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Mechanism of Action of Sulforaphane as a Superoxide Radical Anion
and Hydrogen Peroxide Scavenger by Double Hydrogen Transfer: A
Model for Iron Superoxide Dismutase
Ajit Kumar Prasad and P.C. Mishra*
Department of Physics, Banaras Hindu University, Varanasi 221 005, India
*
SSupporting Information
ABSTRACT: The mechanism of action of sulforaphane as a
scavenger of superoxide radical anion (O2
•−) and hydrogen
peroxide (H2O2) was investigated using density functional
theory (DFT) in both gas phase and aqueous media. Iron
superoxide dismutase (Fe-SOD) involved in scavenging
superoxide radical anion from biological media was modeled by a complex consisting of the ferric ion (Fe3+) attached to
three histidine rings. Reactions related to scavenging of superoxide radical anion by sulforaphane were studied using DFT in the
presence and absence of Fe-SOD represented by this model in both gas phase and aqueous media. The scavenging action of
sulforaphane toward both superoxide radical anion and hydrogen peroxide was found to involve the unusual mechanism of
double hydrogen transfer. It was found that sulforaphane alone, without Fe-SOD, cannot scavenge superoxide radical anion in gas
phase or aqueous media efficiently as the corresponding reaction barriers are very high. However, in the presence of Fe-SOD
represented by the above-mentioned model, the scavenging reactions become barrierless, and so sulforaphane scavenges
superoxide radical anion by converting it to hydrogen peroxide efficiently. Further, sulforaphane was found to scavenge hydrogen
peroxide also very efficiently by converting it into water. Thus, the mechanism of action of sulforaphane as an excellent
antioxidant has been unravelled.
■INTRODUCTION
Reactive oxygen species (ROS) and reactive nitrogen oxide
species (RNOS) are mostly free radicals that are produced in
biological systems during metabolic activities, and these can
also be formed due to exposure of biological systems to
radiation or pollution. Because of their high chemical
reactivities, ROS and RNOS can cause oxidation and nitration
of DNA, proteins, and enzymes and thus give rise to several
diseases including cancer.
1−4
Superoxide radical anion (O2
•−),
hydroxyl radical (OH•), hydrogen peroxide (H2O2), nitrogen
dioxide (NO2
•), peroxynitrite (ONOO−), etc. belong to the
ROS or RNOS family.
5
The superoxide radical anion is
intrinsically poorly reactive, but in the presence of other
chemical species, it can produce other highly reactive agents.
For example, its reaction with nitric oxide (NO•) produces
peroxynitrite (ONOO−), both these species being poorly
reactive, but when this reaction occurs in the presence of CO2,
the complex nitrosoperoxycarbonate is formed, which disso-
ciates rapidly into two highly reactive species, that is, nitrogen
dioxide (NO2
•) and carbonate radical anion (CO3
•−).
6
Thus,
superoxide radical anion can play crucial roles in causing DNA
damage, depolymerize polysaccharides, peroxidize lipids, kill
mammalian cells, and inactivate enzymes.
7−14
Generation of
superoxide radical anion signals the first sign of oxidative
burst.
15
Superoxide radical anion is produced in the human
body by one-electron reduction of molecular oxygen through
the involvement of various oxidative enzymes.
16,17
The enzyme
xanthine oxidase catalyzes production of superoxide radical
anion,
18
while the enzyme superoxide dismutase (SOD)
catalyzes its conversion to H2O2.
19,20
SOD can have different
forms depending on the presence of cation of iron, zinc,
manganese, or copper in it. The enzymes glutathione
peroxidase and catalase serve as strong endogenous antiox-
idants as the former catalyzes conversion of H2O2into water,
while the latter catalyzes its conversion into water and
oxygen.
21−24
Certain nonenzymatic exogenous antioxidants, which can be
taken as components of diet, include ascorbic acid, α-
tocopherol, vitamin A, lycopene, etc.
25
Epidemiological studies
have shown that an increased dietary intake of fruits and
vegetables strongly decreases the risk of several chronic diseases
such as cardiovascular diseases, neurological disorders, diabetes,
cancer, etc.
26,27
Several plant metabolites including polyphe-
nolics, glucosinolates, and allyl sulfides play crucial roles in the
prevention of several diseases.
28,29
Sulforaphane is also a
naturally occurring sulfur-containing compound that is present
in certain vegetables such as broccoli sprouts, cabbage, Brussels
sprouts, cauliflower, bok choy, collards, mustard, turnip, etc.
30
Several studies have revealed that sulforaphane possesses strong
antioxidant and anticancer properties. It has been shown that
sulforaphane inhibits histone deacetylase (HDAC) activity in
human colon, prostate, breast, cervical, and ovarian cancers as
also in human leukemia cells.
31−33
In other studies,
combinations of sulforaphane with cytotoxic drugs, for example,
Received: February 13, 2015
Revised: May 28, 2015
Published: May 28, 2015
Article
pubs.acs.org/JPCB
© 2015 American Chemical Society 7825 DOI: 10.1021/acs.jpcb.5b01496
J. Phys. Chem. B 2015, 119, 7825−7836
cisplatin, gemcitabine, doxorubicin, etc., have been found to
enhance effectiveness of treatment of cancer.
34
Sulforaphane is
highly effective in reducing the androgen receptor (AR) protein
level by decreasing secretion of prostate-specific antigen (PSA),
which is an AR-regulated gene product in human prostate
cancer cells.
35
Sulforaphane has numerous other highly useful
medical applications.
36−41
Electronic and vibrational properties of sulforaphane have
been studied using a semiempirical method.
42
Yuan et al.
43
have
shown experimentally that sulforaphane can scavenge both
superoxide radical and hydroxyl radical. Niu et al.
44
have shown
that benzynes are capable of removing two vicinal hydrogen
atoms from a hydrocarbon in a concerted manner, which causes
alkane to alkene conversion. To the best of our knowledge,
double hydrogen abstraction by superoxide radical anion or
hydrogen peroxide from sulforaphane or any other antioxidant
has not yet been studied theoretically. The purpose of the
present study is to show that both superoxide radical anion and
hydrogen peroxide can be scavenged through double hydrogen
abstraction from sulforaphane, the reactions in the former case
being catalyzed by an SOD. Other antioxidants scavenge other
ROS through single hydrogen abstraction or addition reaction
mechanisms.
45
The novelty of the present work is that it is the
first theoretical study having the following three important
features: (i) it explains the mechanism of scavenging action of
sulforaphane toward superoxide radical anion and hydrogen
peroxide, (ii) it shows double hydrogen transfer from an
antioxidant, that is, sulforaphane, to both superoxide radical
anion and hydrogen peroxide, and (iii) it presents a simple
model for iron superoxide dismutase (Fe-SOD) that explains
well the catalytic activity of the enzyme.
2. COMPUTATIONAL DETAILS
All the calculations reported here were performed at the
B3LYP/6-311+G(d) and M06-2X/6-311+G(d) levels of
theory. Geometries of all the reactant molecules, reactant
complexes (RCs), transition states (TSs), and product
complexes (PCs) were optimized in gas phase at the B3LYP/
6-311+G(d) and M06-2X/6-311+G(d) levels of density
functional theory.
46−50
Solvent effect of aqueous media was
treated by single point energy calculations using the gas phase
optimized geometries and employing the integral equation
formalism of the polarizable continuum model (IEF-PCM)
51,52
at the same level of theory at which the gas phase result was
obtained. However, there were certain exceptions as follows: (i)
In certain cases, geometry optimization calculations at the M06-
2X/6-311+G(d) level could not be completed due to internal
errors or lack of convergence. In these cases, as indicated in the
corresponding sections, single point energy calculations were
performed at the M06-2X/6-311+G(d) level using the
geometries optimized at the B3LYP/6-311+G(d) level. And,
(ii) In the study of reactions involving sulforaphane and
hydrogen peroxide, geometries of the RCs, TSs, and PCs were
optimized in both gas phase and aqueous media at both the
B3LYP/6-311+G(d) and M06-2X/6-311+G(d) levels of
density functional theory.
Reactions were considered to initiate from optimized
geometries of RCs. Intrinsic reaction coordinate (IRC)
calculations
53
were performed using optimized TSs to confirm
that hydrogen transfer actually took place from the considered
sites of sulforaphane. Forward Gibbs barrier energies were
calculated as differences of Gibbs free energies of the
corresponding TSs and RCs, while reverse Gibbs barrier
energies were obtained as differences of Gibbs free energies of
the corresponding TSs and PCs at 298.15 K. Vibrational
frequency analysis was performed for each optimized geometry.
TSs were found to be associated with one imaginary vibrational
frequency each, whereas all RCs and PCs were characterized by
all real vibrational frequencies. Genuineness of the calculated
transition states was confirmed by visually examining the
vibrational modes related to the imaginary frequencies and
applying the condition that these connected the corresponding
reactant and product complexes properly. Rate constants were
calculated using the transition states theory.
54−56
The Windows
version of the Gaussian 09 suite of programs (G09W, ver. B.01)
was used for all the calculations.
57
The optimized geometries
and vibrational modes were visualized using the GaussView
program (ver. 5.0).
58
3. RESULTS AND DISCUSSION
3.1. Scavenging Superoxide Radical Anions (O2
•−).
(i). Without Iron Superoxide Dismutase (Fe-SOD). Superoxide
radical anion reacts with other molecules to form hydrogen
peroxide.
59,60
Our calculations indicated that double hydrogen
abstraction from sulforaphane by superoxide radical anion can
occur much more easily than single hydrogen abstraction, and
therefore we investigated only the former mechanism. For
example, when a transition state search was made by placing
superoxide radical anion near the (C7,C9) pair of carbon atoms
(Figure 1) such that one of its oxygen atoms was nearer to one
of the hydrogen atoms of the methyl (C9H3) group and the
initial C9H and OH distances were ∼1.3 Å each. When the
transition state search was continued for 10 steps, the H atom
that was to be abstracted from the C9 site came back near it so
that the C9H and OH distances changed to ∼1.11 and 1.66 Å.
Thus, the transition state search involving a single hydrogen
abstraction from the C9 site (Figure 1) did not succeed. Similar
observations were made in other cases involving single
hydrogen abstraction by superoxide radical anion also.
By double hydrogen abstraction from an alkyl chain, an
organic molecule would get converted from the alkane to the
alkene form. This transformation is an interesting aspect of
chemical synthesis.
44
Double hydrogen transfer occurring from
sulforaphane can be represented as follows
−+ ⎯→⎯⎯⎯ − +
•− Δ
−
S
HO SH HO
nn2
G
222
n
b
(1)
where ΔGnbis the forward Gibbs barrier energy of the reaction,
S−Hnis the sulforaphane molecule with nhydrogen atoms, and
S−Hn−2is a form of the molecule with (n−2) hydrogen atoms,
the two hydrogen atoms having been abstracted from two
Figure 1. Optimized geometry of sulforaphane at the M06-2X/6-
311+G (d) level of theory and adopted atomic numbering scheme.
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neighboring carbon sites of sulforaphane, and superoxide
radical anion (O2
•−) is converted to hydrogen peroxide
(H2O2) due to the reaction. Pairs of hydrogen atoms can be
abstracted from the pairs of neighboring sites of sulforaphane,
that is, (C4,C5), (C5,C6), (C6,C7), and (C7,C9) (Figure 1).
Gibbs free energies of RCs, TSs, and PCs corresponding to
double hydrogen abstraction by superoxide radical anion from
different pairs of neighboring carbon sites of sulforaphane
obtained using B3LYP and M06-2X functionals along with the
6-311+G(d) basis set in gas phase and aqueous media are
presented in Supporting Information, Table S1. The calculated
Gibbs interaction energies of RCs as well as forward and
reverse Gibbs barrier energies corresponding to double
hydrogen abstraction by superoxide radical anion from
Table 1
serial no. pair of carbon sites interaction energy of RC
a
forward/reverse barrier
b
B3LYP/6-311+G(d) M06-2X/6-311+G(d)
c
1 (C4,C5) −23.33(7.04) forward 40.99(40.51) 45.59(43.01)
−29.29(0.95) reverse 16.41(16.79) 21.03(19.44)
2 (C5,C6) −20.99(9.08) forward 38.31(41.69) 40.53(43.67)
−28.63(3.04) reverse 20.97(19.84) 13.07(12.18)
3 (C6,C7) −23.34(7.67) forward 36.50(39.24) 34.82(38.49)
−26.72(3.33) reverse 4.83(2.86) 6.51(4.51)
4 (C7,C9) −20.46(10.19) forward 45.49(43.27) 49.87(46.31)
−26.28(4.32) reverse 0.69(−1.44) 2.10(−0.70)
d
a
Gibbs interaction energies of reactant complexes: upper values: B3LYP/6-311+G(d) level, lower values: M06-2X/6-311+G(d) level, outside
parentheses: gas phase, inside parentheses: aqueous media.
b
Forward and reverse Gibbs barrier heights (kcal/mol) corresponding to double
hydrogen abstraction by superoxide radical anion from sulforaphane at two different levels of theory in gas and aqueous media. Atomic numbering is
given in Figure 1. Barrier energies in aqueous media are given in parentheses.
c
Barrier energies for double hydrogen abstraction from the (C6,C7)
sites were obtained by geometry optimization at the M06-2X/6-311+G(d) level while those from the other pairs of sites were obtained by single
point energy calculations at the M06-2X/6-311+G(d) level using the geometries optimized at the B3LYP/6-311+G(d) level.
d
Negative reverse
barrier energy implies that the corresponding energy lies above the barrier.
Figure 2. Optimized geometries of reactant complexes (RC1−RC4) shown in (a−d), respectively, involved in double hydrogen abstraction from
different sites of sulforaphane by superoxide radical anion (O2
•−) in aqueous media at the M06-2X/6-311+G(d) level of theory. Certain optimized
interatomic distances (Å) are given.
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sulforaphane in gas and aqueous media are presented in Table
1. The gas phase barrier energies for double hydrogen
abstraction from the (C6,C7) sites given in this table were
obtained by geometry optimization at both the B3LYP/6-
311+G(d) and M06-2X/6-311+G(d) levels, while those from
the other sites were obtained by single point energy calculations
attheM06-2X/6-311+G(d)levelusingthegeometries
optimized at the B3LYP/6-311+G(d) level. The Gibbs barrier
energies in aqueous media were obtained by single point energy
calculations at the same levels at which the gas phase barrier
energies were obtained.
The Gibbs interaction energies of RCs (defined in each case
as Gibbs free energy of the complex −sum of Gibbs free
energies of the individual reactants) in gas phase and aqueous
media obtained at both the levels of theory mentioned above
and included in Table 1 reveal the following information. The
calculated gas phase Gibbs interaction energies are all negative,
while those in aqueous media are all positive. When superoxide
radical anion is solvated in aqueous media, it gets highly
stabilized or trapped due to the negative charge and associated
polarization of the aqueous medium. For this reason, the
second term in the above definition of Gibbs interaction energy
in aqueous media becomes highly negative making the
corresponding Gibbs interaction energies positive. Positive
Gibbs interaction energies imply that the reactants under
ambient conditions would not likely make stable complexes,
making the probability of reactions highly reduced.
The optimized structures of reactant complexes (RC1, RC2,
RC3, and RC4) and transition states (TS1, TS2, TS3, and TS4)
involved in double hydrogen transfer from sulforaphane to
superoxide radical anion are shown in Figures 2a−d and 3a−d,
respectively. In these figures, the corresponding imaginary
vibrational frequencies (ν) are also presented. The optimized
structures of product complexes (PC1, PC2, PC3, and PC4)
formed by double hydrogen abstraction from sulforaphane by
superoxide radical anion are presented in Figure 4a−d. Certain
optimized geometrical parameters at the M06-2X/6-311+G(d)
level of theory are also given in Figures 2a−d, 3a−d, and 4a−d.
It is noted that in one case, that is, in the product complex PC2
corresponding to double hydrogen abstraction from the
(C5,C6) pair of carbon sites (Figure 4b), the OSCH3portion
of sulforaphane gets dissociated from the rest of the molecule.
The forward Gibbs barrier energies for double hydrogen
abstraction by superoxide radical anion from the different pairs
of carbon sites of sulforaphane in gas phase and aqueous media
presented in Table 1 and obtained at the B3LYP/6-311+G(d)
or M06-2X/6-311+G(d) level of theory lie in the range
between ∼35 and ∼50 kcal/mol. All these calculated Gibbs
forward barrier energies in both gas phase and aqueous media
are very high (Table 1). The calculated reverse barrier energies
Figure 3. Optimized geometries of transition states (TS1−TS4) shown in (a−d), respectively, involved in double hydrogen abstraction from
different sites of sulforaphane by superoxide radical anion (O2
•−) in aqueous media at the M06-2X/6-311+G(d) level of theory. Certain optimized
interatomic distances (Å) are given. Imaginary vibrational frequency (ν,cm
−1) in each case is given.
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for double hydrogen abstraction presented in Table 1 are quite
different for the different pairs of carbon sites. These barrier
energies corresponding to double hydrogen abstraction from
the (C4,C5) and (C5,C6) pairs of sites in gas phase and
aqueous media obtained at both the levels of theory mentioned
above lie in the range from ∼12 to ∼21 kcal/mol (Table 1).
The corresponding reverse Gibbs barrier energies for the
reaction from the (C6,C7) pair of sites lie in the range from
∼2.9 to ∼6.5 kcal/mol, while those for the reaction involving
the (C6,C7) pair of sites lie in the range from −1.44 to 2.1
kcal/mol (Table 1). The two negative reverse Gibbs barrier
energies (Table 1) imply that the energies under consideration
lie above the Gibbs barrier due to which the reaction cannot
occur. All other reverse Gibbs barrier energies are positive but
appreciably or much smaller than the corresponding forward
Gibbs barrier energies due to which the product complexes
would be appreciably or highly endothermic. The calculated
rate constants for double hydrogen abstraction from the
different pairs of sites of sulforaphane at the B3LYP/6-
311+G(d) and M06-2X/6-311+G(d) levels of theory using
the forward Gibbs barrier energies given in Table 1 (not given)
have very small values (<1 ×10−13 M−1s−1). This along with
the fact that the reverse Gibbs barrier energies are appreciably
or much smaller than the forward ones (even small negative)
shows that the double hydrogen transfer reactions between
sulforaphane and superoxide radical anion would not take place.
(ii). With Iron Superoxide Dismutase. Epidemiological
studies have demonstrated that SOD neutralizes superoxide
radical anion by charge transfer and that superoxide radical is
converted to hydrogen peroxide by taking two hydrogens from
an antioxidant molecule.
21,22,59,60
The active site of Fe-SOD
consists of an iron cation (Fe3+) bonded to three histidines, one
aspartic acid, and a water molecule.
61
However, we excluded
aspartic acid and water molecules from our model of Fe-SOD
and considered only Fe3+ bonded to three histidines for
convenience in computations expecting that the main role of
the enzyme would be revealed by it.
20,62
We studied the
catalytic role of Fe-SOD here, but we hope that, broadly
speaking, similar results would be found if other metal SODs
are considered in place of Fe-SOD. We studied formation of
hydrogen peroxide due to the reaction between superoxide
radical anion and sulforaphane in the presence of this model of
Fe-SOD. We will discuss this reaction in this section, while
reduction of hydrogen peroxide into water will be discussed in
the next section. Double hydrogen abstraction from sulfor-
aphane in biological media can be represented as follows.
−+ ⎯→⎯⎯⎯⎯⎯⎯ − +
•− ‐
−
S
HO SH HO
nn2
Fe SOD
222 (2)
The right-hand side of the above scheme represents the
product complex in the presence of Fe-SOD. We optimized the
structure of the Fe-SOD model at the B3LYP/6-311+G(d) and
M06-2X/6-311+G(d) levels of theory and, subsequently, the
Figure 4. Optimized geometries of different product complexes (PC1−PC4) shown in (a−d), respectively, obtained by double hydrogen abstraction
from the different sites of sulforaphane by superoxide radical anion (O2
•−) in aqueous media at the M06-2X/6-311+G(d) level of theory. Certain
optimized geometrical parameters (Å, deg) are given.
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structure of the complex of superoxide radical anion, and the
Fe-SOD model was also optimized at the same two levels in gas
phase. The optimized structure of the Fe-SOD model at the
M06-2X/6-311+G(d) level of theory is presented in Figure S1
(Supporting Information). We attempted to optimize RCs,
TSs, and PCs corresponding to single or double hydrogen
abstraction from the different sites or pairs of neighboring sites
of sulforaphane by superoxide radical anion in the presence of
Fe-SOD. With regard to the search for TSs, it was noted that
transfer of two corresponding hydrogens from all the four pairs
of neighboring sites of sulforaphane under the influence of Fe-
SOD occurred with some delay. Thus, in all the cases, one of
the hydrogens was abstracted generally only after a few cycles
of calculations, while the other got abstracted much later. The
total energies of RCs and TSs changed slowly, and their
gradients with respect to the geometrical parameters continued
to be appreciable. Thus, none of the RCs and TSs could be
located despite repeated attempts. This indicated that all the
reactions might be barrierless and that the Gibbs barrier
energies did not exist.
The optimized geometries of two product complexes at the
B3LYP/6-311+G(d) level (single point energy calculation at
the M06-2X/6-311+G(d) level) in gas phase in the presence of
Fe-SOD model from the (C4,C5) and (C5,C6) sites, that is,
PC1′and PC2′, are shown in Figure 5a,b, while those of the
other two product complexes in the presence of Fe-SOD, that
is, PC3′and PC4′, at the same level of theory in gas phase are
shown in Figure 6a,b. The results shown in these figures are
discussed herein later. Certain optimized geometrical parame-
ters are also given in all these figures (Figures 5a,b and 6a,b). As
discussed earlier, the OSCH3group was dissociated from
sulforaphane in PC2′when the reactions occurred without Fe-
SOD, but it does not take place in the presence of Fe-SOD.
During the process of double hydrogen abstraction from the C7
and C9 sites of sulforaphane, the geometry of the sulforaphane
moiety is strongly changed since the C7 and C9 sites get
bonded. It is also noted that the Fe-SOD model does not
participate in bonding with any other moiety, and thus its role
as a catalyst is validated.
The lowest total energy points in aqueous media obtained by
searches for RCs (labeled A) and TSs (labeled B) as well as
those corresponding to optimized geometries of PCs (labeled
C) on the potential energy surfaces relevant to double
hydrogen transfer from the (C4,C5), (C5,C6), (C6,C7), and
(C7,C9) pairs of sites of sulforaphane plotted versus the OO
distance in the superoxide radical anion are shown in Figures
S2, S3, S4, and S5 (Supporting Information), respectively. The
Figure 5. Optimized geometries of product complexes of (a) PC1′and
(b) PC2′obtained by double hydrogen abstraction from different sites
of sulforaphane by superoxide radical anion (O2
•−) in the presence of
SOD in aqueous media obtained at the M06-2X/6-311+G(d) level of
theory. Certain optimized geometrical parameters (Å, deg) are given.
Figure 6. Optimized geometries of different product complexes (a)
PC3′and (b) PC4′, respectively, obtained by double hydrogen
abstraction from the different sites of sulforaphane by superoxide
radical anion (O2
•−) in aqueous media at the M06-2X/6-311+G(d)
level of theory. Certain optimized geometrical parameters (Å, deg) are
given.
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corresponding values of two OC and OH distances for each of
the points A, B, and C are given near these points (O = the two
oxygen atoms of O2
•−, C = the corresponding two carbon
atoms from where hydrogens are being abstracted, and H =
corresponding two hydrogens being abstracted). The relative
total energies given in these figures were obtained by M06-2X/
6-311+G(d) level calculations in aqueous media employing the
geometries obtained at the B3LYP/6-311+G(d) level. These
figures show that in each case, the point C lies lowest, the point
B lies higher than C, while the point A lies higher than B in
total energy. These figures, particularly as the points B lie much
below the corresponding points A, confirm that RCs and TSs
would not exist as appropriate total energy extrema, and so the
reactions under consideration would be barrierless, leading
directly to the formation of PCs from the reactants under the
influence of Fe-SOD.
It is quite clear that the reactions involving double hydrogen
transfer from sulforaphane to superoxide radical anion leading
to the formation of hydrogen peroxide and alkene forms of
sulforaphane are strongly catalyzed by Fe-SOD such that large
positive barrier energies in aqueous media are changed to no
barrier energies. This drastic change in the nature of reactions
can be ascribed to delocalization of charges over the entire
system consisting of the iron cation and the histidine rings of
the Fe-SOD model, superoxide radical anion, or H2O2to which
it is converted by double hydrogen transfer and sulforaphane in
alkane or alkene forms. Mulliken charges in its different
complexes are presented in Tables S2−S7 (Supporting
Information). Isolated sulforaphane is electrically neutral
though certain of its sites; for example, C4, C7, S8, O8, and
C9 carry large net charges (Table S2, Supporting Information).
In the complex of superoxide radical anion with the Fe-SOD
model (Table S3, Supporting Information), the former carries a
small positive charge (0.222) instead of −1, Fe3+ looks like
neutral Fe (net charge 0.29), and the three rings carry a net
charge of ∼0.5 each.
In the product complex (PC1′) formed by double hydrogen
abstraction by superoxide radical anion from the (C4,C5) sites
of sulforaphane in the presence of the Fe-SOD model in
aqueous media, the Mulliken net charges are as follows (Table
S4, Supporting Information): Sulforaphane is almost electrically
neutral here also though some of its sites carry large positive or
negative charges, H2O2carries a net positive charge of ∼0.4,
overall the three rings carry a net charge of ∼1.5, while the iron
cation is almost electrically neutral. Thus, a large part of the
positive charge of the iron cation is transferred to the three
rings in PC1′. In the product complex (PC2′) formed by
double hydrogen abstraction by superoxide radical anion from
the (C5,C6) sites of sulforaphane in the presence of Fe-SOD in
aqueous media, the Mulliken charges are as follows (Table S5,
Supporting Information): Sulforaphane carries a positive charge
of ∼0.3, H2O2carries a positive charge of ∼0.2, the iron center
carries a charge of ca. −0.4, thus becoming negatively charged
instead of remaining positively charged, while overall the three
rings carry a net charge of ∼1.9.
In the product complex (PC3′) where two hydrogens were
abstracted from the (C6,C7) pair of sites of sulforaphane
(Table S6, Supporting Information), sulforaphane carries a
positive charge of ∼0.6, H2O2carries a positive charge of 0.28,
the iron center carries a charge of ca. −0.7, thus becoming
appreciably negatively charged instead of being positively
charged, and overall the three rings carry a charge of ∼1.8. In
the product complex (PC4′) formed by double hydrogen
abstraction by superoxide radical anion from the (C7,C9) pair
of sites of sulforaphane in the presence of Fe-SOD in aqueous
media, the Mulliken charge distribution is as follows (Table S7,
Supporting Information): Sulforaphane carries a positive charge
of ∼0.35, H2O2carries a net positive charge of ∼0.2, the iron
center carries a charge of ca. −0.28, while overall the three rings
carry an appreciable positive charge of ∼1.7. There are two
notable points that emerge from the above analysis of the net
Mulliken charge distributions. First, the iron center that
originally carried a net charge of +3 becomes almost electrically
neutral or even carries significant negative charges. Second,
overall the three rings carry a net positive charge lying in the
range from 1.5 to 1.9. The net charges in each component of
the product complex are strongly delocalized due to which
there is no single isolated entity like superoxide radical anion
that carries a large positive or negative charge, which can
drastically polarize the aqueous medium locally. In this
situation, the effect of polarization of aqueous media by
individual components would be absent. This seems to be the
main reason for Fe-SOD being a strong catalyst. To the best of
our knowledge, no such model has been employed earlier for
this enzyme. Models of other metal SODs can be obtained by
replacing the iron cation of Fe-SOD by other metal cations.
3.2. Scavenging of Hydrogen Peroxide. As shown in
Section 3.1, sulforaphane scavenges superoxide radical anion by
double hydrogen transfer in aqueous media in the presence of
superoxide dismutase barrierlessly, and hydrogen peroxide is
formed as the product consequent to this reaction. Though, as
such, hydrogen peroxide is poorly reactive, it can be readily
converted into hydroxyl radicals (OH•)bytheFenton
mechanism, and these radicals are highly reactive and can
damage most biomolecules.
21,22,63,64
Therefore, scavenging of
superoxide radical anion by sulforaphane by converting it to
hydrogen peroxide would be fully beneficial only if it can also
convert hydrogen peroxide to a harmless form, for example,
water. This aspect was studied in this section.
Double hydrogen abstraction by hydrogen peroxide from
sulforaphane can be represented as follows
−+ ⎯→⎯⎯⎯⎯ − +
Δ
−
′
S
HHO SH 2H
O
nn22
G
22
n
b
(3)
where ΔGnb′is the forward Gibbs barrier energy required for
the reaction, S−Hnrepresents sulforaphane having nhydrogen
atoms, while Hn−2represents the form of sulforaphane from
which two hydrogen atoms have been abstracted by hydrogen
peroxide.
Gibbs free energies of RCs, TSs, and PCs corresponding to
double hydrogen abstraction by hydrogen peroxide from the
different pairs of neighboring carbon sites of sulforaphane
obtained at the B3LYP/6-311+G(d) and M06-2X/6-311+G(d)
levels of theory in gas phase and aqueous media are presented
in Table S8. The calculated forward and reverse Gibbs barrier
energies for double hydrogen abstraction by hydrogen peroxide
from the different pairs of sites of sulforaphane obtained at two
different levels of theory in gas phase and aqueous media are
presented in Table 2. Optimized geometries of reactant
complexes between H2O2and sulforaphane at the M06-2X/6-
311+G(d) level of theory are shown in Figure 7, while those of
transition states involved in the corresponding double hydro-
gen transfer reactions are shown in Figure 8. The products
formed due to these reactions are shown in Figure 9. It is found
that the forward Gibbs barrier energies for double hydrogen
abstraction by hydrogen peroxide from the pairs of sites
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(C4,C5), (C5,C6), and (C6,C7) obtained at the B3LYP/6-
311+G(d) and M06-2X/6-311+G(d) levels of theory in both
gas phase and aqueous media (Table 2) are negative or small
positive, the largest forward Gibbs barrier energy being 1.62
kcal/mol (Table 2). However, the forward Gibbs barrier
energies corresponding to double hydrogen abstraction from
the pair of sites (C7,C9) in both gas phase and aqueous media
obtained at both B3LYP/6-311+G(d) and M06-2X/6-
311+G(d) levels of theory are very large lying between ∼21.7
and ∼31.4 kcal/mol. The reason for high Gibbs barrier energies
in this case appears to be the fact that double hydrogen
abstraction from the (C7,C9) pair of sites requires S8 to make
three double bonds (Figure 1). The reverse Gibbs barrier
energies for double hydrogen abstraction by hydrogen peroxide
from the pairs of sites (C4,C5), (C5,C6), and (C6,C7) lie in
the range ∼42.6 to ∼56.3 kcal/mol in gas phase and in the
range ∼46 to 54 kcal/mol in aqueous media (Table 2). For
double hydrogen transfer from the (C7,C9) pair of sites of
sulforaphane, the calculated reverse Gibbs barrier energies are
smaller than the forward ones, and therefore, this reaction
would be endothermic.
The structures of transition states involved in the four double
hydrogen transfer reactions between hydrogen peroxide and
sulforaphane in aqueous media optimized at the M06-2X/6-
311+G(d) level of theory and denoted by TS1′−TS4′along
with the corresponding imaginary vibrational frequencies (ν′)
are presented in Figure 8. Certain optimized interatomic
distances (Å) are also given in each case in this figure. We
observed that in each of these reactions, a hydrogen peroxide
molecule positions itself near sulforaphane such that its oxygen
atoms can interact effectively with selected sites of the latter.
Table 2
serial
no. pair of
carbon sites forward/reverse
barrier
a
B3LYP/
6-311+G(d) M06-2X/
6-311+G(d)
1 (C4,C5) forward −5.69(−8.83) 1.57(−0.52)
reverse 50.48(48.59) 50.59(46.77)
2 (C5,C6) forward 1.62(−0.46) −0.14(−1.76)
reverse 47.56(49.02) 42.56(45.85)
3 (C6,C7) forward −3.83(−0.08) −8.61(−5.14)
reverse 56.25(53.97) 50.67(50.51)
4 (C7,C9) forward 21.74(14.61) 31.42(26.22)
reverse 18.39(10.16) 26.09(19.55)
a
Forward and reverse Gibbs barrier energies (kcal/mol) correspond-
ing to double hydrogen abstraction by hydrogen peroxide (H2O2)
from sulforaphane at two different levels of theory in gas and aqueous
media. Atomic numbering is given in Figure 1. Barrier energies in
aqueous media are given in parentheses.
Figure 7. Optimized geometries of reactant complexes (RC1′−RC4′) shown in (a−d), respectively, involved in double hydrogen abstraction from
the different pairs of sites of sulforaphane by hydrogen peroxide (H2O2) in aqueous media obtained at the M06-2X/6-311+G (d) level of theory.
Certain optimized interatomic distances (Å) are given.
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These interactions dissociate H2O2into two OH groups each of
which, in turn, abstracts a hydrogen atom from a nearby site of
sulforaphane resulting in the formation of two water molecules.
A graphical comparison of the Gibbs barrier energies involved
in double hydrogen abstraction by hydrogen peroxide from the
different pairs of sites of sulforaphane in aqueous media
optimized at the B3LYP/6-311+G(d) and M06-2X/6-
311+G(d) levels of theory is made in Figure S6 (Supporting
Information). It clearly shows that the Gibbs barrier energies
for double hydrogen abstraction from sulforaphane by
hydrogen peroxide in aqueous media from the (C4,C5),
(C5,C6), and (C6,C7) pairs of sites are small, or the reactions
are barrierless, while the double hydrogen transfer reactions
from the (C7,C9) pair sites involves a much higher barrier
energy. The calculated rate constants for double hydrogen
abstraction from the different pairs of carbon sites obtained at
the B3LYP/6-311+G(d) and M06-2X/6-311+G(d) levels of
theory using the forward Gibbs barriers energies given in Table
2 are presented in Table 3. Negative barrier energies were taken
to be zero implying barrierless reactions for calculating rate
constants. We note that double hydrogen transfer from the
three pairs of sites (C4,C5), (C5,C6), and (C6,C7), in
conformity with the calculated barrier energies, would occur
with very high rate constants (∼1×1012 M−1s−1), while those
from the sites (C7,C9) would occur very slowly.
The optimized structures of the product complexes resulting
from double hydrogen abstraction by hydrogen peroxide from
sulforaphane denoted by PC1″−PC4″are presented in Figure
9. Each of these complexes includes two water molecules and a
form of sulforaphane that has lost two hydrogen atoms from
vicinal sites. Note that the bonds between carbon atoms that
have lost one hydrogen atom each due to the reactions under
consideration acquire typical double (CC) bond lengths
(1.326−1.332 Å), and thus sulforaphane transforms from the
alkane to the alkene form. The results discussed above clearly
show that sulforaphane can scavenge both superoxide radical
anion (in the presence of Fe-SOD) and hydrogen peroxide
(without involving any catalyst) very efficiently. Since super-
oxide radical anion is a deadly oxidizing agent that plays crucial
roles in DNA damage, sulforaphane is a very efficient and
valuable antioxidant.
4. CONCLUSION
We arrive at the following conclusions from this study:
1. Sulforaphane scavenges both superoxide radical anion
(which is converted to hydrogen peroxide) and hydrogen
peroxide (which is converted to water) by double
hydrogen transfer barrierlessly or with low barrier
energies. Presence of Fe-SOD is required for scavenging
Figure 8. Optimized geometries of transition states (TS1′−TS4′) shown in (a−d), respectively, involved in double hydrogen abstraction from the
different pairs of sites of sulforaphane by hydrogen peroxide (H2O2) in aqueous media obtained at the M06-2X/6-311+G(d) level of theory. Certain
optimized interatomic distances (Å) are given. Imaginary vibrational frequency (ν′,cm
−1) in each case is given.
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superoxide radical anion, but hydrogen peroxide is
scavenged without any additional agent.
2. The calculated Gibbs interaction energies of reactant
complexes between sulforaphane and superoxide radical
anion as well as forward and reverse Gibbs barrier
energies for double hydrogen transfer from the former to
the latter show that the reactions under consideration
would be very unlikely to occur in the absence of a
catalyst. The fact that superoxide radical anion gets
trapped in water due to its negative charge and associated
polarization of the medium plays an important role in
making the reactions unlikely to occur. The presence of
superoxide dismutase changes this situation drastically as
the charges get widely spread on the different
components preventing superoxide radical anion from
getting trapped due to local polarization of the aqueous
medium. It results in barrierless scavenging of superoxide
radical anion by sulforaphane in aqueous media in the
presence of Fe-SOD.
3. We employed a model for Fe-SOD that consists of Fe3+
cation bonded to three histidine rings. The model works
well in conformity with the known catalytic role of SOD.
■ASSOCIATED CONTENT
*
SSupporting Information
Tabulated Gibbs free energies and Mulliken charge distribu-
tions, Gibbs barrier heights of hydrogen abstraction sites,
variation of total energy as a function of OO bond distance,
optimized geometry of SOD model. The Supporting
Information is available free of charge on the ACS Publications
website at DOI: 10.1021/acs.jpcb.5b01496.
Figure 9. Optimized geometries of product complexes (PC1″−PC4″) shown in (a−d), respectively, formed by double hydrogen abstraction from
the different pairs of sites of sulforaphane by hydrogen peroxide in aqueous media obtained at the M06-2X/6-311+G(d) level of theory. Certain
optimized interatomic distances (Å) are given.
Table 3
serial no. hydrogen abstraction sites B3LYP/6-311+G(d)
a
M06-2X/6-311+G(d)
a
1 (C4,C5) 6.2 ×1012 (6.2 ×1012) 4.4 ×1011 (6.2 ×1012)
2 (C5,C6) 4.0 ×1011 (6.2 ×1012) 6.2 ×1012 (6.2 ×1012)
3 (C6,C7) 6.2 ×1012 (6.2 ×1011) 6.2 ×1012 (6.2 ×1012)
4 (C7,C9) 7.0 ×10−4(0.1 ×10−3) 5.6 ×10−11 (3.6 ×10−7)
a
Rate constants (M−1s−1) for double hydrogen abstraction by hydrogen peroxide from the different pairs of sites of sulforaphane obtained at two
different levels of theory in gas phase and aqueous media. Results obtained in aqueous media are given in parentheses. Transition states involved in
these reactions are shown in Figure 8, while the products due to these reactions are shown in Figure 9.
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■AUTHOR INFORMATION
Corresponding Author
*E-mail: pcmishra_in@yahoo.com.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
One of the authors (P.C.M.) thanks the National Academy of
Sciences, India (NASI), for the award of a NASI Senior
Scientist Fellowship. The authors are thankful to the University
Grants Commission (New Delhi) for financial support.
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The Journal of Physical Chemistry B Article
DOI: 10.1021/acs.jpcb.5b01496
J. Phys. Chem. B 2015, 119, 7825−7836
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