The FeII(citrate) Fenton reaction under physiological conditions

Article abstract of DOI:10.1016/j.jinorgbio.2020.111018

The Fenton reaction of Fe^II(citrate) in the presence and absence of bicarbonate (HCO₃ ^?) is studied. It is found that the rate constant of the Fenton reaction (k_obs) increases with increasing [citrate]. k_obs also increase with increasing [HCO₃ ^?]; this effect is most significant at biological citrate concentrations. Methane and ethane gases are formed from (CH₃)₂SO when the Fenton reaction is carried out in the presence of large [citrate] due to the reaction of the citrate radical, (^? ₂OC)CH₂C(OH)(CO₂ ^?)CH(CO₂ ^?)[rad]/(^? ₂OC)CH₂C(O)(CO₂ ^?)CH₂(CO₂ ^?)[rad] with (CH₃)₂SO. In the absence of citrate (CH₃)₂SO₂ is the main product of the Fenton reaction. However, in the presence of 0.10 mM citrate, no (CH₃)₂SO₂ is formed, some (CH₃)SOOH is formed, along with a low yield of beta-ketoglutaric acid. Formation of (CH₃)SOOH and beta-ketoglutaric acid are due to the citrate radical and Fe^IV(citrate). In the presence of bicarbonate formation of abundant beta-ketoglutaric acid confirms the formation of carbonate radical anion (CO₃[rad]^?). Thus, bicarbonate affects the mechanism and kinetics of the reaction dramatically. Hydroxyl radicals (OH[rad]) are not formed in the presence of bicarbonate and probably also not in its absence. These results point out that hydroxyl radicals, formed by the Fenton reaction, do not initiate oxidative stress in biological systems.

Full text of DOI:10.1016/j.jinorgbio.2020.111018

Journal Pre-proof
The FeII(citrate) Fenton reaction under physiological conditions
Erzsébet Illés, Shanti G. Patra, Vered Marks, Amir Mizrahi, Dan
Meyerstein
PII:
S0162-0134(20)30046-5
DOI:
https://doi.org/10.1016/j.jinorgbio.2020.111018
JIB 111018
Reference:
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
29 November 2019
1 February 2020
1 February 2020
Please cite this article as: E. Illés, S.G. Patra, V. Marks, et al., The FeII(citrate) Fenton
reaction under physiological conditions, Journal of Inorganic Biochemistry (2020),
https://doi.org/10.1016/j.jinorgbio.2020.111018
This is a PDF file of an article that has undergone enhancements after acceptance, such
as the addition of a cover page and metadata, and formatting for readability, but it is
not yet the definitive version of record. This version will undergo additional copyediting,
typesetting and review before it is published in its final form, but we are providing this
version to give early visibility of the article. Please note that, during the production
process, errors may be discovered which could affect the content, and all legal disclaimers
that apply to the journal pertain.
© 2020 Published by Elsevier.

Journal Pre-proof
The FeII(citrate) Fenton reaction under physiological
conditions
Erzsébet Illés,a Shanti G. Patra,a Vered Marks,a Amir Mizrahi,b and Dan Meyerstein*a,c
a
Department of Chemical Sciences, The Center for Radical Reactions and the Schlesinger Family
Center for Compact Accelerators, Radiation Sources and Applications, Ariel University, Ariel, Israel.
b Department of Chemistry, Nuclear Research Centre Negev, Beer-Sheva, Israel.
cDepartment of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
A B S T R A C T
─
The Fenton reaction of FeII(citrate) in the presence and absence of bicarbonate (HCO3 ) is
studied. It is found that the rate constant of the Fenton reaction (kobs) increases with
─
increasing [citrate]. kobs also increase with increasing [HCO3 ]; this effect is most significant
at biological citrate concentrations. Methane and ethane gases are formed from (CH3)2SO
when the Fenton reaction is carried out in the presence of large [citrate] due to the reaction of
─
─ •
─
─ •
the citrate radical, (─ OC)CH2C(OH)(CO2 )CH(CO2 ) /(─ OC)CH2C(O)(CO2 )CH2(CO2 )
2
2
with (CH3)2SO. In the absence of citrate (CH3)2SO2 is the main product of the Fenton
reaction. However, in the presence of 0.10 mM citrate, no (CH3)2SO2 is formed, some
(CH3)SOOH is formed, along with a low yield of beta-ketoglutaric acid. Formation of
(CH3)SOOH and beta-ketoglutaric acid are due to the citrate radical and FeIV(citrate). In the
presence of bicarbonate formation of abundant beta-ketoglutaric acid confirms the formation
•─
of carbonate radical anion (CO3 ). Thus, bicarbonate affects the mechanism and kinetics of
the reaction dramatically. Hydroxyl radicals (OH•) are not formed in the presence of
bicarbonate and probably also not in its absence. These results point out that hydroxyl
radicals, formed by the Fenton reaction, do not initiate oxidative stress in biological systems.
Keywords: Fenton reaction; FeII(citrate); bicarbonate; biological oxidative stress; FeIV=O;
carbonate anion radical
——————————————
*Corresponding author
Dan Meyerstein
Email-danm@ariel.ac.il, danmeyer@bgu.ac.il
1

Journal Pre-proof
1. Introduction
The Fenton reaction[1] is of major importance in causing oxidative stress[2–4]
and in advanced oxidation processes[5–7]. Originally the Fenton reaction is the
2+
reaction between Fe(H2O)6 and H2O2[1], but over the years, many other Fenton like
reactions have been studied by changing the metal ion, water ligand, and peroxide.
However, the mechanism of the Fenton reactions is still in debate. In 1932 two
mechanisms were proposed: Haber and Weiss[8] proposed that the mechanism
involves the formation of hydroxyl radicals (OH•) via:
(1)
Fe(H2O)62+ + H2O2 Fe(H2O)63+ + OH• + OH─

While Bray and Gorin[9] proposed that the mechanism of the Fenton reaction is:
Fe(H2O)62+ + H2O2 (H2O)5Fe=O2+ + 2H2O
(2)

The debate about which of these mechanisms is correct lasted for decades. It was
shown that these reactions could not proceed via the outer-sphere mechanism and the
minimal mechanism has to be written as follows:[10,11]
(3)
MnLm + H2O2 Lm-1Mn(O2H) + H3O+ + L

H+
 Lm-1Mn+2=O + H2O
(a)
(b)
(c)
(4)
Lm-1Mn(O2H)  Lm-1Mn+1(OH) + OH•
RH
 Lm-1Mn+1(OH) + R• + H2O
It is now clear that for M = Fe both mechanisms (4a) and (4b) are correct, thus for
Fe(H2O)62+ reaction (4b) is correct at pH < 4 whereas reaction (4a) is correct in neutral
2+
solutions[12]. For [H2O2] >> [Fe(H2O)6 ] it was shown that at least in acidic solutions
reaction (5) contributes to the process:[13,14]
2

Journal Pre-proof
(5)
FeII(H2O)62+ + FeIII(H2O)5(HO2─)2+  FeIII(H2O)63+ + (H2O)5FeIV=O2+
Furthermore, different mechanisms are reported for other central cations and other
ligands. For example, central cations with a high redox potential (e.g., Al,[15] and
Co[16]), the mechanism is as follows:
(6) (H2O)6-l-1Mn(O2H─)l(H2O2)n-l

(H2O)6-l-1Mn(O2H─)l-1(O2H•)(OH─)n-l + OH•
Whereas for central cations with somewhat lower oxidation potential (Mo[17]) it is:
(7) MoVO43─ + H2O2

O3MoV(O22─) + H2O
MoVIO42─ + O2•─ + H2O
(8) O3MoV(O22─)3─ + H2O2

Finally, it was shown, theoretically, that transient complexes of the types:
(H2O)6-l-kLlMn(O2H─) or (H2O)6-l-1LlMn(O2H─)k are expected to decompose via the
oxidation of an L ligand if that is mechanistically and/or thermodynamically
favorable[18]. This was proposed for carboxylate ligands and carbonate. Indeed, it was
•─
demonstrated experimentally that carbonate radical anion (CO3 ) are formed in the
2+
2+
Fenton like reactions of Co(H2O)6
bicarbonate.[14,19]
and Fe(H2O)6
in the presence of
This short discussion points out that one cannot assume a priori that under
given conditions, the product of the Fenton like reaction would be a strong oxidizing
agent. Oxidative stress in biological systems is commonly induced by the reactions of
iron in the mobile pool of non-transferrin bound iron[20,21]. FeIII(citrate), and
FeII(citrate) are the main iron-containing species in this pool[22,23]. The Fenton
reaction in the presence of citrate was studied often. The following are some relevant
systems in which this reaction was studied: a. In biological systems[21,24–27]. b. In
advanced oxidation processes[28–30]. In all these studies, it is proposed that OH•
radicals are the product of the reaction of FeII(citrate) with peroxides. One study raise
the question of whether the products are OH• radicals or FeIV=O2+aq[27]. In none of
these studies, the role of bicarbonate in the process was considered. It seemed,
therefore, of interest to check the mechanism of the Fenton like reaction in solutions
containing citrate and bicarbonate at concentrations present in the body.
3

Journal Pre-proof
2. Experimental
2.1. Materials, instruments, and methods
The chemicals used were all of the analytical grade, purchased from the
following companies; Iron(II) perchlorate, Sigma-Aldrich; hydrogen peroxide (H2O2),
J.T.Baker; sodium bicarbonate, J.T.Baker; trisodium citrate, Alfa Aesar; dimethyl
sulfoxide, (CH3)2SO, TCI and D2O, Tzamal D-Chem Laboratories Ltd.
UV-Vis kinetic measurements were performed using an Applied Photophysics
SX20 stopped-flow instrument. The kinetics were followed at 270 nm due to the
1
formation of FeIII complex[31]. The H NMR spectra were recorded on a 400 MHz
Bruker AvanceIII spectrometer. GC determinations of methane and ethane were
performed using an Agilent 7890B GC System with FID and TCD detectors and a GS
Gaspro column. Pulse radiolysis of the aqueous solutions was done using 1.5 μs pulses
from the linear electron accelerator of Ariel University. The pH was measured using a
Schott Instrument Lab 850 pH meter.
All experiments were performed using the citrate as the buffer. Its capacity is
enough to keep the pH at 7.2[32,33]. HClO4 was used for adjusting the pH to 7.2 ±
0.1. A 7.8 mM H2O2 stock solution was prepared from 30 wt. % H2O2. Low
concentrations of the reactants were used in order to minimize the ionic strength
effect. All the solutions were purged in glass syringes with argon during the
preparation and before mixing. Stock solutions of iron(II) perchlorate in buffered
Milli-Q H2O (Millipore) were prepared freshly before each set of experiments. The
exact amounts of iron(II) perchlorate crystals were added to the saturated argon
solutions while the argon purging was running to avoid any contact of the iron(II) with
oxygen. IronII(citrate) complexes were prepared in one syringe by adding trisodium
citrate to the Milli-Q water, adjusting the pH with HClO4 solution, purging the
solution with Ar, and then finishing by the addition of the deaerated iron(II) stock
solution. In the case of individual 1H NMR and GC measurements dimethyl-sulfoxide,
(CH3)2SO, was added to the iron(II) solution at the end of the preparation.
Concentrated sodium bicarbonate solutions were prepared and added to the diluted
H2O2 solutions, to form solutions containing the desired compositions. Then the pH
was set to the required pH 7.2 by adding HClO4.
At this pH, CO2 was also present in the solutions. Thus, while O2 is removed
─
by purging an inert gas, it decreases the HCO3 concentration considerably. To
4

Journal Pre-proof
overcome this problem, Ar was passed through a gas-washing bottle containing a
─
solution of HCO3 at the same concentration. This method and its effectiveness were
previously reported.[14] In all the kinetic measurements, the FeII (with or without
citrate) and the H2O2 (with or without bicarbonate) solutions in different
concentrations were mixed in a 1:1 volumetric ratio. Thus, the pH in the kinetic runs
was always pH of 7.2 ± 0.1. The concentrations stated in the manuscript are those in
the final solutions. Kinetic experiments were performed at 25 ± 0.10 °C. The
experiments were repeated at least three times in order to check the reproducibility.
Dimethyl sulfoxide, (CH3)2SO, was added to the solutions in order to analyze
the products formed by the Fenton reaction, and different products were monitored by
1H NMR and gas chromatography (GC). 1H NMR spectra of the products of oxidation
of (CH3)2SO via the Fenton reaction were measured in the absence and presence of
bicarbonate. It is known that (CH3)2SO reacts with FeIV=Oaq by oxygen atom transfer
forming dimethyl sulfone, (CH3)2SO2[12], while OH· generates methyl-sulfinic acid,
(CH3SOOH) and ethane (via methyl radicals).[12,14,34] As the concentrations of the
products of the Fenton reaction are too low to measure by NMR, the reactions were
performed in bulk. The final solution (reaction mixture) was then acidified and
concentrated using a rotary evaporator at room temperature.
•─
Further, in order to investigate the reactions of CO3 with (CH3)2SO and
citrate, the solutions were irradiated in a 60Co γ source with a dose rate of ~4 Gy/min.
The Milli-Q H2O (Millipore) was used for all the experimental measurements, while
1
10% D2O was added to record H NMR spectra. Fenton reactions were performed for
[H2O2] > [FeII]. The FeII and the H2O2 concentrations were 0.020 and 0.39 mM. The
(CH3)2SO concentration was 12.5 mM or 25 mM in all experiments. This means high
excess over FeII and H2O2 in all cases.
3. Results and discussion
3.1. Rate constant (kobs) dependence on the citrate concentration
The effect of [citrate] on the observed rate constant of the Fenton reaction is
determined; the results are summed up in Fig. 1. The results demonstrate that the rate of the
Fenton reaction depends on [citrate]. Even at the physiological concentration of 0.1 mM
5

Journal Pre-proof
citrate[35], the observed rate constant is considerably higher than that in the absence of
citrate. The effect of the citrate on the rate constant is due to the formation of the Fe(Cit)─;
Fig. 1. Observed rate constant dependence on the citrate concentration during the Fenton-like
reaction of FeII(citrate) complex with H2O2. The solutions contained: 0.020 mM FeII, 0.10-
0.80 mM trisodium citrate, 0.39 mM H2O2. The pH was 7.2, adjusted with HClO4.
4─
Fe(CitH)0 and Fe(Cit)2 complexes (where CitH, Cit are di and tri negative citrate ligand
respectively) that lower the redox potentials of the corresponding FeIII/II and FeIV/II couples.
The stability constants of the citrate complexes are poorly known; see results reported in the
following references.[36–42] Therefore it is impossible to determine the rate constants of the
reactions of each component in the solution with H2O2. The measured rate constant
((4.8±0.6)×103 M-1s-1) of the Fenton reaction of FeII(citrate) matches well with the previously
reported ((4.9±0.3)×103 M-1s-1) value[32].
─
3.2. Rate constant (kobs) dependence on the HCO3 concentration
The effect of bicarbonate on the reaction rate of the Fenton reaction of the FeII(citrate)
complex with H2O2 at pH 7.2 at [citrate] = 0.10; 0.50 and 2.0 mM was studied. These
concentrations of citrate were chosen in order to determine the effect under conditions
where the citrate affects the kinetics differently. The results are summed up in Fig. 2.
6

Journal Pre-proof
Fig. 2 Observed rate constant dependence on the bicarbonate concentration at different
citrate concentrations during the Fenton-like reaction of the FeII(citrate) complexes with
H2O2. The solutions contained: 0.020 mM FeII, 0.10-2.0 mM trisodium citrate, 0.39 mM
H2O2, 0.0-8.0 mM NaHCO3. The pH was 7.2, adjusted with HClO4.
The presence of bicarbonate unambiguously increases the rate constant of the
─
reaction considerably. The dependence of the observed rate constant on [HCO3 ] is
affected by the citrate concentration. Under near-physiological conditions, in the
2+
presence of 0.10 mM citrate, where some Fe(H2O)6 is present in the solution, the
slope of the linear line is the largest, 17,040 mol-1dm3s-1. In the absence of citrate, the
slope of the linear line is considerably larger, 105,400 mol-1dm3s-1 [14]. Thus, the
reaction rate is much faster in the presence of only bicarbonate. The effect of [citrate]
on the results is attributed to its effect on the concentrations of the following
complexes in the solutions: Fe(CO3)0, Fe(HCO3)+, Fe(Cit)─, Fe(Cit)(CO3)3─
Fe(Cit)(HCO3)2─, Fe(CitH)0, Fe(CitH)(HCO3)─, Fe(CitH)(CO3)2─, and Fe(Cit)24─. In
this context, it should be noted that Adam et. al.[25] have shown that with the addition
of ascorbate to the FeII(citrate) complex the reaction rate increases.
Also, the stability constants of Fe(CO3)0 and Fe(HCO3)+ are poorly known; see
discussion by Illés et. al.[14] The stability constants of the mixed complexes
Fe(Cit)(HCO3)2─; Fe(CitH)(CO3)2─; Fe(Cit)(CO3)3─ and Fe(CitH)(HCO3) are
7

Journal Pre-proof
unknown. Therefore, it is impossible to determine the rate constants of the reactions
of each component in the solution with H2O2. The nature of the reactive oxidizing
species in these reactions is unclear at present. In principle, the formation of the
corresponding (FeIII +OH•), FeIV, and/or FeII(CO42─) [19] complexes can be envisaged.
─
Clearly with the increase in [HCO3 ], the concentrations of all the complexes
containing bicarbonate/carbonate as a ligand increases: This lowers the redox potential
of the central FeII ion and increases the chance to form per-carbonate complexes.
3.3. Gas-chromatographic measurements of methane end ethane after the Fenton
reaction
The remaining question is what the products of these reactions are. For this purpose, it was
decided to add (CH3)2SO to the solutions as it is known that OH• radicals react with
(CH3)2SO to form methyl radicals via:
(9) (CH3)2SO + OH•  (CH3)2S(OH)=O• k9 = 7.0x109 mol-1dm3s-1 [43]
•
(10) (CH3)2S(OH)=O•  CH3SOOH + CH3 k10 = 1.5x107 s-1 [43]
It should be pointed out that also other radicals react with (CH3)2SO to form methyl
radicals.[44,45] The methyl radicals thus formed react to form ethane and methane via:
•
(11) CH3 + (CH3)2SO  CH4 + CH2(CH3)SO• k11 = 100 mol-1dm3s-1 [46]
•
(12) CH3 + RH  CH4 + R• k12 = depending on RH (Where RH is an aliphatic
compound.)[47]
(13) 2CH3•  C2H6 k13 = 1.6x109 mol-1dm3s-1 [47]
On the other hand, FeIVaq reacts with (CH3)2SO to form (CH3)2SO2[12]. Therefore, the
yields of methane, ethane, and (CH3)2SO2 under different conditions were determined.
The Fenton reaction in the presence of 0.10 mM FeII(citrate), where over 90%
of the reaction proceeds via the citrate complex, Fig. 1, no methane and ethane
formation takes place, Fig. 3. However, when higher citrate concentrations are used,
1.0 mM, methane, and ethane formation are observed. It is assumed that the citrate
─
─ •
─
─ •
radical, (─ OC)CH2C(OH)(CO2 )CH(CO2 ) /(─ OC)-CH2C(O)(CO2 )CH2(CO2 ) [48]
2
2
reacts with (CH3)2SO to yield methyl radicals that form methane and some ethane.
8

Journal Pre-proof
Fig. 3 Methane (tr = 3.1) and ethane (tr = 4.5) determination by GC in (CH3)2SO, 25 mM,
containing solutions after Fenton reaction under different conditions. The initial
concentrations were [FeII] = 0.020 mM, [H2O2] = 0.39 mM in all of the samples. The pH was
2.2 in the acidic solution, and 7.2 in the neutral solutions.
During the Fenton reaction of FeII with H2O2 in neutral pH (pH 7.2), no methane or
ethane formation was observed, Fig. 4. The presence of bicarbonate does not enhance
Fig. 4 Methane (tr = 3.1) and ethane (tr = 4.5) determination by GC in (CH3)2SO, 25 mM,
containing solutions after Fenton reaction under different conditions. The initial
concentrations were [FeII] = 0.020 mM, [H2O2] = 0.39 mM in all of the samples. The pH was
2.2 in the acidic solution, and 7.2 in the neutral solutions.
9

Journal Pre-proof
the methane and ethane formation in the Fenton-like reaction of the FeII(citrate) complexes,
Fig. 4. These experiments point out that there is no OH• radical formation during the Fenton
reaction of FeII(citrate) complexes and in the presence of bicarbonate in these reactions.
However, it must be checked whether the active intermediate in the presence of bicarbonate
is the CO3•─ anion radical.
3.4. Yields of methane and ethane in γ irradiated solutions
Two experiments to check the nature of the reactive intermediate were performed: 1.
60
The yields of methane and ethane gas are measured after Co γ irradiation of samples
in the presence of (CH3)2SO and citrate. 2. The final organic products after the Fenton
reaction under different conditions were determined by 1H NMR analysis.
N2O saturated solutions containing either 1.0 M citrate or 0.0050 M (CH3)2SO
60
or 1.0 M Na2CO3 or mixtures of these compounds were irradiated in the Co γ source
to a dose of 1.0 kGy (This dose produces ca. 6x10-4 M of radicals in the sample). The
yields of methane and ethane obtained in these experiments are summed up in Fig. 5.
Fig. 5 Methane and ethane determination by GC of (CH3)2SO, 0.0050 M, citrate, 1.0 M
and/or Na2CO3, 1.0 M containing N2O saturated solutions after irradiation by a 60Co γ source,
total dose 1.0 kGy. The pH was not adjusted.
In order to analyze the results presented in Fig. 5 one has to note that when N2O
neutral solutions are irradiated the only reactive radical formed is the OH• radical[43].
10

Journal Pre-proof
The OH• radicals thus formed react with the solutes present via reactions (9), (10),
(14), and (15):
(14) citrate + OH• 
─
─ •
─
─ •
(─ OC)CH2C(OH)(CO2 )CH(CO2 ) /(─ OC)CH2C(O)(CO2 )CH2(CO2 ) + H2O
2
2
k14 = 5.0x107 mol-1dm3s-1 [49]
k15 = 3.0x108 mol-1dm3s-1 [47]
(15) CO32─ + OH•  CO3•─ + H2O
Thus, at the concentrations used in these experiments k9[(CH3)2SO] = 3.5x107 s-
2-
1, k14[(citrate] = 5.0x107 s-1 and k15[(CO3 ] = 3.0x108 s-1. When citrate solutions are
irradiated, no methane and ethane are formed as expected. The yields of methane and
ethane in the (CH3)2SO irradiated solution are as expected according to reactions (11)
& (13). The total yield of methyl radicals ([CH4] + 2[C2H6]) in the solutions
containing both (CH3)2SO and citrate is ~19 % of that observed in solutions containing
only (CH3)2SO. This value is considerably smaller than the yield of methyl radicals
calculated via 100k9[(CH3)2SO]/(k9[(CH3)2SO] + k14[citrate]) = 41 %. The decrease in
the yield of methyl radicals vs. the calculated one is attributed to reactions (16) and
(17).
•
─
(16) CH3 + (─ OC)CH2C(OH)(CO2 )CH(CO2─)• 
2
─
─ •
(─ OC)CH2C(OH)(CO2 )CH(CH3)(CO2 )
2
•
─ •
─ •
(17) CH3 + (─ OC)CH2C(O)(CO2)CH2(CO2 ) (─ OC)CH2C(OCH3)(CO2)CH2(CO2 )
2
2
The contribution of the latter reactions inhibits the verification of the suggestion that the
─
─ •
─
─ •
radicals (─ OC)CH2C(OH)(CO2 )CH(CO2 ) /(─ OC)CH2C(O)(CO2 )CH2(CO2 ) react with
2
2
(CH3)2SO to form methyl radicals, as suggested above. The yield of methyl radicals, ([CH4] +
2[C2H6]), in the solutions containing all three components is ~14.5 % of the value obtained in
the (CH3)2SO solutions. This value is considerably larger than that calculated via
100k9[(CH3)2SO]/(k9[(CH3)2SO] + k14([citrate] + k15[CO32─]) = 9 %. This is attributed to the
reaction (18) that was reported earlier[44].
•
2─
(18) CO3•─ + (CH3)2SO  CH3 + CH3SOO─ + CO3 [44]
11

Journal Pre-proof
The same conclusion is supported by the yield of methyl radicals in solutions containing
(CH3)2SO and carbonate that is ~ 60 % larger than that calculates assuming that all the
methyl radicals are formed via reaction (9).
1
3.5. Analysis of the products of oxidation of (CH3)2SO via the Fenton reaction by H
NMR
In order to get more information on the oxidation products in these Fenton
reactions, the oxidation products of (CH3)2SO were determined, the results are
summed up in Fig. 6. In the absence of citrate (CH3)2SO2 is the main product, as
expected[12].
1
Fig. 6 H NMR spectra after the Fenton reaction in the presence of (CH3)2SO, 25 mM). The
─
experimental conditions were the following: [citrate] = 0.10 mM, [HCO3 ] = 0 or 4 mM,
[FeII] = 0.020 mM, [H2O2] = 0.39 mM, the pH was 6.4 in the solution without citrate and 7.2
in the solutions with citrate.
When the Fenton reaction is performed in the presence of 0.10 mM citrate, some
(CH3)SOOH and a low yield of beta-ketoglutaric acid (1,3-acetone-dicarboxylic acid)
are obtained. But (CH3)2SO2 is not formed. Since it is not known that FeIV(citrate)
always oxidizes (CH3)2SO to (CH3)2SO2 we can not state that FeIV(citrate) is not
formed in the reaction. Maybe FeIV(citrate) is formed but oxidizes the citrate ligand to
beta-ketoglutaric acid with a low yield. However, as proposed above the citrate
─
─ •
─
─ •
radicals,
(─ OC)CH2C(OH)(CO2 )CH(CO2 ) /(─ OC)CH2C(O)(CO2 )CH2(CO2 ) ,
2
2
probably react with (CH3)2SO to form methyl radicals and (CH3)SOOH. When 4.0
12

Journal Pre-proof
─
mM HCO3 is present, the products change again, and the only product observed is
beta-ketoglutaric acid in considerably higher yield than in the absence of bicarbonate.
As the addition of bicarbonate does not shift anodically the potential of the reactive
intermediate formed, this result suggests that the bicarbonate changes the mechanism
probably by forming a per-carbonate as the active intermediate.
In order to verify that the formation of the beta-ketoglutaric acid is due only to
the presence of bicarbonate and that the (CH3)2SO is not involved, the experiment is
repeated in the absence of (CH3)2SO. The results summed up in Fig. 7 clearly points
out that indeed the formation of the beta-ketoglutaric acid is due to the presence of
bicarbonate.
1
Fig. 7 H NMR spectra after Fenton reaction. The experimental conditions were the
─
following: [citrate] = 0.10 mM, [HCO3 ] = 0 or 4 mM, [FeII] = 0.020 mM, [H2O2] = 0.39
mM, the pH of the solutions was 7.2.
4. Conclusions
The results obtained in this study point out that the presence of low
─
concentrations of HCO3 , i.e., physiological conditions, affect the mechanism and
kinetics of the Fenton reaction of FeII(citrate) dramatically. Under these conditions
clearly, OH• radicals are not formed. OH•, radicals are probably not formed also in this
Fenton reaction in the absence of bicarbonate. These results reiterate our
conclusion[14] that the source of oxidative stress in biological systems is not hydroxyl
radicals.
The results obtained in this study do not enable a definite conclusion
concerning the nature of the active oxidizing species formed in this system. As no
13

Journal Pre-proof
hydroxyl radicals are formed in the absence of bicarbonate, it is reasonable to suggest
that the Fenton reaction in the presence of citrate proceeds via:
(19) FeII(citrate)─ + H2O2

FeIV(citrate)+aq + 2OH─
Whether one should write FeIV(citrate)+ or (citrate)FeIV=O─ is not clear at present.
aq
aq
In the presence of bicarbonate, the reaction rates increase dramatically; this can, in
principle, be due to the following:
1. Reaction 19 is not an outer sphere process [11] and should be written as:
(19) FeII(citrate)─ + H2O2
and in the presence of bicarbonate:
(20) FeII(citrate)(HCO3)2─ + H2O2

(citrate)FeII(H2O2)─  FeIV(citrate)+aq + 2OH─

(citrate)FeII(H2O2)(HCO3)2─
FeIV(citrate)(HCO3)aq + 2OH─

and as the bicarbonate/carbonate ligands lower the redox potential of the central
cation[50] it accelerates the reaction rate. The mechanism of decomposition of the
FeIV(citrate)(HCO3)aq thus formed is not clear at present: It can decompose via the
.-
formation of CO3 or via oxidation of the citrate ligand.
2. Alternatively, the (citrate)FeII(H2O2)(HCO3)2─ formed as an intermediate in
reaction (21) can isomerize via:
(21) (citrate)FeII(H2O2)(HCO3)2─

(citrate)FeII(CO4)3─ + H3O+
in analogy to many earlier reports, see, for example, reference [19]. The
(citrate)FeII(CO4)3─ thus formed is expected to decompose via: [19]
(22) (citrate)FeII(CO4)3─ + H2O

FeIII(citrate) + CO3•─ + 2OH─
The latter mechanism seems to fit better the experimental results, although the
other mechanisms cannot be ruled out.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We are indebted to the Pazy Foundation for a grant that enabled this study.
Notes and references
[1] H.J.H. Fenton, LXXIII.—Oxidation of tartaric acid in presence of iron, J. Chem. Soc.,
14

Journal Pre-proof
Trans. 65 (1894) 899–910. doi:10.1039/CT8946500899.
[2] A. Friedman, J. Galazka-Friedman, Mössbauer spectroscopy and the understanding of
the role of iron in neurodegeneration, Hyperfine Interact. 238 (2017) 62.
doi:10.1007/s10751-017-1433-7.
[3] H. Pi, J.D. Helmann, Ferrous iron efflux systems in bacteria, Metallomics. 9 (2017)
840–851. doi:10.1039/C7MT00112F.
[4] P. Poprac, K. Jomova, M. Simunkova, V. Kollar, C.J. Rhodes, M. Valko, Targeting
Free Radicals in Oxidative Stress-Related Human Diseases, Trends Pharmacol. Sci. 38
(2017) 592–607. doi:10.1016/j.tips.2017.04.005.
[5] L. Clarizia, D. Russo, I. Di Somma, R. Marotta, R. Andreozzi, Homogeneous photo-
Fenton processes at near neutral pH: A review, Appl. Catal. B Environ. 209 (2017)
358–371. doi:10.1016/j.apcatb.2017.03.011.
[6] A. Mirzaei, Z. Chen, F. Haghighat, L. Yerushalmi, Removal of pharmaceuticals from
water by homo/heterogonous Fenton-type processes – A review, Chemosphere. 174
(2017) 665–688. doi:10.1016/j.chemosphere.2017.02.019.
[7] G. Zhen, X. Lu, H. Kato, Y. Zhao, Y.-Y. Li, Overview of pretreatment strategies for
enhancing sewage sludge disintegration and subsequent anaerobic digestion: Current
advances, full-scale application and future perspectives, Renew. Sustain. Energy Rev.
69 (2017) 559–577. doi:10.1016/j.rser.2016.11.187.
[8] F. Haber, J. Weiss, Über die Katalyse des Hydroperoxydes, Naturwissenschaften. 20
(1932) 948–950. doi:10.1007/BF01504715.
[9] W.C. Bray, M.H. Gorin, The Interaction of Ozone and Hydrogen Peroxide in Aqueous
Solution, J. Am. Chem. Soc. 60 (1938) 82–87. doi:10.1021/ja01268a027.
[10] M. Masarwa, H. Cohen, D. Meyerstein, D.L. Hickman, A. Bakac, J.H. Espenson,
Reactions of low-valent transition-metal complexes with hydrogen peroxide. Are they
“Fenton-like” or not? 1. The case of Cu+aq and Cr2+aq, J. Am. Chem. Soc. 110 (1988)
4293–4297. doi:10.1021/ja00221a031.
[11] S. Goldstein, D. Meyerstein, G. Czapski, The Fenton reagents, Free Radic. Biol. Med.
15 (1993) 435–445. doi:10.1016/0891-5849(93)90043-T.
[12] H. Bataineh, O. Pestovsky, A. Bakac, pH-induced mechanistic changeover from
hydroxyl radicals to iron(iv) in the Fenton reaction, Chem. Sci. 3 (2012) 1594.
doi:10.1039/c2sc20099f.
[13] S. Rachmilovich-Calis, A. Masarwa, N. Meyerstein, D. Meyerstein, R. van Eldik, New
Mechanistic Aspects of the Fenton Reaction, Chem. - A Eur. J. 15 (2009) 8303–8309.
15

Journal Pre-proof
doi:10.1002/chem.200802572.
[14] E. Illés, A. Mizrahi, V. Marks, D. Meyerstein, Carbonate-radical-anions, and not
hydroxyl radicals, are the products of the Fenton reaction in neutral solutions
containing bicarbonate, Free Radic. Biol. Med. 131 (2019) 1–6.
doi:10.1016/j.freeradbiomed.2018.11.015.
[15] M.L. Kuznetsov, Y.N. Kozlov, D. Mandelli, A.J.L. Pombeiro, G.B. Shul’pin,
Mechanism of Al3+ -Catalyzed Oxidations of Hydrocarbons: Dramatic Activation of
H2O2 toward O−O Homolysis in Complex [Al(H2O)4(OOH)(H2O2)]2+ Explains the
Formation of HO• Radicals, Inorg. Chem. 50 (2011) 3996–4005.
doi:10.1021/ic102476x.
[16] A. Burg, I. Shusterman, H. Kornweitz, D. Meyerstein, Three H2O2 molecules are
involved in the “Fenton-like” reaction between Co(H2O)62+ and H2O2, Dalton Trans.
43 (2014) 9111. doi:10.1039/c4dt00401a.
[17] I. Popivker, I. Zilbermann, E. Maimon, H. Cohen, D. Meyerstein, The “Fenton like”
reaction of MoO43− involves two H2O2 molecules, Dalton Trans. 42 (2013) 16666.
doi:10.1039/c3dt52333k.
[18] H. Kornweitz, A. Burg, D. Meyerstein, Plausible Mechanisms of the Fenton-Like
Reactions, M = Fe(II) and Co(II), in the Presence of 2– Substrates: Are OH• Radicals
Formed in the Process?, J. Phys. Chem. A. 119 (2015) 4200–4206.
doi:10.1021/jp512826f.
[19] A. Burg, D. Shamir, I. Shusterman, H. Kornweitz, D. Meyerstein, The role of
carbonate as a catalyst of Fenton-like reactions in AOP processes: CO3•− as the active
intermediate, Chem. Commun. 50 (2014) 13096–13099. doi:10.1039/C4CC05852F.
[20] R.W. Evans, R. Rafique, A. Zarea, C. Rapisarda, R. Cammack, P.J. Evans, J.B. Porter,
R.C. Hider, Nature of non-transferrin-bound iron: studies on iron citrate complexes
and thalassemic sera, JBIC J. Biol. Inorg. Chem. 13 (2007) 57–74.
doi:10.1007/s00775-007-0297-8.
[21] M.A. Farooq, T.M. Pracheil, Z. Dong, F. Xiao, Z. Liu, Mitochondrial DNA Instability
in Cells Lacking Aconitase Correlates with Iron Citrate Toxicity, Oxid. Med. Cell.
Longev. 2013 (2013) 1–10. doi:10.1155/2013/493536.
[22] P.M. May, P.W. Linder, D.R. Williams, Computer simulation of metal-ion equilibria
in biofluids: models for the low-molecular-weight complex distribution of calcium(II),
magnesium(II), manganese(II), iron(III), copper(II), zinc(II), and lead(II) ions in
human blood plasma, J. Chem. Soc. Dalt. Trans. 2 (1977) 588.
16

Journal Pre-proof
doi:10.1039/dt9770000588.
[23] M. Grootveld, J.D. Bell, B. Halliwell, O.I. Aruoma, A. Bomford, P.J. Sadler, Non-
transferrin-bound iron in plasma or serum from patients with idiopathic
hemochromatosis. Characterization by high performance liquid chromatography and
nuclear magnetic resonance spectroscopy., J. Biol. Chem. 264 (1989) 4417–22.
http://www.jbc.org/content/264/8/4417.long.
[24] M.D. Engelmann, R.T. Bobier, T. Hiatt, I.F. Cheng, Variability of the Fenton reaction
characteristics of the EDTA, DTPA, and citrate complexes of iron, BioMetals. 16
(2003) 519–527. doi:10.1023/A:1023480617038.
[25] F.I. Adam, P.L. Bounds, R. Kissner, W.H. Koppenol, Redox Properties and Activity of
Iron–Citrate Complexes: Evidence for Redox Cycling, Chem. Res. Toxicol. 28 (2015)
604–614. doi:10.1021/tx500377b.
[26] F. Shi, P. Zhang, Y. Mao, C. Wang, M. Zheng, Z. Zhao, The nitroxide Tempo inhibits
hydroxyl radical production from the Fenton-like reaction of iron(II)-citrate with
hydrogen peroxide, Biochem. Biophys. Res. Commun. 483 (2017) 159–164.
doi:10.1016/j.bbrc.2016.12.174.
[27] W.H. Koppenol, R.H. Hider, Iron and redox cycling. Do’s and don’ts, Free Radic.
Biol. Med. 133 (2019) 3–10. doi:10.1016/j.freeradbiomed.2018.09.022.
[28] B. Costa e Silva, J.A. de Lima Perini, R.F.P. Nogueira, Influence of
dihydroxybenzenes on paracetamol and ciprofloxacin degradation and iron(III)
reduction in Fenton processes, Environ. Sci. Pollut. Res. 24 (2017) 6157–6164.
doi:10.1007/s11356-016-6402-1.
[29] M. Popescu, E. Rosales, C. Sandu, J. Meijide, M. Pazos, G. Lazar, M.A. Sanromán,
Soil flushing and simultaneous degradation of organic pollutants in soils by
electrokinetic-Fenton treatment, Process Saf. Environ. Prot. 108 (2017) 99–107.
doi:10.1016/j.psep.2016.03.012.
[30] C. Kantar, O. Oral, O. Urken, N.A. Oz, Role of complexing agents on oxidative
degradation of chlorophenolic compounds by pyrite-Fenton process: Batch and column
experiments, J. Hazard. Mater. 373 (2019) 160–167.
doi:10.1016/j.jhazmat.2019.03.065.
[31] C.J. Miller, A.L. Rose, T.D. Waite, Importance of Iron Complexation for Fenton-
Mediated Hydroxyl Radical Production at Circumneutral pH, Front. Mar. Sci. 3 (2016)
1–13. doi:10.3389/fmars.2016.00134.
[32] J.D. Rush, Z. Maskos, W.H. Koppenol, Reactions of iron(II) nucleotide complexes
17

Journal Pre-proof
with hydrogen peroxide, FEBS Lett. 261 (1990) 121–123. doi:10.1016/0014-
5793(90)80651-X.
[33] N.A. Kim, D.G. Lim, J.Y. Lim, K.H. Kim, S.H. Jeong, Fundamental analysis of
recombinant human epidermal growth factor in solution with biophysical methods,
Drug Dev. Ind. Pharm. 41 (2015) 300–306. doi:10.3109/03639045.2013.859152.
[34] O. Pestovsky, S. Stoian, E.L. Bominaar, X. Shan, E. Münck, L. Que, A. Bakac,
Aqueous FeIV=O: Spectroscopic Identification and Oxo-Group Exchange, Angew.
Chemie Int. Ed. 44 (2005) 6871–6874. doi:10.1002/anie.200502686.
[35] K.D. Weaver, M. Gabriꢀeviꢁ, D.S. Anderson, P. Adhikari, T.A. Mietzner, A.L.
Crumbliss, Role of Citrate and Phosphate Anions in the Mechanism of Iron(III)
Sequestration by Ferric Binding Protein: Kinetic Studies of the Formation of the
Holoprotein of Wild-Type FbpA and Its Engineered Mutants, Biochemistry. 49 (2010)
6021–6032. doi:10.1021/bi902231c.
[36] R.E. Hamm, C.M. Shull, D.M. Grant, Citrate Complexes with Iron(II) and Iron(III) 1,
J. Am. Chem. Soc. 76 (1954) 2111–2114. doi:10.1021/ja01637a021.
[37] C.F. Timberlake, 975. Iron–malate and iron–citrate complexes, J. Chem. Soc. (1964)
5078–5085. doi:10.1039/JR9640005078.
[38] T.B. Field, J.L. McCourt, W.A.E. McBryde, Composition and Stability of Iron and
Copper Citrate Complexes in Aqueous Solution, Can. J. Chem. 52 (1974) 3119–3124.
doi:10.1139/v74-458.
[39] P. Amico, P.G. Daniele, V. Cucinotta, E. Rizzarelli, S. Sammartano, Equilibrium study
of iron(II) and manganese(II) complexes with citrate ion in aueous solution: Relevance
to coordination of citrate to the active site of aconitase and to gastrointestinal
absorption of some essential metal ions, Inorganica Chim. Acta. 36 (1979) 1–7.
doi:10.1016/S0020-1693(00)89363-9.
[40] M. Mamczák, V.V. Palchevskii, Complexing properties of bivalent and trivalent iron
in the system citric acid — iron ( II ) Perchlorate — iron ( III ) Perchlorate — water,
Chem. Zvesti. 36 (1982) 333–343.
https://www.chempap.org/file_access.php?file=363a333.pdf.
[41] J.R. Duffield, J.R. Johns, F. Marsicano, D.R. Williams, Chemical speciation modelling
and thermodynamic database compilation—III. Modelling solid formation and
dissolution, Polyhedron. 10 (1991) 1121–1129. doi:10.1016/S0277-5387(00)81379-0.
[42] L.-C. Königsberger, E. Königsberger, P.M. May, G.T. Hefter, Complexation of
iron(III) and iron(II) by citrate. Implications for iron speciation in blood plasma, J.
18

Journal Pre-proof
Inorg. Biochem. 78 (2000) 175–184. doi:10.1016/S0162-0134(99)00222-6.
[43] D. Veltwisch, E. Janata, K.-D. Asmus, Primary processes in the reaction of OH·-
radicals with sulphoxides, J. Chem. Soc., Perkin Trans. 2. (1980) 146–153.
doi:10.1039/P29800000146.
[44] R. Herscu‐ Kluska, A. Masarwa, M. Saphier, H. Cohen, D. Meyerstein, Mechanism of
the Reaction of Radicals with Peroxides and Dimethyl Sulfoxide in Aqueous Solution,
Chem. - A Eur. J. 14 (2008) 5880–5889. doi:10.1002/chem.200800218.
[45] A. Lerner, M.Sc. Thesis, Ben-Gurion University, 2018.
[46] I. Rusonik, H. Polat, H. Cohen, D. Meyerstein, Reaction of Methyl Radicals with
Metal Powders Immersed in Aqueous Solutions, Eur. J. Inorg. Chem. 2003 (2003)
4227–4233. doi:10.1002/ejic.200300403.
[47] NDRL/NIST Solution Kinetics Database on the Web, (n.d.).
https://kinetics.nist.gov/solution/.
[48] S. Al Gharib, J.-L. Marignier, A.K. El Omar, A. Naja, S. Le Caer, M. Mostafavi, J.
Belloni, Key Role of the Oxidized Citrate-Free Radical in the Nucleation Mechanism
of the Metal Nanoparticle Turkevich Synthesis, J. Phys. Chem. C. 123 (2019) 22624–
22633. doi:10.1021/acs.jpcc.9b06090.
[49] S. Al Gharib, J.-L. Marignier, A.K. El Omar, A. Naja, S. Le Caer, M. Mostafavi, J.
Belloni, Key Role of the Oxidized Citrate-Free Radical in the Nucleation Mechanism
of the Metal Nanoparticle Turkevich Synthesis, J. Phys. Chem. C. 123 (2019) 22624–
22633. doi:10.1021/acs.jpcc.9b06090.
[50] S.G. Patra, E. Illés, A. Mizrahi, D. Meyerstein, Cobalt Carbonate as an Electrocatalyst
for Water Oxidation, Chem. – A Eur. J. (2019) chem.201904051.
doi:10.1002/chem.201904051.
19

Journal Pre-proof
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
20

Journal Pre-proof
Graphical abstract
Synopsis
The kinetics and mechanism of the Fenton reaction of FeII(citrate) are significantly affected
by bicarbonate. The products of oxidation of (CH3)2SO by the Fenton reaction are analyzed.
Biological oxidative stress is caused by FeIV=O, citrate radical and/or carbonate radical anion
•─
(CO3 ) but not by the hydroxyl radical (OH•).
21

Journal Pre-proof
Highlights
 The Fenton reaction is crucial in causing oxidative stress under biological conditions.
 The Fenton reaction of FeII(citrate) in the presence of bicarbonate is studied.
 Bicarbonate affects the mechanism and kinetics of the Fenton reaction dramatically.
 Hydroxyl radicals (OH•) are not formed in the FeII(citrate) Fenton reaction.
 OH• radicals do not initiate oxidative stress in biological systems.
22