[VV(NO)(HIDA)(HIDAH)]– (17) and requires only
8.5 kcal/mol.
2. Initial N–N bond formation followed by reduction of NO.
In this mechanism of the NO-to-N2O conversion, the N–N bond
is formed between two unreduced NO molecules. The N–N
bond formation may occur without an involvement of the metal
complex (see Supporting Information), or with participation of
one or two molecules of the vanadium species.
The HNO2 reduction step is barrierless with the negative
Gibbs free energy of activation –0.7 kcal/mol. The negative G
value may be explained by the fact that the total energy rather
than the Gibbs free energy was minimized during the geometry
optimization. Indeed, in terms of enthalpy at 0 K, the activation
barrier is positive (2.5 kcal/mol). The overall activation barrier for
this mechanism relative to the initial complex 1 is 14.3 kcal/mol.
The formation of NO within this mechanism is only slightly
endergonic by 3.2 kcal/mol relative to 1. Thus, the
computational data demonstrate a significant effect of the
medium acidity on the nitrite reduction that is in agreement with
the experimental data.
Formation of N2O. The further conversion of NO into N2O
– the principal nitrogen containing product of the nitrite reduction
detected experimentally – should include two main processes,
i.e. the NO reduction and the preceding or following N–N bond
formation.
(i) Mononuclear mechanism involving the V(IV) complex 2.
Dimerization of NO may be assisted by one molecule of the
V(IV) complex 2 (Scheme 5). Adduct 2•NO reacts with another
3
NO
molecule
giving
the
doublet
complex
2[VV(ONNO)•(HIDA)(HIDAH)]– (11). Spin density in 11 is
delocalized among the ONNO ligand and it is virtually zero at
the V atom (Figure 5). Therefore, 11 should be described as the
V(V) complex bearing the ONNO•– anion-radical ligand. Thus,
dimerization of NO in the presence of 2 is accompanied by the
single electron reduction of the ONNO dimer by the V(IV) atom.
This process requires only 10.1 kcal/mol.
1. Initial reduction of NO followed by the N–N bond
formation. There are two reducing agents in the reaction mixture
which, in principle, could reduce NO to NO–, i.e. the amavadin
–
homologue and the NO2 anion (or HNO2 acid), see also
Supporting Information.
Reduction of NO by the V(IV) complex 2. The reduction of
NO by V(IV) requires the coordination of nitric oxide to the metal
centre (inner sphere ET) or, at least, a close interaction between
NO and V which could permit an outer sphere ET. Equilibrium
structures
for
one
inner
sphere
V(IV)
complex
3[VIV(NO•)(HIDA)(HIDAH)]– (37) and an outer sphere complex
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kcal/mol and results in the decoordination of the ONNOH• ligand
to give the outer sphere complex [VV(HIDA)(HIDAH)](HONNO)•
(14) (see Supporting Information for computational details). The
obtained HONNO• radical in complex 14 is reduced by NO2– or 2
to give [VV(ONNOH)(HIDA)(HIDAH)]– 13 with the G values of –
11.6 or –15.6 kcal/mol, respectively. Complex 13 is transformed
to N2O and 6 (see Scheme 7).
O
N
N
O
2–
2–
•
N
N
=
N
O
HO 2–
O
V
O
O
O
HO
N
HO
V
HO
N
O
O
O
O
V
V
O
N
N
O
V
O
N
O
N
N
O
N
O
0
V
V
V
O
O
O
O
–N2O
O
O
O
O
O
5
12
TS5
(12.6)a (8.6)b
2 •••NO
(0.0)
(–31.7)a (–35.7)b
(12.3)a (8.3)b
The mechanisms based on the protonation of 11 to 14 or
on the oxidation of 11 to 12 by 2 are the most favourable one
with the overall activation barrier of 11.8 kcal/mol relative to the
initial level 20•••NO. Meanwhile other mechanisms based on the
–
NO2
or
2
–NO2
or
–16
0
–2
2
–
N
O
N
O
–
•
–
N
O
–
N
•
=
H
–
N
O
N
N
direct oxidation of 11 by NO2 or HNO2 have only slightly higher
H
O
O
HO
HO
N
HO
N
O
O
V
overall activation barrier 12.3–12.6 kcal/mol and, therefore, may
be realized concurrently.
O
O
HO
N
O
0
HNO2
IV
V
V
2 •••NO
O
N
O
N
O
O
N
O
N
O
O
N
V
V
V
V
0
–NO2
O
O
–2
O
(ii) Mechanism involving two molecules of the V(IV)
complex. In this mechanism, complex 32•••NO may dimerize,
giving 15 (Scheme 6). Both trans- and cis-isomers of 15 in both
triplet and closed shell singlet spin states have been optimized.
The calculations indicate that the singlet trans-isomer trans-15 is
the most stable among these four structures (see for the
energies Table S1 in Supporting Information). The G energy of
dimerization of 32•••NO is 12.3 kcal/mol. Since trans-15 is a
closed shell structure, both V atoms have the oxidation state +V.
Hence, dimerization of 32•••NO is accompanied by the two
electron reduction of the ONNO ligand by the V(IV) centres.
O
O
O
O
O
O
3
2•••NO
(1.7)
11
13
TS6
(–3.5)d
(–7.5)e
(3.5)c
(5.3)d (1.3)e
(12.3)c
(11.8)
–
NO2
H+
H2O
or
–NO2
or
–16
2
–N2O
ONNOH•
O
H
O
–
=
N
N
H
HO
N
O
O
V
H
HO
–
HO
O
N
OH
O
V
O
O
O
V
V
O
N
O
N
O
O
O
O
N
N
O
V
V
–H2O
14
(8.1)
O
O
–N2O
6
O
O
TS7
(25.4)c
V
(–46.8)e
2–
O
(–35.8)c (–42.8)d
O
OH
O
N
N
O
O
V
V
–
O
N
•
N
•
V
N
N
O
N
O
V
V
O
2
O
O
N
O
O
OH
O
O
OH
HO
O
HO
N
HO
O
O
16
+
Scheme 5. Mononuclear mechanisms of the NO-to-N2O conversion based on
the initial N–N bond formation. Gibbs free energies are indicated relative to
V
N
2 2
IV
2 H
N
O
N
O
N
N
O
2
2
V
V
N
0
N
O
+
–2 2
a
–
b
c
20•••NO in kcal/mol. For NO2 as oxidant. For 2 as oxidant. For the
HONNOH
O
O
O
O
3
O
V
OH
HO
HO
N
O
0
2
•••NO
oxidation of 11 by HNO2. d For the oxidation of 14 by NO2
.
e For the oxidation
–
O
O
2•••NO
(3.4)
O
N
(–14.0)
V
(0.0)
V
O
N
N
O
O
V
of 14 by 2.
O
O
–H
2
O
O
trans-15
17
(4.7)
(15.8)
N2O
2–
=
O
O
O
V
O
N
N
O
V
O
OH
O
2–
OH
OH
O
O
N
V
V
O
N
O
O
N
O
V
V
+
N
N
O
N
O
–N2O
O
O
O
HO
N
5
16
O
V
(–35.7)
O
N
V
O
O
O
TS8
(37.5)
Scheme 6. Binuclear mechanisms of the NO-to-N2O conversion based on the
initial N–N bond formation (relative Gibbs free energies are indicated in
parentheses in kcal/mol).
Figure 5. Spin density distribution in complex 11.
Complex 11 may then undergo the second single electron
–
Complex trans-15 may undergo direct decomposition to
reduction either by NO2 /HNO2 or by V(IV) in 2 to give
the
V(V)
species
[VV(=O)(HIDA)(HIDAH)]2–
(5),
[VV(ONNO)(HIDA)(HIDAH)]2–
(12)
or
[VV(HIDA)(HIDAH)] (16) and N2O via TS8. However, the
activation energy of this step is high (21.7 kcal/mol) allowing its
exclusion from consideration.
[VV(ONNOH)(HIDA)(HIDAH)]– (13) depending on acidity of the
medium. Such a reduction is almost energetically neutral for
NO2 (G = 0.5 kcal/mol) or clearly exergonic for HNO2 and 2
–
Alternatively, trans-15 may be doubly protonated to give
17. The estimated energy of the protonation is –11.0 kcal/mol
(see Supporting Information for details). The hyponitrous acid
HONNOH may be liberated from 17 (G = –18.8 kcal/mol) and
then easily decomposed to N2O and water. The overall
activation barrier for this mechanism is 15.8 kcal/mol relative to
20•••NO and it appears to be slightly less favourable than the
mechanism based on one molecule of the V(IV) complex.
(G = –8.4 and –3.6 kcal/mol). Nitrous oxide N2O may be
directly formed from 12 or 13 upon monomolecular O–N bond
cleavage via TS5 or TS6, respectively, with the activation
barriers of 0.3 and 8.8 kcal/mol, correspondingly. In the case of
13, the N2O formation could also occur via TS7 with the
assistance of one water molecule which plays the role of a
proton shuttle. However, the water assisted step is 13.2 kcal/mol
less favourable than the monomolecular pathway due to the
entropic factor.
Alternatively, the ONNO•– ligand in 11 may be initially
protonated. The protonation is slightly exergonic by –3.7
This article is protected by copyright. All rights reserved.

10.1002/chem.201705385
Chemistry - A European Journal
FULL PAPER
Conclusions
respectively. The endergonic character of the NO formation by
3.15 kcal/mol and the low activation barrier for the NO
transformation to N2O do not allow an efficient accumulation of
NO in the reaction mixture explaining why nitric oxide was not
detected experimentally.
Some transition metal compounds are able to reduce
nitrite to N2O, including some minerals,[43-45] and herein is
revealed that amavadin (a metallobiomolecule) mediates a
similar reaction, in contrast with other metallobiomolecules
(nitrite reductases) concerning final product. However, the
formation of N2O by living organisms is known with other
substrates.[67]
Investigations of the past decades showed that amavadin
and homologues compounds exhibit interesting chemical
properties being efficient catalysts or mediators of a number of
redox reaction. In this work, we demonstrate that two chemical
processes with environmental and biological significance occur
in
a simple chemical system bearing amavadin (or its
homologues), nitrite and water.
First, these vanadium species mediate the homogeneous
water oxidation to molecular oxygen. This process usually
requires very strong oxidants such as Ce4+, HSO5 , ClO– or IO4
–
–
.
[66] However, in the presence of amavadin or its homologues,
The biological function of amavadin in organisms is still
unknown. Meanwhile, results of this work suggest that this
complex exhibits the nitrite reductase type activity and,
therefore, may be involved in the nitrogen metabolism of
Amanita muscaria and other fungi bearing amavadin. Moreover,
it can be associated with the molecular oxygen production under
suitable biological conditions or biological thiols (see Supporting
Information). The nitrite reductase, peroxidase and catalase type
activities of amavadin uncovered in this and previous works[4,68-
even significantly weaker oxidants such as nitrite become active
towards water oxidation. Vanadium atom in amavadin and its
homologues can experience only the single electron oxidation to
V(V), the highest oxidation state for this metal. However, in
accord with DFT calculations, the N-oxyiminodicarboxylate
ligands in these complexes are non-innocent and may also
undergo a relatively facile single electron oxidation due to the
presence of the electron rich NO group. Such ligand centred
oxidation allows the accumulation of an additional oxidative
strength which is sufficient for the oxidation of water[15] and is
likely associated with an unusual 51V NMR spectrum.[16]
70]
suggest that this vanadium metallobiomolecule may play a
multiple role in organisms depending on the cellular
environment, including a different protein moiety. However, due
to the high coordination number of metal centre and being an
anion, amavadin favours interactions with cationic side-chains of
the proteins, as observed for its homologue [V(HIDA)2]2- by
ammonium ion.[71] Additionally, if amavadin participates in the
metabolic reduction of nitrite, the reaction should occur at higher
pH than the tested in this work, since the metabolic pH is less
acid, with plausible cooperation of suitable amino acid residues.
These results suggest that homologues of amavadin could
be employed to reduce nitrite contamination in aqueous
environments, e.g., a bell jar, opaque to the light radiation, with
the complex immobilized into an acidic moiety in the interior side
The
second
process
occurring
in
the
amavadin(homologue)/nitrite/H2O system is the reduction of
nitrite with the V(IV) species. Experimental observations and
results of theoretical DFT calculations indicate that such a
reduction occurs via formation of NO which is then converted to
the final N2O product. The most plausible mechanism is shown
in Scheme 7. The formation of NO is strongly pH dependent and
efficiently occurs with involvement of three protons, two of them
–
protonating the amavadin model molecule and the NO2 ion and
the third proton being directly involved in the N–O bond
cleavage capturing the releasing OH– anion. The transformation
of nitric oxide into N2O includes (i) dimerization of NO which is
assisted by the V(IV) centre in the amavadin model molecule
and accompanied by the single electron reduction of ONNO by
and
a cylinder to store N2O (which has technological
applications and it is not easy to prepare)[41] arising from the
nitrite reduction could be used for that purpose.
V(IV),
(ii)
protonation
of
the
ONNO•–
ligand
in
Finally, studies with other oxidants using amavadin and its
homologues as mediators should be a profitable field concerning
new approaches on chemical synthesis and catalysis.
2[VV(ONNO)•(HIDA)(HIDAH)]– 11 (iii) reduction of HONNO• in
[VV(HIDA)(HIDAH)](HONNO)• 14 by a V(IV) complex or NO2
–
and
(iv)
N–O
bond
cleavage/proton
transfer
in
[VV(ONNOH)(HIDA)(HIDAH)]– 13 to give N2O and the hydroxo
complex [VV(OH)(HIDA)(HIDAH)]– 6.
Experimental Section
The proligands, H3HIDPA, and H3HIDA (Chart 1), were
synthesized according to Bayer and Kneiffel.[72] The solutions of the
complexes, usually 0.01 M, were prepared in situ with deoxygenated
distilled water (to be sure that the presence of molecular oxygen results
only from the oxidation of water), oxovanadium(IV) sulfate pentahydrate
and H3HIDPA or H3HIDA. The initial pH of these solutions was ca. 1.72.
All reactions and operations were carried out at room temperature. Some
experiments were carried out in absence of light radiation by covering the
samples with an aluminium foil or using some coloured filters (see
Supporting Information).
NO formation
N
–
HNO2
HO
O
OH
O
O
–
HNO
2
HO
N
–
HO
N
TS3
(13.5)
O
O
O
O
O
O
O
V
IV
IV
IV
O
N
O
N
N
O
N
O
N
N
O
V
V
V
V
O
O
O
+
+H
O
O
O
O
O
O
–H
O
2
0
2
•••NO
2
O
2•••HNO
(14.3)
2
1
(3.2)
(2.9)
(0.0)
Absorption spectra were recorded with Perkin-Elmer L35 and
Jasco V-560 spectrophotometers in the wavelength range 380-850 nm
using quartz cells (b = 1 cm). The pH measurements were performed
with a Metrohm 827 pH lab. Water oxidation was detected and quantified
by the oxygen meter with SG9-SevenGo proTm equipment calibrated
NO-to-N2O conversion
N
O
–
N
O
–
N
ONNOH•
•
H
TS6
(8.5)
(4.5)
O
N
N
–
2
a
b
HO
OH
V
or
NO
O
HO
N
HO
O
O
HO
N
O
O
HO
N
–
O
O
+
2
V
2
with a saturated solution of distilled water in air (see Supporting
V
H
V
O
N
N
O
V
N
O
O
N
O
N
O
N
2
O
N
V
V
V
V
V
0
O
–16
or
–NO
Information). Preliminary tests for the identification of the reduction
product of nitrite were carried out (see Supporting Information). For the
final identification of the gaseous products from nitrite reduction, three
experiments were carried out with complexes [V(HIDPA)2]2- and
[V(HIDA)2]2- fully covered with an aluminium foil, and another similar
experiment was carried out just for [V(HIDPA)2]2- but uncovered and it
was irradiated in continuous mode using a 450 W xenon lamp in a
Fluorolog (Horiba Jobin Yvon spectrofluorometer) with incident light
bandpass of 515.0±14.7 nm (this range is based on tests with coloured
filters, see Supporting Information). The identification of the gaseous
O
–2 2
O
–N2O
O
O
O
O
O
O
O
O
O
O
0
2
6
2
•••NO
14
(11.3)
13
(–0.3)
11
a
b
(–39.6)
a
(3.2)
(15.0)
b
(–43.6)
(–4.3)
Scheme 7. The most plausible mechanism of the nitrite reduction with
amavadin model 1 (relative Gibbs free energies are indicated in parentheses
in kcal/mol). a For NO2– a oxidant. b For 2 as oxidant.
The overall activation energies of the NO formation and
the NO-to-N2O conversion are 14.3 and 11.8 kcal/mol,
This article is protected by copyright. All rights reserved.

10.1002/chem.201705385
Chemistry - A European Journal
FULL PAPER
[8] P.M. Reis, J.A.L. Silva, A.F. Palavra, J.J.R.F. da Silva, T. Kitamura, Y.
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Reis, A.M.F. Palavra, Patent WO2004/037416-A2, May 6, 2004.
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Catal. 2005, 235, 333-340.
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[14] T. Hubregtse, PhD Thesis, Technical University of Delft, The Netherlands,
2007.
[15] M. Domarus, M. L. Kutnesov, J. Marçalo, A. J. L. Pombeiro, J. A. L. da
Silva, Angew. Chem., Int. Ed. 2016, 128, 1511-1514.
species on the samples was carried out using an ABB EL 3020 infrared
analyser (for details see Supporting Information).
Computational Details
The full geometry optimization of all structures and transition state
has been carried out at the DFT/HF hybrid level of theory using the
B3LYP* functional[73–76] with the Gaussian-09 program package.[77] It was
shown that while the popular pure functionals, such as BP86,
underestimate the relative stability of high spin states and many hybrid
functionals with a high contribution of the Hartree-Fock term overestimate
the relative stability of high spin states, the modified B3LYP functional
(B3LYP*) correctly describes the relative energies of the low and high spin
states in a number of transition metal complexes.[75,76] Since in the
proposed mechanisms the relative stability of the high and low spin states
is a crucial issue, especially the B3LYP* functional was selected for the
calculations. This functional was previously employed by some of us with
success for the theoretical treatment of water oxidation mediated by
amavadin.[15] The B3LYP* functional was constructed using the keywords
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blyp,
IOp(3/76=1000001500),
IOp(3/77=0720008500)
and
IOp(3/78=0810010000) in Gaussian.
[18] Photoinduced water oxidation with Na2S2O8 catalysed by a vanadium
The relativistic Stuttgart pseudopotential that described 10 core
electrons (MDF10) and the appropriate contracted basis set[78] were
employed for the V atoms, whereas the standard basis set 6-31G* was
applied for all other atoms. Single point calculations with the 6-311+G**
basis set for all non-metal atoms were then performed. The MP4(SDQ)/6-
31G* level was used for the estimates of the NO dimerization energy.
To test the effect of the functional on the rate limiting activation
barrier, the calculations of the key species with the M06 functional[79] has
also been performed. The activation barriers for the NO formation
calculated with the B3LYP* and M06 functionals differ by less than 2
kcal/mol (14.3 and 16.2 kcal/mol, respectively).
No symmetry operations have been applied for any of the
calculated structures. Restricted approximations for the structures with
closed electron shells and unrestricted methods for the structures with
open electron shells have been employed. The geometry optimization of
all structures was carried out for water solution using the SMD model
with default parameters.[80]
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frequencies) or saddle points (only one imaginary frequency) and to
estimate the thermodynamic parameters, the latter being calculated at
25°C. The nature of all transition states was investigated by the analysis
of vectors associated with the imaginary frequency and by the
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All energies discussed in the text are Gibbs free energies if not
stated otherwise. The computational details for the reaction steps with
participation of a proton are discussed in Supporting Information.
Acknowledgements
The authors are indebted to Fundação para a Ciência e a
Tecnologia (FCT), Portugal, for financial support (project
UID/QUI/00100/2013) and to Professor Carlos Henriques for the
access to the infrared analyser and to Dr. Esther Bailón-García
for the help and support regarding the IR analysis.
Keywords: Amanita muscaria; DFT calculations; Nitric
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Entry for the Table of Contents (Please choose one layout)
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Direct nitrite reduction to N2O is
unusual to reactions mediated by
biomolecules, as well as is
Lúcia Dias, Nihel Bekhti,
Maxim L. Kuznetsov*, José A.
B. Ferreira, Maria C. Bacariza,
José Armando L. da Silva*
promotor of the water oxidation
Page No. – Page No.
-
NO2
AmavadinRed
O2
N2O
Nitrite reduction in aqueous solution
mediated by amavadin homologues:
N2O formation and water oxidation
AmavadinOx
H2O
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